Differentiation of Xylella fastidiosa Strains via Multilocus Sequence Analysis of Environmentally Mediated Genes (MLSA-E)

PubMed Central

Parker, Jennifer K.; Havird, Justin C.

2012-01-01

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops. PMID:22194287

Differentiation of Xylella fastidiosa strains via multilocus sequence analysis of environmentally mediated genes (MLSA-E).

PubMed

Parker, Jennifer K; Havird, Justin C; De La Fuente, Leonardo

2012-03-01

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops.

Pathogenic Leptospira Species in Insectivorous Bats, China, 2015.

PubMed

Han, Hui-Ju; Wen, Hong-Ling; Liu, Jian-Wei; Qin, Xiang-Rong; Zhao, Min; Wang, Li-Jun; Luo, Li-Mei; Zhou, Chuan-Min; Zhu, Ye-Lei; Qi, Rui; Li, Wen-Qian; Yu, Hao; Yu, Xue-Jie

2018-06-01

PCR amplification of the rrs2 gene indicated that 50% (62/124) of insectivorous bats from eastern China were infected with Leptospira borgpetersenii, L. kirschneri, and several potentially new Leptospira species. Multilocus sequence typing defined 3 novel sequence types in L. kirschneri, suggesting that bats are major carriers of Leptospira.

Development of Multilocus Sequence Typing (MLST) for Mycoplasma synoviae.

PubMed

El-Gazzar, Mohamed; Ghanem, Mostafa; McDonald, Kristina; Ferguson-Noel, Naola; Raviv, Ziv; Slemons, Richard D

2017-03-01

Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification, and facilitating prevention and control as well as eradication efforts. Currently, a segment of the variable lipoprotein hemagglutinin A (vlhA) gene (420 bp) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, seven genes were selected for the MLST assay. An internal segment (450-711 bp) from each of the seven genes was successfully amplified and sequenced from 58 different MS strains and field isolates (n = 30) or positive clinical samples (n = 28). The collective sequence of all seven gene segments (3960 bp total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay and, coincidentally, all the samples were typed into 30 sequence types using the vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiologic information than was the tree generated by the vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control, and eradication efforts.

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

PubMed Central

2009-01-01

Background Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. Results We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. Conclusion Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population. PMID:19573249

16S rRNA Gene Sequencing, Multilocus Sequence Analysis, and Mass Spectrometry Identification of the Proposed New Species “Clostridium neonatale”

PubMed Central

Bouvet, Philippe; Ferraris, Laurent; Dauphin, Brunhilde; Popoff, Michel-Robert; Butel, Marie Jose

2014-01-01

In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, “Clostridium neonatale.” To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies. PMID:25232167

Diversity of the Cronobacter Genus as Revealed by Multilocus Sequence Typing

PubMed Central

Joseph, S.; Sonbol, H.; Hariri, S.; Desai, P.; McClelland, M.

2012-01-01

Cronobacter (previously known as Enterobacter sakazakii) is a diverse bacterial genus consisting of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. universalis, C. muytjensii, C. dublinensis, and C. condimenti. In this study, we have used a multilocus sequence typing (MLST) approach employing the alleles of 7 genes (atpD, fusA, glnS, gltB, gyrB, infB, and ppsA; total length, 3,036 bp) to investigate the phylogenetic relationship of 325 Cronobacter species isolates. Strains were chosen on the basis of their species, geographic and temporal distribution, source, and clinical outcome. The earliest strain was isolated from milk powder in 1950, and the earliest clinical strain was isolated in 1953. The existence of seven species was supported by MLST. Intraspecific variation ranged from low diversity in C. sakazakii to extensive diversity within some species, such as C. muytjensii and C. dublinensis, including evidence of gene conversion between species. The predominant species from clinical sources was found to be C. sakazakii. C. sakazakii sequence type 4 (ST4) was the predominant sequence type of cerebral spinal fluid isolates from cases of meningitis. PMID:22785185

Population structure of Lactobacillus helveticus isolates from naturally fermented dairy products based on multilocus sequence typing.

PubMed

Sun, Zhihong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Yu, Jie; Bilige, Menghe; Zhang, Heping; Chen, Yongfu

2015-05-01

Lactobacillus helveticus is an economically important lactic acid bacterium used in industrial dairy fermentation. In the present study, the population structure of 245 isolates of L. helveticus from different naturally fermented dairy products in China and Mongolia were investigated using an multilocus sequence typing scheme with 11 housekeeping genes. A total of 108 sequence types were detected, which formed 8 clonal complexes and 27 singletons. Results from Structure, SplitsTree, and ClonalFrame software analyses demonstrated the presence of 3 subpopulations in the L. helveticus isolates used in our study, namely koumiss, kurut-tarag, and panmictic lineages. Most L. helveticus isolates from particular ecological origins had specific population structures. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Taxonomic evaluation of species in the Streptomyces hirsutus clade using multi-locus sequence analysis and proposals to reclassify several species in this clade

USDA-ARS?s Scientific Manuscript database

Previous phylogenetic analyses of species of Streptomyces based on 16S rRNA gene sequences resulted in a statistically well-supported clade (100% bootstrap value) containing 8 species that exhibited very similar gross morphology in producing open looped (Retinaculum-Apertum) to spiral (Spira) chains...

Molecular Epidemiologic Analysis of Enterococcus faecalis Isolates in Cuba by Multilocus Sequence Typing

PubMed Central

Kobayashi, Nobumichi; Nagashima, Shigeo

2009-01-01

We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6′)-aph(2″), aph(3′), ant(6), ant(3″)(9), aph(2″)-Id, aph(2″)-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6′)-Ie-aph(2″)-Ia, aph(3′), ant(6), ant(3″)(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits. PMID:19857135

Multilocus Sequence Typing Analysis of Staphylococcus lugdunensis Implies a Clonal Population Structure

PubMed Central

Chassain, Benoît; Lemée, Ludovic; Didi, Jennifer; Thiberge, Jean-Michel; Brisse, Sylvain; Pons, Jean-Louis

2012-01-01

Staphylococcus lugdunensis is recognized as one of the major pathogenic species within the genus Staphylococcus, even though it belongs to the coagulase-negative group. A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 87 S. lugdunensis isolates from various clinical and geographic sources by DNA sequence analysis of seven housekeeping genes (aroE, dat, ddl, gmk, ldh, recA, and yqiL). The number of alleles ranged from four (gmk and ldh) to nine (yqiL). Allelic profiles allowed the definition of 20 different sequence types (STs) and five clonal complexes. The 20 STs lacked correlation with geographic source. Isolates recovered from hematogenic infections (blood or osteoarticular isolates) or from skin and soft tissue infections did not cluster in separate lineages. Penicillin-resistant isolates clustered mainly in one clonal complex, unlike glycopeptide-tolerant isolates, which did not constitute a distinct subpopulation within S. lugdunensis. Phylogenies from the sequences of the seven individual housekeeping genes were congruent, indicating a predominantly mutational evolution of these genes. Quantitative analysis of the linkages between alleles from the seven loci revealed a significant linkage disequilibrium, thus confirming a clonal population structure for S. lugdunensis. This first MLST scheme for S. lugdunensis provides a new tool for investigating the macroepidemiology and phylogeny of this unusually virulent coagulase-negative Staphylococcus. PMID:22785196

Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation.

PubMed

Mulet, M; Gomila, M; Ramírez, A; Cardew, S; Moore, E R B; Lalucat, J; García-Valdés, E

2017-02-01

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes.

PubMed

Nakano, V; Ignacio, A; Llanco, L; Bueris, V; Sircili, M P; Avila-Campos, M J

2017-04-01

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic Characterization of Atypical Enteropathogenic Escherichia coli Isolates from Ewes' Milk, Sheep Farm Environments, and Humans by Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

PubMed Central

Otero, Verónica; Rodríguez-Calleja, José-María; Otero, Andrés; García-López, María-Luisa

2013-01-01

A collection of 81 isolates of enteropathogenic Escherichia coli (EPEC) was obtained from samples of bulk tank sheep milk (62 isolates), ovine feces (4 isolates), sheep farm environment (water, 4 isolates; air, 1 isolate), and human stool samples (9 isolates). The strains were considered atypical EPEC organisms, carrying the eae gene without harboring the pEAF plasmid. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 19 sequence types (ST) were detected, with none of them having been previously reported for atypical EPEC. The most frequent ST included 41 strains isolated from milk and human stool samples. Genetic typing by pulsed-field gel electrophoresis (PFGE) resulted in 57 patterns which grouped in 24 clusters. Comparison of strains isolated from the different samples showed phylogenetic relationships between milk and human isolates and also between milk and water isolates. The results obtained show a possible risk for humans due to the presence of atypical EPEC in ewes' milk and suggest a transmission route for this emerging pathogen through contaminated water. PMID:23872571

Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

PubMed

Marco, Jorge D; Bhutto, Abdul M; Soomro, Farooq R; Baloch, Javed H; Barroso, Paola A; Kato, Hirotomo; Uezato, Hiroshi; Katakura, Ken; Korenaga, Masataka; Nonaka, Shigeo; Hashiguchi, Yoshihisa

2006-08-01

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Multilocus inference of species trees and DNA barcoding.

PubMed

Mallo, Diego; Posada, David

2016-09-05

The unprecedented amount of data resulting from next-generation sequencing has opened a new era in phylogenetic estimation. Although large datasets should, in theory, increase phylogenetic resolution, massive, multilocus datasets have uncovered a great deal of phylogenetic incongruence among different genomic regions, due both to stochastic error and to the action of different evolutionary process such as incomplete lineage sorting, gene duplication and loss and horizontal gene transfer. This incongruence violates one of the fundamental assumptions of the DNA barcoding approach, which assumes that gene history and species history are identical. In this review, we explain some of the most important challenges we will have to face to reconstruct the history of species, and the advantages and disadvantages of different strategies for the phylogenetic analysis of multilocus data. In particular, we describe the evolutionary events that can generate species tree-gene tree discordance, compare the most popular methods for species tree reconstruction, highlight the challenges we need to face when using them and discuss their potential utility in barcoding. Current barcoding methods sacrifice a great amount of statistical power by only considering one locus, and a transition to multilocus barcodes would not only improve current barcoding methods, but also facilitate an eventual transition to species-tree-based barcoding strategies, which could better accommodate scenarios where the barcode gap is too small or inexistent.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

STBase: one million species trees for comparative biology.

PubMed

McMahon, Michelle M; Deepak, Akshay; Fernández-Baca, David; Boss, Darren; Sanderson, Michael J

2015-01-01

Comprehensively sampled phylogenetic trees provide the most compelling foundations for strong inferences in comparative evolutionary biology. Mismatches are common, however, between the taxa for which comparative data are available and the taxa sampled by published phylogenetic analyses. Moreover, many published phylogenies are gene trees, which cannot always be adapted immediately for species level comparisons because of discordance, gene duplication, and other confounding biological processes. A new database, STBase, lets comparative biologists quickly retrieve species level phylogenetic hypotheses in response to a query list of species names. The database consists of 1 million single- and multi-locus data sets, each with a confidence set of 1000 putative species trees, computed from GenBank sequence data for 413,000 eukaryotic taxa. Two bodies of theoretical work are leveraged to aid in the assembly of multi-locus concatenated data sets for species tree construction. First, multiply labeled gene trees are pruned to conflict-free singly-labeled species-level trees that can be combined between loci. Second, impacts of missing data in multi-locus data sets are ameliorated by assembling only decisive data sets. Data sets overlapping with the user's query are ranked using a scheme that depends on user-provided weights for tree quality and for taxonomic overlap of the tree with the query. Retrieval times are independent of the size of the database, typically a few seconds. Tree quality is assessed by a real-time evaluation of bootstrap support on just the overlapping subtree. Associated sequence alignments, tree files and metadata can be downloaded for subsequent analysis. STBase provides a tool for comparative biologists interested in exploiting the most relevant sequence data available for the taxa of interest. It may also serve as a prototype for future species tree oriented databases and as a resource for assembly of larger species phylogenies from precomputed trees.

Class 1 integrons characterization and multilocus sequence typing of Salmonella spp. from swine production chains in Chiang Mai and Lamphun provinces, Thailand.

PubMed

Boonkhot, Phacharaporn; Tadee, Pakpoom; Yamsakul, Panuwat; Pocharoen, Chairoj; Chokesajjawatee, Nipa; Patchanee, Prapas

2015-05-01

Pigs and pork products are well known as an important source of Salmonella, one of the major zoonotic foodborne pathogens. The emergence and spread of antimicrobial resistance is becoming a major public health concern worldwide. Integrons are genetic elements known to have a role in the acquisition and expression of genes conferring antibiotic resistance. This study focuses on the prevalence of class 1 integrons-carrying Salmonella, the genetic diversity of strains of those organisms obtained from swine production chains in Chiang Mai and Lamphun provinces, Thailand, using multilocus sequence typing (MLST) and comparison of genetic diversity of sequence types of Salmonella from this study with pulsotypes identified in previous study. In 175 Salmonella strains, the overall prevalence of class 1 integrons-carrying-Salmonella was 14%. The gene cassettes array pattern "dfrA12-orfF-aadA2" was the most frequently observed. Most of the antimicrobial resistance identified was not associated with related gene cassettes harbored by Salmonella. Six sequence types were generated from 30 randomly selected strains detected by MLST. Salmonella at the human-animal-environment interface was confirmed. Linkages both in the farm to slaughterhouse contamination route and the horizontal transmission of resistance genes were demonstrated. To reduce this problem, the use of antimicrobials in livestock should be controlled by veterinarians. Education and training of food handlers as well as promotion of safe methods of food consumption are important avenues for helping prevent foodborne illness.

Streptococcus mutans clonal variation revealed by multilocus sequence typing.

PubMed

Nakano, Kazuhiko; Lapirattanakul, Jinthana; Nomura, Ryota; Nemoto, Hirotoshi; Alaluusua, Satu; Grönroos, Lisa; Vaara, Martti; Hamada, Shigeyuki; Ooshima, Takashi; Nakagawa, Ichiro

2007-08-01

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.

Development of Mycoplasma synoviae (MS) core genome multilocus sequence typing (cgMLST) scheme.

PubMed

Ghanem, Mostafa; El-Gazzar, Mohamed

2018-05-01

Mycoplasma synoviae (MS) is a poultry pathogen with reported increased prevalence and virulence in recent years. MS strain identification is essential for prevention, control efforts and epidemiological outbreak investigations. Multiple multilocus based sequence typing schemes have been developed for MS, yet the resolution of these schemes could be limited for outbreak investigation. The cost of whole genome sequencing became close to that of sequencing the seven MLST targets; however, there is no standardized method for typing MS strains based on whole genome sequences. In this paper, we propose a core genome multilocus sequence typing (cgMLST) scheme as a standardized and reproducible method for typing MS based whole genome sequences. A diverse set of 25 MS whole genome sequences were used to identify 302 core genome genes as cgMLST targets (35.5% of MS genome) and 44 whole genome sequences of MS isolates from six countries in four continents were used for typing applying this scheme. cgMLST based phylogenetic trees displayed a high degree of agreement with core genome SNP based analysis and available epidemiological information. cgMLST allowed evaluation of two conventional MLST schemes of MS. The high discriminatory power of cgMLST allowed differentiation between samples of the same conventional MLST type. cgMLST represents a standardized, accurate, highly discriminatory, and reproducible method for differentiation between MS isolates. Like conventional MLST, it provides stable and expandable nomenclature, allowing for comparing and sharing the typing results between different laboratories worldwide. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Pneumocystis jirovecii multilocus gene sequencing: findings and implications.

PubMed

Matos, Olga; Esteves, Francisco

2010-08-01

Pneumocystis jirovecii pneumonia (PcP) remains a major cause of respiratory illness among immunocompromised patients, especially patients infected with HIV, but it has also been isolated from immunocompetent persons. This article discusses the application of multilocus genotyping analysis to the study of the genetic diversity of P. jirovecii and its epidemiological and clinical parameters, and the important concepts achieved to date with these approaches. The multilocus typing studies performed until now have shown that there is an important genetic diversity of stable and ubiquitous P. jirovecii genotypes; infection with P. jirovecii is not necessarily clonal, recombination between some P. jirovecii multilocus genotypes has been suggested. P. jirovecii-specific multilocus genotypes can be associated with severity of PcP. Patients infected with P. jirovecii, regardless of the form of infection they present with, are part of a common human reservoir for future infections. The CYB, DHFR, DHPS, mtLSU rRNA, SOD and the ITS loci are suitable genetic targets to be used in further epidemiological studies focused on the identification and characterization of P. jirovecii haplotypes correlated with drug resistance and PcP outcome.

Delineation of the Species Haemophilus influenzae by Phenotype, Multilocus Sequence Phylogeny, and Detection of Marker Genes▿ †

PubMed Central

Nørskov-Lauritsen, Niels; Overballe, Merete D.; Kilian, Mogens

2009-01-01

To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of Haemophilus influenzae by comparative analysis of H. influenzae reference strains with closely related bacteria including strains assigned to Haemophilus haemolyticus, cryptic genospecies biotype IV, and the never formally validated species “Haemophilus intermedius”. Multilocus sequence phylogeny based on six housekeeping genes separated a cluster encompassing the type and the reference strains of H. influenzae from 31 more distantly related strains. Comparison of 16S rRNA gene sequences supported this delineation but was obscured by a conspicuously high number of polymorphic sites in many of the strains that did not belong to the core group of H. influenzae strains. The division was corroborated by the differential presence of genes encoding H. influenzae adhesion and penetration protein, fuculokinase, and Cu,Zn-superoxide dismutase, whereas immunoglobulin A1 protease activity or the presence of the iga gene was of limited discriminatory value. The existence of porphyrin-synthesizing strains (“H. intermedius”) closely related to H. influenzae was confirmed. Several chromosomally encoded hemin biosynthesis genes were identified, and sequence analysis showed these genes to represent an ancestral genotype rather than recent transfers from, e.g., Haemophilus parainfluenzae. Strains previously assigned to H. haemolyticus formed several separate lineages within a distinct but deeply branching cluster, intermingled with strains of “H. intermedius” and cryptic genospecies biotype IV. Although H. influenzae is phenotypically more homogenous than some other Haemophilus species, the genetic diversity and multicluster structure of strains traditionally associated with H. influenzae make it difficult to define the natural borders of that species. PMID:19060144

Comprehensive Phylogenetic Analysis of Bovine Non-aureus Staphylococci Species Based on Whole-Genome Sequencing

PubMed Central

Naushad, Sohail; Barkema, Herman W.; Luby, Christopher; Condas, Larissa A. Z.; Nobrega, Diego B.; Carson, Domonique A.; De Buck, Jeroen

2016-01-01

Non-aureus staphylococci (NAS), a heterogeneous group of a large number of species and subspecies, are the most frequently isolated pathogens from intramammary infections in dairy cattle. Phylogenetic relationships among bovine NAS species are controversial and have mostly been determined based on single-gene trees. Herein, we analyzed phylogeny of bovine NAS species using whole-genome sequencing (WGS) of 441 distinct isolates. In addition, evolutionary relationships among bovine NAS were estimated from multilocus data of 16S rRNA, hsp60, rpoB, sodA, and tuf genes and sequences from these and numerous other single genes/proteins. All phylogenies were created with FastTree, Maximum-Likelihood, Maximum-Parsimony, and Neighbor-Joining methods. Regardless of methodology, WGS-trees clearly separated bovine NAS species into five monophyletic coherent clades. Furthermore, there were consistent interspecies relationships within clades in all WGS phylogenetic reconstructions. Except for the Maximum-Parsimony tree, multilocus data analysis similarly produced five clades. There were large variations in determining clades and interspecies relationships in single gene/protein trees, under different methods of tree constructions, highlighting limitations of using single genes for determining bovine NAS phylogeny. However, based on WGS data, we established a robust phylogeny of bovine NAS species, unaffected by method or model of evolutionary reconstructions. Therefore, it is now possible to determine associations between phylogeny and many biological traits, such as virulence, antimicrobial resistance, environmental niche, geographical distribution, and host specificity. PMID:28066335

Using multi-locus allelic sequence data to estimate genetic divergence among four Lilium (Liliaceae) cultivars

PubMed Central

Shahin, Arwa; Smulders, Marinus J. M.; van Tuyl, Jaap M.; Arens, Paul; Bakker, Freek T.

2014-01-01

Next Generation Sequencing (NGS) may enable estimating relationships among genotypes using allelic variation of multiple nuclear genes simultaneously. We explored the potential and caveats of this strategy in four genetically distant Lilium cultivars to estimate their genetic divergence from transcriptome sequences using three approaches: POFAD (Phylogeny of Organisms from Allelic Data, uses allelic information of sequence data), RAxML (Randomized Accelerated Maximum Likelihood, tree building based on concatenated consensus sequences) and Consensus Network (constructing a network summarizing among gene tree conflicts). Twenty six gene contigs were chosen based on the presence of orthologous sequences in all cultivars, seven of which also had an orthologous sequence in Tulipa, used as out-group. The three approaches generated the same topology. Although the resolution offered by these approaches is high, in this case there was no extra benefit in using allelic information. We conclude that these 26 genes can be widely applied to construct a species tree for the genus Lilium. PMID:25368628

First isolation of Actinobacillus genomospecies 2 in Japan.

PubMed

Murakami, Miyuki; Shimonishi, Yoshimasa; Hobo, Seiji; Niwa, Hidekazu; Ito, Hiroya

2016-05-03

We describe here the first isolation of Actinobacillus genomospecies 2 in Japan. The isolate was found in a septicemic foal and characterized by phenotypic and genetic analyses, with the latter consisting of 16S rDNA nucleotide sequence analysis plus multilocus sequence analysis using three housekeeping genes, recN, rpoA and thdF, that have been proposed for use as a genomic tool in place of DNA-DNA hybridization.

Taxonomic evaluation of Streptomyces albus and related species using multilocus sequence analysis and proposals to emend the description of Streptomyces albus and describe Streptomyces pathocidini sp. nov.

PubMed Central

Doroghazi, J. R.; Ju, K.-S.; Metcalf, W. W.

2014-01-01

In phylogenetic analyses of the genus Streptomyces using 16S rRNA gene sequences, Streptomyces albus subsp. albus NRRL B-1811T forms a cluster with five other species having identical or nearly identical 16S rRNA gene sequences. Moreover, the morphological and physiological characteristics of these other species, including Streptomyces almquistii NRRL B-1685T, Streptomyces flocculus NRRL B-2465T, Streptomyces gibsonii NRRL B-1335T and Streptomyces rangoonensis NRRL B-12378T are quite similar. This cluster is of particular taxonomic interest because Streptomyces albus is the type species of the genus Streptomyces. The related strains were subjected to multilocus sequence analysis (MLSA) utilizing partial sequences of the housekeeping genes atpD, gyrB, recA, rpoB and trpB and confirmation of previously reported phenotypic characteristics. The five strains formed a coherent cluster supported by a 100 % bootstrap value in phylogenetic trees generated from sequence alignments prepared by concatenating the sequences of the housekeeping genes, and identical tree topology was observed using various different tree-making algorithms. Moreover, all but one strain, S. flocculus NRRL B-2465T, exhibited identical sequences for all of the five housekeeping gene loci sequenced, but NRRL B-2465T still exhibited an MLSA evolutionary distance of 0.005 from the other strains, a value that is lower than the 0.007 MLSA evolutionary distance threshold proposed for species-level relatedness. These data support a proposal to reclassify S. almquistii, S. flocculus, S. gibsonii and S. rangoonensis as later heterotypic synonyms of S. albus with NRRL B-1811T as the type strain. The MLSA sequence database also demonstrated utility for quickly and conclusively confirming that numerous strains within the ARS Culture Collection had been previously misidentified as subspecies of S. albus and that Streptomyces albus subsp. pathocidicus should be redescribed as a novel species, Streptomyces pathocidini sp. nov., with the type strain NRRL B-24287T. PMID:24277863

Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed.

PubMed

Tanaka, Mami; Endo, Shoko; Kotake, Fumihito; Al-Saari, Nurhidayu; Amin, A K M Rohul; Feng, Gao; Mino, Sayaka; Doi, Hidetaka; Ogura, Yoshitoshi; Hayashi, Tetsuya; Suda, Wataru; Hattori, Masahira; Yumoto, Isao; Sawabe, Toko; Sawabe, Tomoo; Araki, Toshiyoshi

2017-01-01

A novel strain Vibrio aphrogenes sp. nov. strain CA-1004T isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using polyphasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis clade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis clade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004T as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004T was separate from four known Rumoiensis clade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non-motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis clade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis clade, with CA-1004T as the type strain (JCM 31643T = DSM 103759T).

Enterobacter muelleri sp. nov., isolated from the rhizosphere of Zea mays.

PubMed

Kämpfer, Peter; McInroy, John A; Glaeser, Stefanie P

2015-11-01

A beige-pigmented, oxidase-negative bacterial strain (JM-458T), isolated from a rhizosphere sample, was studied using a polyphasic taxonomic approach. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of strain JM-458T with sequences of the type strains of closely related species of the genus Enterobacter showed that it shared highest sequence similarity with Enterobacter mori (98.7 %), Enterobacter hormaechei (98.3 %), Enterobacter cloacae subsp. dissolvens, Enterobacter ludwigii and Enterobacter asburiae (all 98.2 %). 16S rRNA gene sequence similarities to all other Enterobacter species were below 98 %. Multilocus sequence analysis based on concatenated partial rpoB, gyrB, infB and atpD gene sequences showed a clear distinction of strain JM-458T from its closest related type strains. The fatty acid profile of the strain consisted of C16 : 0, C17 : 0 cyclo, iso-C15 : 0 2-OH/C16 : 1ω7c and C18 : 1ω7c as major components. DNA-DNA hybridizations between strain JM-458T and the type strains of E. mori, E. hormaechei and E. ludwigii resulted in relatedness values of 29 % (reciprocal 25 %), 24 % (reciprocal 43 %) and 16 % (reciprocal 17 %), respectively. DNA-DNA hybridization results together with multilocus sequence analysis results and differential biochemical and chemotaxonomic properties showed that strain JM-458T represents a novel species of the genus Enterobacter, for which the name Enterobacter muelleri sp. nov. is proposed. The type strain is JM-458T ( = DSM 29346T = CIP 110826T = LMG 28480T = CCM 8546T).

Investigation of genetic diversity and epidemiological characteristics of Pasteurella multocida isolates from poultry in southwest China by population structure, multi-locus sequence typing and virulence-associated gene profile analysis.

PubMed

Li, Zhangcheng; Cheng, Fangjun; Lan, Shimei; Guo, Jianhua; Liu, Wei; Li, Xiaoyan; Luo, Zeli; Zhang, Manli; Wu, Juan; Shi, Yang

2018-04-25

Fowl cholera caused by Pasteurella multocida has always been a disease of global importance for poultry production. The aim of this study was to obtain more information about the epidemiology of avian P. multocida infection in southwest China and the genetic characteristics of clinical isolates. P. multocida isolates were characterized by biochemical and molecular-biological methods. The distributions of the capsular serogroups, the phenotypic antimicrobial resistance profiles, lipopolysaccharide (LPS) genotyping and the presence of 19 virulence genes were investigated in 45 isolates of P. multocida that were associated with clinical disease in poultry. The genetic diversity of P. multocida strains was performed by 16S rRNA and rpoB gene sequence analysis as well as multilocus sequence typing (MLST). The results showed that most (80.0%) of the P. multocida isolates in this study represented special P. multocida subspecies, and 71.1% of the isolates showed multiple-drug resistance. 45 isolates belonged to capsular types: A (100%) and two LPS genotypes: L1 (95.6%) and L3 (4.4%). MLST revealed two new alleles (pmi77 and gdh57) and one new sequence type (ST342). ST129 types dominated in 45 P. multocida isolates. Isolates belonging to ST129 were with the genes ompH+plpB+ptfA+tonB, whereas ST342 included isolates with fur+hgbA+tonB genes. Population genetic analysis and the MLST results revealed that at least one new ST genotype was present in the avian P. multocida in China. These findings provide novel insights into the epidemiological characteristics of avian P. multocida isolates in southwest China.

Multilocus sequence typing scheme for the Mycobacterium abscessus complex.

PubMed

Macheras, Edouard; Konjek, Julie; Roux, Anne-Laure; Thiberge, Jean-Michel; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby E; Bodmer, Thomas; Jarlier, Vincent; Cambau, Emmanuelle; Brisse, Sylvain; Caro, Valérie; Rastogi, Nalin; Gaillard, Jean-Louis; Heym, Beate

2014-01-01

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST).

PubMed

Dan, Tong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Menghe, Bilige; Zhang, Heping; Sun, Zhihong

2015-05-20

Lactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). MLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (I A (S)) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type. The MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.

A Wide Variety of Clostridium perfringens Type A Food-Borne Isolates That Carry a Chromosomal cpe Gene Belong to One Multilocus Sequence Typing Cluster

PubMed Central

Xiao, Yinghua; Wagendorp, Arjen; Moezelaar, Roy; Abee, Tjakko

2012-01-01

Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)5 fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods. PMID:22865060

Biogeography of Burkholderia pseudomallei in the Torres Strait Islands of Northern Australia

PubMed Central

Baker, Anthony; Mayo, Mark; Owens, Leigh; Burgess, Graham; Norton, Robert; McBride, William John Hannan; Currie, Bart J.

2013-01-01

It has been hypothesized that biogeographical boundaries are a feature of Burkholderia pseudomallei ecology, and they impact the epidemiology of melioidosis on a global scale. This study examined the relatedness of B. pseudomallei sourced from islands in the Torres Strait of Northern Australia to determine if the geography of isolated island communities is a determinant of the organisms' dispersal. Environmental sampling on Badu Island in the Near Western Island cluster recovered a single clone. An additional 32 clinical isolates from the region were sourced. Isolates were characterized using multilocus sequence typing and a multiplex PCR targeting the flagellum gene cluster. Gene cluster analysis determined that 69% of the isolates from the region encoded the ancestral Burkholderia thailandensis-like flagellum and chemotaxis gene cluster, a proportion significantly lower than that reported from mainland Australia and consistent with observations of isolates from southern Papua New Guinea. A goodness-of-fit test indicated that there was geographic localization of sequence types throughout the archipelago, with the exception of Thursday Island, the economic and cultural hub of the region. Sequence types common to mainland Australia and Papua New Guinea were identified. These findings demonstrate for the first time an environmental reservoir for B. pseudomallei in the Torres Strait, and multilocus sequence typing suggests that the organism is not randomly distributed throughout this region and that seawater may provide a barrier to dispersal of the organism. Moreover, these findings support an anthropogenic dispersal hypothesis for the spread of B. pseudomallei throughout this region. PMID:23698533

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household.

PubMed

Staples, M; Graham, R M A; Doyle, C J; Smith, H V; Jennison, A V

2012-05-01

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters. © 2012 QUEENSLAND HEALTH. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Species-Level Phylogeny and Polyploid Relationships in Hordeum (Poaceae) Inferred by Next-Generation Sequencing and In Silico Cloning of Multiple Nuclear Loci.

PubMed

Brassac, Jonathan; Blattner, Frank R

2015-09-01

Polyploidization is an important speciation mechanism in the barley genus Hordeum. To analyze evolutionary changes after allopolyploidization, knowledge of parental relationships is essential. One chloroplast and 12 nuclear single-copy loci were amplified by polymerase chain reaction (PCR) in all Hordeum plus six out-group species. Amplicons from each of 96 individuals were pooled, sheared, labeled with individual-specific barcodes and sequenced in a single run on a 454 platform. Reference sequences were obtained by cloning and Sanger sequencing of all loci for nine supplementary individuals. The 454 reads were assembled into contigs representing the 13 loci and, for polyploids, also homoeologues. Phylogenetic analyses were conducted for all loci separately and for a concatenated data matrix of all loci. For diploid taxa, a Bayesian concordance analysis and a coalescent-based dated species tree was inferred from all gene trees. Chloroplast matK was used to determine the maternal parent in allopolyploid taxa. The relative performance of different multilocus analyses in the presence of incomplete lineage sorting and hybridization was also assessed. The resulting multilocus phylogeny reveals for the first time species phylogeny and progenitor-derivative relationships of all di- and polyploid Hordeum taxa within a single analysis. Our study proves that it is possible to obtain a multilocus species-level phylogeny for di- and polyploid taxa by combining PCR with next-generation sequencing, without cloning and without creating a heavy load of sequence data. © The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.

First isolation of Actinobacillus genomospecies 2 in Japan

PubMed Central

MURAKAMI, Miyuki; SHIMONISHI, Yoshimasa; HOBO, Seiji; NIWA, Hidekazu; ITO, Hiroya

2015-01-01

We describe here the first isolation of Actinobacillus genomospecies 2 in Japan. The isolate was found in a septicemic foal and characterized by phenotypic and genetic analyses, with the latter consisting of 16S rDNA nucleotide sequence analysis plus multilocus sequence analysis using three housekeeping genes, recN, rpoA and thdF, that have been proposed for use as a genomic tool in place of DNA-DNA hybridization. PMID:26668165

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

PubMed

Reid, S D; Green, N M; Buss, J K; Lei, B; Musser, J M

2001-06-19

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing

PubMed Central

Didi, Jennifer; Lemée, Ludovic; Gibert, Laure; Pons, Jean-Louis

2014-01-01

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis. PMID:25078912

Molecular characterization and epidemiology of cefoxitin resistance among Enterobacteriaceae lacking inducible chromosomal ampC genes from hospitalized and non-hospitalized patients in Algeria: description of new sequence type in Klebsiella pneumoniae isolates.

PubMed

Gharout-Sait, Alima; Touati, Abdelaziz; Guillard, Thomas; Brasme, Lucien; de Champs, Christophe

2015-01-01

In this study, 922 consecutive non-duplicate clinical isolates of Enterobacteriaceae obtained from hospitalized and non-hospitalized patients at Bejaia, Algeria were analyzed for AmpC-type β-lactamases production. The ampC genes and their genetic environment were characterized using polymerase chain reaction (PCR) and sequencing. Plasmid incompatibility groups were determined by using PCR-based replicon typing. Phylogenetic grouping and multilocus sequence typing were determined for molecular typing of the plasmid-mediated AmpC (pAmpC) isolates. Of the isolates, 15 (1.6%) were identified as AmpC producers including 14 CMY-4-producing isolates and one DHA-1-producing Klebsiella pneumoniae. All AmpC-producing isolates co-expressed the broad-spectrum TEM-1 β-lactamase and three of them co-produced CTX-M and/or SHV-12 ESBL. Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to groups D and B1. Multilocus sequence typing analysis of K. pneumoniae isolates identified four different sequence types (STs) with two new sequences: ST1617 and ST1618. Plasmid replicon typing indicates that blaCMY-4 gene was located on broad host range A/C plasmid, while LVPK replicon was associated with blaDHA-1. All isolates carrying blaCMY-4 displayed the transposon-like structures ISEcp1/ΔISEcp1-blaCMY-blc-sugE. Our study showed that CMY-4 was the main pAmpC in the Enterobacteriaceae isolates in Algeria. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

The multilocus sequence typing network: mlst.net.

PubMed

Aanensen, David M; Spratt, Brian G

2005-07-01

The unambiguous characterization of strains of a pathogen is crucial for addressing questions relating to its epidemiology, population and evolutionary biology. Multilocus sequence typing (MLST), which defines strains from the sequences at seven house-keeping loci, has become the method of choice for molecular typing of many bacterial and fungal pathogens (and non-pathogens), and MLST schemes and strain databases are available for a growing number of prokaryotic and eukaryotic organisms. Sequence data are ideal for strain characterization as they are unambiguous, meaning strains can readily be compared between laboratories via the Internet. Laboratories undertaking MLST can quickly progress from sequencing the seven gene fragments to characterizing their strains and relating them to those submitted by others and to the population as a whole. We provide the gateway to a number of MLST schemes, each of which contain a set of tools for the initial characterization of strains, and methods for relating query strains to other strains of the species, including clustering based on differences in allelic profiles, phylogenetic trees based on concatenated sequences, and a recently developed method (eBURST) for identifying clonal complexes within a species and displaying the overall structure of the population. This network of MLST websites is available at http://www.mlst.net.

STBase: One Million Species Trees for Comparative Biology

PubMed Central

McMahon, Michelle M.; Deepak, Akshay; Fernández-Baca, David; Boss, Darren; Sanderson, Michael J.

2015-01-01

Comprehensively sampled phylogenetic trees provide the most compelling foundations for strong inferences in comparative evolutionary biology. Mismatches are common, however, between the taxa for which comparative data are available and the taxa sampled by published phylogenetic analyses. Moreover, many published phylogenies are gene trees, which cannot always be adapted immediately for species level comparisons because of discordance, gene duplication, and other confounding biological processes. A new database, STBase, lets comparative biologists quickly retrieve species level phylogenetic hypotheses in response to a query list of species names. The database consists of 1 million single- and multi-locus data sets, each with a confidence set of 1000 putative species trees, computed from GenBank sequence data for 413,000 eukaryotic taxa. Two bodies of theoretical work are leveraged to aid in the assembly of multi-locus concatenated data sets for species tree construction. First, multiply labeled gene trees are pruned to conflict-free singly-labeled species-level trees that can be combined between loci. Second, impacts of missing data in multi-locus data sets are ameliorated by assembling only decisive data sets. Data sets overlapping with the user’s query are ranked using a scheme that depends on user-provided weights for tree quality and for taxonomic overlap of the tree with the query. Retrieval times are independent of the size of the database, typically a few seconds. Tree quality is assessed by a real-time evaluation of bootstrap support on just the overlapping subtree. Associated sequence alignments, tree files and metadata can be downloaded for subsequent analysis. STBase provides a tool for comparative biologists interested in exploiting the most relevant sequence data available for the taxa of interest. It may also serve as a prototype for future species tree oriented databases and as a resource for assembly of larger species phylogenies from precomputed trees. PMID:25679219

Vibrio chromosomes share common history.

PubMed

Kirkup, Benjamin C; Chang, LeeAnn; Chang, Sarah; Gevers, Dirk; Polz, Martin F

2010-05-10

While most gamma proteobacteria have a single circular chromosome, Vibrionales have two circular chromosomes. Horizontal gene transfer is common among Vibrios, and in light of this genetic mobility, it is an open question to what extent the two chromosomes themselves share a common history since their formation. Single copy genes from each chromosome (142 genes from chromosome I and 42 genes from chromosome II) were identified from 19 sequenced Vibrionales genomes and their phylogenetic comparison suggests consistent phylogenies for each chromosome. Additionally, study of the gene organization and phylogeny of the respective origins of replication confirmed the shared history. Thus, while elements within the chromosomes may have experienced significant genetic mobility, the backbones share a common history. This allows conclusions based on multilocus sequence analysis (MLSA) for one chromosome to be applied equally to both chromosomes.

Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr)

USDA-ARS?s Scientific Manuscript database

A Multilocus Sequence Typing (MLST) method based on allelic variation of 7 chromosomal loci was developed for characterizing genotypes within the genus Bradyrhizobium. With the method 29 distinct multilocus genotypes (GTs) were identified among 191 culture collection soybean strains. The occupancy ...

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China.

PubMed

Yang, Xiaojuan; Yu, Shubo; Wu, Qingping; Zhang, Jumei; Wu, Shi; Rong, Dongli

2018-01-01

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes ( sea , seb , sec , sed , see , seg , seh , sei , sej ), the exfoliative toxin genes ( eta and etb ), the toxic shock syndrome toxin-1 gene ( tst ), and the Panton-Valentine leucocidin-encoding gene ( pvl ). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes ( sea - see ), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl , eta , etb , and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China

PubMed Central

Yang, Xiaojuan; Yu, Shubo; Wu, Qingping; Zhang, Jumei; Wu, Shi; Rong, Dongli

2018-01-01

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, sej), the exfoliative toxin genes (eta and etb), the toxic shock syndrome toxin-1 gene (tst), and the Panton-Valentine leucocidin-encoding gene (pvl). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes (sea–see), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl, eta, etb, and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies. PMID:29662467

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses.

PubMed

Li, Xiang; Tambong, James; Yuan, Kat Xiaoli; Chen, Wen; Xu, Huimin; Lévesque, C André; De Boer, Solke H

2018-01-01

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA-DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses

PubMed Central

Li, Xiang; Tambong, James; Yuan, Kat (Xiaoli); Chen, Wen; Xu, Huimin; Lévesque, C. André; De Boer, Solke H.

2018-01-01

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA–DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov. PMID:29160202

Lactobacillus cypricasei Lawson et al. 2001 is a later heterotypic synonym of Lactobacillus acidipiscis Tanasupawat et al. 2000.

PubMed

Naser, Sabri M; Vancanneyt, Marc; Hoste, Bart; Snauwaert, Cindy; Swings, Jean

2006-07-01

The applicability of a multilocus sequence analysis (MLSA)-based identification system for lactobacilli was evaluated. Two housekeeping genes that code for the phenylalanyl-tRNA synthase alpha-subunit (pheS) and RNA polymerase alpha-subunit (rpoA) were sequenced and analysed for members of the Lactobacillus salivarius species group. The type strains of Lactobacillus acidipiscis and Lactobacillus cypricasei were investigated further using a third gene that encodes the alpha-subunit of ATP synthase (atpA). The MLSA data revealed close relatedness between L. acidipiscis and L. cypricasei, with 99.8-100 % pheS, rpoA and atpA gene sequence similarities. Comparison of the 16S rRNA gene sequences of the type strains of the two species confirmed the close relatedness (99.8 % gene sequence similarity) between the two taxa. Similar phenotypes and high DNA-DNA binding values in the range of 84 to 97.5 % confirmed that L. acidipiscis and L. cypricasei are synonymous species. On the basis of the present study, it is proposed that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis.

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting▿

PubMed Central

Rademaker, Jan L. W.; Herbet, Hélène; Starrenburg, Marjo J. C.; Naser, Sabri M.; Gevers, Dirk; Kelly, William J.; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E. T.

2007-01-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene. PMID:17890345

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

PubMed

Rademaker, Jan L W; Herbet, Hélène; Starrenburg, Marjo J C; Naser, Sabri M; Gevers, Dirk; Kelly, William J; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E T

2007-11-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

Imbalanced presence of Borrelia burgdorferi s.l. multilocus sequence types in clinical manifestations of Lyme borreliosis.

PubMed

Coipan, E Claudia; Jahfari, Setareh; Fonville, Manoj; Oei, G Anneke; Spanjaard, Lodewijk; Takumi, Katsuhisa; Hovius, Joppe W R; Sprong, Hein

2016-08-01

In this study we used typing based on the eight multilocus sequence typing scheme housekeeping genes (MLST) and 5S-23S rDNA intergenic spacer (IGS) to explore the population structure of Borrelia burgdorferi sensu lato isolates from patients with Lyme borreliosis (LB) and to test the association between the B. burgdorferi s.l. sequence types (ST) and the clinical manifestations they cause in humans. Isolates of B. burgdorferi from 183 LB cases across Europe, with distinct clinical manifestations, and 257 Ixodes ricinus lysates from The Netherlands, were analyzed for this study alone. For completeness, we incorporated in our analysis also 335 European B. burgdorferi s.l. MLST profiles retrieved from literature. Borrelia afzelii and Borrelia bavariensis were associated with human cases of LB while Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana were associated with questing I. ricinus ticks. B. afzelii was associated with acrodermatitis chronica atrophicans, while B. garinii and B. bavariensis were associated with neuroborreliosis. The samples in our study belonged to 251 different STs, of which 94 are newly described, adding to the overall picture of the genetic diversity of Borrelia genospecies. The fraction of STs that were isolated from human samples was significantly higher for the genospecies that are known to be maintained in enzootic cycles by mammals (B. afzelii, B. bavariensis, and Borrelia spielmanii) than for genospecies that are maintained by birds (B. garinii and B. valaisiana) or lizards (B. lusitaniae). We found six multilocus sequence types that were significantly associated to clinical manifestations in humans and five IGS haplotypes that were associated with the human LB cases. While IGS could perform just as well as the housekeeping genes in the MLST scheme for predicting the infectivity of B. burgdorferi s.l., the advantage of MLST is that it can also capture the differential invasiveness of the various STs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

[Standard algorithm of molecular typing of Yersinia pestis strains].

PubMed

Eroshenko, G A; Odinokov, G N; Kukleva, L M; Pavlova, A I; Krasnov, Ia M; Shavina, N Iu; Guseva, N P; Vinogradova, N A; Kutyrev, V V

2012-01-01

Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad. Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number. A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established. The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

Nonencapsulated or nontypeable Haemophilus influenzae are more likely than their encapsulated or serotypeable counterparts to have mutations in their fucose operon.

PubMed

Shuel, Michelle L; Karlowsky, Kathleen E; Law, Dennis K S; Tsang, Raymond S W

2011-12-01

Population biology of Haemophilus influenzae can be studied by multilocus sequence typing (MLST), and isolates are assigned sequence types (STs) based on nucleotide sequence variations in seven housekeeping genes, including fucK. However, the ST cannot be assigned if one of the housekeeping genes is absent or cannot be detected by the current protocol. Occasionally, strains of H. influenzae have been reported to lack the fucK gene. In this study, we examined the prevalence of this mutation among our collection of H. influenzae isolates. Of the 704 isolates studied, including 282 encapsulated and 422 nonencapsulated isolates, nine were not typeable by MLST owing to failure to detect the fucK gene. All nine fucK-negative isolates were nonencapsulated and belonged to various biotypes. DNA sequencing of the fucose operon region confirmed complete deletion of genes in the operon in seven of the nine isolates, while in the remaining two isolates, some of the genes were found intact or in parts. The significance of these findings is discussed.

Multilocus genotyping of Giardia duodenalis in Brazilian children.

PubMed

Scalia, Luana A M; Fava, Natália M N; Soares, Rodrigo M; Limongi, Jean E; da Cunha, Maria Júlia R; Pena, Isabella F; Kalapothakis, Evanguedes; Cury, Márcia C

2016-06-01

Giardia duodenalis is a parasite of several mammalian species, including humans, distributed worldwide. This research aimed to identify the molecular assemblages/sub-assemblages of G. duodenalis and to determine the intra-assemblage genetic variation of the different genes of assemblages A and B in pre-school children in the cities of Araguari and Uberlândia, Minas Gerais, Brazil. The molecular characterization followed β-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) protocols. Of 226 stool samples, G. duodenalis cysts were found in 45 (19.9%). The tpi gene was amplified in 34 samples: 16 assemblage A, 14 B and four mixed samples A/B. The gdh gene was amplified in 32 samples, including 14 A, 16 B and two A/B. For the bg gene, 19 samples were sequenced: nine assemblage A, five B, three E, and two mixed, A/E and B/E. Animal-specific assemblage E were identified by bg, but were not confirmed for other genes. Twelve samples were characterized by full agreement of the three genes. Two new multilocus genotyping (MLGs) for assemblage A and two new MLGs for assemblage B were also described. These findings substantiate the importance of using more than one gene protocol since the sensitivity and genetic variability changes with the locus used.Access numbers: The GenBank access numbers for the nucleotide sequences reported in this article are: JQ794877-JQ794890, JX033113-JX033118. © The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[Development of Staphylococcus Haemolyticus multilocus sequencing scheme and its use for molecular-epidemiologic analysis of strains isolated in hospitals in Russian federation in 2009-2010].

PubMed

Voronina, O L; Kunda, M S; Dmitrenko, O A; Lunin, V G; Gintsburg, A L

2011-01-01

Development of Staphylococcus haemolyticus strain typing method based on multilocus sequencing for resolving problems of molecular epidemiology. 102 strains of coagulase negative staphylococci (CNS) isolated in hospitals of various specialization in N. Novgorod and Moscow were studied. Species identification of strain was performed by using tuf gene fragment sequencing, S. haemolyticus strain differentiation--by MLST results. eBURST approach was used for cluster analysis of MLST data; structural changes in tagatose-6-phosphate kinase were studied by using InterProScan platform and SWISS-MODEL site programs; MLST scheme gene allele variability analysis was performed by using MEGA4.0 program package. In the 102 strains sampled CNS was detected in 28 strains of the S. haemolyticus species. The MLST scheme developed for the first time for S. haemolyticus including mvaK, rphE, tphK, gtr, arcC, triA, aroE genes allowed the differentiation of the sampled strains by 11 genotypes. Strains with ST 3, 8, 6, 1, 4, 5 and 11 differed by highest epidemiologic significance. Cluster and phylogenetic analysis of the data obtained showed a high adaptive ability of the nosocomial S. haemolyticus strains. Multiresistance to antibacterial preparations was detected in the analyzed strains. The MLST method developed was effective in the differentiation of S. haemolyticus strains that circulate in hospitals and threaten both neonates and hospitalized adult patients.

Antimicrobial susceptibility, tetracycline and erythromycin resistance genes, and multilocus sequence typing of Streptococcus suis isolates from diseased pigs in China.

PubMed

Chen, Lei; Song, Yajing; Wei, Zigong; He, Hongkui; Zhang, Anding; Jin, Meilin

2013-01-01

Streptococcus suis (S. suis) is an emerging zoonotic pathogen causing significant economic losses in the swine industry. Here, we investigated the antimicrobial susceptibility, associated antibiotic-resistant determinants and sequence type (ST) of S. suis isolates from diseased pigs in China from 2008 to 2010. Serotype 2 was the most frequently observed strain (n=95) among the 106 S. suis strains collected, followed by serotypes 3 (n=3), 5 (n=3), 4 (n=2), 7 (n=1), 11 (n=1) and 28 (n=1). Multilocus sequence typing analysis revealed that ST1 (n=21) and ST7 (n=74) were the predominant STs, and serotype 2 was found to be significantly correlated with ST7 (P=0.017, Fisher's exact test) and CC1 (P=0.024, Fisher's exact test). The antimicrobial susceptibility results indicated that the antibiotic resistance rate was highest for tetracycline (99.1%), followed by azithromycin (68.9%), erythromycin (67.9%), clindamycin (67.9%), trimethoprim/sulfamethoxazole (16%), levofloxacin (2.8%), chloramphenicol (1.9%), cefaclor (0.9%) and ceftriaxone (0.9%). Antibiotic-resistant genes tet(M), tet(O), tet(O/W/32/O), tet(O/32/O), tet(S), tet(W), tet(L), tet(40), erm(B), mef(A/E) and msr(D) could be detected, and several tandem organizations of antibiotic resistance genes were also found in this study. In conclusion, S. suis strains isolated from diseased pigs in China were less diverse and multi-drug resistant.

Determining Clostridium difficile intra-taxa diversity by mining multilocus sequence typing databases.

PubMed

Muñoz, Marina; Ríos-Chaparro, Dora Inés; Patarroyo, Manuel Alfonso; Ramírez, Juan David

2017-03-14

Multilocus sequence typing (MLST) is a highly discriminatory typing strategy; it is reproducible and scalable. There is a MLST scheme for Clostridium difficile (CD), a gram positive bacillus causing different pathologies of the gastrointestinal tract. This work was aimed at describing the frequency of sequence types (STs) and Clades (C) reported and evalute the intra-taxa diversity in the CD MLST database (CD-MLST-db) using an MLSA approach. Analysis of 1778 available isolates showed that clade 1 (C1) was the most frequent worldwide (57.7%), followed by C2 (29.1%). Regarding sequence types (STs), it was found that ST-1, belonging to C2, was the most frequent. The isolates analysed came from 17 countries, mostly from the United Kingdom (UK) (1541 STs, 87.0%). The diversity of the seven housekeeping genes in the MLST scheme was evaluated, and alleles from the profiles (STs), for identifying CD population structure. It was found that adk and atpA are conserved genes allowing a limited amount of clusters to be discriminated; however, different genes such as drx, glyA and particularly sodA showed high diversity indexes and grouped CD populations in many clusters, suggesting that these genes' contribution to CD typing should be revised. It was identified that CD STs reported to date have a mostly clonal population structure with foreseen events of recombination; however, one group of STs was not assigned to a clade being highly different containing at least nine well-supported clusters, suggesting a greater amount of clades for CD. This study shows the usefulness of CD-MLST-db as a tool for studying CD distribution and population structure, identifying the need for reviewing the usefulness of sodA as housekeeping gene within the MLST scheme and suggesting the existence of a greater amount of CD clades. The study also shows the plausible exchange of genetic material between STs, contributing towards intra-taxa genetic diversity.

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study▿†

PubMed Central

Wang, He; Xiao, Meng; Kong, Fanrong; Chen, Sharon; Dou, Hong-Tao; Sorrell, Tania; Li, Ruo-Yu; Xu, Ying-Chun

2011-01-01

Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria. PMID:21389150

Antimicrobial susceptibility and multilocus sequence typing of Mycoplasma capricolum subsp. capricolum

PubMed Central

Tatay-Dualde, Juan; Prats-van der Ham, Miranda; Paterna, Ana; Sánchez, Antonio; Corrales, Juan Carlos; Contreras, Antonio; Tola, Sebastiana; Gómez-Martin, Ángel

2017-01-01

Mycoplasma capricolum subsp. capricolum is one of the causative agents of contagious agalactia (CA). Nevertheless, there is still a lack of information about its antimicrobial susceptibility and genetic characteristics. Therefore, the aim of this work was to study the antimicrobial and genetic variability of different Mycoplasma capricolum subsp. capricolum field isolates. For this purpose, the growth inhibition effect of 18 antimicrobials and a multilocus sequence typing (MLST) scheme based on five housekeeping genes (fusA, glpQ, gyrB, lepA and rpoB) were performed on 32 selected field isolates from Italy and Spain.The results showed a wide range of growth inhibitory effects for almost all the antimicrobials studied. Macrolides presented lower efficacy inhibiting Mcc growth than in previous works performed on other CA-causative mycoplasmas. Erythromycin was not able to inhibit the growth of any of the studied strains, contrary to doxycycline, which inhibited the growth of all of them from low concentrations. On the other hand, the study of the concatenated genes revealed a high genetic variability among the different Mcc isolates. Hence, these genetic variations were greater than the ones reported in prior works on other mycoplasma species. PMID:28346546

Molecular epidemiology and multilocus sequence analysis of potentially zoonotic Giardia spp. from humans and dogs in Jamaica.

PubMed

Lee, Mellesia F; Cadogan, Paul; Eytle, Sarah; Copeland, Sonia; Walochnik, Julia; Lindo, John F

2017-01-01

Giardia spp. are the causative agents of intestinal infections in a wide variety of mammals including humans and companion animals. Dogs may be reservoirs of zoonotic Giardia spp.; however, the potential for transmission between dogs and humans in Jamaica has not been studied. Conventional PCR was used to screen 285 human and 225 dog stool samples for Giardia targeting the SSU rDNA gene followed by multilocus sequencing of the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and β-giardin (bg) genes. Prevalence of human infections based on PCR was 6.7 % (19/285) and canine infections 19.6 % (44/225). Nested PCR conducted on all 63 positive samples revealed the exclusive presence of assemblage A in both humans and dogs. Sub-assemblage A-II was responsible for 79.0 % (15/19) and 70.5 % (31/44) of the infections in humans and dogs, respectively, while sub-assemblage A-I was identified at a rate of 15.8 % (3/19) and 29.5 % (13/44) in humans and dogs, respectively. The predominance of a single circulating assemblage among both humans and dogs in Jamaica suggests possible zoonotic transmission of Giardia infections.

Multi-locus analysis of Giardia duodenalis from nonhuman primates kept in zoos in China: geographical segregation and host-adaptation of assemblage B isolates.

PubMed

Karim, Md Robiul; Wang, Rongjun; Yu, Fuchang; Li, Tongyi; Dong, Haiju; Li, Dezhong; Zhang, Longxian; Li, Junqiang; Jian, Fuchun; Zhang, Sumei; Rume, Farzana Islam; Ning, Changshen; Xiao, Lihua

2015-03-01

Only a few studies based on single locus characterization have been conducted on the molecular epidemiology of Giardia duodenalis in nonhuman primates (NHPs). The present study was conducted to examine the occurrence and genotype identity of G. duodenalis in NHPs based on multi-locus analysis of the small-subunit ribosomal RNA (SSU rRNA), triose phosphate isomerase (tpi), glutamate dehydrogenase (gdh), and beta-giardin (bg) genes. Fecal specimens were collected from 496 animals of 36 NHP species kept in seven zoos in China and screened for G. duodenalis by tpi-based PCR. G. duodenalis was detected in 92 (18.6%) specimens from 18 NHP species, belonging to assemblage A (n=4) and B (n=88). In positive NHP species, the infection rates ranged from 4.8% to 100%. In tpi sequence analysis, the assemblage A included subtypes A1, A2 and one novel subtype. Multi-locus analysis of the tpi, gdh, and bg genes detected 11 (8 known and 3 new), 6 (3 known and 3 new) and 9 (2 known and 7 new) subtypes in 88, 47 and 35 isolates in assemblage B, respectively. Thirty-two assemblage B isolates with data at all three loci yielded 15 multi-locus genotypes (MLGs), including 2 known and 13 new MLGs. Phylogenetic analysis of concatenated sequences of assemblage B showed that MLGs found here were genetically different from those of humans, NHPs, rabbit and guinea pig in Italy and Sweden. It further indicated that assemblage B isolates in ring-tailed lemurs and squirrel monkeys might be genetically different from those in other NHPs. These data suggest that NHPs are mainly infected with G. duodenalis assemblage B and there might be geographical segregation and host-adaptation in assemblage B in NHPs. Copyright © 2014 Elsevier B.V. All rights reserved.

A distinct alleles and genetic recombination of pmrCAB operon in species of Acinetobacter baumannii complex isolates.

PubMed

Kim, Dae Hun; Ko, Kwan Soo

2015-07-01

To investigate pmrCAB sequence divergence in 5 species of Acinetobacter baumannii complex, a total of 80 isolates from a Korean hospital were explored. We evaluated nucleotide and amino acid polymorphisms of pmrCAB operon, and phylogenetic trees were constructed for each gene of prmCAB operon. Colistin and polymyxin B susceptibility was determined for all isolates, and multilocus sequence typing was also performed for A. baumannii isolates. Our results showed that each species of A. baumannii complex has divergent pmrCAB operon sequences. We identified a distinct pmrCAB allele allied with Acinetobacter nosocomialis in gene trees. Different grouping in each gene tree suggests sporadic recombination or emergence of pmrCAB genes among Acinetobacter species. Sequence polymorphisms among Acinetobacter species might not be associated with colistin resistance. We revealed that a distinct pmrCAB allele may be widespread across the continents such as North America and Asia and that sporadic genetic recombination or emergence of pmrCAB genes might occur. Copyright © 2015 Elsevier Inc. All rights reserved.

Insertion sequence ISRP10 inactivation of the oprD gene in imipenem-resistant Pseudomonas aeruginosa clinical isolates.

PubMed

Sun, Qinghui; Ba, Zhaofen; Wu, Guoying; Wang, Wei; Lin, Shuxiang; Yang, Hongjiang

2016-05-01

Carbapenem resistance mechanisms were investigated in 32 imipenem-resistant Pseudomonas aeruginosa clinical isolates recovered from hospitalised children. Sequence analysis revealed that 31 of the isolates had an insertion sequence element ISRP10 disrupting the porin gene oprD, demonstrating that ISRP10 inactivation of oprD conferred imipenem resistance in the majority of the isolates. Multilocus sequence typing (MLST) was used to discriminate the isolates. In total, 11 sequence types (STs) were identified including 3 novel STs, and 68.3% (28/41) of the tested strains were characterised as clone ST253. In combination with random amplified polymorphic DNA (RAPD) analysis, the imipenem-resistant isolates displayed a relatively high degree of genetic variability and were unlikely associated with nosocomial infections. Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Characterization of Encapsulated and Noncapsulated Haemophilus influenzae and Determination of Phylogenetic Relationships by Multilocus Sequence Typing

PubMed Central

Meats, Emma; Feil, Edward J.; Stringer, Suzanna; Cody, Alison J.; Goldstein, Richard; Kroll, J. Simon; Popovic, Tanja; Spratt, Brian G.

2003-01-01

A multilocus sequence typing (MLST) scheme has been developed for the unambiguous characterization of encapsulated and noncapsulated Haemophilus influenzae isolates. The sequences of internal fragments of seven housekeeping genes were determined for 131 isolates, comprising a diverse set of 104 serotype a, b, c, d, e, and f isolates and 27 noncapsulated isolates. Many of the encapsulated isolates had previously been characterized by multilocus enzyme electrophoresis (MLEE), and the validity of the MLST scheme was established by the very similar clustering of isolates obtained by these methods. Isolates of serotypes c, d, e, and f formed monophyletic groups on a dendrogram constructed from the differences in the allelic profiles of the isolates, whereas there were highly divergent lineages of both serotype a and b isolates. Noncapsulated isolates were distinct from encapsulated isolates and, with one exception, were within two highly divergent clusters. The relationships between the major lineages of encapsulated H. influenzae inferred from MLEE data could not be discerned on a dendrogram constructed from differences in the allelic profiles, but were apparent on a tree reconstructed from the concatenated nucleotide sequences. Recombination has not therefore completely eliminated phylogenetic signal, and in support of this, for encapsulated isolates, there was significant congruence between many of the trees reconstructed from the sequences of the seven individual loci. Congruence was less apparent for noncapsulated isolates, suggesting that the impact of recombination is greater among noncapsulated than encapsulated isolates. The H. influenzae MLST scheme is available at www.mlst.net, it allows any isolate to be compared with those in the MLST database, and (for encapsulated isolates) it assigns isolates to their phylogenetic lineage, via the Internet. PMID:12682154

Pan-genome multilocus sequence typing and outbreak-specific reference-based single nucleotide polymorphism analysis to resolve two concurrent Staphylococcus aureus outbreaks in neonatal services.

PubMed

Roisin, S; Gaudin, C; De Mendonça, R; Bellon, J; Van Vaerenbergh, K; De Bruyne, K; Byl, B; Pouseele, H; Denis, O; Supply, P

2016-06-01

We used a two-step whole genome sequencing analysis for resolving two concurrent outbreaks in two neonatal services in Belgium, caused by exfoliative toxin A-encoding-gene-positive (eta+) methicillin-susceptible Staphylococcus aureus with an otherwise sporadic spa-type t209 (ST-109). Outbreak A involved 19 neonates and one healthcare worker in a Brussels hospital from May 2011 to October 2013. After a first episode interrupted by decolonization procedures applied over 7 months, the outbreak resumed concomitantly with the onset of outbreak B in a hospital in Asse, comprising 11 neonates and one healthcare worker from mid-2012 to January 2013. Pan-genome multilocus sequence typing, defined on the basis of 42 core and accessory reference genomes, and single-nucleotide polymorphisms mapped on an outbreak-specific de novo assembly were used to compare 28 available outbreak isolates and 19 eta+/spa-type t209 isolates identified by routine or nationwide surveillance. Pan-genome multilocus sequence typing showed that the outbreaks were caused by independent clones not closely related to any of the surveillance isolates. Isolates from only ten cases with overlapping stays in outbreak A, including four pairs of twins, showed no or only a single nucleotide polymorphism variation, indicating limited sequential transmission. Detection of larger genomic variation, even from the start of the outbreak, pointed to sporadic seeding from a pre-existing exogenous source, which persisted throughout the whole course of outbreak A. Whole genome sequencing analysis can provide unique fine-tuned insights into transmission pathways of complex outbreaks even at their inception, which, with timely use, could valuably guide efforts for early source identification. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Multilocus sequence typing of Scedosporium apiospermum and Pseudallescheria boydii isolates from cystic fibrosis patients.

PubMed

Bernhardt, A; Sedlacek, L; Wagner, S; Schwarz, C; Würstl, B; Tintelnot, K

2013-12-01

Scedosporium and Pseudallescheria species are the second most common lung-colonising fungi in cystic fibrosis (CF) patients. For epidemiological reasons it is important to trace sources of infection, routes of transmission and to determine whether these fungi are transient or permanent colonisers of the respiratory tract. Molecular typing methods like multilocus sequence typing (MLST) help provide this data. Clinical isolates of the P. boydii complex (including S. apiospermum and P. boydii) from CF patients in different regions of Germany were studied using MLST. Five gene loci, ACT, CAL, RPB2, BT2 and SOD2, were analysed. The S. apiospermum isolates from 34 patients were assigned to 32 sequence types (STs), and the P. boydii isolates from 14 patients to 8 STs. The results revealed that patients can be colonised by individual strains for years. The MLST scheme developed for S. apiospermum and P. boydii is a highly effective tool for epidemiologic studies worldwide. The MLST data are accessible at http://mlst.mycologylab.org/. Copyright © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Beta-hemolytic Streptococcus dysgalactiae strains isolated from horses are a genetically distinct population within the Streptococcus dysgalactiae taxon.

PubMed

Pinho, Marcos D; Erol, Erdal; Ribeiro-Gonçalves, Bruno; Mendes, Catarina I; Carriço, João A; Matos, Sandra C; Preziuso, Silvia; Luebke-Becker, Antina; Wieler, Lothar H; Melo-Cristino, Jose; Ramirez, Mario

2016-08-17

The pathogenic role of beta-hemolytic Streptococcus dysgalactiae in the equine host is increasingly recognized. A collection of 108 Lancefield group C (n = 96) or L (n = 12) horse isolates recovered in the United States and in three European countries presented multilocus sequence typing (MLST) alleles, sequence types and emm types (only 56% of the isolates could be emm typed) that were, with few exceptions, distinct from those previously found in human Streptococcus dysgalactiae subsp. equisimilis. Characterization of a subset of horse isolates by multilocus sequence analysis (MLSA) and 16S rRNA gene sequence showed that most equine isolates could also be differentiated from S. dysgalactiae strains from other animal species, supporting the existence of a horse specific genomovar. Draft genome information confirms the distinctiveness of the horse genomovar and indicates the presence of potentially horse-specific virulence factors. While this genomovar represents most of the isolates recovered from horses, a smaller MLST and MLSA defined sub-population seems to be able to cause infections in horses, other animals and humans, indicating that transmission between hosts of strains belonging to this group may occur.

Dissemination of metallo-β-lactamase-producing Pseudomonas aeruginosa of sequence type 235 in Asian countries.

PubMed

Kim, Moon Jung; Bae, Il Kwon; Jeong, Seok Hoon; Kim, So Hyun; Song, Jae Hoon; Choi, Jae Young; Yoon, Sang Sun; Thamlikitkul, Visanu; Hsueh, Po-Ren; Yasin, Rohani Md; Lalitha, M K; Lee, Kyungwon

2013-12-01

To investigate the epidemiological traits of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa (MPPA) clinical isolates collected by the Asian Network for Surveillance of Resistant Pathogens (ANSORP). A total of 16 MPPA clinical isolates were collected from six Asian countries in 2000 to 2009 by ANSORP. The MBL gene was detected by PCR amplification. The genetic organization of the class 1 integron carrying the MBL gene cassette was investigated by PCR mapping and sequencing. Southern blotting, repetitive sequence-based PCR and multilocus sequence typing (MLST) experiments were performed to characterize the isolates. PCR and sequencing experiments detected the blaVIM-2 (n = 12), blaVIM-3 (n = 1), blaIMP-6 (n = 2) and blaIMP-26 (n = 1) genes. The MBL genes were located on the chromosome in all isolates except one. Furthermore, all the MBL genes were located in a class 1 integron. All the MPPA isolates from Malaysia, Thailand, Sri Lanka and Korea were identified as sequence type (ST) 235 by MLST. Three VIM-2-producing isolates from India were identified as ST773, and one isolate harbouring VIM-3 from Taiwan was identified as ST298. P. aeruginosa ST235 might play a role in dissemination of MBL genes in Asian countries.

Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance.

PubMed

Bojarska, A; Molska, E; Janas, K; Skoczyńska, A; Stefaniuk, E; Hryniewicz, W; Sadowy, E

2016-06-01

The purpose of this study was to perform an analysis of Streptococcus suis human invasive isolates, collected in Poland by the National Reference Centre for Bacterial Meningitis. Isolates obtained from 21 patients during 2000-2013 were investigated by phenotypic tests, multilocus sequence typing (MLST), analysis of the TR9 locus from the multilocus variable number tandem repeat (VNTR) analysis (MLVA) scheme and pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA. Determinants of virulence and antimicrobial resistance were detected by polymerase chain reaction (PCR) and analysed by sequencing. All isolates represented sequence type 1 (ST1) and were suggested to be serotype 2. PFGE and analysis of the TR9 locus allowed the discrimination of four and 17 types, respectively. Most of the isolates were haemolysis- and DNase-positive, and around half of them formed biofilm. Genes encoding suilysin, extracellular protein factor, fibronectin-binding protein, muramidase-released protein, surface antigen one, enolase, serum opacity factor and pili were ubiquitous in the studied group, while none of the isolates carried sequences characteristic for the 89K pathogenicity island. All isolates were susceptible to penicillin, cefotaxime, imipenem, moxifloxacin, chloramphenicol, rifampicin, gentamicin, linezolid, vancomycin and daptomycin. Five isolates (24 %) were concomitantly non-susceptible to erythromycin, clindamycin and tetracycline, and harboured the tet(O) and erm(B) genes; for one isolate, lsa(E) and lnu(B) were additionally detected. Streptococcus suis isolated in Poland from human invasive infections belongs to a globally distributed clonal complex of this pathogen, enriched in virulence markers. This is the first report of the lsa(E) and lnu(B) resistance genes in S. suis.

Development and evaluation of a multi-locus sequence typing scheme for Mycoplasma synoviae.

PubMed

Dijkman, R; Feberwee, A; Landman, W J M

2016-08-01

Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans

PubMed Central

Carter, Stuart D.; Birtles, Richard J.; Brown, Jennifer M.; Hart, C. Anthony; Evans, Nicholas J.

2016-01-01

ABSTRACT Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used “-like” suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. IMPORTANCE The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens. PMID:27208135

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans.

PubMed

Clegg, Simon R; Carter, Stuart D; Birtles, Richard J; Brown, Jennifer M; Hart, C Anthony; Evans, Nicholas J

2016-08-01

Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used "-like" suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens. Copyright © 2016 Clegg et al.

Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food

PubMed Central

Wu, Shi; Wu, Qingping; Zhang, Jumei; Chen, Moutong; Guo, Weipeng

2016-01-01

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen. PMID:26909076

Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food.

PubMed

Wu, Shi; Wu, Qingping; Zhang, Jumei; Chen, Moutong; Guo, Weipeng

2016-01-01

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen.

Multi-virulence-locus sequence typing of Staphylococcus lugdunensis generates results consistent with a clonal population structure and is reliable for epidemiological typing.

PubMed

Didi, Jennifer; Lemée, Ludovic; Gibert, Laure; Pons, Jean-Louis; Pestel-Caron, Martine

2014-10-01

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Computational analysis of gene-gene interactions using multifactor dimensionality reduction.

PubMed

Moore, Jason H

2004-11-01

Understanding the relationship between DNA sequence variations and biologic traits is expected to improve the diagnosis, prevention and treatment of common human diseases. Success in characterizing genetic architecture will depend on our ability to address nonlinearities in the genotype-to-phenotype mapping relationship as a result of gene-gene interactions, or epistasis. This review addresses the challenges associated with the detection and characterization of epistasis. A novel strategy known as multifactor dimensionality reduction that was specifically designed for the identification of multilocus genetic effects is presented. Several case studies that demonstrate the detection of gene-gene interactions in common diseases such as atrial fibrillation, Type II diabetes and essential hypertension are also discussed.

A multilocus perspective on the speciation history of a North American aridland toad (Anaxyrus punctatus).

PubMed

Bryson, Robert W; Jaeger, Jef R; Lemos-Espinal, Julio A; Lazcano, David

2012-09-01

Interpretations of phylogeographic patterns can change when analyses shift from single gene-tree to multilocus coalescent analyses. Using multilocus coalescent approaches, a species tree and divergence times can be estimated from a set of gene trees while accounting for gene-tree stochasticity. We utilized the conceptual strengths of a multilocus coalescent approach coupled with complete range-wide sampling to examine the speciation history of a broadly distributed, North American warm-desert toad, Anaxyrus punctatus. Phylogenetic analyses provided strong support for three major lineages within A. punctatus. Each lineage broadly corresponded to one of three desert regions. Early speciation in A. punctatus appeared linked to late Miocene-Pliocene development of the Baja California peninsula. This event was likely followed by a Pleistocene divergence associated with the separation of the Chihuahuan and Sonoran Deserts. Our multilocus coalescent-based reconstruction provides an informative contrast to previous single gene-tree estimates of the evolutionary history of A. punctatus. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular Analysis of VanA Outbreak of Enterococcus faecium in Two Warsaw Hospitals: The Importance of Mobile Genetic Elements

PubMed Central

Wardal, Ewa; Markowska, Katarzyna; Żabicka, Dorota; Wróblewska, Marta; Giemza, Małgorzata; Mik, Ewa; Połowniak-Pracka, Hanna; Woźniak, Agnieszka; Hryniewicz, Waleria; Sadowy, Ewa

2014-01-01

Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections. Two outbreaks of this pathogen from neighboring Warsaw hospitals have been analyzed in this study. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA, multilocus VNTR analysis (MLVA), and multilocus sequence typing (MLST) revealed a clonal variability of isolates which belonged to three main lineages (17, 18, and 78) of nosocomial E. faecium. All isolates were multidrug resistant and carried several resistance, virulence, and plasmid-specific genes. Almost all isolates shared the same variant of Tn1546 transposon, characterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene. In the majority of cases, this transposon was located on 50 kb or 100 kb pRUM-related plasmids, which lacked, however, the axe-txe toxin-antitoxin genes. 100 kb plasmid was easily transferred by conjugation and was found in various clonal backgrounds in both institutions, while 50 kb plasmid was not transferable and occurred solely in MT159/ST78 strains that disseminated clonally in one institution. Although molecular data indicated the spread of VRE between two institutions or a potential common source of this alert pathogen, epidemiological investigations did not reveal the possible route by which outbreak strains disseminated. PMID:25003118

Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria.

PubMed

Delamuta, Jakeline Renata Marçon; Ribeiro, Renan Augusto; Menna, Pâmela; Bangel, Eliane Villamil; Hungria, Mariangela

2012-04-01

Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.

Combination of multilocus sequence typing and pulsed-field gel electrophoresis reveals an association of molecular clonality with the emergence of extensive-drug resistance (XDR) in Salmonella.

PubMed

Cao, Yongzhong; Shen, Yongxiu; Cheng, Lingling; Zhang, Xiaorong; Wang, Chao; Wang, Yan; Zhou, Xiaohui; Chao, Guoxiang; Wu, Yantao

2018-03-01

Salmonellae is one of the most important foodborne pathogens and becomes resistant to multiple antibiotics, which represents a significant challenge to food industry and public health. However, a molecular signature that can be used to distinguish antimicrobial resistance profile, particularly multi-drug resistance or extensive-drug resistance (XDR). In the current study, 168 isolates from the chicken and pork production chains and ill chickens were characterized by serotyping, antimicrobial susceptibility test, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The results showed that these isolates belonged to 13 serotypes, 14 multilocus sequence types (STs), 94 PFGE genotypes, and 70 antimicrobial resistant profiles. S. Enteritidis, S. Indiana, and S. Derby were the predominant serotypes, corresponding to the ST11, ST17, and ST40 clones, respectively and the PFGE Cluster A, Cluster E, and Cluster D, respectively. Among the ST11-S. Enteritidis (Cluster A) and the ST40-S. Derby (Cluster D) clones, the majority of isolates were resistant to 4-8 antimicrobial agents, whereas in the ST17S. Indiana (Cluster E) clone, isolates showed extensive-drug resistance (XDR) to 9-16 antimicrobial agents. The bla TEM-1-like gene was prevalent in the ST11 and ST17 clones corresponding to high ampicillin resistance. The bla TEM-1-like , bla CTX-M , bla OXA-1-like , sul1, aaC4, aac(6')-1b, dfrA17, and floR gene complex was highly prevalent among isolates of ST17, corresponding to an XDR phenotype. These results demonstrated the association of the resistant phenotypes and genotypes with ST clone and PFGE cluster. Our results also indicated that the newly identified gene complex comprising bla TEM-1-like , bla CTX-M , bla OXA-1-like , sul1, aaC4, aac(6')-1b, dfrA17, and floR, was responsible for the emergence of the ST17S. Indiana XDR clone. ST17 could be potentially used as a molecular signature to distinguish S. Indiana XDR clone. Copyright © 2017 Elsevier GmbH. All rights reserved.

Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex.

PubMed

O'Donnell, Kerry; Sutton, Deanna A; Fothergill, Annette; McCarthy, Dora; Rinaldi, Michael G; Brandt, Mary E; Zhang, Ning; Geiser, David M

2008-08-01

Members of the species-rich Fusarium solani species complex (FSSC) are responsible for approximately two-thirds all fusarioses of humans and other animals. In addition, many economically important phytopathogenic species are nested within this complex. Due to their increasing clinical relevance and because most of the human pathogenic and plant pathogenic FSSC lack Latin binomials, we have extended the multilocus haplotype nomenclatural system introduced in a previous study (D. C. Chang, G. B. Grant, K. O'Donnell, K. A. Wannemuehler, J. Noble-Wang, C. Y. Rao, L. M. Jacobson, C. S. Crowell, R. S. Sneed, F. M. T. Lewis, J. K. Schaffzin, M. A. Kainer, C. A. Genese, E. C. Alfonso, D. B. Jones, A. Srinivasan, S. K. Fridkin, and B. J. Park, JAMA 296:953-963, 2006) to all 34 species within the medically important FSSC clade 3 to facilitate global epidemiological studies. The typing scheme is based on polymorphisms in portions of the following three genes: the internal transcribed spacer region and domains D1 plus D2 of the nuclear large-subunit rRNA, the translation elongation factor 1 alpha gene (EF-1alpha), and the second largest subunit of RNA polymerase II gene (RPB2). Of the 251 isolates subjected to multilocus DNA sequence typing, 191 sequence types were differentiated, and these were distributed among three strongly supported clades designated 1, 2, and 3. All of the mycosis-associated isolates were restricted to FSSC clade 3, as previously reported (N. Zhang, K. O'Donnell, D. A. Sutton, F. A Nalim, R. C. Summerbell, A. A. Padhye, and D. M. Geiser, J. Clin. Microbiol. 44:2186-2190, 2006), and these represent at least 20 phylogenetically distinct species. Analyses of the combined DNA sequence data by use of two separate phylogenetic methods yielded the most robust hypothesis of evolutionary relationships and genetic diversity within the FSSC to date. The in vitro activities of 10 antifungals tested against 19 isolates representing 18 species that span the breadth of the FSSC phylogeny show that members of this complex are broadly resistant to these drugs.

Development of a multilocus-based approach for sponge (phylum Porifera) identification: refinement and limitations.

PubMed

Yang, Qi; Franco, Christopher M M; Sorokin, Shirley J; Zhang, Wei

2017-02-02

For sponges (phylum Porifera), there is no reliable molecular protocol available for species identification. To address this gap, we developed a multilocus-based Sponge Identification Protocol (SIP) validated by a sample of 37 sponge species belonging to 10 orders from South Australia. The universal barcode COI mtDNA, 28S rRNA gene (D3-D5), and the nuclear ITS1-5.8S-ITS2 region were evaluated for their suitability and capacity for sponge identification. The highest Bit Score was applied to infer the identity. The reliability of SIP was validated by phylogenetic analysis. The 28S rRNA gene and COI mtDNA performed better than the ITS region in classifying sponges at various taxonomic levels. A major limitation is that the databases are not well populated and possess low diversity, making it difficult to conduct the molecular identification protocol. The identification is also impacted by the accuracy of the morphological classification of the sponges whose sequences have been submitted to the database. Re-examination of the morphological identification further demonstrated and improved the reliability of sponge identification by SIP. Integrated with morphological identification, the multilocus-based SIP offers an improved protocol for more reliable and effective sponge identification, by coupling the accuracy of different DNA markers.

Development of a multilocus-based approach for sponge (phylum Porifera) identification: refinement and limitations

PubMed Central

Yang, Qi; Franco, Christopher M. M.; Sorokin, Shirley J.; Zhang, Wei

2017-01-01

For sponges (phylum Porifera), there is no reliable molecular protocol available for species identification. To address this gap, we developed a multilocus-based Sponge Identification Protocol (SIP) validated by a sample of 37 sponge species belonging to 10 orders from South Australia. The universal barcode COI mtDNA, 28S rRNA gene (D3–D5), and the nuclear ITS1-5.8S-ITS2 region were evaluated for their suitability and capacity for sponge identification. The highest Bit Score was applied to infer the identity. The reliability of SIP was validated by phylogenetic analysis. The 28S rRNA gene and COI mtDNA performed better than the ITS region in classifying sponges at various taxonomic levels. A major limitation is that the databases are not well populated and possess low diversity, making it difficult to conduct the molecular identification protocol. The identification is also impacted by the accuracy of the morphological classification of the sponges whose sequences have been submitted to the database. Re-examination of the morphological identification further demonstrated and improved the reliability of sponge identification by SIP. Integrated with morphological identification, the multilocus-based SIP offers an improved protocol for more reliable and effective sponge identification, by coupling the accuracy of different DNA markers. PMID:28150727

Cryptic Diversity of Malassezia pachydermatis from Healthy and Diseased Domestic Animals.

PubMed

Puig, Laura; Castellá, Gemma; Cabañes, F Javier

2016-10-01

Malassezia pachydermatis is part of the normal cutaneous microbiota of wild and domestic carnivores. However, under certain conditions this yeast can overproliferate and cause several diseases in its host, mainly otitis and dermatitis in dogs. The aim of this study was to conduct a molecular characterization of M. pachydermatis isolates from healthy and diseased domestic animals, in order to assess the molecular diversity and phylogenetic relationship within this species. The large subunit (LSU) and the internal transcribed spacer (ITS) of ribosomal RNA, chitin synthase 2 (CHS2) and β-tubulin genes from sixteen strains isolated from dogs, cats, a goat, a pig and a horse were sequenced. A different number of types of sequences were identified for each target gene, including some types described for the first time. Five sequence types were characterized for the LSU, eleven for the ITS region, nine for CHS2 and eight for β-tubulin. A multilocus analysis was performed including the four genes, and the resulting phylogenetic tree revealed fifteen genotypes. Genotypes were distributed in two well-supported clades. One clade comprised strains isolated from different domestic animals and a strongly supported cluster constituted by strains isolated from cats. The second clade included strains isolated mainly from dogs and an outlier strain isolated from a horse. No apparent association could be observed between the health status of the animal hosts and concrete strains. The multilocus phylogenetic analysis is a useful tool to assess the intraspecific variation within this species and could help understand the ecology, epidemiology and speciation process of M. pachydermatis.

Multilocus sequence analysis of phytopathogenic species of the genus Streptomyces

USDA-ARS?s Scientific Manuscript database

The identification and classification of species within the genus Streptomyces is difficult because there are presently 576 validly described species and this number increases every year. The value of the application of multilocus sequence analysis scheme to the systematics of Streptomyces species h...

Characterisation of nasal Staphylococcus delphini and Staphylococcus pseudintermedius isolates from healthy donkeys in Tunisia.

PubMed

Gharsa, H; Slama, K Ben; Gómez-Sanz, E; Gómez, P; Klibi, N; Zarazaga, M; Boudabous, A; Torres, C

2015-07-01

Staphylococcus intermedius group (SIG) bacteria can colonise the nares of some animals but are also emerging pathogens in humans and animals. To analyse SIG nasal carriage in healthy donkeys destined for food consumption in Tunisia and to characterise recovered isolates. Nasal swabs from 100 healthy donkeys were tested for SIG recovery, and isolates were identified by biochemical and molecular methods. Antimicrobial susceptibility of isolates was tested and detection of antimicrobial resistance and virulence genes was performed. Isolates were typed at the clonal level by multilocus sequence typing and SmaI pulsed-field gel electrophoresis. Staphylococcus delphini and Staphylococcus pseudintermedius (included in SIG) were obtained in 19% and 2% of the tested samples, respectively, and one isolate per sample was characterised. All isolates were meticillin susceptible and mecA negative. Most S. delphini and S. pseudintermedius isolates showed susceptibility to all antimicrobials tested, with the exception of 2 isolates resistant to tetracycline (tet(M) gene) or fusidic acid. The following toxin genes were identified (percentage of isolates): lukS-I (100%), lukF-I (9.5%), siet (100%), se-int (90%), seccanine (19%) and expA (9.5%). Thirteen different pulsed-field gel electrophoresis profiles were identified among the 21 SIG isolates. Additionally, the following 9 different sequence types (STs) were detected by multilocus sequence typing, 6 of them new: ST219 (6 isolates), ST12 (5 isolates), ST220 (3 isolates), ST13, ST50, ST193, ST196, ST218 and ST221 (one isolate each). Staphylococcus delphini and S. pseudintermedius are common nasal colonisers of donkeys, generally susceptible to the antimicrobials tested; nevertheless, these SIG isolates contain virulence genes, including the recently described exfoliative gene (expA) and several enterotoxin genes, with potential implications for public health. This is the first description of S. delphini in Tunisia. The Summary is available in Chinese - see Supporting information. © 2014 EVJ Ltd.

Zinc Resistance within Swine-Associated Methicillin-Resistant Staphylococcus aureus Isolates in the United States Is Associated with Multilocus Sequence Type Lineage

PubMed Central

Hau, Samantha J.; Frana, Timothy; Sun, Jisun; Davies, Peter R.

2017-01-01

ABSTRACT Zinc resistance in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 398 (ST398) is primarily mediated by the czrC gene colocated with the mecA gene, encoding methicillin resistance, within the type V staphylococcal cassette chromosome mec (SCCmec) element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of zinc in feed as an antidiarrheal agent has the potential to contribute to the emergence and spread of methicillin-resistant S. aureus (MRSA) in swine, through increased selection pressure to maintain the SCCmec element in isolates obtained from pigs. In this study, we report the prevalence of the czrC gene and phenotypic zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and U.S. swine-associated LA-MRSA ST398 isolates. We demonstrated that the prevalence of zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact and swine-associated LA-MRSA ST398 isolates, as well as prevalences from previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively, our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance determinants. IMPORTANCE Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is more associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance markers. This information is important for public health professionals, veterinarians, producers, and consumers. PMID:28526788

Zinc Resistance within Swine-Associated Methicillin-Resistant Staphylococcus aureus Isolates in the United States Is Associated with Multilocus Sequence Type Lineage.

PubMed

Hau, Samantha J; Frana, Timothy; Sun, Jisun; Davies, Peter R; Nicholson, Tracy L

2017-08-01

Zinc resistance in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 398 (ST398) is primarily mediated by the czrC gene colocated with the mecA gene, encoding methicillin resistance, within the type V staphylococcal cassette chromosome mec (SCC mec ) element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of zinc in feed as an antidiarrheal agent has the potential to contribute to the emergence and spread of methicillin-resistant S. aureus (MRSA) in swine, through increased selection pressure to maintain the SCC mec element in isolates obtained from pigs. In this study, we report the prevalence of the czrC gene and phenotypic zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and U.S. swine-associated LA-MRSA ST398 isolates. We demonstrated that the prevalence of zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact and swine-associated LA-MRSA ST398 isolates, as well as prevalences from previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively, our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance determinants. IMPORTANCE Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is more associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance markers. This information is important for public health professionals, veterinarians, producers, and consumers. Copyright © 2017 American Society for Microbiology.

Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances.

PubMed

Xu, Jianping

2006-06-01

Microbial ecology examines the diversity and activity of micro-organisms in Earth's biosphere. In the last 20 years, the application of genomics tools have revolutionized microbial ecological studies and drastically expanded our view on the previously underappreciated microbial world. This review first introduces the basic concepts in microbial ecology and the main genomics methods that have been used to examine natural microbial populations and communities. In the ensuing three specific sections, the applications of the genomics in microbial ecological research are highlighted. The first describes the widespread application of multilocus sequence typing and representational difference analysis in studying genetic variation within microbial species. Such investigations have identified that migration, horizontal gene transfer and recombination are common in natural microbial populations and that microbial strains can be highly variable in genome size and gene content. The second section highlights and summarizes the use of four specific genomics methods (phylogenetic analysis of ribosomal RNA, DNA-DNA re-association kinetics, metagenomics, and micro-arrays) in analysing the diversity and potential activity of microbial populations and communities from a variety of terrestrial and aquatic environments. Such analyses have identified many unexpected phylogenetic lineages in viruses, bacteria, archaea, and microbial eukaryotes. Functional analyses of environmental DNA also revealed highly prevalent, but previously unknown, metabolic processes in natural microbial communities. In the third section, the ecological implications of sequenced microbial genomes are briefly discussed. Comparative analyses of prokaryotic genomic sequences suggest the importance of ecology in determining microbial genome size and gene content. The significant variability in genome size and gene content among strains and species of prokaryotes indicate the highly fluid nature of prokaryotic genomes, a result consistent with those from multilocus sequence typing and representational difference analyses. The integration of various levels of ecological analyses coupled to the application and further development of high throughput technologies are accelerating the pace of discovery in microbial ecology.

Refugial isolation and divergence in the Narrowheaded Gartersnake species complex (Thamnophis rufipunctatus) as revealed by multilocus DNA sequence data

USGS Publications Warehouse

Wood, Dustin A.; Vandergast, A.G.; Espinal, A. Lemos; Fisher, R.N.; Holycross, A.T.

2011-01-01

Glacial–interglacial cycles of the Pleistocene are hypothesized as one of the foremost contributors to biological diversification. This is especially true for cold-adapted montane species, where range shifts have had a pronounced effect on population-level divergence. Gartersnakes of the Thamnophis rufipunctatus species complex are restricted to cold headwater streams in the highlands of the Sierra Madre Occidental and southwestern USA. We used coalescent and multilocus phylogenetic approaches to test whether genetic diversification of this montane-restricted species complex is consistent with two prevailing models of range fluctuation for species affected by Pleistocene climate changes. Our concatenated nuDNA and multilocus species analyses recovered evidence for the persistence of multiple lineages that are restricted geographically, despite a mtDNA signature consistent with either more recent connectivity (and introgression) or recent expansion (and incomplete lineage sorting). Divergence times estimated using a relaxed molecular clock and fossil calibrations fall within the Late Pleistocene, and zero gene flow scenarios among current geographically isolated lineages could not be rejected. These results suggest that increased climate shifts in the Late Pleistocene have driven diversification and current range retraction patterns and that the differences between markers reflect the stochasticity of gene lineages (i.e. ancestral polymorphism) rather than gene flow and introgression. These results have important implications for the conservation of T. rufipunctatus (sensu novo), which is restricted to two drainage systems in the southwestern US and has undergone a recent and dramatic decline.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

PubMed

Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

Genetic diversity of Flavobacterium psychrophilum isolates from three Oncorhynchus spp. in the United States, as revealed by multilocus sequence typing

USDA-ARS?s Scientific Manuscript database

Flavobacterium psychrophilum is an important pathogen of salmonids worldwide. Multilocus sequence typing (MLST) has identified a recombinogenic population structure from which emerged a few epidemic clonal complexes particularly threatening for salmonid aquaculture. To date, MLST genotypes for this ...

Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics.

PubMed

Bybee, Seth M; Bracken-Grissom, Heather; Haynes, Benjamin D; Hermansen, Russell A; Byers, Robert L; Clement, Mark J; Udall, Joshua A; Wilcox, Edward R; Crandall, Keith A

2011-01-01

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach.

Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics

PubMed Central

Bybee, Seth M.; Bracken-Grissom, Heather; Haynes, Benjamin D.; Hermansen, Russell A.; Byers, Robert L.; Clement, Mark J.; Udall, Joshua A.; Wilcox, Edward R.; Crandall, Keith A.

2011-01-01

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach. PMID:22002916

Coagulase-Negative Staphylococci in Human Milk From Mothers of Preterm Compared With Term Neonates.

PubMed

Soeorg, Hiie; Metsvaht, Tuuli; Eelmäe, Imbi; Metsvaht, Hanna Kadri; Treumuth, Sirli; Merila, Mirjam; Ilmoja, Mari-Liis; Lutsar, Irja

2017-05-01

Human milk is the preferred nutrition for neonates and a source of bacteria. Research aim: The authors aimed to characterize the molecular epidemiology and genetic content of staphylococci in the human milk of mothers of preterm and term neonates. Staphylococci were isolated once per week in the 1st month postpartum from the human milk of mothers of 20 healthy term and 49 preterm neonates hospitalized in the neonatal intensive care unit. Multilocus variable-number tandem-repeats analysis and multilocus sequence typing were used. The presence of the mecA gene, icaA gene of the ica-operon, IS 256, and ACME genetic elements was determined by PCR. The human milk of mothers of preterm compared with term neonates had higher counts of staphylococci but lower species diversity. The human milk of mothers of preterm compared with term neonates more often contained Staphylococcus epidermidis mecA (32.7% vs. 2.6%), icaA (18.8% vs. 6%), IS 256 (7.9% vs. 0.9%), and ACME (15.4% vs. 5.1%), as well as Staphylococcus haemolyticus mecA (90.5% vs. 10%) and IS 256 (61.9% vs. 10%). The overall distribution of multilocus variable-number tandem-repeats analysis (MLVA) types and sequence types was similar between the human milk of mothers of preterm and term neonates, but a few mecA-IS 256-positive MLVA types colonized only mothers of preterm neonates. Maternal hospitalization within 1 month postpartum and the use of an arterial catheter or antibacterial treatment in the neonate increased the odds of harboring mecA-positive staphylococci in human milk. Limiting exposure of mothers of preterm neonates to the hospital could prevent human milk colonization with more pathogenic staphylococci.

Defining a Core Genome Multilocus Sequence Typing Scheme for the Global Epidemiology of Vibrio parahaemolyticus

PubMed Central

Jolley, Keith A.; Reed, Elizabeth; Martinez-Urtaza, Jaime

2017-01-01

ABSTRACT Vibrio parahaemolyticus is an important human foodborne pathogen whose transmission is associated with the consumption of contaminated seafood, with a growing number of infections reported over recent years worldwide. A multilocus sequence typing (MLST) database for V. parahaemolyticus was created in 2008, and a large number of clones have been identified, causing severe outbreaks worldwide (sequence type 3 [ST3]), recurrent outbreaks in certain regions (e.g., ST36), or spreading to other regions where they are nonendemic (e.g., ST88 or ST189). The current MLST scheme uses sequences of 7 genes to generate an ST, which results in a powerful tool for inferring the population structure of this pathogen, although with limited resolution, especially compared to pulsed-field gel electrophoresis (PFGE). The application of whole-genome sequencing (WGS) has become routine for trace back investigations, with core genome MLST (cgMLST) analysis as one of the most straightforward ways to explore complex genomic data in an epidemiological context. Therefore, there is a need to generate a new, portable, standardized, and more advanced system that provides higher resolution and discriminatory power among V. parahaemolyticus strains using WGS data. We sequenced 92 V. parahaemolyticus genomes and used the genome of strain RIMD 2210633 as a reference (with a total of 4,832 genes) to determine which genes were suitable for establishing a V. parahaemolyticus cgMLST scheme. This analysis resulted in the identification of 2,254 suitable core genes for use in the cgMLST scheme. To evaluate the performance of this scheme, we performed a cgMLST analysis of 92 newly sequenced genomes, plus an additional 142 strains with genomes available at NCBI. cgMLST analysis was able to distinguish related and unrelated strains, including those with the same ST, clearly showing its enhanced resolution over conventional MLST analysis. It also distinguished outbreak-related from non-outbreak-related strains within the same ST. The sequences obtained from this work were deposited and are available in the public database (http://pubmlst.org/vparahaemolyticus). The application of this cgMLST scheme to the characterization of V. parahaemolyticus strains provided by different laboratories from around the world will reveal the global picture of the epidemiology, spread, and evolution of this pathogen and will become a powerful tool for outbreak investigations, allowing for the unambiguous comparison of strains with global coverage. PMID:28330888

Multilocus sequence analysis for assessment of phylogenetic diversity and biogeography in Thalassospira bacteria from diverse marine environments.

PubMed

Lai, Qiliang; Liu, Yang; Yuan, Jun; Du, Juan; Wang, Liping; Sun, Fengqin; Shao, Zongze

2014-01-01

Thalassospira bacteria are widespread and have been isolated from various marine environments. Less is known about their genetic diversity and biogeography, as well as their role in marine environments, many of them cannot be discriminated merely using the 16S rRNA gene. To address these issues, in this report, the phylogenetic analysis of 58 strains from seawater and deep sea sediments were carried out using the multilocus sequence analysis (MLSA) based on acsA, aroE, gyrB, mutL, rpoD and trpB genes, and the DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) based on genome sequences. The MLSA analysis demonstrated that the 58 strains were clearly separated into 15 lineages, corresponding to seven validly described species and eight potential novel species. The DDH and ANI values further confirmed the validity of the MLSA analysis and eight potential novel species. The MLSA interspecies gap of the genus Thalassospira was determined to be 96.16-97.12% sequence identity on the basis of the combined analyses of the DDH and MLSA, while the ANIm interspecies gap was 95.76-97.20% based on the in silico DDH analysis. Meanwhile, phylogenetic analyses showed that the Thalassospira bacteria exhibited distribution pattern to a certain degree according to geographic regions. Moreover, they clustered together according to the habitats depth. For short, the phylogenetic analyses and biogeography of the Thalassospira bacteria were systematically investigated for the first time. These results will be helpful to explore further their ecological role and adaptive evolution in marine environments.

Multilocus Sequence Analysis for Assessment of Phylogenetic Diversity and Biogeography in Thalassospira Bacteria from Diverse Marine Environments

PubMed Central

Yuan, Jun; Du, Juan; Wang, Liping; Sun, Fengqin; Shao, Zongze

2014-01-01

Thalassospira bacteria are widespread and have been isolated from various marine environments. Less is known about their genetic diversity and biogeography, as well as their role in marine environments, many of them cannot be discriminated merely using the 16S rRNA gene. To address these issues, in this report, the phylogenetic analysis of 58 strains from seawater and deep sea sediments were carried out using the multilocus sequence analysis (MLSA) based on acsA, aroE, gyrB, mutL, rpoD and trpB genes, and the DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) based on genome sequences. The MLSA analysis demonstrated that the 58 strains were clearly separated into 15 lineages, corresponding to seven validly described species and eight potential novel species. The DDH and ANI values further confirmed the validity of the MLSA analysis and eight potential novel species. The MLSA interspecies gap of the genus Thalassospira was determined to be 96.16–97.12% sequence identity on the basis of the combined analyses of the DDH and MLSA, while the ANIm interspecies gap was 95.76–97.20% based on the in silico DDH analysis. Meanwhile, phylogenetic analyses showed that the Thalassospira bacteria exhibited distribution pattern to a certain degree according to geographic regions. Moreover, they clustered together according to the habitats depth. For short, the phylogenetic analyses and biogeography of the Thalassospira bacteria were systematically investigated for the first time. These results will be helpful to explore further their ecological role and adaptive evolution in marine environments. PMID:25198177

Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Genome-Wide Typing of Clostridium difficile.

PubMed

Bletz, Stefan; Janezic, Sandra; Harmsen, Dag; Rupnik, Maja; Mellmann, Alexander

2018-06-01

Clostridium difficile , recently renamed Clostridioides difficile , is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for C. difficile Initially, we determined the breadth of the C. difficile population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with C. difficile clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping ( n = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole C. difficile population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange. Copyright © 2018 American Society for Microbiology.

Core Genome Multilocus Sequence Typing Scheme for High-Resolution Typing of Enterococcus faecium

PubMed Central

de Been, Mark; Pinholt, Mette; Top, Janetta; Bletz, Stefan; van Schaik, Willem; Brouwer, Ellen; Rogers, Malbert; Kraat, Yvette; Bonten, Marc; Corander, Jukka; Westh, Henrik; Harmsen, Dag

2015-01-01

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism (SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks. PMID:26400782

Core Genome Multilocus Sequence Typing Scheme for High- Resolution Typing of Enterococcus faecium.

PubMed

de Been, Mark; Pinholt, Mette; Top, Janetta; Bletz, Stefan; Mellmann, Alexander; van Schaik, Willem; Brouwer, Ellen; Rogers, Malbert; Kraat, Yvette; Bonten, Marc; Corander, Jukka; Westh, Henrik; Harmsen, Dag; Willems, Rob J L

2015-12-01

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism(SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks.

Novel type of VanB2 teicoplanin-resistant hospital-associated Enterococcus faecium.

PubMed

Santona, Antonella; Paglietti, Bianca; Al-Qahtani, Ahmed A; Bohol, Marie Fe F; Senok, Abiola; Deligios, Massimo; Rubino, Salvatore; Al-Ahdal, Mohammed N

2014-08-01

Seven high-risk clones of vancomycin-resistant Enterococcus faecium (VREF) belonging to clonal complex 17 were identified using multilocus sequence typing (MLST) among clinical isolates from Saudi Arabia. Among these isolates, a new hospital-associated sequence type (ST795), VanB(2)-type teicoplanin-resistant strain was detected. Its unusual phenotype resulted from a new combination of mutations in the ddl, vanS and vanW genes, which confirmed the trend of evolution in VanB-type resistance. Furthermore, characteristics of adaptation and persistence in the hospital environment of ST795 were emphasised by the presence of genes and clusters recognised to be specific for hospital-associated VREF. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Dissemination of IMP-6 metallo-β-lactamase-producing Pseudomonas aeruginosa sequence type 235 in Korea.

PubMed

Seok, Yoonmi; Bae, Il Kwon; Jeong, Seok Hoon; Kim, Soo Hyun; Lee, Hyukmin; Lee, Kyungwon

2011-12-01

To investigate the epidemiological traits of Pseudomonas aeruginosa clinical isolates producing metallo-β-lactamases (MBLs) in Korea. A total of 386 non-duplicate P. aeruginosa clinical isolates were collected from Korea in 2009. Detection of MBL genes was performed by PCR. The genetic organization of class 1 integrons carrying the MBL gene cassette was investigated by PCR mapping and sequencing. The epidemiological relationships of the isolates were investigated by multilocus sequence typing and PFGE. Of 386 P. aeruginosa isolates, 30 (7.8%) isolates carried the bla(IMP-6) gene and 1 (0.3%) isolate carried the bla(VIM-2) gene. A probe specific for the bla(IMP-6) gene was hybridized to an ∼950 kbp I-CeuI-macrorestriction fragment from all 30 isolates and a probe specific for the bla(VIM-2) gene also hybridized to an ∼500 kbp I-CeuI-macrorestriction fragment from 1 isolate (BDC10). All 31 MBL-producing isolates shared an identical sequence type (ST), ST235, and they carried the same bla(OXA-50) allelic type, bla(OXA-50g). All MBL-producing isolates showed similar XbaI-macrorestriction patterns (similarity >85%), irrespective of MBL genotype. P. aeruginosa ST235 carrying the chromosomally located bla(IMP-6) gene is widely disseminated in Korea.

Taxonomic evaluation of putative Streptomyces scabiei strains held in the ARS (NRRL) Culture Collection using multi-locus sequence analysis

USDA-ARS?s Scientific Manuscript database

Multi-locus sequence analysis has been demonstrated to be a useful tool for identification of Streptomyces species and was previously applied to phylogenetically differentiate the type strains of species pathogenic on potatoes (Solanum tuberosum L.). The ARS Culture Collection (NRRL) contains 43 str...

Genetic Diversity among Clostridium botulinum Strains Harboring bont/A2 and bont/A3 Genes

PubMed Central

Raphael, Brian H.; Joseph, Lavin A.; Meno, Sarah R.; Fernández, Rafael A.; Maslanka, Susan E.

2012-01-01

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE. PMID:23042179

Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains.

PubMed

Macheras, Edouard; Roux, Anne-Laure; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby; Bodmer, Thomas; Cambau, Emmanuelle; Gaillard, Jean-Louis; Heym, Beate

2011-02-01

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Multilocus Sequence Analysis and rpoB Sequencing of Mycobacterium abscessus (Sensu Lato) Strains▿

PubMed Central

Macheras, Edouard; Roux, Anne-Laure; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby; Bodmer, Thomas; Cambau, Emmanuelle; Gaillard, Jean-Louis; Heym, Beate

2011-01-01

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536T, M. massiliense CIP 108297T, and M. bolletii CIP 108541T) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies. PMID:21106786

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii.

PubMed

Tanigawa, Kana; Watanabe, Koichi

2011-03-01

Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.

Adherent and Invasive Escherichia coli Is Associated with Granulomatous Colitis in Boxer Dogs

PubMed Central

Simpson, Kenneth W.; Dogan, Belgin; Rishniw, Mark; Goldstein, Richard E.; Klaessig, Suzanne; McDonough, Patrick L.; German, Alex J.; Yates, Robin M.; Russell, David G.; Johnson, Susan E.; Berg, Douglas E.; Harel, Josee; Bruant, Guillaume; McDonough, Sean P.; Schukken, Ynte H.

2006-01-01

The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a “splash”-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohn's disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohn's disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile. PMID:16861666

Population Genetic Analyses of the Fungal Pathogen Colletotrichum fructicola on Tea-Oil Trees in China

PubMed Central

Li, He; Zhou, Guo-Ying; Liu, Jun-Ang; Xu, Jianping

2016-01-01

The filamentous fungus Colletotrichum fructicola is found in all five continents and is capable of causing severe diseases in a number of economically important plants such as avocado, fig, cocoa, pear, and tea-oil trees. However, almost nothing is known about its patterns of genetic variation and epidemiology on any of its host plant species. Here we analyzed 167 isolates of C. fructicola obtained from the leaves of tea-oil tree Camellia oleifera at 15 plantations in seven Chinese provinces. Multilocus sequence typing was conducted for all isolates based on DNA sequences at fragments of four genes: the internal transcribed spacers of the nuclear ribosomal RNA gene cluster (539 bp), calmodulin (633 bp), glutamine synthetase (711 bp), and glyceraldehyde-3-phosphate dehydrogenase (190 bp), yielding 3.52%, 0.63%, 8.44%, and 7.89% of single nucleotide polymorphic sites and resulting in 15, 5, 12 and 11 alleles respectively at the four gene fragments in the total sample. The combined allelic information from all four loci identified 53 multilocus genotypes with the most frequent represented by 21 isolates distributed in eight tea-oil plantations in three provinces, consistent with long-distance clonal dispersal. However, despite evidence for clonal dispersal, statistically significant genetic differentiation among geographic populations was detected. In addition, while no evidence of recombination was found within any of the four gene fragments, signatures of recombination were found among the four gene fragments in most geographic populations, consistent with sexual mating of this species in nature. Our study provides the first insights into the population genetics and epidemiology of the important plant fungal pathogen C. fructicola. PMID:27299731

Multilocus sequence typing of Xylella fastidiosa causing Pierce's disease and oleander leaf scorch in the United States.

PubMed

Yuan, Xiaoli; Morano, Lisa; Bromley, Robin; Spring-Pearson, Senanu; Stouthamer, Richard; Nunney, Leonard

2010-06-01

Using a modified multilocus sequence typing (MLST) scheme for the bacterial plant pathogen Xylella fastidiosa based on the same seven housekeeping genes employed in a previously published MLST, we studied the genetic diversity of two subspecies, X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi, which cause Pierce's disease and oleander leaf scorch, respectively. Typing of 85 U.S. isolates (plus one from northern Mexico) of X. fastidiosa subsp. fastidiosa from 15 different plant hosts and 21 isolates of X. fastidiosa subsp. sandyi from 4 different hosts in California and Texas supported their subspecific status. Analysis using the MLST genes plus one cell-surface gene showed no significant genetic differentiation based on geography or host plant within either subspecies. Two cases of homologous recombination (with X. fastidiosa subsp. multiplex, the third U.S. subspecies) were detected in X. fastidiosa subsp. fastidiosa. Excluding recombination, MLST site polymorphism in X. fastidiosa subsp. fastidiosa (0.048%) and X. fastidiosa subsp. sandyi (0.000%) was substantially lower than in X. fastidiosa subsp. multiplex (0.240%), consistent with the hypothesis that X. fastidiosa subspp. fastidiosa and sandyi were introduced into the United States (probably just prior to 1880 and 1980, respectively). Using whole-genome analysis, we showed that MLST is more effective at genetic discrimination at the specific and subspecific level than other typing methods applied to X. fastidiosa. Moreover, MLST is the only technique effective in detecting recombination.

Genomic insights into the taxonomic status of the Bacillus cereus group

PubMed Central

Liu, Yang; Lai, Qiliang; Göker, Markus; Meier-Kolthoff, Jan P.; Wang, Meng; Sun, Yamin; Wang, Lei; Shao, Zongze

2015-01-01

The identification and phylogenetic relationships of bacteria within the Bacillus cereus group are controversial. This study aimed at determining the taxonomic affiliations of these strains using the whole-genome sequence-based Genome BLAST Distance Phylogeny (GBDP) approach. The GBDP analysis clearly separated 224 strains into 30 clusters, representing eleven known, partially merged species and accordingly 19–20 putative novel species. Additionally, 16S rRNA gene analysis, a novel variant of multi-locus sequence analysis (nMLSA) and screening of virulence genes were performed. The 16S rRNA gene sequence was not sufficient to differentiate the bacteria within this group due to its high conservation. The nMLSA results were consistent with GBDP. Moreover, a fast typing method was proposed using the pycA gene, and where necessary, the ccpA gene. The pXO plasmids and cry genes were widely distributed, suggesting little correlation with the phylogenetic positions of the host bacteria. This might explain why classifications based on virulence characteristics proved unsatisfactory in the past. In summary, this is the first large-scale and systematic study of the taxonomic status of the bacteria within the B. cereus group using whole-genome sequences, and is likely to contribute to further insights into their pathogenicity, phylogeny and adaptation to diverse environments. PMID:26373441

Heavy ion mutagenesis: linear energy transfer effects and genetic linkage

NASA Technical Reports Server (NTRS)

Kronenberg, A.; Gauny, S.; Criddle, K.; Vannais, D.; Ueno, A.; Kraemer, S.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

1995-01-01

We have characterized a series of 69 independent mutants at the endogenous hprt locus of human TK6 lymphoblasts and over 200 independent S1-deficient mutants of the human x hamster hybrid cell line AL arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/microns). We find that large deletions are common. The entire hprt gene (> 44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entire hprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency of hprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of the hprt locus to mutation is likely due to tight linkage to a gene that is required for viability.

Reclassification of Borrelia spp. isolated in South Korea using Multilocus Sequence Typing.

PubMed

Park, Kyung-Hee; Choi, Yeon-Joo; Kim, Jeoungyeon; Park, Hye-Jin; Song, Dayoung; Jang, Won-Jong

2018-05-31

Using Borrelia isolated from South Korea, we evaluated by MLST and three intergenic genes (16S rRNA, ospA, and 5S-23S IGS) typing to analyze the relationship between host and vector and molecular background. Using the MLST analysis, we identified B. afzelii, B. yangtzensis, B. garinii, and B. bavariensis. This study was first report of the identification of B. yangtzensis using the MLST in South Korea.

Characterization of Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast of Mandarin in Montenegro.

PubMed

Ivanović, Žarko; Perović, Tatjana; Popović, Tatjana; Blagojević, Jovana; Trkulja, Nenad; Hrnčić, Snježana

2017-02-01

Citrus blast caused by bacterium Pseudomonas syringae is a very important disease of citrus occuring in many areas of the world, but with few data about genetic structure of the pathogen involved. Considering the above fact, this study reports genetic characterization of 43 P. syringae isolates obtained from plant tissue displaying citrus blast symptoms on mandarin ( Citrus reticulata ) in Montenegro, using multilocus sequence analysis of gyrB , rpoD , and gap1 gene sequences. Gene sequences from a collection of 54 reference pathotype strains of P. syringae from the Plant Associated and Environmental Microbes Database (PAMDB) was used to establish a genetic relationship with our isolates obtained from mandarin. Phylogenetic analyses of gyrB , rpoD , and gap1 gene sequences showed that P. syringae pv. syringae causes citrus blast in mandarin in Montenegro, and belongs to genomospecies 1. Genetic homogeneity of isolates suggested that the Montenegrian population might be clonal which indicates a possible common source of infection. These findings may assist in further epidemiological studies of this pathogen and for determining mandarin breeding strategies for P. syringae control.

Characterization of Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast of Mandarin in Montenegro

PubMed Central

Ivanović, Žarko; Perović, Tatjana; Popović, Tatjana; Blagojević, Jovana; Trkulja, Nenad; Hrnčić, Snježana

2017-01-01

Citrus blast caused by bacterium Pseudomonas syringae is a very important disease of citrus occuring in many areas of the world, but with few data about genetic structure of the pathogen involved. Considering the above fact, this study reports genetic characterization of 43 P. syringae isolates obtained from plant tissue displaying citrus blast symptoms on mandarin (Citrus reticulata) in Montenegro, using multilocus sequence analysis of gyrB, rpoD, and gap1 gene sequences. Gene sequences from a collection of 54 reference pathotype strains of P. syringae from the Plant Associated and Environmental Microbes Database (PAMDB) was used to establish a genetic relationship with our isolates obtained from mandarin. Phylogenetic analyses of gyrB, rpoD, and gap1 gene sequences showed that P. syringae pv. syringae causes citrus blast in mandarin in Montenegro, and belongs to genomospecies 1. Genetic homogeneity of isolates suggested that the Montenegrian population might be clonal which indicates a possible common source of infection. These findings may assist in further epidemiological studies of this pathogen and for determining mandarin breeding strategies for P. syringae control. PMID:28167885

Streptococcus oriloxodontae sp. nov., isolated from the oral cavities of elephants.

PubMed

Shinozaki-Kuwahara, Noriko; Saito, Masanori; Hirasawa, Masatomo; Takada, Kazuko

2014-11-01

Two strains were isolated from oral cavity samples of healthy elephants. The isolates were Gram-positive, catalase-negative, coccus-shaped organisms that were tentatively identified as a streptococcal species based on the results of biochemical tests. Comparative 16S rRNA gene sequence analysis suggested classification of these organisms in the genus Streptococcus with Streptococcus criceti ATCC 19642(T) and Streptococcus orisuis NUM 1001(T) as their closest phylogenetic neighbours with 98.2 and 96.9% gene sequence similarity, respectively. When multi-locus sequence analysis using four housekeeping genes, groEL, rpoB, gyrB and sodA, was carried out, similarity of concatenated sequences of the four housekeeping genes from the new isolates and Streptococcus mutans was 89.7%. DNA-DNA hybridization experiments suggested that the new isolates were distinct from S. criceti and other species of the genus Streptococcus. On the basis of genotypic and phenotypic differences, it is proposed that the novel isolates are classified in the genus Streptococcus as representatives of Streptococcus oriloxodontae sp. nov. The type strain of S. oriloxodontae is NUM 2101(T) ( =JCM 19285(T) =DSM 27377(T)). © 2014 IUMS.

Trichomonas vaginalis Metronidazole Resistance Is Associated with Single Nucleotide Polymorphisms in the Nitroreductase Genes ntr4Tv and ntr6Tv

PubMed Central

Paulish-Miller, Teresa E.; Augostini, Peter; Schuyler, Jessica A.; Smith, William L.; Mordechai, Eli; Adelson, Martin E.; Gygax, Scott E.; Secor, William E.

2014-01-01

Metronidazole resistance in the sexually transmitted parasite Trichomonas vaginalis is a problematic public health issue. We have identified single nucleotide polymorphisms (SNPs) in two nitroreductase genes (ntr4Tv and ntr6Tv) associated with resistance. These SNPs were associated with one of two distinct T. vaginalis populations identified by multilocus sequence typing, yet one SNP (ntr6Tv A238T), which results in a premature stop codon, was associated with resistance independent of population structure and may be of diagnostic value. PMID:24550324

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia

PubMed Central

2014-01-01

Background Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Results Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Conclusions Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics. PMID:24912963

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

PubMed

Harrison, Lucas B; Hanson, Nancy D

2017-06-01

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Copyright © 2017 Harrison and Hanson.

Multilocus sequence typing (MLST) for lineage assignment and high resolution diversity studies in Trypanosoma cruzi.

PubMed

Yeo, Matthew; Mauricio, Isabel L; Messenger, Louisa A; Lewis, Michael D; Llewellyn, Martin S; Acosta, Nidia; Bhattacharyya, Tapan; Diosque, Patricio; Carrasco, Hernan J; Miles, Michael A

2011-06-01

Multilocus sequence typing (MLST) is a powerful and highly discriminatory method for analysing pathogen population structure and epidemiology. Trypanosoma cruzi, the protozoan agent of American trypanosomiasis (Chagas disease), has remarkable genetic and ecological diversity. A standardised MLST protocol that is suitable for assignment of T. cruzi isolates to genetic lineage and for higher resolution diversity studies has not been developed. We have sequenced and diplotyped nine single copy housekeeping genes and assessed their value as part of a systematic MLST scheme for T. cruzi. A minimum panel of four MLST targets (Met-III, RB19, TcGPXII, and DHFR-TS) was shown to provide unambiguous assignment of isolates to the six known T. cruzi lineages (Discrete Typing Units, DTUs TcI-TcVI). In addition, we recommend six MLST targets (Met-II, Met-III, RB19, TcMPX, DHFR-TS, and TR) for more in depth diversity studies on the basis that diploid sequence typing (DST) with this expanded panel distinguished 38 out of 39 reference isolates. Phylogenetic analysis implies a subdivision between North and South American TcIV isolates. Single Nucleotide Polymorphism (SNP) data revealed high levels of heterozygosity among DTUs TcI, TcIII, TcIV and, for three targets, putative corresponding homozygous and heterozygous loci within DTUs TcI and TcIII. Furthermore, individual gene trees gave incongruent topologies at inter- and intra-DTU levels, inconsistent with a model of strict clonality. We demonstrate the value of systematic MLST diplotyping for describing inter-DTU relationships and for higher resolution diversity studies of T. cruzi, including presence of recombination events. The high levels of heterozygosity will facilitate future population genetics analysis based on MLST haplotypes.

Genetic variation among Flavobacterium psychrophilum isolates from wild and farmed salmonids in Norway and Chile.

PubMed

Apablaza, P; Løland, A D; Brevik, Ø J; Ilardi, P; Battaglia, J; Nylund, A

2013-04-01

To aim of the study was to describe the genetic relationship between isolates of Flavobacterium psychrophilum with a main emphasis of samples from Chile and Norway. The isolates have been obtained from farmed salmonids in Norway and Chile, and from wild salmonids in Norway, but isolates from North America and European countries are also included in the analysis. The study is based on phylogenetic analysis of 16S rRNA and seven housekeeping genes (HG), gyrB, atpA, dnaK, trpB, fumC, murG and tuf, and the use of a multilocus sequence typing (MLST) system, based on nucleotide polymorphism in the HG, as an alternative to the phylogenies. The variation within the selected genes was limited, and the phylogenetic analysis gave little resolution between the isolates. The MLST gave a much better resolution resulting in 53 sequence types where the same sequences types could be found in Chile, North America and European countries, and in different host species. Multilocus sequence typing give a relatively good separation of different isolates of Fl. psychrophilum and show that there are no distinct geographical or host-specific isolates in the studied material from Chile, North America and Europe. Nor was it possible to separate between isolates from ulcers and systemic infections vs isolates from the surface of healthy salmonids. This study shows a wide geographical distribution of Fl. psychrophilum, indicating that the bacterium has a large potential for transmission over long distances, and between different salmonid hosts species. This knowledge will be important for future management of salmonids diseases connected to Fl. psychrophilum. © 2013 The Society for Applied Microbiology.

Microbial genomic taxonomy

PubMed Central

2013-01-01

A need for a genomic species definition is emerging from several independent studies worldwide. In this commentary paper, we discuss recent studies on the genomic taxonomy of diverse microbial groups and a unified species definition based on genomics. Accordingly, strains from the same microbial species share >95% Average Amino Acid Identity (AAI) and Average Nucleotide Identity (ANI), >95% identity based on multiple alignment genes, <10 in Karlin genomic signature, and > 70% in silico Genome-to-Genome Hybridization similarity (GGDH). Species of the same genus will form monophyletic groups on the basis of 16S rRNA gene sequences, Multilocus Sequence Analysis (MLSA) and supertree analysis. In addition to the established requirements for species descriptions, we propose that new taxa descriptions should also include at least a draft genome sequence of the type strain in order to obtain a clear outlook on the genomic landscape of the novel microbe. The application of the new genomic species definition put forward here will allow researchers to use genome sequences to define simultaneously coherent phenotypic and genomic groups. PMID:24365132

Listeria monocytogenes sequence type 1 is predominant in ruminant rhombencephalitis

PubMed Central

Dreyer, Margaux; Aguilar-Bultet, Lisandra; Rupp, Sebastian; Guldimann, Claudia; Stephan, Roger; Schock, Alexandra; Otter, Arthur; Schüpbach, Gertraud; Brisse, Sylvain; Lecuit, Marc; Frey, Joachim; Oevermann, Anna

2016-01-01

Listeria (L.) monocytogenes is an opportunistic pathogen causing life-threatening infections in diverse mammalian species including humans and ruminants. As little is known on the link between strains and clinicopathological phenotypes, we studied potential strain-associated virulence and organ tropism in L. monocytogenes isolates from well-defined ruminant cases of clinical infections and the farm environment. The phylogeny of isolates and their virulence-associated genes were analyzed by multilocus sequence typing (MLST) and sequence analysis of virulence-associated genes. Additionally, a panel of representative isolates was subjected to in vitro infection assays. Our data suggest the environmental exposure of ruminants to a broad range of strains and yet the strong association of sequence type (ST) 1 from clonal complex (CC) 1 with rhombencephalitis, suggesting increased neurotropism of ST1 in ruminants, which is possibly related to its hypervirulence. This study emphasizes the importance of considering clonal background of L. monocytogenes isolates in surveillance, epidemiological investigation and disease control. PMID:27848981

Genomic Definition of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae Clonal Groups

PubMed Central

Bialek-Davenet, Suzanne; Criscuolo, Alexis; Ailloud, Florent; Passet, Virginie; Jones, Louis; Delannoy-Vieillard, Anne-Sophie; Garin, Benoit; Le Hello, Simon; Arlet, Guillaume; Nicolas-Chanoine, Marie-Hélène; Decré, Dominique

2014-01-01

Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected. PMID:25341126

Genes with minimal phylogenetic information are problematic for coalescent analyses when gene tree estimation is biased.

PubMed

Xi, Zhenxiang; Liu, Liang; Davis, Charles C

2015-11-01

The development and application of coalescent methods are undergoing rapid changes. One little explored area that bears on the application of gene-tree-based coalescent methods to species tree estimation is gene informativeness. Here, we investigate the accuracy of these coalescent methods when genes have minimal phylogenetic information, including the implementation of the multilocus bootstrap approach. Using simulated DNA sequences, we demonstrate that genes with minimal phylogenetic information can produce unreliable gene trees (i.e., high error in gene tree estimation), which may in turn reduce the accuracy of species tree estimation using gene-tree-based coalescent methods. We demonstrate that this problem can be alleviated by sampling more genes, as is commonly done in large-scale phylogenomic analyses. This applies even when these genes are minimally informative. If gene tree estimation is biased, however, gene-tree-based coalescent analyses will produce inconsistent results, which cannot be remedied by increasing the number of genes. In this case, it is not the gene-tree-based coalescent methods that are flawed, but rather the input data (i.e., estimated gene trees). Along these lines, the commonly used program PhyML has a tendency to infer one particular bifurcating topology even though it is best represented as a polytomy. We additionally corroborate these findings by analyzing the 183-locus mammal data set assembled by McCormack et al. (2012) using ultra-conserved elements (UCEs) and flanking DNA. Lastly, we demonstrate that when employing the multilocus bootstrap approach on this 183-locus data set, there is no strong conflict between species trees estimated from concatenation and gene-tree-based coalescent analyses, as has been previously suggested by Gatesy and Springer (2014). Copyright © 2015 Elsevier Inc. All rights reserved.

Multilocus sequence analysis of Echinococcus granulosus strains isolated from humans and animals in Iran.

PubMed

Nikmanesh, Bahram; Mirhendi, Hossein; Mahmoudi, Shahram; Rokni, Mohammad Bagher

2017-12-01

Echinococcus granulosus is now considered a complex consisting of at least four species and ten genotypes. Different molecular targets have been described for molecular characterization of E. granulosus; however, in almost all studies only one or two of the targets have been used, and only limited data is available on the utilization of multiple loci. Therefore, we investigated the genetic diversity among 64 strains isolated from 138 cyst specimens of human and animal isolates, using a set of nuclear and mitochondrial genes; i.e., cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), ATPase subunit 6 (atp6), 12S rRNA (12S), and Actin II (act II). In comparison to the use of molecular reference targets (nad1 + cox1), using singular target (act II or 12S or atp6) yielded lower discriminatory power. Act II and 12S genes could accurately discriminate the G6 genotype, but they were not able to differentiate between G1 and G3 genotypes. As the G1 and G3 genotypes belong to the E. granulosus sensu stricto, low intra-species variation was observed for act II and 12S. The atp6 gene could identify the G3 genotype but could not differentiate G6 and G1 genotypes. Using concatenated sequence of five genes (cox1 + nad1 + atp6 + 12S + act II), genotypes were identified accurately, and markedly higher resolution was obtained in comparison with the use of reference markers (nad1 + cox1) only. Application of multilocus sequence analysis (MLSA) to large-scale studies could provide valuable epidemiological data to make efficient control and management measures for cystic echinococcosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of New Single Nucleotide Polymorphism-Based Markers for Inter- and Intraspecies Discrimination of Obligate Bacterial Parasites (Pasteuria spp.) of Invertebrates ▿ †

PubMed Central

Mauchline, Tim H.; Knox, Rachel; Mohan, Sharad; Powers, Stephen J.; Kerry, Brian R.; Davies, Keith G.; Hirsch, Penny R.

2011-01-01

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of “cryptic” SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms. PMID:21803895

Identification of new single nucleotide polymorphism-based markers for inter- and intraspecies discrimination of obligate bacterial parasites (Pasteuria spp.) of invertebrates.

PubMed

Mauchline, Tim H; Knox, Rachel; Mohan, Sharad; Powers, Stephen J; Kerry, Brian R; Davies, Keith G; Hirsch, Penny R

2011-09-01

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of "cryptic" SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.

An empirical evaluation of two-stage species tree inference strategies using a multilocus dataset from North American pines

PubMed Central

2014-01-01

Background As it becomes increasingly possible to obtain DNA sequences of orthologous genes from diverse sets of taxa, species trees are frequently being inferred from multilocus data. However, the behavior of many methods for performing this inference has remained largely unexplored. Some methods have been proven to be consistent given certain evolutionary models, whereas others rely on criteria that, although appropriate for many parameter values, have peculiar zones of the parameter space in which they fail to converge on the correct estimate as data sets increase in size. Results Here, using North American pines, we empirically evaluate the behavior of 24 strategies for species tree inference using three alternative outgroups (72 strategies total). The data consist of 120 individuals sampled in eight ingroup species from subsection Strobus and three outgroup species from subsection Gerardianae, spanning ∼47 kilobases of sequence at 121 loci. Each “strategy” for inferring species trees consists of three features: a species tree construction method, a gene tree inference method, and a choice of outgroup. We use multivariate analysis techniques such as principal components analysis and hierarchical clustering to identify tree characteristics that are robustly observed across strategies, as well as to identify groups of strategies that produce trees with similar features. We find that strategies that construct species trees using only topological information cluster together and that strategies that use additional non-topological information (e.g., branch lengths) also cluster together. Strategies that utilize more than one individual within a species to infer gene trees tend to produce estimates of species trees that contain clades present in trees estimated by other strategies. Strategies that use the minimize-deep-coalescences criterion to construct species trees tend to produce species tree estimates that contain clades that are not present in trees estimated by the Concatenation, RTC, SMRT, STAR, and STEAC methods, and that in general are more balanced than those inferred by these other strategies. Conclusions When constructing a species tree from a multilocus set of sequences, our observations provide a basis for interpreting differences in species tree estimates obtained via different approaches that have a two-stage structure in common, one step for gene tree estimation and a second step for species tree estimation. The methods explored here employ a number of distinct features of the data, and our analysis suggests that recovery of the same results from multiple methods that tend to differ in their patterns of inference can be a valuable tool for obtaining reliable estimates. PMID:24678701

An empirical evaluation of two-stage species tree inference strategies using a multilocus dataset from North American pines.

PubMed

DeGiorgio, Michael; Syring, John; Eckert, Andrew J; Liston, Aaron; Cronn, Richard; Neale, David B; Rosenberg, Noah A

2014-03-29

As it becomes increasingly possible to obtain DNA sequences of orthologous genes from diverse sets of taxa, species trees are frequently being inferred from multilocus data. However, the behavior of many methods for performing this inference has remained largely unexplored. Some methods have been proven to be consistent given certain evolutionary models, whereas others rely on criteria that, although appropriate for many parameter values, have peculiar zones of the parameter space in which they fail to converge on the correct estimate as data sets increase in size. Here, using North American pines, we empirically evaluate the behavior of 24 strategies for species tree inference using three alternative outgroups (72 strategies total). The data consist of 120 individuals sampled in eight ingroup species from subsection Strobus and three outgroup species from subsection Gerardianae, spanning ∼47 kilobases of sequence at 121 loci. Each "strategy" for inferring species trees consists of three features: a species tree construction method, a gene tree inference method, and a choice of outgroup. We use multivariate analysis techniques such as principal components analysis and hierarchical clustering to identify tree characteristics that are robustly observed across strategies, as well as to identify groups of strategies that produce trees with similar features. We find that strategies that construct species trees using only topological information cluster together and that strategies that use additional non-topological information (e.g., branch lengths) also cluster together. Strategies that utilize more than one individual within a species to infer gene trees tend to produce estimates of species trees that contain clades present in trees estimated by other strategies. Strategies that use the minimize-deep-coalescences criterion to construct species trees tend to produce species tree estimates that contain clades that are not present in trees estimated by the Concatenation, RTC, SMRT, STAR, and STEAC methods, and that in general are more balanced than those inferred by these other strategies. When constructing a species tree from a multilocus set of sequences, our observations provide a basis for interpreting differences in species tree estimates obtained via different approaches that have a two-stage structure in common, one step for gene tree estimation and a second step for species tree estimation. The methods explored here employ a number of distinct features of the data, and our analysis suggests that recovery of the same results from multiple methods that tend to differ in their patterns of inference can be a valuable tool for obtaining reliable estimates.

Genetic affinities of Helicobacter pylori isolates from ethnic Arabs in Kuwait

PubMed Central

2010-01-01

Helicobacter pylori is one of the most genetically diverse of bacterial species, and since the 5'-end of cagA gene and the middle allele of vacA gene of H. pylori from different populations exhibit considerable polymorphisms, these sequence diversities were used to gain insights into the genetic affinities of this gastric pathogen from different populations. Because the genetic affinity of Arab strains from the Arabian Gulf is not known, we carried out genetic analysis based on sequence diversities of the cagA and the vacA genes of H. pylori from 9 ethnic Arabs in Kuwait. The analysis showed that the Kuwaiti isolates are closely related to the Indo-European group of strains, although some strains have a tendency to form a separate cluster close to the Indo- European group, but clearly distinct from East Asian strains. However, these results need to be confirmed by analyses of neutral markers (house-keeping genes in a multi-locus sequence typing [MLST]) platform. The profiling of virulence-associated genes may have resulted from ecologically distinct populations due to human migration and geographical separation over long periods of time. PMID:20602767

Molecular typing of methicillin-resistant Staphylococcus aureus: Comparison of PCR-based open reading frame typing, multilocus sequence typing, and Staphylococcus protein A gene typing.

PubMed

Ogihara, Shinji; Saito, Ryoichi; Sawabe, Etsuko; Kozakai, Takahiro; Shima, Mari; Aiso, Yoshibumi; Fujie, Toshihide; Nukui, Yoko; Koike, Ryuji; Hagihara, Michio; Tohda, Shuji

2018-04-01

The recently developed PCR-based open reading frame typing (POT) method is a useful molecular typing tool. Here, we evaluated the performance of POT for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) isolates and compared its performance to those of multilocus sequence typing (MLST) and Staphylococcus protein A gene typing (spa typing). Thirty-seven MRSA isolates were collected between July 2012 and May 2015. MLST, spa typing, and POT were performed, and their discriminatory powers were evaluated using Simpson's index analysis. The MRSA isolates were classified into 11, 18, and 33 types by MLST, spa typing, and POT, respectively. The predominant strains identified by MLST, spa typing, and POT were ST8 and ST764, t002, and 93-191-127, respectively. The discriminatory power of MLST, spa typing, and POT was 0.853, 0.875, and 0.992, respectively, indicating that POT had the highest discriminatory power. Moreover, the results of MLST and spa were available after 2 days, whereas that of POT was available in 5 h. Furthermore, POT is rapid and easy to perform and interpret. Therefore, POT is a superior molecular typing tool for monitoring nosocomial transmission of MRSA. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Evaluation of two multi-locus sequence typing schemes for commensal Escherichia coli from dairy cattle in Washington State.

PubMed

Ahmed, Sara; Besser, Thomas E; Call, Douglas R; Weissman, Scott J; Jones, Lisa P; Davis, Margaret A

2016-05-01

Multi-locus sequence typing (MLST) is a useful system for phylogenetic and epidemiological studies of multidrug-resistant Escherichiacoli. Most studies utilize a seven-locus MLST, but an alternate two-locus typing method (fumC and fimH; CH typing) has been proposed that may offer a similar degree of discrimination at lower cost. Herein, we compare CH typing to the standard seven-locus method for typing commensal E. coli isolates from dairy cattle. In addition, we evaluated alternative combinations of eight loci to identify combinations that maximize discrimination and congruence with standard seven-locus MLST among commensal E. coli while minimizing the cost. We also compared both methods when used for typing uropathogenic E. coli (UPEC). CH typing was less discriminatory for commensal E. coli than the standard seven-locus method (Simpson's Index of Diversity=0.933 [0.902-0.964] and 0.97 [0.96-0.979], respectively). Combining fimH with housekeeping gene loci improved discriminatory power for commensal E. coli from cattle but resulted in poor congruence with MLST. We found that a four-locus typing method including the housekeeping genes adk, purA, gyrB and recA could be used to minimize cost without sacrificing discriminatory power or congruence with Achtman seven-locus MLST when typing commensal E. coli. Copyright © 2016 Elsevier B.V. All rights reserved.

Epidemiological characterization of a nosocomial outbreak of extended spectrum β-lactamase Escherichia coli ST-131 confirms the clinical value of core genome multilocus sequence typing.

PubMed

Woksepp, Hanna; Ryberg, Anna; Berglind, Linda; Schön, Thomas; Söderman, Jan

2017-12-01

Enhanced precision of epidemiological typing in clinically suspected nosocomial outbreaks is crucial. Our aim was to investigate whether single nucleotide polymorphism (SNP) analysis and core genome (cg) multilocus sequence typing (MLST) of whole genome sequencing (WGS) data would more reliably identify a nosocomial outbreak, compared to earlier molecular typing methods. Sixteen isolates from a nosocomial outbreak of ESBL E. coli ST-131 in southeastern Sweden and three control strains were subjected to WGS. Sequences were explored by SNP analysis and cgMLST. cgMLST clearly differentiated between the outbreak isolates and the control isolates (>1400 differences). All clinically identified outbreak isolates showed close clustering (≥2 allele differences), except for two isolates (>50 allele differences). These data confirmed that the isolates with >50 differing genes did not belong to the nosocomial outbreak. The number of SNPs within the outbreak was ≤7, whereas the two discrepant isolates had >700 SNPs. Two of the ESBL E. coli ST-131 isolates did not belong to the clinically identified outbreak. Our results illustrate the power of WGS in terms of resolution, which may avoid overestimation of patients belonging to outbreaks as judged from epidemiological data and previously employed molecular methods with lower discriminatory ability. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

PubMed Central

Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

Defining a Core Genome Multilocus Sequence Typing Scheme for the Global Epidemiology of Vibrio parahaemolyticus.

PubMed

Gonzalez-Escalona, Narjol; Jolley, Keith A; Reed, Elizabeth; Martinez-Urtaza, Jaime

2017-06-01

Vibrio parahaemolyticus is an important human foodborne pathogen whose transmission is associated with the consumption of contaminated seafood, with a growing number of infections reported over recent years worldwide. A multilocus sequence typing (MLST) database for V. parahaemolyticus was created in 2008, and a large number of clones have been identified, causing severe outbreaks worldwide (sequence type 3 [ST3]), recurrent outbreaks in certain regions (e.g., ST36), or spreading to other regions where they are nonendemic (e.g., ST88 or ST189). The current MLST scheme uses sequences of 7 genes to generate an ST, which results in a powerful tool for inferring the population structure of this pathogen, although with limited resolution, especially compared to pulsed-field gel electrophoresis (PFGE). The application of whole-genome sequencing (WGS) has become routine for trace back investigations, with core genome MLST (cgMLST) analysis as one of the most straightforward ways to explore complex genomic data in an epidemiological context. Therefore, there is a need to generate a new, portable, standardized, and more advanced system that provides higher resolution and discriminatory power among V. parahaemolyticus strains using WGS data. We sequenced 92 V. parahaemolyticus genomes and used the genome of strain RIMD 2210633 as a reference (with a total of 4,832 genes) to determine which genes were suitable for establishing a V. parahaemolyticus cgMLST scheme. This analysis resulted in the identification of 2,254 suitable core genes for use in the cgMLST scheme. To evaluate the performance of this scheme, we performed a cgMLST analysis of 92 newly sequenced genomes, plus an additional 142 strains with genomes available at NCBI. cgMLST analysis was able to distinguish related and unrelated strains, including those with the same ST, clearly showing its enhanced resolution over conventional MLST analysis. It also distinguished outbreak-related from non-outbreak-related strains within the same ST. The sequences obtained from this work were deposited and are available in the public database (http://pubmlst.org/vparahaemolyticus). The application of this cgMLST scheme to the characterization of V. parahaemolyticus strains provided by different laboratories from around the world will reveal the global picture of the epidemiology, spread, and evolution of this pathogen and will become a powerful tool for outbreak investigations, allowing for the unambiguous comparison of strains with global coverage. Copyright © 2017 Gonzalez-Escalona et al.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination.

PubMed

Danet, Jean Luc; Balakishiyeva, Gulnara; Cimerman, Agnès; Sauvion, Nicolas; Marie-Jeanne, Véronique; Labonne, Gérard; Lavina, Amparo; Batlle, Assumpcio; Krizanac, Ivana; Skoric, Dijana; Ermacora, Paolo; Serçe, Cigdem Ulubas; Caglayan, Kadriye; Jarausch, Wolfgang; Foissac, Xavier

2011-02-01

The genetic diversity of three temperate fruit tree phytoplasmas 'Candidatus Phytoplasma prunorum', 'Ca. P. mali' and 'Ca. P. pyri' has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of 'Ca P. prunorum' the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of 'Ca. P. prunorum', the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese 'Ca. P. pyri' isolates showed that they shared some alleles with 'Ca. P. prunorum', supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Characterization and Pathogenicity of Alternaria vanuatuensis, a New Record from Allium Plants in Korea and China.

PubMed

Li, Mei Jia; Deng, Jian Xin; Paul, Narayan Chandra; Lee, Hyang Burm; Yu, Seung Hun

2014-12-01

Alternaria from different Allium plants was characterized by multilocus sequence analysis. Based on sequences of the β-tubulin (BT2b), the Alternaria allergen a1 (Alt a1), and the RNA polymerase II second largest subunit (RPB2) genes and phylogenetic data analysis, isolates were divided into two groups. The two groups were identical to representative isolates of A. porri (EGS48-147) and A. vanuatuensis (EGS45-018). The conidial characteristics and pathogenicity of A. vanuatuensis also well supported the molecular characteristics. This is the first record of A. vanuatuensis E. G. Simmons & C. F. Hill from Korea and China.

A Novel HURRAH Protocol Reveals High Numbers of Monomorphic MHC Class II Loci and Two Asymmetric Multi-Locus Haplotypes in the Père David's Deer

PubMed Central

Wan, Qiu-Hong; Zhang, Pei; Ni, Xiao-Wei; Wu, Hai-Long; Chen, Yi-Yan; Kuang, Ye-Ye; Ge, Yun-Fa; Fang, Sheng-Guo

2011-01-01

The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated “HURRAH” based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ∼ DRB1 ∼ DRB3 ∼ DQA1 ∼ DQB2 (H1) and DRA1*02 ∼ DRB2 ∼ DRB4 ∼ DQA2 ∼ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens. PMID:21267075

Brucella papionis sp. nov., isolated from baboons (Papio spp.)

PubMed Central

Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S.; Brew, Simon D.; Perrett, Lorraine L.; Koylass, Mark S.; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C.; Dick, Edward J.; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E.

2014-01-01

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60T and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60T and F8/08-61 could be distinguished clearly from all known species of the genus Brucellaand their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucellasuggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60T ( = NCTC 13660T = CIRMBP 0958T). PMID:25242540

Latitudinal Clines of the Human Vitamin D Receptor and Skin Color Genes.

PubMed

Tiosano, Dov; Audi, Laura; Climer, Sharlee; Zhang, Weixiong; Templeton, Alan R; Fernández-Cancio, Monica; Gershoni-Baruch, Ruth; Sánchez-Muro, José Miguel; El Kholy, Mohamed; Hochberg, Zèev

2016-05-03

The well-documented latitudinal clines of genes affecting human skin color presumably arise from the need for protection from intense ultraviolet radiation (UVR) vs. the need to use UVR for vitamin D synthesis. Sampling 751 subjects from a broad range of latitudes and skin colors, we investigated possible multilocus correlated adaptation of skin color genes with the vitamin D receptor gene (VDR), using a vector correlation metric and network method called BlocBuster. We discovered two multilocus networks involving VDR promoter and skin color genes that display strong latitudinal clines as multilocus networks, even though many of their single gene components do not. Considered one by one, the VDR components of these networks show diverse patterns: no cline, a weak declining latitudinal cline outside of Africa, and a strong in- vs. out-of-Africa frequency pattern. We confirmed these results with independent data from HapMap. Standard linkage disequilibrium analyses did not detect these networks. We applied BlocBuster across the entire genome, showing that our networks are significant outliers for interchromosomal disequilibrium that overlap with environmental variation relevant to the genes' functions. These results suggest that these multilocus correlations most likely arose from a combination of parallel selective responses to a common environmental variable and coadaptation, given the known Mendelian epistasis among VDR and the skin color genes. Copyright © 2016 Tiosano et al.

Sequence-based characterization of  Listeria monocytogenes  strains isolated from domestic retail meat in the Tokyo metropolitan area of Japan.

PubMed

Yoshikawa, Yuko; Ochiai, Yoshitsugu; Mochizuki, Mariko; Takano, Takashi; Hondo, Ryo; Ueda, Fukiko

2018-05-31

To assess the level of Listeria monocytogenes contamination of domestic retail meat in Tokyo, Japan, we compared isolates from 2004 to 2007 with those isolated before 2003. The overall prevalence of L. monocytogenes among these samples significantly diminished over time (1998-2003, 28.0%; 2004-2007, 17.6%) reflecting a significant decrease in the frequency of contamination of beef. Serotype 1/2a was isolated most frequently, reflecting a change in the predominant serotype in pork from 1/2c to 1/2a. We performed a simple genetic subtyping method based on three genes, iap, sigB, and actA, as well as traditional multilocus sequence typing to classify the allele types (ATs). No extensive variation among sequence types was detected; however, increased genetic diversity among the ATs of the three genes in the 2004-2007 isolates was evident. We identified AT 26 of the iap gene, not previously reported in Japanese isolates, and six ATs of the sigB gene, including four with nonsense mutations not currently registered in L. monocytogenes DNA databases. sigB is an evolutionally conserved gene that plays a role in the stress response. Our results indicate that the sigB gene may be relatively unstable among L. monocytogenes strains circulating in Japan.

Shaking the Tree: Multi-locus Sequence Typing Usurps Current Onchocercid (Filarial Nematode) Phylogeny

PubMed Central

Lefoulon, Emilie; Bourret, Jérôme; Junker, Kerstin; Guerrero, Ricardo; Cañizales, Israel; Kuzmin, Yuriy; Satoto, Tri Baskoro T.; Cardenas-Callirgos, Jorge Manuel; de Souza Lima, Sueli; Raccurt, Christian; Mutafchiev, Yasen; Gavotte, Laurent; Martin, Coralie

2015-01-01

During the past twenty years, a number of molecular analyses have been performed to determine the evolutionary relationships of Onchocercidae, a family of filarial nematodes encompassing several species of medical or veterinary importance. However, opportunities for broad taxonomic sampling have been scarce, and analyses were based mainly on 12S rDNA and coxI gene sequences. While being suitable for species differentiation, these mitochondrial genes cannot be used to infer phylogenetic hypotheses at higher taxonomic levels. In the present study, 48 species, representing seven of eight subfamilies within the Onchocercidae, were sampled and sequences of seven gene loci (nuclear and mitochondrial) analysed, resulting in the hitherto largest molecular phylogenetic investigation into this family. Although our data support the current hypothesis that the Oswaldofilariinae, Waltonellinae and Icosiellinae subfamilies separated early from the remaining onchocercids, Setariinae was recovered as a well separated clade. Dirofilaria, Loxodontofilaria and Onchocerca constituted a strongly supported clade despite belonging to different subfamilies (Onchocercinae and Dirofilariinae). Finally, the separation between Splendidofilariinae, Dirofilariinae and Onchocercinae will have to be reconsidered. PMID:26588229

Multilocus sequence typing of Lactococcus lactis from naturally fermented milk foods in ethnic minority areas of China.

PubMed

Xu, Haiyan; Sun, Zhihong; Liu, Wenjun; Yu, Jie; Song, Yuqin; Lv, Qiang; Zhang, Jiachao; Shao, Yuyu; Menghe, Bilige; Zhang, Heping

2014-05-01

To determine the genetic diversity and phylogenetic relationships among Lactococcus lactis isolates, 197 strains isolated from naturally homemade yogurt in 9 ethnic minority areas of 6 provinces of China were subjected to multilocus sequence typing (MLST). The MLST analysis was performed using internal fragment sequences of 12 housekeeping genes (carB, clpX, dnaA, groEL, murC, murE, pepN, pepX, pyrG, recA, rpoB, and pheS). Six (dnaA) to 8 (murC) different alleles were detected for these genes, which ranged from 33.62 (clpX) to 41.95% (recA) GC (guanine-cytosine) content. The nucleotide diversity (π) ranged from 0.00362 (murE) to 0.08439 (carB). Despite this limited allelic diversity, the allele combinations of each strain revealed 72 different sequence types, which denoted significant genotypic diversity. The dN/dS ratios (where dS is the number of synonymous substitutions per synonymous site, and dN is the number of nonsynonymous substitutions per nonsynonymous site) were lower than 1, suggesting potential negative selection for these genes. The standardized index of association of the alleles IA(S)=0.3038 supported the clonality of Lc. lactis, but the presence of network structure revealed by the split decomposition analysis of the concatenated sequence was strong evidence for intraspecies recombination. Therefore, this suggests that recombination contributed to the evolution of Lc. lactis. A minimum spanning tree analysis of the 197 isolates identified 14 clonal complexes and 23 singletons. Phylogenetic trees were constructed based on the sequence types, using the minimum evolution algorithm, and on the concatenated sequence (6,192 bp), using the unweighted pair-group method with arithmetic mean, and these trees indicated that the evolution of our Lc. lactis population was correlated with geographic origin. Taken together, our results demonstrated that MLST could provide a better understanding of Lc. lactis genome evolution, as well as useful information for future studies on global Lc. lactis structure and genetic evolution, which will lay the foundation for screening Lc. lactis as starter cultures in fermented dairy products. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Validation of Minim typing for fast and accurate discrimination of extended-spectrum, beta-lactamase-producing Klebsiella pneumoniae isolates in tertiary care hospital.

PubMed

Brhelova, Eva; Kocmanova, Iva; Racil, Zdenek; Hanslianova, Marketa; Antonova, Mariya; Mayer, Jiri; Lengerova, Martina

2016-09-01

Minim typing is derived from the multi-locus sequence typing (MLST). It targets the same genes, but sequencing is replaced by high resolution melt analysis. Typing can be performed by analysing six loci (6MelT), four loci (4MelT) or using data from four loci plus sequencing the tonB gene (HybridMelT). The aim of this study was to evaluate Minim typing to discriminate extended-spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KLPN) isolates at our hospital. In total, 380 isolates were analyzed. The obtained alleles were assigned according to both the 6MelT and 4MelT typing scheme. In 97 isolates, the tonB gene was sequenced to enable HybridMelT typing. We found that the presented method is suitable to quickly monitor isolates of ESBL-KLPN; results are obtained in less than 2 hours and at a lower cost than MLST. We identified a local ESBL-KLPN outbreak and a comparison of colonizing and invasive isolates revealed a long term colonization of patients with the same strain. Copyright © 2016 Elsevier Inc. All rights reserved.

Strategies for high-altitude adaptation revealed from high-quality draft genome of non-violacein producing Janthinobacterium lividum ERGS5:01.

PubMed

Kumar, Rakshak; Acharya, Vishal; Singh, Dharam; Kumar, Sanjay

2018-01-01

A light pink coloured bacterial strain ERGS5:01 isolated from glacial stream water of Sikkim Himalaya was affiliated to Janthinobacterium lividum based on 16S rRNA gene sequence identity and phylogenetic clustering. Whole genome sequencing was performed for the strain to confirm its taxonomy as it lacked the typical violet pigmentation of the genus and also to decipher its survival strategy at the aquatic ecosystem of high elevation. The PacBio RSII sequencing generated genome of 5,168,928 bp with 4575 protein-coding genes and 118 RNA genes. Whole genome-based multilocus sequence analysis clustering, in silico DDH similarity value of 95.1% and, the ANI value of 99.25% established the identity of the strain ERGS5:01 (MCC 2953) as a non-violacein producing J. lividum . The genome comparisons across genus Janthinobacterium revealed an open pan-genome with the scope of the addition of new orthologous cluster to complete the genomic inventory. The genomic insight provided the genetic basis of freezing and frequent freeze-thaw cycle tolerance and, for industrially important enzymes. Extended insight into the genome provided clues of crucial genes associated with adaptation in the harsh aquatic ecosystem of high altitude.

Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii.

PubMed

Xu, Jianping; Yan, Zhun; Guo, Hong

2009-06-01

The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii. The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii, but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.

Latitudinal Clines of the Human Vitamin D Receptor and Skin Color Genes

PubMed Central

Tiosano, Dov; Audi, Laura; Climer, Sharlee; Zhang, Weixiong; Templeton, Alan R.; Fernández-Cancio, Monica; Gershoni-Baruch, Ruth; Sánchez-Muro, José Miguel; El Kholy, Mohamed; Hochberg, Zèev

2016-01-01

The well-documented latitudinal clines of genes affecting human skin color presumably arise from the need for protection from intense ultraviolet radiation (UVR) vs. the need to use UVR for vitamin D synthesis. Sampling 751 subjects from a broad range of latitudes and skin colors, we investigated possible multilocus correlated adaptation of skin color genes with the vitamin D receptor gene (VDR), using a vector correlation metric and network method called BlocBuster. We discovered two multilocus networks involving VDR promoter and skin color genes that display strong latitudinal clines as multilocus networks, even though many of their single gene components do not. Considered one by one, the VDR components of these networks show diverse patterns: no cline, a weak declining latitudinal cline outside of Africa, and a strong in- vs. out-of-Africa frequency pattern. We confirmed these results with independent data from HapMap. Standard linkage disequilibrium analyses did not detect these networks. We applied BlocBuster across the entire genome, showing that our networks are significant outliers for interchromosomal disequilibrium that overlap with environmental variation relevant to the genes’ functions. These results suggest that these multilocus correlations most likely arose from a combination of parallel selective responses to a common environmental variable and coadaptation, given the known Mendelian epistasis among VDR and the skin color genes. PMID:26921301

Ideal discrimination of discrete clinical endpoints using multilocus genotypes.

PubMed

Hahn, Lance W; Moore, Jason H

2004-01-01

Multifactor Dimensionality Reduction (MDR) is a method for the classification and prediction of discrete clinical endpoints using attributes constructed from multilocus genotype data. Empirical studies with both real and simulated data suggest that MDR has good power for detecting gene-gene interactions in the absence of independent main effects. The purpose of this study is to develop an objective, theory-driven approach to evaluate the strengths and limitations of MDR. To accomplish this goal, we borrow concepts from ideal observer analysis used in visual perception to evaluate the theoretical limits of classifying and predicting discrete clinical endpoints using multilocus genotype data. We conclude that MDR ideally discriminates between low risk and high risk subjects using attributes constructed from multilocus genotype data. We also how that the classification approach used once a multilocus attribute is constructed is similar to that of a naive Bayes classifier. This study provides a theoretical foundation for the continued development, evaluation, and application of the MDR as a data mining tool in the domain of statistical genetics and genetic epidemiology.

Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans.

PubMed

Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; van Essen-Zandbergen, Alieda; Brouwer, Michael; Mevius, Dik

2015-09-01

The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation in traY and excA genes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Analysis of blaCTX-M-Carrying Plasmids from Escherichia coli Isolates Collected in the BfT-GermVet Study ▿

PubMed Central

Schink, Anne-Kathrin; Kadlec, Kristina; Schwarz, Stefan

2011-01-01

In this study, 417 Escherichia coli isolates from defined disease conditions of companion and farm animals collected in the BfT-GermVet study were investigated for the presence of extended-spectrum β-lactamase (ESBL) genes. Three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates. The E. coli isolates 168 and 246, of canine and porcine origins, respectively, harbored blaCTX-M-1, and the canine isolate 913 harbored blaCTX-M-15, as confirmed by PCR and sequence analysis. The isolates 168 and 246 belonged to the novel multilocus sequence typing (MLST) types ST1576 and ST1153, respectively, while isolate 913 had the MLST type ST410. The ESBL genes were located on structurally related IncN plasmids in isolates 168 and 246 and on an IncF plasmid in isolate 913. The blaCTX-M-1 upstream regions of plasmids pCTX168 and pCTX246 were similar, whereas the downstream regions showed structural differences. The genetic environment of the blaCTX-M-15 gene on plasmid pCTX913 differed distinctly from that of both blaCTX-M-1 genes. Detailed sequence analysis showed that the integration of insertion sequences, as well as interplasmid recombination events, accounted for the structural variability in the blaCTX-M gene regions. PMID:21685166

Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy.

PubMed

Fischer, Sebastian; Greipel, Leonie; Klockgether, Jens; Dorda, Marie; Wiehlmann, Lutz; Cramer, Nina; Tümmler, Burkhard

2017-05-01

Early antimicrobial chemotherapy can prevent or at least delay chronic cystic fibrosis (CF) airways infections with Pseudomonas aeruginosa. During a 10-year study period P. aeruginosa was detected for the first time in 54 CF patients regularly seen at the CF centre Hannover. Amplicon sequencing of 34 loci of the P. aeruginosa core genome was performed in baseline and post-treatment isolates of the 15 CF patients who had remained P. aeruginosa - positive after the first round of antipseudomonal chemotherapy. Deep sequencing uncovered coexisting alternative nucleotides at in total 33 of 55,284 examined genome positions including six non-synonymous polymorphisms in the lasR gene, a key regulator of quorum sensing. After early treatment 42 of 50 novel nucleotide substitutions had emerged in exopolysaccharide biosynthesis, efflux pump and porin genes. Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Novel Molecular Method for Identification of Streptococcus pneumoniae Applicable to Clinical Microbiology and 16S rRNA Sequence-Based Microbiome Studies

PubMed Central

Scholz, Christian F. P.; Poulsen, Knud

2012-01-01

The close phylogenetic relationship of the important pathogen Streptococcus pneumoniae and several species of commensal streptococci, particularly Streptococcus mitis and Streptococcus pseudopneumoniae, and the recently demonstrated sharing of genes and phenotypic traits previously considered specific for S. pneumoniae hamper the exact identification of S. pneumoniae. Based on sequence analysis of 16S rRNA genes of a collection of 634 streptococcal strains, identified by multilocus sequence analysis, we detected a cytosine at position 203 present in all 440 strains of S. pneumoniae but replaced by an adenosine residue in all strains representing other species of mitis group streptococci. The S. pneumoniae-specific sequence signature could be demonstrated by sequence analysis or indirectly by restriction endonuclease digestion of a PCR amplicon covering the site. The S. pneumoniae-specific signature offers an inexpensive means for validation of the identity of clinical isolates and should be used as an integrated marker in the annotation procedure employed in 16S rRNA-based molecular studies of complex human microbiotas. This may avoid frequent misidentifications such as those we demonstrate to have occurred in previous reports and in reference sequence databases. PMID:22442329

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

PubMed

Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T

2011-07-01

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

Sub-typing of extended-spectrum-β-lactamase-producing isolates from a nosocomial outbreak: application of a 10-loci generic Escherichia coli multi-locus variable number tandem repeat analysis.

PubMed

Karami, Nahid; Helldal, Lisa; Welinder-Olsson, Christina; Ahrén, Christina; Moore, Edward R B

2013-01-01

Extended-spectrum β-lactamase producing Escherichia coli (ESBL-E. coli) were isolated from infants hospitalized in a neonatal, post-surgery ward during a four-month-long nosocomial outbreak and six-month follow-up period. A multi-locus variable number tandem repeat analysis (MLVA), using 10 loci (GECM-10), for 'generic' (i.e., non-STEC) E. coli was applied for sub-species-level (i.e., sub-typing) delineation and characterization of the bacterial isolates. Ten distinct GECM-10 types were detected among 50 isolates, correlating with the types defined by pulsed-field gel electrophoresis (PFGE), which is recognized to be the 'gold-standard' method for clinical epidemiological analyses. Multi-locus sequence typing (MLST), multiplex PCR genotyping of bla CTX-M, bla TEM, bla OXA and bla SHV genes and antibiotic resistance profiling, as well as a PCR assay specific for detecting isolates of the pandemic O25b-ST131 strain, further characterized the outbreak isolates. Two clusters of isolates with distinct GECM-10 types (G06-04 and G07-02), corresponding to two major PFGE types and the MLST-based sequence types (STs) 131 and 1444, respectively, were confirmed to be responsible for the outbreak. The application of GECM-10 sub-typing provided reliable, rapid and cost-effective epidemiological characterizations of the ESBL-producing isolates from a nosocomial outbreak that correlated with and may be used to replace the laborious PFGE protocol for analyzing generic E. coli.

Development of a Multilocus Sequence Typing (MLST) scheme for Treponema pallidum subsp. pertenue: Application to yaws in Lihir Island, Papua New Guinea

PubMed Central

Godornes, Charmie; Giacani, Lorenzo; Barry, Alyssa E.; Mitja, Oriol

2017-01-01

Background Yaws is a neglected tropical disease, caused by Treponema pallidum subsp. pertenue. The disease causes chronic lesions, primarily in young children living in remote villages in tropical climates. As part of a global yaws eradication campaign initiated by the World Health Organization, we sought to develop and evaluate a molecular typing method to distinguish different strains of T. pallidum subsp. pertenue for disease control and epidemiological purposes. Methods and principal findings Published genome sequences of strains of T. pallidum subsp. pertenue and pallidum were compared to identify polymorphic genetic loci among the strains. DNA from a number of existing historical Treponema isolates, as well as a subset of samples from yaws patients collected in Lihir Island, Papua New Guinea, were analyzed using these targets. From these data, three genes (tp0548, tp0136 and tp0326) were ultimately selected to give a high discriminating capability among the T. pallidum subsp. pertenue samples tested. Intragenic regions of these three target genes were then selected to enhance the discriminating capability of the typing scheme using short readily amplifiable loci. This 3-gene multilocus sequence typing (MLST) method was applied to existing historical human yaws strains, the Fribourg-Blanc simian isolate, and DNA from 194 lesion swabs from yaws patients on Lihir Island, Papua New Guinea. Among all samples tested, fourteen molecular types were identified, seven of which were found in patient samples and seven among historical isolates or DNA. Three types (JG8, TD6, and SE7) were predominant on Lihir Island. Conclusions This MLST approach allows molecular typing and differentiation of yaws strains. This method could be a useful tool to complement epidemiological studies in regions where T. pallidum subsp. pertenue is prevalent with the overall goals of improving our understanding of yaws transmission dynamics and helping the yaws eradication campaign to succeed. PMID:29281641

Genetic characterization of Anaplasma marginale strains from Tunisia using single and multiple gene typing reveals novel variants with an extensive genetic diversity.

PubMed

Ben Said, Mourad; Ben Asker, Alaa; Belkahia, Hanène; Ghribi, Raoua; Selmi, Rachid; Messadi, Lilia

2018-05-12

Anaplasma marginale, which is responsible for bovine anaplasmosis in tropical and subtropical regions, is a tick-borne obligatory intraerythrocytic bacterium of cattle and wild ruminants. In Tunisia, information about the genetic diversity and the phylogeny of A. marginale strains are limited to the msp4 gene analysis. The purpose of this study is to investigate A. marginale isolates infecting 16 cattle located in different bioclimatic areas of northern Tunisia with single gene analysis and multilocus sequence typing methods on the basis of seven partial genes (dnaA, ftsZ, groEL, lipA, secY, recA and sucB). The single gene analysis confirmed the presence of different and novel heterogenic A. marginale strains infecting cattle from the north of Tunisia. The concatenated sequence analysis showed a phylogeographical resolution at the global level and that most of the Tunisian sequence types (STs) formed a separate cluster from a South African isolate and from all New World isolates and strains. By combining the characteristics of each single locus with those of the multi-loci scheme, these results provide a more detailed understanding on the diversity and the evolution of Tunisian A. marginale strains. Copyright © 2018 Elsevier GmbH. All rights reserved.

Comparative genomics of Burkholderia multivorans, a ubiquitous pathogen with a highly conserved genomic structure

PubMed Central

Cooper, Vaughn S.; Hatcher, Philip J.; Verheyde, Bart; Carlier, Aurélien; Vandamme, Peter

2017-01-01

The natural environment serves as a reservoir of opportunistic pathogens. A well-established method for studying the epidemiology of such opportunists is multilocus sequence typing, which in many cases has defined strains predisposed to causing infection. Burkholderia multivorans is an important pathogen in people with cystic fibrosis (CF) and its epidemiology suggests that strains are acquired from non-human sources such as the natural environment. This raises the central question of whether the isolation source (CF or environment) or the multilocus sequence type (ST) of B. multivorans better predicts their genomic content and functionality. We identified four pairs of B. multivorans isolates, representing distinct STs and consisting of one CF and one environmental isolate each. All genomes were sequenced using the PacBio SMRT sequencing technology, which resulted in eight high-quality B. multivorans genome assemblies. The present study demonstrated that the genomic structure of the examined B. multivorans STs is highly conserved and that the B. multivorans genomic lineages are defined by their ST. Orthologous protein families were not uniformly distributed among chromosomes, with core orthologs being enriched on the primary chromosome and ST-specific orthologs being enriched on the second and third chromosome. The ST-specific orthologs were enriched in genes involved in defense mechanisms and secondary metabolism, corroborating the strain-specificity of these virulence characteristics. Finally, the same B. multivorans genomic lineages occur in both CF and environmental samples and on different continents, demonstrating their ubiquity and evolutionary persistence. PMID:28430818

Identification and characterization of Burkholderia multivorans CCA53.

PubMed

Akita, Hironaga; Kimura, Zen-Ichiro; Yusoff, Mohd Zulkhairi Mohd; Nakashima, Nobutaka; Hoshino, Tamotsu

2017-07-06

A lignin-degrading bacterium, Burkholderia sp. CCA53, was previously isolated from leaf soil. The purpose of this study was to determine phenotypic and biochemical features of Burkholderia sp. CCA53. Multilocus sequence typing (MLST) analysis based on fragments of the atpD, gltD, gyrB, lepA, recA and trpB gene sequences was performed to identify Burkholderia sp. CCA53. The MLST analysis revealed that Burkholderia sp. CCA53 was tightly clustered with B. multivorans ATCC BAA-247 T . The quinone and cellular fatty acid profiles, carbon source utilization, growth temperature and pH were consistent with the characteristics of B. multivorans species. Burkholderia sp. CCA53 was therefore identified as B. multivorans CCA53.

Characterization and Pathogenicity of Alternaria vanuatuensis, a New Record from Allium Plants in Korea and China

PubMed Central

Li, Mei Jia; Deng, Jian Xin; Paul, Narayan Chandra

2014-01-01

Alternaria from different Allium plants was characterized by multilocus sequence analysis. Based on sequences of the β-tubulin (BT2b), the Alternaria allergen a1 (Alt a1), and the RNA polymerase II second largest subunit (RPB2) genes and phylogenetic data analysis, isolates were divided into two groups. The two groups were identical to representative isolates of A. porri (EGS48-147) and A. vanuatuensis (EGS45-018). The conidial characteristics and pathogenicity of A. vanuatuensis also well supported the molecular characteristics. This is the first record of A. vanuatuensis E. G. Simmons & C. F. Hill from Korea and China. PMID:25606017

Pantoea hericii sp. nov., Isolated from the Fruiting Bodies of Hericium erinaceus.

PubMed

Rong, Chengbo; Ma, Yuanwei; Wang, Shouxian; Liu, Yu; Chen, Sanfeng; Huang, Bin; Wang, Jing; Xu, Feng

2016-06-01

Three Gram-negative, facultatively anaerobic bacterial isolates were obtained from the fruiting bodies of the edible mushroom Hericium erinaceus showing symptoms of soft rot disease in Beijing, China. Sequences of partial 16S rRNA gene placed these isolates in the genus Pantoea. Multilocus sequence analysis based on the partial sequences of atpD, gyrB, infB and rpoB revealed P. eucalypti and P. anthophila as their closest phylogenetic relatives and indicated that these isolates constituted a possible novel species. DNA-DNA hybridization studies confirmed the classification of these isolates as a novel species and phenotypic tests allowed for differentiation from the closest phylogenetic neighbours. The name Pantoea hericii sp. nov. [Type strain LMG 28847(T) = CGMCC 1.15224(T) = JZB 2120024(T)] is proposed.

Major clades of Agaricales: a multilocus phylogenetic overview.

Treesearch

P. Brandon Matheny; Judd M. Curtis; Valerie Hofstetter; M. Catherine Aime; Jean-Marc Moncalvo; Zai-Wei Ge; Zhu-Liang Yang; Joseph F. Ammirati; Timothy J. Baroni; Neale L. Bougher; Karen W. Lodge Hughes; Richard W. Kerrigan; Michelle T. Seidl; Aanen; Matthew Duur K. DeNitis; Graciela M. Daniele; Dennis E. Desjardin; Bradley R. Kropp; Lorelei L. Norvell; Andrew Parker; Else C. Vellinga; Rytas Vilgalys; David S. Hibbett

2006-01-01

An overview of the phylogeny of the Agaricales is presented based on a multilocus analysis of a six-gene region supermatrix. Bayesian analyses of 5611 nucleotide characters of rpb1, rpb1-intron 2, rpb2 and 18S, 25S, and 5.8S ribosomal RNA genes recovered six major clades, which are recognized informally and labeled the Agaricoid, Tricholomatoid, Marasmioid, Pluteoid,...

Sinorhizobium meliloti strains TII7 and A5 by Multilocus Sequence Typing (MLST) have chromsomes identical with Rm1021 and form an effective and ineffective symbiosis with Medicago truncatula line Jemalong A17, respectively

USDA-ARS?s Scientific Manuscript database

The strains TII7 and A5 formed an effective and ineffective symbiosis with Medicago truncatula Jemalong A17, respectively. Both were shown to have identical chromsomes with strains Rm1021 and RCR2011 using a Multilocus Sequence Typing method. The 2260 bp segments of DNA stretching from the 3’ end ...

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

PubMed

Chawjiraphan, Wireeya; Sonthayanon, Piengchan; Chanket, Phanita; Benjathummarak, Surachet; Kerdsin, Anusak; Kalambhaheti, Thareerat

2016-11-01

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.

First Description of KPC-2-Producing Escherichia coli and ST15 OXA-48-Positive Klebsiella pneumoniae in Tunisia.

PubMed

Ben Tanfous, Farah; Alonso, Carla Andrea; Achour, Wafa; Ruiz-Ripa, Laura; Torres, Carmen; Ben Hassen, Assia

2017-04-01

The aim of this study was to investigate the molecular features among Klebsiella pneumoniae and Escherichia coli strains showing a resistant/intermediate-resistant phenotype to ertapenem (R/IR-ERT), implicated in colonization/infection in patients of the Hematology and Graft Units of the National Bone Marrow Transplant Center of Tunisia (3-year period, 2011-2014). The major carbapenemase, extended-spectrum beta-lactamase, and plasmidic AmpC beta-lactamase genes were analyzed and characterized by PCR and sequencing. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequencing typing. The bla OXA-48 and bla KPC carbapenemase genes were detected among R/IR-ERT isolates. All R/IR-ERT K. pneumoniae strains (n = 19) had bla OXA-48 gene, and 14/19 strains also harbored the bla CTX-M-15 gene. Eight different PFGE patterns were detected among these K. pneumoniae isolates, and they showed eight different sequences types, ST11 and ST15 being the most prevalent ones. Two out of three R/IR-ERT E. coli isolates carried bla OXA-48 and one coproduced the bla CTX-M-15 gene. One E. coli strain, ascribed to the new sequence type ST5700, harbored the bla KPC-2 gene. E. coli isolates were not clonally related and belonged to different sequence types (ST5700, ST227, and ST58). To our knowledge, this is the first report in Tunisia of either KPC-2 carbapenemase in E. coli or OXA-48 carbapenemase in K. pneumoniae of lineage ST15.

Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli.

PubMed

Iguchi, Atsushi; Shirai, Hiroki; Seto, Kazuko; Ooka, Tadasuke; Ogura, Yoshitoshi; Hayashi, Tetsuya; Osawa, Kayo; Osawa, Ro

2011-01-01

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci.

Comparative Analysis of Genome Sequences Covering the Seven Cronobacter Species

PubMed Central

Cummings, Craig A.; Shih, Rita; Degoricija, Lovorka; Rico, Alain; Brzoska, Pius; Hamby, Stephen E.; Masood, Naqash; Hariri, Sumyya; Sonbol, Hana; Chuzhanova, Nadia; McClelland, Michael; Furtado, Manohar R.; Forsythe, Stephen J.

2012-01-01

Background Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages. Methodology/Principal Findings We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes. Conclusions/Significance Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types. PMID:23166675

Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and students.

PubMed

Roberts, Marilyn C; Soge, Olusegun O; Horst, Jeremy A; Ly, Kiet A; Milgrom, Peter

2011-10-01

Methicillin-resistant Staphylococcus aureus (MRSA) isolated from frequently touched dental school clinic surfaces were compared with MRSA isolated nasal cultures of dental students. Sixty-one dental students and 95 environmental surfaces from 7 clinics were sampled using SANICULT (Starplex Scientific Inc, Etobicoke, Ontario, Canada) swabs. Antimicrobial susceptibility testing was performed, and pulsed-field gel electrophoresis analysis, the mecA gene, multilocus sequence type, and SCCmec type were determined by polymerase chain reaction and sequencing. Thirteen (21%) dental students and 8 (8.4%) surfaces were MRSA positive. Three MRSA strains were SCCmec type IV, whereas 3 were nontypeable isolates and Panton-Valentine leukocidin positive (PVL+), and none were USA300. One surface and 1 student isolate shared the same multilocus sequence type ST 8 and were 75% related. Two groups of students carried the same MRSA strains. The MRSA-positive samples were from 4 of 7 dental clinics. In addition, 21% of the dental students carried MRSA, which is > 10 times higher than the general public and twice as frequent as in other university students. This is the first study to characterize MRSA from dental clinic surfaces and dental students and suggests that both may be reservoirs for MRSA. Further studies are needed to verify this premise. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Serotypes, antibiotic susceptibilities, and multi-locus sequence type profiles of Streptococcus agalactiae isolates circulating in Beijing, China.

PubMed

Wang, Ping; Tong, Jing-jing; Ma, Xiu-hua; Song, Feng-li; Fan, Ling; Guo, Cui-mei; Shi, Wei; Yu, Sang-jie; Yao, Kai-hu; Yang, Yong-hong

2015-01-01

To investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of Streptococcus agalactiae (S. agalactiae) in Beijing to provide references for the prevention and treatment of S. agalactiae infections. All isolates were identified using the CAMP test and the latex-agglutination assay and serotyped using a Strep-B-Latex kit, after which they were assessed for antibiotic susceptibility, macrolide-resistance genes, and MLST profiles. In total, 56 S. agalactiae isolates were identified in 863 pregnant women (6.5%). Serotypes Ia, Ib, II, III, and V were identified, among which types III (32.1%), Ia (17.9%), Ib (16.1%), and V (14.3%) were the predominant serotypes. All isolates were susceptible to penicillin and ceftriaxone. The nonsusceptiblity rates measured for erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, tetracycline, and levofloxacin were 85.7%, 92.9%, 98.2%, 30.4%, 73.2%, 91%, and 39.3%, respectively. We identified 14 sequence types (STs) for the 56 isolates, among which ST19 (30.4%) was predominant. The rate of fluoroquinolone resistance was higher in serotype III than in the other serotypes. Among the 44 erythromycin-resistant isolates, 32 (72.7%) carried ermB. S. agalactiae isolates of the serotypes Ia, Ib, III, and V are common in Beijing. Among the S. agalactiae isolates, the macrolide and clindamycin resistance rates are extremely high. Most of the erythromycin-resistant isolates carry ermB.

Diverse Mesorhizobium bacteria nodulate native Astragalus and Oxytropis in arctic and subarctic areas in Eurasia.

PubMed

Ampomah, Osei Yaw; Mousavi, Seyed Abdollah; Lindström, Kristina; Huss-Danell, Kerstin

2017-01-01

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. 15 N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen. Copyright © 2016 Elsevier GmbH. All rights reserved.

Benchmarking of Methods for Genomic Taxonomy

DOE PAGES

Larsen, Mette V.; Cosentino, Salvatore; Lukjancenko, Oksana; ...

2014-02-26

One of the first issues that emerges when a prokaryotic organism of interest is encountered is the question of what it is—that is, which species it is. The 16S rRNA gene formed the basis of the first method for sequence-based taxonomy and has had a tremendous impact on the field of microbiology. Nevertheless, the method has been found to have a number of shortcomings. In this paper, we trained and benchmarked five methods for whole-genome sequence-based prokaryotic species identification on a common data set of complete genomes: (i) SpeciesFinder, which is based on the complete 16S rRNA gene; (ii) Reads2Typemore » that searches for species-specific 50-mers in either the 16S rRNA gene or the gyrB gene (for the Enterobacteraceae family); (iii) the ribosomal multilocus sequence typing (rMLST) method that samples up to 53 ribosomal genes; (iv) TaxonomyFinder, which is based on species-specific functional protein domain profiles; and finally (v) KmerFinder, which examines the number of cooccurring k-mers (substrings of k nucleotides in DNA sequence data). The performances of the methods were subsequently evaluated on three data sets of short sequence reads or draft genomes from public databases. In total, the evaluation sets constituted sequence data from more than 11,000 isolates covering 159 genera and 243 species. Our results indicate that methods that sample only chromosomal, core genes have difficulties in distinguishing closely related species which only recently diverged. Finally, the KmerFinder method had the overall highest accuracy and correctly identified from 93% to 97% of the isolates in the evaluations sets.« less

spads 1.0: a toolbox to perform spatial analyses on DNA sequence data sets.

PubMed

Dellicour, Simon; Mardulyn, Patrick

2014-05-01

SPADS 1.0 (for 'Spatial and Population Analysis of DNA Sequences') is a population genetic toolbox for characterizing genetic variability within and among populations from DNA sequences. In view of the drastic increase in genetic information available through sequencing methods, spads was specifically designed to deal with multilocus data sets of DNA sequences. It computes several summary statistics from populations or groups of populations, performs input file conversions for other population genetic programs and implements locus-by-locus and multilocus versions of two clustering algorithms to study the genetic structure of populations. The toolbox also includes two MATLAB and r functions, GDISPAL and GDIVPAL, to display differentiation and diversity patterns across landscapes. These functions aim to generate interpolating surfaces based on multilocus distance and diversity indices. In the case of multiple loci, such surfaces can represent a useful alternative to multiple pie charts maps traditionally used in phylogeography to represent the spatial distribution of genetic diversity. These coloured surfaces can also be used to compare different data sets or different diversity and/or distance measures estimated on the same data set. © 2013 John Wiley & Sons Ltd.

Complex dissemination of the diversified mcr-1-harbouring plasmids in Escherichia coli of different sequence types

PubMed Central

Lin, Jingxia; Wang, Xiuna; Deng, Xianbo; Feng, Youjun

2016-01-01

The emergence of the mobilized colistin resistance gene, representing a novel mechanism for bacterial drug resistance, challenges the last resort against the severe infections by Gram-negative bacteria with multi-drug resistances. Very recently, we showed the diversity in the mcr-1-carrying plasmid reservoirs from the gut microbiota. Here, we reported that a similar but more complex scenario is present in the healthy swine populations, Southern China, 2016. Amongst the 1026 pieces of Escherichia coli isolates from 3 different pig farms, 302 E. coli isolates were determined to be positive for the mcr-1 gene (30%, 302/1026). Multi-locus sequence typing assigned no less than 11 kinds of sequence types including one novel Sequence Type to these mcr-1-positive strains. PCR analyses combined with the direct DNA sequencing revealed unexpected complexity of the mcr-1-harbouring plasmids whose backbones are at least grouped into 6 types four of which are new. Transcriptional analyses showed that the mcr-1 promoter of different origins exhibits similar activity. It seems likely that complex dissemination of the diversified mcr-1-bearing plasmids occurs amongst the various ST E. coli inhabiting the healthy swine populations, in Southern China. PMID:27741523

Application of MLST and Pilus Gene Sequence Comparisons to Investigate the Population Structures of Actinomyces naeslundii and Actinomyces oris

PubMed Central

Henssge, Uta; Do, Thuy; Gilbert, Steven C.; Cox, Steven; Clark, Douglas; Wickström, Claes; Ligtenberg, A. J. M.; Radford, David R.; Beighton, David

2011-01-01

Actinomyces naeslundii and Actinomyces oris are members of the oral biofilm. Their identification using 16S rRNA sequencing is problematic and better achieved by comparison of metG partial sequences. A. oris is more abundant and more frequently isolated than A. naeslundii. We used a multi-locus sequence typing approach to investigate the genotypic diversity of these species and assigned A. naeslundii (n = 37) and A. oris (n = 68) isolates to 32 and 68 sequence types (ST), respectively. Neighbor-joining and ClonalFrame dendrograms derived from the concatenated partial sequences of 7 house-keeping genes identified at least 4 significant subclusters within A. oris and 3 within A. naeslundii. The strain collection we had investigated was an under-representation of the total population since at least 3 STs composed of single strains may represent discrete clusters of strains not well represented in the collection. The integrity of these sub-clusters was supported by the sequence analysis of fimP and fimA, genes coding for the type 1 and 2 fimbriae, respectively. An A. naeslundii subcluster was identified with both fimA and fimP genes and these strains were able to bind to MUC7 and statherin while all other A. naeslundii strains possessed only fimA and did not bind to statherin. An A. oris subcluster harboured a fimA gene similar to that of Actinomyces odontolyticus but no detectable fimP failed to bind significantly to either MUC7 or statherin. These data are evidence of extensive genotypic and phenotypic diversity within the species A. oris and A. naeslundii but the status of the subclusters identified here will require genome comparisons before their phylogenic position can be unequivocally established. PMID:21738661

Application of MLST and pilus gene sequence comparisons to investigate the population structures of Actinomyces naeslundii and Actinomyces oris.

PubMed

Henssge, Uta; Do, Thuy; Gilbert, Steven C; Cox, Steven; Clark, Douglas; Wickström, Claes; Ligtenberg, A J M; Radford, David R; Beighton, David

2011-01-01

Actinomyces naeslundii and Actinomyces oris are members of the oral biofilm. Their identification using 16S rRNA sequencing is problematic and better achieved by comparison of metG partial sequences. A. oris is more abundant and more frequently isolated than A. naeslundii. We used a multi-locus sequence typing approach to investigate the genotypic diversity of these species and assigned A. naeslundii (n = 37) and A. oris (n = 68) isolates to 32 and 68 sequence types (ST), respectively. Neighbor-joining and ClonalFrame dendrograms derived from the concatenated partial sequences of 7 house-keeping genes identified at least 4 significant subclusters within A. oris and 3 within A. naeslundii. The strain collection we had investigated was an under-representation of the total population since at least 3 STs composed of single strains may represent discrete clusters of strains not well represented in the collection. The integrity of these sub-clusters was supported by the sequence analysis of fimP and fimA, genes coding for the type 1 and 2 fimbriae, respectively. An A. naeslundii subcluster was identified with both fimA and fimP genes and these strains were able to bind to MUC7 and statherin while all other A. naeslundii strains possessed only fimA and did not bind to statherin. An A. oris subcluster harboured a fimA gene similar to that of Actinomyces odontolyticus but no detectable fimP failed to bind significantly to either MUC7 or statherin. These data are evidence of extensive genotypic and phenotypic diversity within the species A. oris and A. naeslundii but the status of the subclusters identified here will require genome comparisons before their phylogenic position can be unequivocally established.

Genetic diversity of Streptococcus equi subsp. zooepidemicus and doxycycline resistance in kennelled dogs.

PubMed

Chalker, Victoria J; Waller, Andrew; Webb, Katy; Spearing, Emma; Crosse, Patricia; Brownlie, Joe; Erles, Kerstin

2012-06-01

The genetic diversity and antibiotic resistance profiles of 38 Streptococcus equi subsp. zooepidemicus isolates were determined from a kennelled canine population during two outbreaks of hemorrhagic pneumonia (1999 to 2002 and 2007 to 2010). Analysis of the szp gene hypervariable region and the 16S-23S rRNA intergenic spacer region and multilocus sequence typing (MLST) indicated a predominant tetO-positive, doxycycline-resistant ST-10 strain during 1999 to 2002 and a predominant tetM-positive doxycycline-resistant ST-62 strain during 2007 to 2010.

Genetic Diversity of Streptococcus equi subsp. zooepidemicus and Doxycycline Resistance in Kennelled Dogs

PubMed Central

Chalker, Victoria J.; Waller, Andrew; Webb, Katy; Spearing, Emma; Crosse, Patricia; Brownlie, Joe

2012-01-01

The genetic diversity and antibiotic resistance profiles of 38 Streptococcus equi subsp. zooepidemicus isolates were determined from a kennelled canine population during two outbreaks of hemorrhagic pneumonia (1999 to 2002 and 2007 to 2010). Analysis of the szp gene hypervariable region and the 16S-23S rRNA intergenic spacer region and multilocus sequence typing (MLST) indicated a predominant tetO-positive, doxycycline-resistant ST-10 strain during 1999 to 2002 and a predominant tetM-positive doxycycline-resistant ST-62 strain during 2007 to 2010. PMID:22495558

ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium

PubMed Central

Bjørkeng, Eva Katrin; Tessema, Girum Tadesse; Lundblad, Eirik Wasmuth; Butaye, Patrick; Willems, Rob; Sollid, Johanna Ericsson; Sundsfjord, Arnfinn; Hegstad, Kristin

2010-01-01

The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrABEnt genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrABEnt genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrAEnt probe (n=76) and partial DNA sequencing of ccrAEnt and ccrBEnt genes (n=38). ccrABEnt genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrABEnt genes were not found. Thirty-eight sequenced ccrABEnt genes from five different enterococcal species showed 94–100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrABEnt flanking chromosomal genes. Expression analysis of ccrABEnt genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrABEnt mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrABEnt genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrABEnt positive and negative isolates, suggesting acquisition or loss of ccrABEnt in E. faecium. In summary, ccrABEnt genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups. PMID:20817645

Enterotoxin gene profile of methicillin-resistant Staphylococcus pseudintermedius isolates from dogs, humans and the environment.

PubMed

Phumthanakorn, Nathita; Fungwithaya, Punpichaya; Chanchaithong, Pattrarat; Prapasarakul, Nuvee

2018-06-01

This study aimed to detect and identify staphylococcal enterotoxin (SE) genes in methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains from different sources, and to investigate the relationship between their sequence types (STs) and SE gene patterns. The profiles of 17 SE genes in 93 MRSP strains isolated from dogs (n=43), humans (n=18) and the environment (n=32) were detected by PCR. Multilocus sequence typing (MLST), SCCmec typing and pulsed-field gel electrophoresis (PFGE) were used to analyse the clonal relatedness between the molecular type and SE gene profiles.Results/Key findings. The human MRSP strains harboured the greatest number of SE genes (12/17; sea, sec, seg, sei, sek, sel, sem, sen, seo, sep, seq and tst-1) compared to those from dogs (5/17; sec, sel, sem, seq and tst-1) and environmental sources (3/17; sec, seq and tst-1). Using MLST and PFGE, different SE gene profiles were found within the same clonal type. We show that isolates of MRSP vary in their virulence gene profiles, depending on the source from which they have been isolated. This insight should encourage the development of appropriate monitoring and mitigation strategies to reduce the transmission of MRSP in veterinary hospitals and households.

Genotyping of Salmonella enterica serovar Typhi strains isolated from 1959 to 2006 in China and analysis of genetic diversity by genomic microarray.

PubMed

Zhang, Haifang; Zhang, Xiaolei; Yan, Meiying; Pang, Bo; Kan, Biao; Xu, Huaxi; Huang, Xinxiang

2011-12-15

To determine the genotype of Salmonella enterica serovar Typhi (S. Typhi) strains in China and analyze their genetic diversity. We collected S. Typhi strains from 1959 to 2006 in five highly endemic Chinese provinces and chose 40 representative strains. Multilocus sequence typing was used to determine the genotypes or sequence types (ST) and microarray-based comparative genomic hybridization (M-CGH) to investigate the differences in gene content among these strains. Forty representative S. Typhi strains belonged to 4 sequence types (ST1, ST2, ST890, and ST892). The predominant S. Typhi genotype (31/40) was ST2 and it had a diverse geographic distribution. We discovered two novel STs - ST890 and ST892. M-CGH showed that 69 genes in these two novel STs were divergent from S. Typhi Ty2, which belongs to ST1. In addition, 5 representative Typhi strains of ST2 isolated from Guizhou province showed differences in divergent genes. We determined two novel sequence types, ST890 and ST892, and found that ST2 was the most prevalent genotype of S. Typhi in China. Genetic diversity was present even within a highly clonal bacterial population.

Reassessment of the taxonomic position of Burkholderia andropogonis and description of Robbsia andropogonis gen. nov., comb. nov.

PubMed

Lopes-Santos, Lucilene; Castro, Daniel Bedo Assumpção; Ferreira-Tonin, Mariana; Corrêa, Daniele Bussioli Alves; Weir, Bevan Simon; Park, Duckchul; Ottoboni, Laura Maria Mariscal; Neto, Júlio Rodrigues; Destéfano, Suzete Aparecida Lanza

2017-06-01

The phylogenetic classification of the species Burkholderia andropogonis within the Burkholderia genus was reassessed using 16S rRNA gene phylogenetic analysis and multilocus sequence analysis (MLSA). Both phylogenetic trees revealed two main groups, named A and B, strongly supported by high bootstrap values (100%). Group A encompassed all of the Burkholderia species complex, whi.le Group B only comprised B. andropogonis species, with low percentage similarities with other species of the genus, from 92 to 95% for 16S rRNA gene sequences and 83% for conserved gene sequences. Average nucleotide identity (ANI), tetranucleotide signature frequency, and percentage of conserved proteins POCP analyses were also carried out, and in the three analyses B. andropogonis showed lower values when compared to the other Burkholderia species complex, near 71% for ANI, from 0.484 to 0.724 for tetranucleotide signature frequency, and around 50% for POCP, reinforcing the distance observed in the phylogenetic analyses. Our findings provide an important insight into the taxonomy of B. andropogonis. It is clear from the results that this bacterial species exhibits genotypic differences and represents a new genus described herein as Robbsia andropogonis gen. nov., comb. nov.

Population and genomic analysis of the genus Halorubrum

PubMed Central

Fullmer, Matthew S.; Soucy, Shannon M.; Swithers, Kristen S.; Makkay, Andrea M.; Wheeler, Ryan; Ventosa, Antonio; Gogarten, J. Peter; Papke, R. Thane

2014-01-01

The Halobacteria are known to engage in frequent gene transfer and homologous recombination. For stably diverged lineages to persist some checks on the rate of between lineage recombination must exist. We surveyed a group of isolates from the Aran-Bidgol endorheic lake in Iran and sequenced a selection of them. Multilocus Sequence Analysis (MLSA) and Average Nucleotide Identity (ANI) revealed multiple clusters (phylogroups) of organisms present in the lake. Patterns of intein and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) presence/absence and their sequence similarity, GC usage along with the ANI and the identities of the genes used in the MLSA revealed that two of these clusters share an exchange bias toward others in their phylogroup while showing reduced rates of exchange with other organisms in the environment. However, a third cluster, composed in part of named species from other areas of central Asia, displayed many indications of variability in exchange partners, from within the lake as well as outside the lake. We conclude that barriers to gene exchange exist between the two purely Aran-Bidgol phylogroups, and that the third cluster with members from other regions is not a single population and likely reflects an amalgamation of several populations. PMID:24782836

Alternative methods of phylogenetic inference for the Patagonian lizard group Liolaemus elongatus-kriegi (Iguania: Liolaemini) based on mitochondrial and nuclear markers.

PubMed

Medina, Cintia Débora; Avila, Luciano Javier; Sites, Jack Walter; Santos, Juan; Morando, Mariana

2018-03-01

We present different approaches to a multi-locus phylogeny for the Liolaemus elongatus-kriegi group, including almost all species and recognized lineages. We sequenced two mitochondrial and five nuclear gene regions for 123 individuals from 35 taxa, and compared relationships resolved from concatenated and species tree methods. The L. elongatus-kriegi group was inferred as monophyletic in three of the five analyses (concatenated mitochondrial, concatenated mitochondrial + nuclear gene trees, and SVD quartet species tree). The mitochondrial gene tree resolved four haploclades, three corresponding to the previously recognized complexes: L. elongatus, L. kriegi and L. petrophilus complexes, and the L. punmahuida group. The BEAST species tree approach included the L. punmahuida group within the L. kriegi complex, but the SVD quartet method placed it as sister to the L. elongatus-kriegi group. BEAST inferred species of the L. elongatus and L. petrophilus complexes as one clade, while SVDquartet inferred these two complexes as monophyletic (although with no statistical support for the L. petrophilus complex). The species tree approach also included the L. punmahuida group as part of the L. elongatus-kriegi group. Our study provides detailed multilocus phylogenetic hypotheses for the L. elongatus-kriegi group, and we discuss possible reasons for differences in the concatenation and species tree methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Phylogenetic Network Analysis Revealed the Occurrence of Horizontal Gene Transfer of 16S rRNA in the Genus Enterobacter

PubMed Central

Sato, Mitsuharu; Miyazaki, Kentaro

2017-01-01

Horizontal gene transfer (HGT) is a ubiquitous genetic event in bacterial evolution, but it seldom occurs for genes involved in highly complex supramolecules (or biosystems), which consist of many gene products. The ribosome is one such supramolecule, but several bacteria harbor dissimilar and/or chimeric 16S rRNAs in their genomes, suggesting the occurrence of HGT of this gene. However, we know little about whether the genes actually experience HGT and, if so, the frequency of such a transfer. This is primarily because the methods currently employed for phylogenetic analysis (e.g., neighbor-joining, maximum likelihood, and maximum parsimony) of 16S rRNA genes assume point mutation-driven tree-shape evolution as an evolutionary model, which is intrinsically inappropriate to decipher the evolutionary history for genes driven by recombination. To address this issue, we applied a phylogenetic network analysis, which has been used previously for detection of genetic recombination in homologous alleles, to the 16S rRNA gene. We focused on the genus Enterobacter, whose phylogenetic relationships inferred by multi-locus sequence alignment analysis and 16S rRNA sequences are incompatible. All 10 complete genomic sequences were retrieved from the NCBI database, in which 71 16S rRNA genes were included. Neighbor-joining analysis demonstrated that the genes residing in the same genomes clustered, indicating the occurrence of intragenomic recombination. However, as suggested by the low bootstrap values, evolutionary relationships between the clusters were uncertain. We then applied phylogenetic network analysis to representative sequences from each cluster. We found three ancestral 16S rRNA groups; the others were likely created through recursive recombination between the ancestors and chimeric descendants. Despite the large sequence changes caused by the recombination events, the RNA secondary structures were conserved. Successive intergenomic and intragenomic recombination thus shaped the evolution of 16S rRNA genes in the genus Enterobacter. PMID:29180992

New lipid-dependent Malassezia species from parrots.

PubMed

Cabañes, F Javier; Coutinho, S Dall' Acqua; Puig, Laura; Bragulat, M Rosa; Castellá, Gemma

2016-01-01

All the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals. In this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov. The isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the β-tubulin gene were also performed. The strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species. These studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots. Copyright © 2016 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

'Streptomyces caelicus', an antibiotic-producing species of the genus Streptomyces, and Streptomyces canchipurensis Li et al. 2015 are later heterotypic synonyms of Streptomyces muensis Ningthoujam et al. 2014.

PubMed

Wink, Joachim; Schumann, Peter; Atasayar, Ewelina; Klenk, Hans-Peter; Zaburannyi, Nestor; Westermann, Martin; Martin, Karin; Glaeser, Stefanie P; Kämpfer, Peter

2017-04-01

'Streptomyces caelicus' DSM 40835 was first reported as the producer of the antibiotic griselimycin by some coworkers of Rhone Poulenc in 1971. The project on isolation of the antibiotic compound was stopped because of the bad solubility and selectivity of the compound towards Mycobacteria. At Sanofi-Aventis, Germany, the project was re-evaluated in 2007 and the gene cluster of griselimycin could be identified, characterized and was patented in 2013. At this time, 'S. caelicus' was an invalid name. During the strain characterization work, it was found that 'S. caelicus' belongs to the group of species of the genus Streptomyces which show an unusual heterogeneity of the 16S rRNA gene sequences. However, high 16S rRNA gene sequence similarities to Streptomyces muensis JCM 17576T and Streptomyces canchipurensis JCM 17575T were obvious. Here, we present a comparative description of 'Streptomyces caelicus' DS 9461 (=DSM 40835=NCCB 100592) with S. muensis and S. canchipurensis by use of a polyphasic taxonomy approach and additional comparison of some housekeeping genes by multilocus sequence analysis (MLSA). An emended description of Streptomyces muensis is provided as a result of this work.

Basfia succiniciproducens gen. nov., sp. nov., a new member of the family Pasteurellaceae isolated from bovine rumen.

PubMed

Kuhnert, Peter; Scholten, Edzard; Haefner, Stefan; Mayor, Désirée; Frey, Joachim

2010-01-01

Gram-negative, coccoid, non-motile bacteria that are catalase-, urease- and indole-negative, facultatively anaerobic and oxidase-positive were isolated from the bovine rumen using an improved selective medium for members of the Pasteurellaceae. All strains produced significant amounts of succinic acid under anaerobic conditions with glucose as substrate. Phenotypic characterization and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, infB and recN genes were performed on seven independent isolates. All four genes showed high sequence similarity to their counterparts in the genome sequence of the patent strain MBEL55E, but less than 95 % 16S rRNA gene sequence similarity to any other species of the Pasteurellaceae. Genetically these strains form a very homogeneous group in individual as well as combined phylogenetic trees, clearly separated from other genera of the family from which they can also be separated based on phenotypic markers. Genome relatedness as deduced from the recN gene showed high interspecies similarities, but again low similarity to any of the established genera of the family. No toxicity towards bovine, human or fish cells was observed and no RTX toxin genes were detected in members of the new taxon. Based on phylogenetic clustering in the MLSA analysis, the low genetic similarity to other genera and the phenotypic distinction, we suggest to classify these bovine rumen isolates as Basfia succiniciproducens gen. nov., sp. nov. The type strain is JF4016(T) (=DSM 22022(T) =CCUG 57335(T)).

Colletotrichum – current status and future directions

PubMed Central

Cannon, P.F.; Damm, U.; Johnston, P.R.; Weir, B.S.

2012-01-01

A review is provided of the current state of understanding of Colletotrichum systematics, focusing on species-level data and the major clades. The taxonomic placement of the genus is discussed, and the evolution of our approach to species concepts and anamorph-teleomorph relationships is described. The application of multilocus technologies to phylogenetic analysis of Colletotrichum is reviewed, and selection of potential genes/loci for barcoding purposes is discussed. Host specificity and its relation to speciation and taxonomy is briefly addressed. A short review is presented of the current status of classification of the species clusters that are currently without comprehensive multilocus analyses, emphasising the orbiculare and destructivum aggregates. The future for Colletotrichum biology will be reliant on consensus classification and robust identification tools. In support of these goals, a Subcommission on Colletotrichum has been formed under the auspices of the International Commission on Taxonomy of Fungi, which will administer a carefully curated barcode database for sequence-based identification of species within the BioloMICS web environment. PMID:23136460

Cephalothrix gen. nov. (Cyanobacteria): towards an intraspecific phylogenetic evaluation by multilocus analyses.

PubMed

da Silva Malone, Camila Francieli; Rigonato, Janaína; Laughinghouse, Haywood Dail; Schmidt, Éder Carlos; Bouzon, Zenilda Laurita; Wilmotte, Annick; Fiore, Marli Fátima; Sant'Anna, Célia Leite

2015-09-01

For more than a decade, the taxonomy of the Phormidiaceae has been problematic, since morphologically similar organisms represent phylogenetically distinct entities. Based on 16S rRNA gene sequence analyses, the polyphyletic genus Phormidium and other gas-vacuolated oscillatorioids appear scattered throughout the cyanobacterial tree of life. Recently, several studies have focused on understanding the oscillatorioid taxa at the generic level. At the specific level, few studies have characterized cyanobacterial strains using combined datasets (morphology, ultrastructure and molecular multilocus analyses). Using a multifaceted approach, we propose a new, well-defined genus, Cephalothrix gen. nov., by analysing seven filamentous strains that are morphologically 'intermediate' between gas-vacuolated taxa and Phormidium. Furthermore, we characterize two novel species: Cephalothrix komarekiana sp. nov. (strains CCIBt 3277, CCIBt 3279, CCIBt 3523, CCALA 155, SAG 75.79 and UTEX 1580) and Cephalothrix lacustris sp. nov. (strain CCIBt 3261). The generic name and specific epithets are proposed under the provisions of the International Code of Nomenclature for Algae, Fungi, and Plants.

aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species.

PubMed

Lescat, Mathilde; Hoede, Claire; Clermont, Olivier; Garry, Louis; Darlu, Pierre; Tuffery, Pierre; Denamur, Erick; Picard, Bertrand

2009-12-29

Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. We identified the gene encoding esterase B as the acetyl-esterase gene (aes) using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR) strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.

Assessing models of speciation under different biogeographic scenarios; An empirical study using multi-locus and RNA-seq analyses

USGS Publications Warehouse

Edwards, Taylor; Tollis, Marc; Hsieh, PingHsun; Gutenkunst, Ryan N.; Liu, Zhen; Kusumi, Kenro; Culver, Melanie; Murphy, Robert W.

2016-01-01

Evolutionary biology often seeks to decipher the drivers of speciation, and much debate persists over the relative importance of isolation and gene flow in the formation of new species. Genetic studies of closely related species can assess if gene flow was present during speciation, because signatures of past introgression often persist in the genome. We test hypotheses on which mechanisms of speciation drove diversity among three distinct lineages of desert tortoise in the genus Gopherus. These lineages offer a powerful system to study speciation, because different biogeographic patterns (physical vs. ecological segregation) are observed at opposing ends of their distributions. We use 82 samples collected from 38 sites, representing the entire species' distribution and generate sequence data for mtDNA and four nuclear loci. A multilocus phylogenetic analysis in *BEAST estimates the species tree. RNA-seq data yield 20,126 synonymous variants from 7665 contigs from two individuals of each of the three lineages. Analyses of these data using the demographic inference package ∂a∂i serve to test the null hypothesis of no gene flow during divergence. The best-fit demographic model for the three taxa is concordant with the *BEAST species tree, and the ∂a∂i analysis does not indicate gene flow among any of the three lineages during their divergence. These analyses suggest that divergence among the lineages occurred in the absence of gene flow and in this scenario the genetic signature of ecological isolation (parapatric model) cannot be differentiated from geographic isolation (allopatric model).

Brucella papionis sp. nov., isolated from baboons (Papio spp.).

PubMed

Whatmore, Adrian M; Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S; Brew, Simon D; Perrett, Lorraine L; Koylass, Mark S; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C; Dick, Edward J; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E

2014-12-01

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) ( = NCTC 13660(T) = CIRMBP 0958(T)). Crown Copyright 2014. Reproduced with the permission of the Controller of Her Majesty's Stationery Office/Queen's Printer for Scotland and AHVLA.

Isolation of Brucella inopinata-Like Bacteria from White's and Denny's Tree Frogs.

PubMed

Kimura, Masanobu; Une, Yumi; Suzuki, Michio; Park, Eun-Sil; Imaoka, Koichi; Morikawa, Shigeru

2017-05-01

Brucella inopinata strain BO1 and B. sp. strain BO2 isolated from human patients, respectively, are genetically different from classical Brucella species. We isolated bacteria of the genus Brucella from two species of wild-caught tropical frogs kept in the facilities in Japan: White's tree frog, which inhabits Oceania, and Denny's tree frog, which inhabits Southeast Asia. Phylogenetic analyses based on 16S rRNA and recA gene sequences and multilocus sequence analysis showed that two isolates of Brucella spp. showed significant similarity to BO1, BO2, and the isolates from other wild-caught frogs. These results suggest that a variety of frog species are susceptible to a novel clade of Brucella bacteria, including B. inopinata.

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda

PubMed Central

Akpaka, Patrick Eberechi; Kissoon, Shivnarine; Wilson, Clyde; Jayaratne, Padman; Smith, Ashley; Golding, George R.

2017-01-01

Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island. PMID:28267763

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda.

PubMed

Akpaka, Patrick Eberechi; Kissoon, Shivnarine; Wilson, Clyde; Jayaratne, Padman; Smith, Ashley; Golding, George R

2017-01-01

Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

PubMed

Vonk, Freek J; Casewell, Nicholas R; Henkel, Christiaan V; Heimberg, Alysha M; Jansen, Hans J; McCleary, Ryan J R; Kerkkamp, Harald M E; Vos, Rutger A; Guerreiro, Isabel; Calvete, Juan J; Wüster, Wolfgang; Woods, Anthony E; Logan, Jessica M; Harrison, Robert A; Castoe, Todd A; de Koning, A P Jason; Pollock, David D; Yandell, Mark; Calderon, Diego; Renjifo, Camila; Currier, Rachel B; Salgado, David; Pla, Davinia; Sanz, Libia; Hyder, Asad S; Ribeiro, José M C; Arntzen, Jan W; van den Thillart, Guido E E J M; Boetzer, Marten; Pirovano, Walter; Dirks, Ron P; Spaink, Herman P; Duboule, Denis; McGlinn, Edwina; Kini, R Manjunatha; Richardson, Michael K

2013-12-17

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

Multilocus Genetic Characterization of Lactobacillus fermentum Isolated from Ready-to-Eat Canned Food.

PubMed

Sulaiman, Irshad M; Jacobs, Emily; Simpson, Steven; Kerdahi, Khalil

2017-06-01

The primary mission of the U.S. Food and Drug Administration is to enforce the Food, Drug, and Cosmetic Act and regulate food, drug, and cosmetic products. Thus, this agency monitors the presence of pathogenic microorganisms in these products, including canned foods, as one of the regulatory action criteria and also ensures that these products are safe for human consumption. This study was carried out to investigate the effectiveness of pathogen control and integrity of ready-to-eat canned food containing Black Bean Corn Poblano Salsa. A total of nine unopened and recalled canned glass jars from the same lot were examined initially by conventional microbiologic protocols that involved a two-step enrichment, followed by streaking on selective agar plates, for the presence of gram-positive and gram-negative bacteria. Of the eight subsamples examined for each sample, all subsamples of one of the containers were found positive for the presence of slow-growing rod-shaped, gram-positive, facultative anaerobic bacteria. The recovered isolates were subsequently sequenced at rRNA and gyrB loci. Afterward, multilocus sequence typing (MLST) was performed characterizing 11 additional known MLST loci (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). Analyses of the nucleotide sequences of rRNA, gyrB, and 11 MLST loci confirmed these gram-positive bacteria recovered from canned food to be Lactobacillus fermentum . Thus, the DNA sequencing of housekeeping MLST genes can provide species identification of L. fermentum and can be used in the canned food monitoring program of public health importance.

Multilocus sequence typing (MLST) analysis of Propionibacterium acnes isolates from radical prostatectomy specimens.

PubMed

Mak, Tim N; Yu, Shu-Han; De Marzo, Angelo M; Brüggemann, Holger; Sfanos, Karen S

2013-05-01

Inflammation is commonly observed in radical prostatectomy specimens, and evidence suggests that inflammation may contribute to prostate carcinogenesis. Multiple microorganisms have been implicated in serving as a stimulus for prostatic inflammation. The pro-inflammatory anaerobe, Propionibacterium acnes, is ubiquitously found on human skin and is associated with the skin disease acne vulgaris. Recent studies have shown that P. acnes can be detected in prostatectomy specimens by bacterial culture or by culture-independent molecular techniques. Radical prostatectomy tissue samples were obtained from 30 prostate cancer patients and subject to both aerobic and anaerobic culture. Cultured species were identified by 16S rDNA gene sequencing. Propionibacterium acnes isolates were typed using multilocus sequence typing (MLST). Our study confirmed that P. acnes can be readily cultured from prostatectomy tissues (7 of 30 cases, 23%). In some cases, multiple isolates of P. acnes were cultured as well as other Propionibacterium species, such as P. granulosum and P. avidum. Overall, 9 of 30 cases (30%) were positive for Propionibacterium spp. MLST analyses identified eight different sequence types (STs) among prostate-derived P. acnes isolates. These STs belong to two clonal complexes, namely CC36 (type I-2) and CC53/60 (type II), or are CC53/60-related singletons. MLST typing results indicated that prostate-derived P. acnes isolates do not fall within the typical skin/acne STs, but rather are characteristic of STs associated with opportunistic infections and/or urethral flora. The MLST typing results argue against the likelihood that prostatectomy-derived P. acnes isolates represent contamination from skin flora. Copyright © 2012 Wiley Periodicals, Inc.

Evaluation of an Optimal Epidemiological Typing Scheme for Legionella pneumophila with Whole-Genome Sequence Data Using Validation Guidelines

PubMed Central

Mentasti, Massimo; Tewolde, Rediat; Aslett, Martin; Harris, Simon R.; Afshar, Baharak; Underwood, Anthony; Harrison, Timothy G.

2016-01-01

Sequence-based typing (SBT), analogous to multilocus sequence typing (MLST), is the current “gold standard” typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila. However, as common sequence types (STs) cause many infections, some investigations remain unresolved. In this study, various whole-genome sequencing (WGS)-based methods were evaluated according to published guidelines, including (i) a single nucleotide polymorphism (SNP)-based method, (ii) extended MLST using different numbers of genes, (iii) determination of gene presence or absence, and (iv) a kmer-based method. L. pneumophila serogroup 1 isolates (n = 106) from the standard “typing panel,” previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI), were tested together with another 229 isolates. Over 98% of isolates were considered typeable using the SNP- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50 genes) to 86.8% (1,455 genes), while only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP based), and all values were higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila. PMID:27280420

Evaluation of an Optimal Epidemiological Typing Scheme for Legionella pneumophila with Whole-Genome Sequence Data Using Validation Guidelines.

PubMed

David, Sophia; Mentasti, Massimo; Tewolde, Rediat; Aslett, Martin; Harris, Simon R; Afshar, Baharak; Underwood, Anthony; Fry, Norman K; Parkhill, Julian; Harrison, Timothy G

2016-08-01

Sequence-based typing (SBT), analogous to multilocus sequence typing (MLST), is the current "gold standard" typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila However, as common sequence types (STs) cause many infections, some investigations remain unresolved. In this study, various whole-genome sequencing (WGS)-based methods were evaluated according to published guidelines, including (i) a single nucleotide polymorphism (SNP)-based method, (ii) extended MLST using different numbers of genes, (iii) determination of gene presence or absence, and (iv) a kmer-based method. L. pneumophila serogroup 1 isolates (n = 106) from the standard "typing panel," previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI), were tested together with another 229 isolates. Over 98% of isolates were considered typeable using the SNP- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50 genes) to 86.8% (1,455 genes), while only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP based), and all values were higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila. Copyright © 2016 David et al.

Correlation between Ureaplasma subgroup 2 and genitourinary tract disease outcomes revealed by an expanded multilocus sequence typing (eMLST) scheme.

PubMed

Zhang, Jun; Kong, Yingying; Ruan, Zhi; Huang, Jun; Song, Tiejun; Song, Jingjuan; Jiang, Yan; Yu, Yunsong; Xie, Xinyou

2014-01-01

The multilocus sequence typing (MLST) scheme of Ureaplasma based on four housekeeping genes (ftsH, rpL22, valS, and thrS) was described in our previous study; here we introduced an expanded MLST (eMLST) scheme with improved discriminatory power, which was developed by adding two putative virulence genes (ureG and mba-np1) to the original MLST scheme. To evaluate the discriminatory power of eMLST, a total of 14 reference strains of Ureaplasma serovars and 269 clinical strains (134 isolated from symptomatic patients and 135 obtained from asymptomatic persons) were investigated. Our study confirmed that all 14 serotype strains could successfully be differentiated into 14 eMLST STs (eSTs), while some of them could not even be differentiated by the MLST, and a total of 136 eSTs were identified among the clinical isolates we investigated. In addition, phylogenetic analysis indicated that two genetically significantly distant clusters (cluster I and II) were revealed and most clinical isolates were located in cluster I. These findings were in accordance with and further support for the concept of two well-known genetic lineages (Ureaplasma parvum and Ureaplasma urealyticum) in our previous study. Interestingly, although both clusters were associated with clinical manifestation, the sub-group 2 of cluster II had pronounced and adverse effect on patients and might be a potential risk factor for clinical outcomes. In conclusion, the eMLST scheme offers investigators a highly discriminative typing tool that is capable for precise epidemiological investigations and clinical relevance of Ureaplasma.

Correlation between Ureaplasma Subgroup 2 and Genitourinary Tract Disease Outcomes Revealed by an Expanded Multilocus Sequence Typing (eMLST) Scheme

PubMed Central

Ruan, Zhi; Huang, Jun; Song, Tiejun; Song, Jingjuan; Jiang, Yan; Yu, Yunsong; Xie, Xinyou

2014-01-01

The multilocus sequence typing (MLST) scheme of Ureaplasma based on four housekeeping genes (ftsH, rpL22, valS, and thrS) was described in our previous study; here we introduced an expanded MLST (eMLST) scheme with improved discriminatory power, which was developed by adding two putative virulence genes (ureG and mba-np1) to the original MLST scheme. To evaluate the discriminatory power of eMLST, a total of 14 reference strains of Ureaplasma serovars and 269 clinical strains (134 isolated from symptomatic patients and 135 obtained from asymptomatic persons) were investigated. Our study confirmed that all 14 serotype strains could successfully be differentiated into 14 eMLST STs (eSTs), while some of them could not even be differentiated by the MLST, and a total of 136 eSTs were identified among the clinical isolates we investigated. In addition, phylogenetic analysis indicated that two genetically significantly distant clusters (cluster I and II) were revealed and most clinical isolates were located in cluster I. These findings were in accordance with and further support for the concept of two well-known genetic lineages (Ureaplasma parvum and Ureaplasma urealyticum) in our previous study. Interestingly, although both clusters were associated with clinical manifestation, the sub-group 2 of cluster II had pronounced and adverse effect on patients and might be a potential risk factor for clinical outcomes. In conclusion, the eMLST scheme offers investigators a highly discriminative typing tool that is capable for precise epidemiological investigations and clinical relevance of Ureaplasma. PMID:25093900

Multilocus sequence analysis of Thermoanaerobacter isolates reveals recombining, but differentiated, populations from geothermal springs of the Uzon Caldera, Kamchatka, Russia

PubMed Central

Wagner, Isaac D.; Varghese, Litty B.; Hemme, Christopher L.; Wiegel, Juergen

2013-01-01

Thermal environments have island-like characteristics and provide a unique opportunity to study population structure and diversity patterns of microbial taxa inhabiting these sites. Strains having ≥98% 16S rRNA gene sequence similarity to the obligately anaerobic Firmicutes Thermoanaerobacter uzonensis were isolated from seven geothermal springs, separated by up to 1600 m, within the Uzon Caldera (Kamchatka, Russian Far East). The intraspecies variation and spatial patterns of diversity for this taxon were assessed by multilocus sequence analysis (MLSA) of 106 strains. Analysis of eight protein-coding loci (gyrB, lepA, leuS, pyrG, recA, recG, rplB, and rpoB) revealed that all loci were polymorphic and that nucleotide substitutions were mostly synonymous. There were 148 variable nucleotide sites across 8003 bp concatenates of the protein-coding loci. While pairwise FST values indicated a small but significant level of genetic differentiation between most subpopulations, there was a negligible relationship between genetic divergence and spatial separation. Strains with the same allelic profile were only isolated from the same hot spring, occasionally from consecutive years, and single locus variant (SLV) sequence types were usually derived from the same spring. While recombination occurred, there was an “epidemic” population structure in which a particular T. uzonensis sequence type rose in frequency relative to the rest of the population. These results demonstrate spatial diversity patterns for an anaerobic bacterial species in a relative small geographic location and reinforce the view that terrestrial geothermal springs are excellent places to look for biogeographic diversity patterns regardless of the involved distances. PMID:23801987

Whole genome sequencing as a tool to investigate a cluster of seven cases of listeriosis in Austria and Germany, 2011-2013.

PubMed

Schmid, D; Allerberger, F; Huhulescu, S; Pietzka, A; Amar, C; Kleta, S; Prager, R; Preußel, K; Aichinger, E; Mellmann, A

2014-05-01

A cluster of seven human cases of listeriosis occurred in Austria and in Germany between April 2011 and July 2013. The Listeria monocytogenes serovar (SV) 1/2b isolates shared pulsed-field gel electrophoresis (PFGE) and fluorescent amplified fragment length polymorphism (fAFLP) patterns indistinguishable from those from five food producers. The seven human isolates, a control strain with a different PFGE/fAFLP profile and ten food isolates were subjected to whole genome sequencing (WGS) in a blinded fashion. A gene-by-gene comparison (multilocus sequence typing (MLST)+) was performed, and the resulting whole genome allelic profiles were compared using SeqSphere(+) software version 1.0. On analysis of 2298 genes, the four human outbreak isolates from 2012 to 2013 had different alleles at ≤6 genes, i.e. differed by ≤6 genes from each other; the dendrogram placed these isolates in between five Austrian unaged soft cheese isolates from producer A (≤19-gene difference from the human cluster) and two Austrian ready-to-eat meat isolates from producer B (≤8-gene difference from the human cluster). Both food products appeared on grocery bills prospectively collected by these outbreak cases after hospital discharge. Epidemiological results on food consumption and MLST+ clearly separated the three cases in 2011 from the four 2012-2013 outbreak cases (≥48 different genes). We showed that WGS is capable of discriminating L. monocytogenes SV1/2b clones not distinguishable by PFGE and fAFLP. The listeriosis outbreak described clearly underlines the potential of sequence-based typing methods to offer enhanced resolution and comparability of typing systems for public health applications. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Emergence of Pseudomonas aeruginosa with class 1 integron carrying blaVIM-2 and blaVIM-4 in the University Clinical Hospital of Bialystok (northeastern Poland).

PubMed

Michalska-Falkowska, Anna; Sacha, Paweł Tomasz; Grześ, Henryk; Hauschild, Tomasz; Wieczorek, Piotr; Ojdana, Dominika; Tryniszewska, Elżbieta Anna

2017-07-11

The effectiveness of carbapenems, considered as last-resort antimicrobials in severe infections, becomes compromised by bacterial resistance. The production of metallo-β-lactamases (MBLs) is the most significant threat to carbapenems activity among Pseudomonas aeruginosa. The aim of this study was to assess the presence and type of MBLs genes in carbapenem-resistant P. aeruginosa clinical strains, to identify the location of MBLs genes and to determine genetic relatedness between MBL-producers using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The first identified MBL-positive (with blaVIM genes) P. aeruginosa strains were isolated from patients hospitalized in the University Clinical Hospital of Bialystok in the period from September 2012 to December 2013. Variants of MBLs genes and variable integron regions were characterized by PCR and sequencing. PFGE was performed after digesting of bacterial genomes by XbaI enzyme. By MLST seven housekeeping genes were analyzed for the determination of sequence type (ST). Three strains carried the blaVIM-2 gene and one harbored the blaVIM-4 gene. The blaVIM genes resided within class 1 integrons. PCR mapping of integrons revealed the presence of four different cassette arrays. Genetic relatedness analysis by PFGE classified VIM-positive strains into four unrelated pulsotypes (A-D). MLST demonstrated the presence of four (ST 111, ST27, and ST17) different sequence type including one previously undescribed new type of ST 2342. Antimicrobial susceptibility testing showed that VIM-positive strains were resistant to carbapenems, cephalosporins, aminoglycosides, and quinolones, intermediate to aztreonam, and susceptible only to colistin. Integrons mapping, PFGE, and MLST results may point to different origin of these strains and independent introduction into hospitalized patients.

[The use of multilocus sequence typing (MLST) and randomly amplified polymorphic DNA (RAPD) for the differentiation between strains of Burkholderia mallei].

PubMed

Antonov, V A; Altukhova, V V; Savchenko, S S; Zamaraev, V S; Iliukhin, V I; Alekseev, V V

2007-01-01

Burkholderia mallei is highly pathogenic microorganism for both humans and animals. In this work, the possibility of the use of the genotyping method for differentiation between strains of B. mallei was studied. A collection of 14 isolates of B. mallei was characterized using randomly amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). RAPD was the best method used for detecting strain differences of B. mallei. It was suggested that this method would be an increasingly useful molecular epidemiological tool.

Frequent gene flow blurred taxonomic boundaries of sections in Lilium L. (Liliaceae)

PubMed Central

Liu, Shih-Hui; Chiang, Tzen-Yuh

2017-01-01

Gene flow between species may last a long time in plants. Reticulation inevitably causes difficulties in phylogenetic reconstruction. In this study, we looked into the genetic divergence and phylogeny of 20 Lilium species based on multilocus analyses of 8 genes of chloroplast DNA (cpDNA), the internally transcribed nuclear ribosomal DNA (nrITS) spacer and 20 loci extracted from the expressed sequence tag (EST) libraries of L. longiflorum Thunb. and L. formosanum Wallace. The phylogeny based on the combined data of the maternally inherited cpDNA and nrITS was largely consistent with the taxonomy of Lilium sections. This phylogeny was deemed the hypothetical species tree and uncovered three groups, i.e., Cluster A consisting of 4 taxa from the sections Pseudolirium and Liriotypus, Cluster B consisting of the 4 taxa from the sections Leucolirion, Archelirion and Daurolirion, and Cluster C comprising 10 taxa mostly from the sections Martagon and Sinomartagon. In contrast, systematic inconsistency occurred across the EST loci, with up to 19 genes (95%) displaying tree topologies deviating from the hypothetical species tree. The phylogenetic incongruence was likely attributable to the frequent genetic exchanges between species/sections, as indicated by the high levels of genetic recombination and the IMa analyses with the EST loci. Nevertheless, multilocus analysis could provide complementary information among the loci on the species split and the extent of gene flow between the species. In conclusion, this study not only detected frequent gene flow among Lilium sections that resulted in phylogenetic incongruence but also reconstructed a hypothetical species tree that gave insights into the nature of the complex relationships among Lilium species. PMID:28841664

Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-Like Eschar.

PubMed

Marston, Chung K; Ibrahim, Hisham; Lee, Philip; Churchwell, George; Gumke, Megan; Stanek, Danielle; Gee, Jay E; Boyer, Anne E; Gallegos-Candela, Maribel; Barr, John R; Li, Han; Boulay, Darbi; Cronin, Li; Quinn, Conrad P; Hoffmaster, Alex R

2016-01-01

Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids (pBCXO1, pBC210 and pBFH_1). This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity.

Assessing Species Boundaries Using Multilocus Species Delimitation in a Morphologically Conserved Group of Neotropical Freshwater Fishes, the Poecilia sphenops Species Complex (Poeciliidae)

PubMed Central

Bagley, Justin C.; Alda, Fernando; Breitman, M. Florencia; Bermingham, Eldredge; van den Berghe, Eric P.; Johnson, Jerald B.

2015-01-01

Accurately delimiting species is fundamentally important for understanding species diversity and distributions and devising effective strategies to conserve biodiversity. However, species delimitation is problematic in many taxa, including ‘non-adaptive radiations’ containing morphologically cryptic lineages. Fortunately, coalescent-based species delimitation methods hold promise for objectively estimating species limits in such radiations, using multilocus genetic data. Using coalescent-based approaches, we delimit species and infer evolutionary relationships in a morphologically conserved group of Central American freshwater fishes, the Poecilia sphenops species complex. Phylogenetic analyses of multiple genetic markers (sequences of two mitochondrial DNA genes and five nuclear loci) from 10/15 species and genetic lineages recognized in the group support the P. sphenops species complex as monophyletic with respect to outgroups, with eight mitochondrial ‘major-lineages’ diverged by ≥2% pairwise genetic distances. From general mixed Yule-coalescent models, we discovered (conservatively) 10 species within our concatenated mitochondrial DNA dataset, 9 of which were strongly supported by subsequent multilocus Bayesian species delimitation and species tree analyses. Results suggested species-level diversity is underestimated or overestimated by at least ~15% in different lineages in the complex. Nonparametric statistics and coalescent simulations indicate genealogical discordance among our gene tree results has mainly derived from interspecific hybridization in the nuclear genome. However, mitochondrial DNA show little evidence for introgression, and our species delimitation results appear robust to effects of this process. Overall, our findings support the utility of combining multiple lines of genetic evidence and broad phylogeographical sampling to discover and validate species using coalescent-based methods. Our study also highlights the importance of testing for hybridization versus incomplete lineage sorting, which aids inference of not only species limits but also evolutionary processes influencing genetic diversity. PMID:25849959

Limited Phylogeographic Signal in Sex-Linked and Autosomal Loci Despite Geographically, Ecologically, and Phenotypically Concordant Structure of mtDNA Variation in the Holarctic Avian Genus Eremophila

PubMed Central

Drovetski, Sergei V.; Raković, Marko; Semenov, Georgy; Fadeev, Igor V.; Red’kin, Yaroslav A.

2014-01-01

Phylogeographic studies of Holarctic birds are challenging because they involve vast geographic scale, complex glacial history, extensive phenotypic variation, and heterogeneous taxonomic treatment across countries, all of which require large sample sizes. Knowledge about the quality of phylogeographic information provided by different loci is crucial for study design. We use sequences of one mtDNA gene, one sex-linked intron, and one autosomal intron to elucidate large scale phylogeographic patterns in the Holarctic lark genus Eremophila. The mtDNA ND2 gene identified six geographically, ecologically, and phenotypically concordant clades in the Palearctic that diverged in the Early - Middle Pleistocene and suggested paraphyly of the horned lark (E. alpestris) with respect to the Temminck's lark (E. bilopha). In the Nearctic, ND2 identified five subclades which diverged in the Late Pleistocene. They overlapped geographically and were not concordant phenotypically or ecologically. Nuclear alleles provided little information on geographic structuring of genetic variation in horned larks beyond supporting the monophyly of Eremophila and paraphyly of the horned lark. Multilocus species trees based on two nuclear or all three loci provided poor support for haplogroups identified by mtDNA. The node ages calculated using mtDNA were consistent with the available paleontological data, whereas individual nuclear loci and multilocus species trees appeared to underestimate node ages. We argue that mtDNA is capable of discovering independent evolutionary units within avian taxa and can provide a reasonable phylogeographic hypothesis when geographic scale, geologic history, and phenotypic variation in the study system are too complex for proposing reasonable a priori hypotheses required for multilocus methods. Finally, we suggest splitting the currently recognized horned lark into five Palearctic and one Nearctic species. PMID:24498139

Assessing species boundaries using multilocus species delimitation in a morphologically conserved group of neotropical freshwater fishes, the Poecilia sphenops species complex (Poeciliidae).

PubMed

Bagley, Justin C; Alda, Fernando; Breitman, M Florencia; Bermingham, Eldredge; van den Berghe, Eric P; Johnson, Jerald B

2015-01-01

Accurately delimiting species is fundamentally important for understanding species diversity and distributions and devising effective strategies to conserve biodiversity. However, species delimitation is problematic in many taxa, including 'non-adaptive radiations' containing morphologically cryptic lineages. Fortunately, coalescent-based species delimitation methods hold promise for objectively estimating species limits in such radiations, using multilocus genetic data. Using coalescent-based approaches, we delimit species and infer evolutionary relationships in a morphologically conserved group of Central American freshwater fishes, the Poecilia sphenops species complex. Phylogenetic analyses of multiple genetic markers (sequences of two mitochondrial DNA genes and five nuclear loci) from 10/15 species and genetic lineages recognized in the group support the P. sphenops species complex as monophyletic with respect to outgroups, with eight mitochondrial 'major-lineages' diverged by ≥2% pairwise genetic distances. From general mixed Yule-coalescent models, we discovered (conservatively) 10 species within our concatenated mitochondrial DNA dataset, 9 of which were strongly supported by subsequent multilocus Bayesian species delimitation and species tree analyses. Results suggested species-level diversity is underestimated or overestimated by at least ~15% in different lineages in the complex. Nonparametric statistics and coalescent simulations indicate genealogical discordance among our gene tree results has mainly derived from interspecific hybridization in the nuclear genome. However, mitochondrial DNA show little evidence for introgression, and our species delimitation results appear robust to effects of this process. Overall, our findings support the utility of combining multiple lines of genetic evidence and broad phylogeographical sampling to discover and validate species using coalescent-based methods. Our study also highlights the importance of testing for hybridization versus incomplete lineage sorting, which aids inference of not only species limits but also evolutionary processes influencing genetic diversity.

Development of a multilocus sequence typing scheme for Ureaplasma.

PubMed

Zhang, J; Kong, Y; Feng, Y; Huang, J; Song, T; Ruan, Z; Song, J; Jiang, Y; Yu, Y; Xie, X

2014-04-01

Ureaplasma is a commensal of the human urogenital tract but is always associated with invasive diseases such as non-gonococcal urethritis and infertility adverse pregnancy outcomes. To better understand the molecular epidemiology and population structure of Ureaplasma, a multilocus sequence typing (MLST) scheme based on four housekeeping genes (ftsH, rpL22, valS, thrS) was developed and validated using 283 isolates, including 14 serovars of reference strains and 269 strains obtained from clinical patients. A total of 99 sequence types (STs) were revealed: the 14 type strains of the Ureaplasma serovars were assigned to 12 STs, and 87 novel and special STs appeared among the clinical isolates. ST1 and ST22 were the predominant STs, which contained 68 and 70 isolates, respectively. Two clonal lineages (CC1 and CC2) were shown by eBURST analysis, and linkage disequilibrium was revealed through a standardized index of association (I A (S)). The neighbor-joining tree results of 14 Ureaplasma serovars showed two genetically significantly distant clusters, which was highly congruent with the species taxonomy of ureaplasmas [Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR)]. Analysis of the biotypes of 269 clinical isolates revealed that all the isolates of CC1 were UPA and those of CC2 were UUR. Additionally, CC2 was found more often in symptomatic patients with vaginitis, tubal obstruction, and cervicitis. In conclusion, this MLST scheme is adequate for investigations of molecular epidemiology and population structure with highly discriminating capacity.

Serotypes, Antibiotic Susceptibilities, and Multi-Locus Sequence Type Profiles of Streptococcus agalactiae Isolates Circulating in Beijing, China

PubMed Central

Ma, Xiu-hua; Song, Feng-li; Fan, Ling; Guo, Cui-mei; Shi, Wei; Yu, Sang-jie; Yao, Kai-hu; Yang, Yong-hong

2015-01-01

Background To investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of Streptococcus agalactiae (S. agalactiae) in Beijing to provide references for the prevention and treatment of S. agalactiae infections. Methods All isolates were identified using the CAMP test and the latex-agglutination assay and serotyped using a Strep-B-Latex kit, after which they were assessed for antibiotic susceptibility, macrolide-resistance genes, and MLST profiles. Results In total, 56 S. agalactiae isolates were identified in 863 pregnant women (6.5%). Serotypes Ia, Ib, II, III, and V were identified, among which types III (32.1%), Ia (17.9%), Ib (16.1%), and V (14.3%) were the predominant serotypes. All isolates were susceptible to penicillin and ceftriaxone. The nonsusceptiblity rates measured for erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, tetracycline, and levofloxacin were 85.7%, 92.9%, 98.2%, 30.4%, 73.2%, 91%, and 39.3%, respectively. We identified 14 sequence types (STs) for the 56 isolates, among which ST19 (30.4%) was predominant. The rate of fluoroquinolone resistance was higher in serotype III than in the other serotypes. Among the 44 erythromycin-resistant isolates, 32 (72.7%) carried ermB. Conclusion S. agalactiae isolates of the serotypes Ia, Ib, III, and V are common in Beijing. Among the S. agalactiae isolates, the macrolide and clindamycin resistance rates are extremely high. Most of the erythromycin-resistant isolates carry ermB. PMID:25781346

Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations.

PubMed

Cholley, Pascal; Stojanov, Milos; Hocquet, Didier; Thouverez, Michelle; Bertrand, Xavier; Blanc, Dominique S

2015-08-01

Reliable molecular typing methods are necessary to investigate the epidemiology of bacterial pathogens. Reference methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are costly and time consuming. Here, we compared our newly developed double-locus sequence typing (DLST) method for Pseudomonas aeruginosa to MLST and PFGE on a collection of 281 isolates. DLST was as discriminatory as MLST and was able to recognize "high-risk" epidemic clones. Both methods were highly congruent. Not surprisingly, a higher discriminatory power was observed with PFGE. In conclusion, being a simple method (single-strand sequencing of only 2 loci), DLST is valuable as a first-line typing tool for epidemiological investigations of P. aeruginosa. Coupled to a more discriminant method like PFGE or whole genome sequencing, it might represent an efficient typing strategy to investigate or prevent outbreaks. Copyright © 2015 Elsevier Inc. All rights reserved.

Environmental Staphylococcus aureus contamination in a Tunisian hospital.

PubMed

Gharsa, Haythem; Dziri, Raoudha; Klibi, Naouel; Chairat, Sarra; Lozano, Carmen; Torres, Carmen; Bellaaj, Ridha; Slama, Karim Ben

2016-12-01

One hundred hospital environment samples were obtained in 2012 in a Tunisian hospital and tested for Staphylococcus aureus recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST), spa-typing, agr-typing and SmaI-pulsed-field gel electrophoresis (PFGE) were performed. Two methicillin-resistant S. aureus (MRSA) isolates typed as: ST247-t052-SCCmecI-agrI were recovered from the intensive care unit (ICU). Ten samples contained methicillin-susceptible S. aureus (MSSA) and these samples were collected in different services, highlighting the presence of the tst gene encoding the toxic shock syndrome toxin as well as the lukED, hla, hlb, hld and hlg v virulence genes in some of the isolates. In conclusion, we have shown that the hospital environment could be a reservoir contributing to dissemination of virulent S. aureus and MRSA.

Comparison of Genotypes and Enterotoxin Genes Between Staphylococcus aureus Isolates from Blood and Nasal Colonizers in a Korean Hospital

PubMed Central

Peck, Kyong Ran; Baek, Jin Yang; Song, Jae-Hoon

2009-01-01

In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness. PMID:19654937

A phylogenomic approach to bacterial subspecies classification: proof of concept in Mycobacterium abscessus.

PubMed

Tan, Joon Liang; Khang, Tsung Fei; Ngeow, Yun Fong; Choo, Siew Woh

2013-12-13

Mycobacterium abscessus is a rapidly growing mycobacterium that is often associated with human infections. The taxonomy of this species has undergone several revisions and is still being debated. In this study, we sequenced the genomes of 12 M. abscessus strains and used phylogenomic analysis to perform subspecies classification. A data mining approach was used to rank and select informative genes based on the relative entropy metric for the construction of a phylogenetic tree. The resulting tree topology was similar to that generated using the concatenation of five classical housekeeping genes: rpoB, hsp65, secA, recA and sodA. Additional support for the reliability of the subspecies classification came from the analysis of erm41 and ITS gene sequences, single nucleotide polymorphisms (SNPs)-based classification and strain clustering demonstrated by a variable number tandem repeat (VNTR) assay and a multilocus sequence analysis (MLSA). We subsequently found that the concatenation of a minimal set of three median-ranked genes: DNA polymerase III subunit alpha (polC), 4-hydroxy-2-ketovalerate aldolase (Hoa) and cell division protein FtsZ (ftsZ), is sufficient to recover the same tree topology. PCR assays designed specifically for these genes showed that all three genes could be amplified in the reference strain of M. abscessus ATCC 19977T. This study provides proof of concept that whole-genome sequence-based data mining approach can provide confirmatory evidence of the phylogenetic informativeness of existing markers, as well as lead to the discovery of a more economical and informative set of markers that produces similar subspecies classification in M. abscessus. The systematic procedure used in this study to choose the informative minimal set of gene markers can potentially be applied to species or subspecies classification of other bacteria.

High frequency of silver resistance genes in invasive isolates of Enterobacter and Klebsiella species.

PubMed

Sütterlin, S; Dahlö, M; Tellgren-Roth, C; Schaal, W; Melhus, Å

2017-07-01

Silver-based products have been marketed as an alternative to antibiotics, and their consumption has increased. Bacteria may, however, develop resistance to silver. To study the presence of genes encoding silver resistance (silE, silP, silS) over time in three clinically important Enterobacteriaceae genera. Using polymerase chain reaction (PCR), 752 bloodstream isolates from the years 1990-2010 were investigated. Age, gender, and ward of patients were registered, and the susceptibility to antibiotics and silver nitrate was tested. Clonality and single nucleotide polymorphism were assessed with repetitive element sequence-based PCR, multi-locus sequence typing, and whole-genome sequencing. Genes encoding silver resistance were detected most frequently in Enterobacter spp. (48%), followed by Klebsiella spp. (41%) and Escherichia coli 4%. Phenotypical resistance to silver nitrate was found in Enterobacter (13%) and Klebsiella (3%) isolates. The lowest carriage rate of sil genes was observed in blood isolates from the neonatology ward (24%), and the highest in blood isolates from the oncology/haematology wards (66%). Presence of sil genes was observed in international high-risk clones. Sequences of the sil and pco clusters indicated that a single mutational event in the silS gene could have caused the phenotypic resistance. Despite a restricted consumption of silver-based products in Swedish health care, silver resistance genes are widely represented in clinical isolates of Enterobacter and Klebsiella species. To avoid further selection and spread of silver-resistant bacteria with a high potential for healthcare-associated infections, the use of silver-based products needs to be controlled and the silver resistance monitored. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Antibiotic Susceptibilities and Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Stools of Pediatric Diarrhea Patients in Surabaya, Indonesia.

PubMed

Bagus Wasito, Eddy; Shigemura, Katsumi; Osawa, Kayo; Fardah, Alpha; Kanaida, Akiho; Raharjo, Dadik; Kuntaman, K; Hadi, Usman; Harijono, Sugeng; Marto Sudarmo, Subijanto; Nakamura, Tatsuya; Shibayama, Keigo; Fujisawa, Masato; Shirakawa, Toshiro

2017-07-24

The purpose of this study was to investigate extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from pediatric (aged 0 to 3 years) diarrhea patients in Surabaya, Indonesia, where this kind of survey is rare; our study included assessment of their antibiotic susceptibilities, as well as ESBL typing, multilocus sequence typing (MLST), and diarrheagenic E. coli (DEC)-typing. ESBL-producing E. coli were detected in 18.8% of all the samples. Many ESBL-producing E. coli had significantly lower susceptibility to gentamicin (p ＜ 0.0001) and the quinolones nalidixic acid (p＝0.004) and ciprofloxacin (p ＜ 0.0001) than non-producers. In ESBL-producing E. coli, 84.0% of strains expressed CTX-M-15 alone or in combination with other ESBL types. MLST revealed that 24.0% of ESBL-producers had sequence type 617, all of which expressed the CTX-M-15 gene; we also detected expression of 3 DEC-related genes: 2 enteroaggregative E. coli genes and 1 enteropathogenic E. coli gene. In conclusion, CTX-M-15-type ESBL-producing E. coli ST617 appear to have spread to Indonesia.

Comparison and Evaluation of the Molecular Typing Methods for Toxigenic Vibrio cholerae in Southwest China.

PubMed

Liao, Feng; Mo, Zhishuo; Chen, Meiling; Pang, Bo; Fu, Xiaoqing; Xu, Wen; Jing, Huaiqi; Kan, Biao; Gu, Wenpeng

2018-01-01

Vibrio cholerae O1 strains taken from the repository of Yunnan province, southwest China, were abundant and special. We selected 70 typical toxigenic V. cholerae (69 O1 and one O139 serogroup strains) isolated from Yunnan province, performed the pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and MLST of virulence gene (V-MLST) methods, and evaluated the resolution abilities for typing methods. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. Seventy V. cholerae obtained 50 PFGE patterns, with a high resolution. The strains could be divided into three groups with predominance of strains isolated during 1980s, 1990s, and 2000s, respectively, showing a good consistency with the epidemiological investigation. We also evaluated two MLST method for V. cholerae , one was used seven housekeeping genes ( adk , gyrB , metE , pntA , mdh , purM , and pyrC ), and all the isolates belonged to ST69; another was used nine housekeeping genes ( cat , chi , dnaE , gyrB , lap , pgm , recA , rstA , and gmd ). A total of seven sequence types (STs) were found by using this method for all the strains; among them, rstA gene had five alleles, recA and gmd have two alleles, and others had only one allele. The virulence gene sequence typing method ( ctxAB , tcpA , and toxR ) showed that 70 strains were divided into nine STs; among them, tcpA gene had six alleles, toxR had five alleles, while ctxAB was identical for all the strains. The latter two sequences based typing methods also had consistency with epidemiology of the strains. PFGE had a higher resolution ability compared with the sequence based typing method, and MLST used seven housekeeping genes showed the lower resolution power than nine housekeeping genes and virulence genes methods. These two sequence typing methods could distinguish some epidemiological special strains in local area.

The Nostoc punctiforme Genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

John C. Meeks

2001-12-31

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple development alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9more » Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.« less

The population structure of Vibrio cholerae from the Chandigarh Region of Northern India.

PubMed

Abd El Ghany, Moataz; Chander, Jagadish; Mutreja, Ankur; Rashid, Mamoon; Hill-Cawthorne, Grant A; Ali, Shahjahan; Naeem, Raeece; Thomson, Nicholas R; Dougan, Gordon; Pain, Arnab

2014-07-01

Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century. Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters. The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.

Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

PubMed

De Meyer, Sofie E; Briscoe, Leah; Martínez-Hidalgo, Pilar; Agapakis, Christina M; de-Los Santos, Paulina Estrada; Seshadri, Rekha; Reeve, Wayne; Weinstock, George; O'Hara, Graham; Howieson, John G; Hirsch, Ann M

2016-08-01

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

Clavibacter michiganensis subsp. phaseoli subsp. nov., pathogenic in bean.

PubMed

González, Ana J; Trapiello, Estefanía

2014-05-01

A yellow Gram-reaction-positive bacterium isolated from bean seeds (Phaseolus vulgaris L.) was identified as Clavibacter michiganensis by 16S rRNA gene sequencing. Molecular methods were employed in order to identify the subspecies. Such methods included the amplification of specific sequences by PCR, 16S amplified rDNA restriction analysis (ARDRA), RFLP and multilocus sequence analysis as well as the analysis of biochemical and phenotypic traits including API 50CH and API ZYM results. The results showed that strain LPPA 982T did not represent any known subspecies of C. michiganensis. Pathogenicity tests revealed that the strain is a bean pathogen causing a newly identified bacterial disease that we name bacterial bean leaf yellowing. On the basis of these results, strain LPPA 982T is regarded as representing a novel subspecies for which the name Clavibacter michiganensis subsp. phaseoli subsp. nov. is proposed. The type strain is LPPA 982T (=CECT 8144T=LMG 27667T).

Antimicrobial Resistance, Virulence Profile, and Molecular Characterization of Listeria monocytogenes Isolated from Ready-to-eat Food in China, 2013-2014.

PubMed

Yan, Shao Fei; Wang, Wei; Bai, Li; Hu, Yu Jie; Dong, Yin Ping; Xu, Jin; Li, Feng Qin

2016-06-01

We aimed to investigate the potential pathogenic profile and antibiotic resistance of Listeria monocytogenes isolated from ready-to-eat food in China. Antimicrobial resistance was determined by broth microdilution following the Clinical and Laboratory Standards Institute protocol. Molecular serotyping, virulence, and resistance genes were identified using PCR. Multi-locus sequence typing was performed on resistant strains. A total of 11.53% (113/980) isolates were resistant, from which 82.3% (93/113) harbored all the virulence genes tested. The resistant strains were subtyped into 18 sequence types (STs), from which ST2, ST5, ST8, and ST9 were involved in listeriosis. This study indicated that several L. monocytogenes isolates from ready-to-eat foods in China have pathogenic potential and are resistant to antibiotics, including antibiotics used as medicines by humans for listeriosis treatment. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Epidemiological information is key when interpreting whole genome sequence data – lessons learned from a large Legionella pneumophila outbreak in Warstein, Germany, 2013

PubMed Central

Petzold, Markus; Prior, Karola; Moran-Gilad, Jacob; Harmsen, Dag; Lück, Christian

2017-01-01

Introduction Whole genome sequencing (WGS) is increasingly used in Legionnaires’ disease (LD) outbreak investigations, owing to its higher resolution than sequence-based typing, the gold standard typing method for Legionella pneumophila, in the analysis of endemic strains. Recently, a gene-by-gene typing approach based on 1,521 core genes called core genome multilocus sequence typing (cgMLST) was described that enables a robust and standardised typing of L. pneumophila. Methods: We applied this cgMLST scheme to isolates obtained during the largest outbreak of LD reported so far in Germany. In this outbreak, the epidemic clone ST345 had been isolated from patients and four different environmental sources. In total 42 clinical and environmental isolates were retrospectively typed. Results: Epidemiologically unrelated ST345 isolates were clearly distinguishable from the epidemic clone. Remarkably, epidemic isolates split up into two distinct clusters, ST345-A and ST345-B, each respectively containing a mix of clinical and epidemiologically-related environmental samples. Discussion/conclusion: The outbreak was therefore likely caused by both variants of the single sequence type, which pre-existed in the environmental reservoirs. The two clusters differed by 40 alleles located in two neighbouring genomic regions of ca 42 and 26 kb. Additional analysis supported horizontal gene transfer of the two regions as responsible for the difference between the variants. Both regions comprise virulence genes and have previously been reported to be involved in recombination events. This corroborates the notion that genomic outbreak investigations should always take epidemiological information into consideration when making inferences. Overall, cgMLST proved helpful in disentangling the complex genomic epidemiology of the outbreak. PMID:29162202

Epidemiological information is key when interpreting whole genome sequence data - lessons learned from a large Legionella pneumophila outbreak in Warstein, Germany, 2013.

PubMed

Petzold, Markus; Prior, Karola; Moran-Gilad, Jacob; Harmsen, Dag; Lück, Christian

2017-11-01

IntroductionWhole genome sequencing (WGS) is increasingly used in Legionnaires' disease (LD) outbreak investigations, owing to its higher resolution than sequence-based typing, the gold standard typing method for Legionella pneumophila, in the analysis of endemic strains. Recently, a gene-by-gene typing approach based on 1,521 core genes called core genome multilocus sequence typing (cgMLST) was described that enables a robust and standardised typing of L. pneumophila . Methods : We applied this cgMLST scheme to isolates obtained during the largest outbreak of LD reported so far in Germany. In this outbreak, the epidemic clone ST345 had been isolated from patients and four different environmental sources. In total 42 clinical and environmental isolates were retrospectively typed. Results : Epidemiologically unrelated ST345 isolates were clearly distinguishable from the epidemic clone. Remarkably, epidemic isolates split up into two distinct clusters, ST345-A and ST345-B, each respectively containing a mix of clinical and epidemiologically-related environmental samples. Discussion/conclusion : The outbreak was therefore likely caused by both variants of the single sequence type, which pre-existed in the environmental reservoirs. The two clusters differed by 40 alleles located in two neighbouring genomic regions of ca 42 and 26 kb. Additional analysis supported horizontal gene transfer of the two regions as responsible for the difference between the variants. Both regions comprise virulence genes and have previously been reported to be involved in recombination events. This corroborates the notion that genomic outbreak investigations should always take epidemiological information into consideration when making inferences. Overall, cgMLST proved helpful in disentangling the complex genomic epidemiology of the outbreak.

Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit).

PubMed

Lima, Luciana; Espinosa-Álvarez, Oneida; Ortiz, Paola A; Trejo-Varón, Javier A; Carranza, Julio C; Pinto, C Miguel; Serrano, Myrna G; Buck, Gregory A; Camargo, Erney P; Teixeira, Marta M G

2015-11-01

Trypanosoma cruzi is a complex of phenotypically and genetically diverse isolates distributed in six discrete typing units (DTUs) designated as TcI-TcVI. Five years ago, T. cruzi isolates from Brazilian bats showing unique patterns of traditional ribosomal and spliced leader PCRs not clustering into any of the six DTUs were designated as the Tcbat genotype. In the present study, phylogenies inferred using SSU rRNA (small subunit of ribosomal rRNA), gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cytb (cytochrome b) genes strongly supported Tcbat as a monophyletic lineage prevalent in Brazil, Panama and Colombia. Providing strong support for Tcbat, sequences from 37 of 47 nuclear and 12 mitochondrial genes (retrieved from a draft genome of Tcbat) and reference strains of all DTUs available in databanks corroborated Tcbat as an independent DTU. Consistent with previous studies, multilocus analysis of most nuclear genes corroborated the evolution of T. cruzi from bat trypanosomes its divergence into two main phylogenetic lineages: the basal TcII; and the lineage clustering TcIV, the clade comprising TcIII and the sister groups TcI-Tcbat. Most likely, the common ancestor of Tcbat and TcI was a bat trypanosome. However, the results of the present analysis did not support Tcbat as the ancestor of all DTUs. Despite the insights provided by reports of TcIII, TcIV and TcII in bats, including Amazonian bats harbouring TcII, further studies are necessary to understand the roles played by bats in the diversification of all DTUs. We also demonstrated that in addition to value as molecular markers for DTU assignment, Cytb, ITS rDNA and the spliced leader (SL) polymorphic sequences suggest spatially structured populations of Tcbat. Phylogenetic and phylogeographical analyses, multiple molecular markers specific to Tcbat, and the degrees of sequence divergence between Tcbat and the accepted DTUs strongly support the definitive classification of Tcbat as a new DTU. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of extended-spectrum cephalosporin resistant Salmonella enterica serovar Heidelberg isolated from food animals, retail meat, and humans in the United States 2009

PubMed Central

Folster, J. P.; Pecic, G.; Singh, A.; Duval, B.; Rickert, R.; Ayers, S.; Abbott, J.; McGlinchey, B.; Bauer-Turpin, J.; Haro, J.; Hise, K.; Zhao, S.; Fedorka-Cray, P. J.; Whichard, J.; McDermott, P. F.

2015-01-01

Salmonella enterica is one of the most common causes of foodborne illness in the United States. Although salmonellosis is usually self-limiting, severe infections typically require antimicrobial treatment and ceftriaxone, an extended-spectrum cephalosporin, is commonly used in both adults and children. Surveillance conducted by the National Antimicrobial Resistance Monitoring System (NARMS) has shown a recent increase in extended-spectrum cephalosporin (ESC) resistance among Salmonella Heidelberg isolated from food animals at slaughter, retail meat, and humans. ESC resistance among Salmonella in the United States is usually mediated by a plasmid-encoded blaCMY β-lactamase. In 2009, we identified 47 ESC resistant blaCMY-positive Heidelberg isolates from humans (n=18), food animals at slaughter (n=16), and retail meats (n=13) associated with a spike in the prevalence of this serovar. Almost 90% (26/29) of the animal and meat isolates were isolated from chicken carcasses or retail chicken meat. We screened NARMS isolates for the presence of blaCMY, determined whether the gene was plasmid-encoded, examined pulsed-field gel electrophoresis patterns to assess the genetic diversities of the isolates, and categorized the blaCMY plasmids by plasmid incompatibility groups and plasmid multi-locus sequence typing. All 47 blaCMY genes were found to be plasmid encoded. Incompatibility/replicon typing demonstrated that 41 were IncI1 plasmids, 40 of which only conferred blaCMY associated resistance. Six were IncA/C plasmids that carried additional resistance genes. Plasmid multi-locus sequence typing (pMLST) of the IncI1-blaCMY plasmids showed that 27 (65.8%) were sequence type (ST) 12, the most common ST among blaCMY-IncI1 plasmids from Heidelberg isolated from humans. Ten plasmids had a new ST profile, ST66, a type very similar to ST12. This work showed that the 2009 increase in ESC resistance among Salmonella Heidelberg was caused mainly by the dissemination of blaCMY on IncI1 and IncA/C plasmids in a variety of genetic backgrounds, and likely not the result of clonal expansion. PMID:22755514

Genomic diversity of necrotic enteritis-associated strains of Clostridium perfringens: a review.

PubMed

Lacey, Jake A; Johanesen, Priscilla A; Lyras, Dena; Moore, Robert J

2016-06-01

The investigation of genomic variation between Clostridium perfringens isolates from poultry has been an important tool to enhance our understanding of the genetic basis of strain pathogenicity and the epidemiology of virulent and avirulent strains within the context of necrotic enteritis (NE). The earliest studies used whole genome profiling techniques such as pulsed-field gel electrophoresis to differentiate isolates and determine their relative levels of relatedness. DNA sequencing has been used to investigate genetic variation in (a) individual genes, such as those encoding the alpha and NetB toxins; (b) panels of housekeeping genes for multi-locus sequence typing and (c) most recently whole genome sequencing to build a more complete picture of genomic differences between isolates. Conclusions drawn from these studies include: differential carriage of large conjugative plasmids accounts for a large proportion of inter-strain differences; plasmid-encoded genes are more highly conserved than chromosomal genes, perhaps indicating a relatively recent origin for the plasmids; isolates from NE-affected birds fall into three distinct sequence-based clades while non-pathogenic isolates from healthy birds tend to be more genomically diverse. Overall, the NE causing strains are closely related to C. perfringens isolates from other birds and other diseases whereas the non-pathogenic poultry strains are generally more remotely related to either the pathogenic strains or the strains from other birds. Genomic analysis has indicated that genes in addition to netB are associated with NE pathogenic isolates. Collectively, this work has resulted in a deeper understanding of the pathogenesis of this important poultry disease.

Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain.

PubMed

Esteves, F; Montes-Cano, M A; de la Horra, C; Costa, M C; Calderón, E J; Antunes, F; Matos, O

2008-04-01

Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.

First report of blaNDM-1-producing Acinetobacter baumannii isolated in Lebanon from civilians wounded during the Syrian war.

PubMed

Rafei, Rayane; Dabboussi, Fouad; Hamze, Monzer; Eveillard, Matthieu; Lemarié, Carole; Mallat, Hassan; Rolain, Jean-Marc; Joly-Guillou, Marie-Laure; Kempf, Marie

2014-04-01

The emergence of carbapenem-resistant Acinetobacter baumannii has been observed worldwide. We describe the first detection of A. baumannii carrying the blaNDM-1 gene in Lebanon, isolated from Syrian patients wounded during the civil war. Four carbapenem-resistant A. baumannii strains isolated in 2012 in the Tripoli Government Hospital, Lebanon, from civilians wounded during the Syrian war, were analysed. Susceptibility was determined by disk diffusion testing, and resistance to carbapenems was confirmed by Etest. The presence of blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, blaOXA-143-like, and blaNDM was investigated by PCR. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and blaOXA-51 sequence-based typing. All isolates harboured the blaNDM-1 gene and were negative for other tested carbapenemases. They all belonged to the sequence type 85 and formed a single cluster by PFGE. Finally, blaOXA-51-like gene sequencing revealed the presence of the blaOXA-94 variant in all four isolates. These findings show that Syria constitutes a reservoir for NDM-1-producing bacteria. These results also highlight the need for effective measures to stop the threatening spread of such strains. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

The utility of multiple molecular methods including whole genome sequencing as tools to differentiate Escherichia coli O157:H7 outbreaks.

PubMed

Berenger, Byron M; Berry, Chrystal; Peterson, Trevor; Fach, Patrick; Delannoy, Sabine; Li, Vincent; Tschetter, Lorelee; Nadon, Celine; Honish, Lance; Louie, Marie; Chui, Linda

2015-01-01

A standardised method for determining Escherichia coli O157:H7 strain relatedness using whole genome sequencing or virulence gene profiling is not yet established. We sought to assess the capacity of either high-throughput polymerase chain reaction (PCR) of 49 virulence genes, core-genome single nt variants (SNVs) or k-mer clustering to discriminate between outbreak-associated and sporadic E. coli O157:H7 isolates. Three outbreaks and multiple sporadic isolates from the province of Alberta, Canada were included in the study. Two of the outbreaks occurred concurrently in 2014 and one occurred in 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA) were employed as comparator typing methods. The virulence gene profiles of isolates from the 2012 and 2014 Alberta outbreak events and contemporary sporadic isolates were mostly identical; therefore the set of virulence genes chosen in this study were not discriminatory enough to distinguish between outbreak clusters. Concordant with PFGE and MLVA results, core genome SNV and k-mer phylogenies clustered isolates from the 2012 and 2014 outbreaks as distinct events. k-mer phylogenies demonstrated increased discriminatory power compared with core SNV phylogenies. Prior to the widespread implementation of whole genome sequencing for routine public health use, issues surrounding cost, technical expertise, software standardisation, and data sharing/comparisons must be addressed.

Migratory White Stork (Ciconia ciconia): A Potential Vector of the OXA-48-Producing Escherichia coli ST38 Clone in Algeria.

PubMed

Bouaziz, Amira; Loucif, Lotfi; Ayachi, Ammar; Guehaz, Karima; Bendjama, Esma; Rolain, Jean-Marc

2018-05-01

The emergence of carbapenemase-producing Enterobacteriaceae is of great concern to public health worldwide. The aim of this study was to screen for the presence of carbapenemase-producing Enterobacteriaceae in white stork (Ciconia ciconia) migratory bird stools, and to investigate their molecular support on β-lactamase production. In March 2015, 32 fecal samples of white stork were collected in the Commune of El Madher Wilaya de Batna, in eastern Algeria. Samples were subjected to selective isolation of carbapenem-resistant Enterobacteriaceae. Representative colonies were screened phenotypically for carbapenemase production. Carbapenemase-producing isolates were subjected to antibiotic susceptibility testing and extended-spectrum β-lactamase (ESBL) coproduction. β-Lactamase determinants were searched for by PCR and sequencing. Three carbapenemase-producing Escherichia coli were obtained. Only one strain was positive for ESBL production. The OXA-48-type carbapenemase-encoding gene was detected in all isolates. Screening for other β-lactamase-encoding genes showed that all isolates coexpress the bla TEM gene, whereas one of them additionally harbored the bla CTX-M-15 ESBL gene. Multilocus sequence typing results showed that two strains belonged to the sequence type 38. This work demonstrated for the first time that the migratory white stork can play an important role in the dissemination of OXA-48-producing E. coli as a potential reservoir and vector.

Difficult identification of Haemophilus influenzae, a typical cause of upper respiratory tract infections, in the microbiological diagnostic routine

PubMed Central

Hinz, Rebecca; Zautner, Andreas Erich; Hagen, Ralf Matthias

2015-01-01

Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region. Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption–ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing. PMID:25883794

Difficult identification of Haemophilus influenzae, a typical cause of upper respiratory tract infections, in the microbiological diagnostic routine.

PubMed

Hinz, Rebecca; Zautner, Andreas Erich; Hagen, Ralf Matthias; Frickmann, Hagen

2015-03-01

Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region. Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.

Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch.

PubMed

den Bakker, Henk C; Manuel, Clyde S; Fortes, Esther D; Wiedmann, Martin; Nightingale, Kendra K

2013-09-01

Twenty Listeria-like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1(T)) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91% of the genomes was 95.16%. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria-like isolates to Listeria fleischmannii LU2006-1(T). Sufficient genetic divergence of the Listeria-like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001(T) ( =BAA-2414(T) =DSM 25391(T)). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp. coloradonensis subsp. nov. genome also encoded a putative mosquitocidal toxin. The presence of these genes suggests possible adaptation to an insect host, and further studies are needed to probe niche adaptation of Listeria fleischmannii.

Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients.

PubMed

Hoshino, Tomonori; Fujiwara, Taku; Kilian, Mogens

2005-12-01

The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.

Population Structure and Antimicrobial Resistance Profiles of Streptococcus suis Serotype 2 Sequence Type 25 Strains.

PubMed

Athey, Taryn B T; Teatero, Sarah; Takamatsu, Daisuke; Wasserscheid, Jessica; Dewar, Ken; Gottschalk, Marcelo; Fittipaldi, Nahuel

2016-01-01

Strains of serotype 2 Streptococcus suis are responsible for swine and human infections. Different serotype 2 genetic backgrounds have been defined using multilocus sequence typing (MLST). However, little is known about the genetic diversity within each MLST sequence type (ST). Here, we used whole-genome sequencing to test the hypothesis that S. suis serotype 2 strains of the ST25 lineage are genetically heterogeneous. We evaluated 51 serotype 2 ST25 S. suis strains isolated from diseased pigs and humans in Canada, the United States of America, and Thailand. Whole-genome sequencing revealed numerous large-scale rearrangements in the ST25 genome, compared to the genomes of ST1 and ST28 S. suis strains, which result, among other changes, in disruption of a pilus island locus. We report that recombination and lateral gene transfer contribute to ST25 genetic diversity. Phylogenetic analysis identified two main and distinct Thai and North American clades grouping most strains investigated. These clades also possessed distinct patterns of antimicrobial resistance genes, which correlated with acquisition of different integrative and conjugative elements (ICEs). Some of these ICEs were found to be integrated at a recombination hot spot, previously identified as the site of integration of the 89K pathogenicity island in serotype 2 ST7 S. suis strains. Our results highlight the limitations of MLST for phylogenetic analysis of S. suis, and the importance of lateral gene transfer and recombination as drivers of diversity in this swine pathogen and zoonotic agent.

Population Structure and Antimicrobial Resistance Profiles of Streptococcus suis Serotype 2 Sequence Type 25 Strains

PubMed Central

Athey, Taryn B. T.; Teatero, Sarah; Takamatsu, Daisuke; Wasserscheid, Jessica; Dewar, Ken; Gottschalk, Marcelo; Fittipaldi, Nahuel

2016-01-01

Strains of serotype 2 Streptococcus suis are responsible for swine and human infections. Different serotype 2 genetic backgrounds have been defined using multilocus sequence typing (MLST). However, little is known about the genetic diversity within each MLST sequence type (ST). Here, we used whole-genome sequencing to test the hypothesis that S. suis serotype 2 strains of the ST25 lineage are genetically heterogeneous. We evaluated 51 serotype 2 ST25 S. suis strains isolated from diseased pigs and humans in Canada, the United States of America, and Thailand. Whole-genome sequencing revealed numerous large-scale rearrangements in the ST25 genome, compared to the genomes of ST1 and ST28 S. suis strains, which result, among other changes, in disruption of a pilus island locus. We report that recombination and lateral gene transfer contribute to ST25 genetic diversity. Phylogenetic analysis identified two main and distinct Thai and North American clades grouping most strains investigated. These clades also possessed distinct patterns of antimicrobial resistance genes, which correlated with acquisition of different integrative and conjugative elements (ICEs). Some of these ICEs were found to be integrated at a recombination hot spot, previously identified as the site of integration of the 89K pathogenicity island in serotype 2 ST7 S. suis strains. Our results highlight the limitations of MLST for phylogenetic analysis of S. suis, and the importance of lateral gene transfer and recombination as drivers of diversity in this swine pathogen and zoonotic agent. PMID:26954687

Molecular epidemiology, phylogeny and evolution of Candida albicans.

PubMed

McManus, Brenda A; Coleman, David C

2014-01-01

A small number of Candida species form part of the normal microbial flora of mucosal surfaces in humans and may give rise to opportunistic infections when host defences are impaired. Candida albicans is by far the most prevalent commensal and pathogenic Candida species. Several different molecular typing approaches including multilocus sequence typing, multilocus microsatellite typing and DNA fingerprinting using C. albicans-specific repetitive sequence-containing DNA probes have yielded a wealth of information regarding the epidemiology and population structure of this species. Such studies revealed that the C. albicans population structure consists of multiple major and minor clades, some of which exhibit geographical or phenotypic enrichment and that C. albicans reproduction is predominantly clonal. Despite this, losses of heterozygosity by recombination, the existence of a parasexual cycle, toleration of a wide range of aneuploidies and the recent description of viable haploid strains have all demonstrated the extensive plasticity of the C. albicans genome. Recombination and gross chromosomal rearrangements are more common under stressful environmental conditions, and have played a significant role in the evolution of this opportunistic pathogen. Surprisingly, Candida dubliniensis, the closest relative of C. albicans exhibits more karyotype variability than C. albicans, but is significantly less adaptable to unfavourable environments. This disparity most likely reflects the evolutionary processes that occurred during or soon after the divergence of both species from their common ancestor. Whilst C. dubliniensis underwent significant gene loss and pseudogenisation, C. albicans expanded gene families considered to be important in virulence. It is likely that technological developments in whole genome sequencing and data analysis in coming years will facilitate its routine use for population structure, epidemiological investigations, and phylogenetic analyses of Candida species. These are likely to reveal more minor C. albicans clades and to enhance our understanding of the population biology of this versatile organism. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Prevalence of Diverse Clones of Vancomycin-Resistant Enterococcus faecium ST78 in a Chinese Hospital.

PubMed

Yang, Jiyong; Jiang, Yufeng; Guo, Ling; Ye, LIyan; Ma, Yanning; Luo, Yanping

2016-06-01

Vancomycin-resistant Enterococcus (VRE) has been identified in China. However, little is known about the spread of VRE isolates. The genetic relatedness of vancomycin-resistant Enterococcus faecium (VREfm) isolates was analyzed by pulsed-field gel electrophoresis (PFGE), their antimicrobial susceptibilities were analyzed by E-test and the VITEK 2 AST-GP67 test Kit, and their sequence types (STs) were investigated by multilocus sequence typing (MLST). S1-PFGE was used for plasmid profiling, and PCR and subsequent sequencing were performed to identify the virulence genes. A total of 96 nonduplicated VREfm isolates were obtained and categorized into 38 PFGE types (type 1-38). The predominant MLST type was ST78, while ST17, ST341, and ST342 were also sporadically identified. All types of clinical VREfm strains harbored the vanA gene; however, they carried plasmids of different sizes. While 92.1%, 71.1%, and 60.5% of VREfm strains carried hyl, scm, and ecbA genes, respectively, all of them were positive for esp, acm, sgrA, pilA, and pilB genes. Clonal VREfm spread was observed, and nonplasmid-mediated horizontal transfer of vancomycin-resistant gene might have conveyed resistance to some vancomycin-susceptible E. faecium strains. E. faecium ST78 carrying vanA gene was the most prevalent clone in this study. The high prevalence of virulence genes, including esp, hyl, acm, scm, ecbA, sgrA, pilA, and pilB, confirmed their important roles in the emergence of VREfm ST78 in nosocomial infections.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system

PubMed Central

Vonk, Freek J.; Casewell, Nicholas R.; Henkel, Christiaan V.; Heimberg, Alysha M.; Jansen, Hans J.; McCleary, Ryan J. R.; Kerkkamp, Harald M. E.; Vos, Rutger A.; Guerreiro, Isabel; Calvete, Juan J.; Wüster, Wolfgang; Woods, Anthony E.; Logan, Jessica M.; Harrison, Robert A.; Castoe, Todd A.; de Koning, A. P. Jason; Pollock, David D.; Yandell, Mark; Calderon, Diego; Renjifo, Camila; Currier, Rachel B.; Salgado, David; Pla, Davinia; Sanz, Libia; Hyder, Asad S.; Ribeiro, José M. C.; Arntzen, Jan W.; van den Thillart, Guido E. E. J. M.; Boetzer, Marten; Pirovano, Walter; Dirks, Ron P.; Spaink, Herman P.; Duboule, Denis; McGlinn, Edwina; Kini, R. Manjunatha; Richardson, Michael K.

2013-01-01

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection. PMID:24297900

Extended-spectrum β-lactamases, transferable quinolone resistance, and virulotyping in extra-intestinal E. coli in Uruguay.

PubMed

Vignoli, Rafael; García-Fulgueiras, Virginia; Cordeiro, Nicolás F; Bado, Inés; Seija, Verónica; Aguerrebere, Paula; Laguna, Gabriel; Araújo, Lucía; Bazet, Cristina; Gutkind, Gabriel; Chabalgoity, José

2016-01-31

To characterize extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli isolates obtained from extra-intestinal samples in three Uruguayan hospitals. Fifty-five ESBL-producing E. coli isolates were studied. Virulence genes, ESBLs, and PMQR genes were detected by polymerase chain reaction. ESBL-producing isolates were compared by pulsed-field gel electrophoresis. Multi-locus sequence typing was also performed on 13 selected isolates. Thirty-seven isolates harbored blaCTX-M-15 (67.3%), eight blaCTX-M-2 (14.6%), five blaCTX-M-14 (9.1%), three carried both blaCTX-M-2 and blaCTX-M-14, one blaCTX-M-9, and one blaCTX-M-8. Among the CTX-M-15 producers, 92% belonged to sequence types ST131 and ST405, and carried aac(6')Ib-cr as well. Isolates harboring blaCTX-M-2, blaCTX-M-14, blaCTX-M-9, or blaCTX-M-8 were found to be genetically unrelated. The successful dissemination of CTX-M-15-producing E.coli isolates seems to be linked to the spreading of high-risk clones and horizontal gene transfer. A trade-off between carrying more antibiotic resistance and less virulence-related genes could partially account for the evolutionary advantages featured by successful clones.

Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis.

PubMed

Carro, Lorena; Spröer, Cathrin; Alonso, Pilar; Trujillo, Martha E

2012-03-01

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species. Copyright © 2011 Elsevier GmbH. All rights reserved.

A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

PubMed Central

Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

2013-01-01

Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis. PMID:23359622

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

PubMed

Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; de Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate; Leão, Sylvia Cardoso

2014-08-01

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

AgdbNet – antigen sequence database software for bacterial typing

PubMed Central

Jolley, Keith A; Maiden, Martin CJ

2006-01-01

Background Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences. Results Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script. Conclusion The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated. PMID:16790057

Dissemination of IMP-6-producing Pseudomonas aeruginosa ST244 in multiple cities in China.

PubMed

Chen, Y; Sun, M; Wang, M; Lu, Y; Yan, Z

2014-07-01

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for nosocomial infections and is currently reported to be a worldwide nosocomial menace. The aim of this study was to investigate the epidemiological traits and the distribution of metallo-β-lactamases (MBLs)-producing P. aeruginosa clinical isolates in ten cities in China between January 2010 and May 2012. Antimicrobial susceptibility was determined by disc diffusion assay and the minimum inhibitory concentrations (MICs) of imipenem and meropenem were also determined by the Etest according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In addition, polymerase chain reaction (PCR) and DNA sequencing were applied to detect bla MBL genes, and their epidemiological relationships were investigated by multilocus sequence typing (MLST). Of 368 P. aeruginosa isolates, MLST analysis identified 138 sequence types (STs), including 122 known and 16 novel STs, and the most frequently detected clone was ST244, followed by ST235. Besides, our study revealed that 25 isolates carried the bla IMP-6 gene and three isolates carried the bla VIM-2 gene, and a probe specific for both genes could be hybridised to an ~1,125-kb fragment in all isolates. Interestingly, all of the bla IMP-6-producing isolates shared an identical ST, ST244, and exhibited a higher level of resistance to several antibiotics. Overall, these observations suggest that P. aeruginosa ST244 carrying the chromosomally located bla IMP-6 gene is widely disseminated in multiple cites in China.

Genetic Relatedness of Staphylococcus haemolyticus in Gut and Skin of Preterm Neonates and Breast Milk of Their Mothers.

PubMed

Soeorg, Hiie; Metsvaht, Hanna Kadri; Keränen, Evamaria Elisabet; Eelmäe, Imbi; Merila, Mirjam; Ilmoja, Mari-Liis; Metsvaht, Tuuli; Lutsar, Irja

2018-04-02

Staphylococcus haemolyticus is a common colonizer and cause of late-onset sepsis (LOS) in preterm neonates. By describing genetic relatedness, we aimed to determine whether mother's breast milk (BM) is a source of S. haemolyticus colonizing neonatal gut and skin and/or causing LOS. S. haemolyticus was isolated from stool and skin swabs of 49 BM-fed preterm neonates admitted to neonatal intensive care unit, 20 healthy BM-fed term neonates and BM of mothers once a week and typed by multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Virulence-related genes were determined by PCR. Compared with term neonates S. haemolyticus colonized more commonly gut (35% vs 89.9%; p<0.001) and skin (50% vs 91.8%; p<0.001) of preterm neonates and mothers' BM (15% vs 38.8%). Isolates from preterm compared with term neonates and their mothers carried more commonly the mecA gene (83.5% vs 5.4%; p<0.001) and IS256 (52.4% vs 2.7%; p<0.001) and belonged to clonal complex 29 (89.1% vs 63%; p=0.014). Only 7 (14.3%) preterm and 3 (15%) term neonates were colonized in gut or on skin with MLVA-types indistinguishable from those in BM. Most frequent MLVA-types belonged to sequence type 3 or 42, comprised 71.1-78.4% of isolates from preterm neonates/mothers and caused all seven LOS episodes. LOS-causing strain colonized the gut of 4/7 and the skin of 5/7 neonates, but not BM, prior to onset of LOS. S. haemolyticus colonizing gut and skin or causing LOS in preterm neonates rarely originate from BM, but are mecA-positive strains adapted to hospital environment.

Comparison of the isolation rates and characteristics of Salmonella isolated from antibiotic-free and conventional chicken meat samples.

PubMed

Park, J-H; Kim, H-S; Yim, J-H; Kim, Y-J; Kim, D-H; Chon, J-W; Kim, H; Om, A-S; Seo, K-H

2017-08-01

Salmonella contamination in chicken samples can cause major health problems in humans. However, not only the effects of antibiotic treatment during growth but also the impacts of the poultry slaughter line on the prevalence of Salmonellae in final chicken meat sold to consumers are unknown. In this study, we compared the isolation rates and antimicrobial resistance of Salmonellae among antibiotic-free, conventional, conventional Korean native retail chicken meat samples, and clonal divergence of Salmonella isolates by multilocus sequence typing. In addition, the distribution of extended-spectrum β-lactamase (ESBL) genes in ESBL-producing Salmonella isolates was analyzed. A total of 72 retail chicken meat samples (n = 24 antibiotic-free broiler [AFB] chickens, n = 24 conventional broiler [CB] chickens, and n = 24 conventional Korean native [CK] chickens) was collected from local retail markets in Seoul, South Korea. The isolation rates of Salmonellae were 66.6% in AFB chickens, 45.8% in CB chickens, and 25% in CK chickens. By analyzing the minimum inhibitory concentrations of β-lactam antibiotics with the disc-diffusion test, we found that 81.2% of Salmonella isolates from AFB chickens, 63.6% of isolates from CB chickens, and 50% of isolates from CK chickens were ESBL producers; all ESBL-positive isolates had the CTX-M-15 genotype. Interestingly, all ESBL-producing Salmonellae were revealed as ST16 by multilocus sequence typing and had the genetic platform of blaCTX-M gene (IS26-ISEcp1-blaCTX-M-15-IS903), which was first reported in Salmonellae around the world. The Salmonella ST33 strain (S. Hadar) isolated in this study has never been reported in South Korea. In conclusion, our findings showed that antibiotic-free retail chicken meat products were also largely contaminated with ESBL-producing Salmonellae and that their ESBL genes and genetic platforms were the same as those isolated from conventional retail chicken meat products. © 2017 Poultry Science Association Inc.

Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission.

PubMed

Wang, Yuanfei; Yang, Wenli; Cama, Vitaliano; Wang, Lin; Cabrera, Lilia; Ortega, Ynes; Bern, Caryn; Feng, Yaoyu; Gilman, Robert; Xiao, Lihua

2014-07-01

Population genetic studies have been used to understand the transmission of pathogens in humans and animals, especially the role of zoonotic infections and evolution and dispersal of virulent subtypes. In this study, we analysed the genetic diversity and population structure of Cryptosporidium meleagridis, the only known Cryptosporidium species that infects both avian and mammalian hosts and is responsible for approximately 10% of human cryptosporidiosis in some areas. A total of 62 C. meleagridis specimens from children, AIDS patients, and birds in Lima, Peru were characterised by sequence analysis of the ssrRNA gene and five minisatellite, microsatellite and polymorphic markers in chromosome 6, including the 60 kDa glycoprotein (gp60), 47 kDa glycoprotein (CP47), a serine repeat antigen (MSC6-5), retinitis pigmentosa GTPase regulator (RPGR) and thrombospondin protein 8 (TSP8). The multilocus sequence analysis identified concurrent infections with Cryptosporidium hominis in four AIDS patients and three children. Unique subtypes of C. meleagridis ranged from eight at the gp60 locus (gene diversity -Hd=0.651), three at the RPGR (Hd=0.556), three at the MSC6-5 locus (Hd=0.242), two at TSP8 (Hd=0.198), to one at CP47 (monomorphic), much lower than that of C. hominis in the same area. Intragenic linkage disequilibrium was strong and complete at all gene loci. Intergenic linkage disequilibrium was highly significant (P<0.001) for all pairs of polymorphic loci. Two major groups of subtypes were seen, with most subtypes belonging to group 1. Within group 1, there was no clear population segregation, and two of the 14 multilocus subtypes of C. meleagridis were found in both AIDS patients and birds. We believe that these results provide the first evidence of a clonal population structure of C. meleagridis and the likely occurrence of cross-species transmission of C. meleagridis between birds and humans. Published by Elsevier Ltd.

AMPLISAS: a web server for multilocus genotyping using next-generation amplicon sequencing data.

PubMed

Sebastian, Alvaro; Herdegen, Magdalena; Migalska, Magdalena; Radwan, Jacek

2016-03-01

Next-generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus-specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post-processing of NGS data. Amplicon Sequence Assignment (AMPLISAS) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. AMPLISAS is designed as a three-step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. AMPLISAS performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies. © 2015 John Wiley & Sons Ltd.

A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance.

PubMed

Thomsen, Martin Christen Frølund; Ahrenfeldt, Johanne; Cisneros, Jose Luis Bellod; Jurtz, Vanessa; Larsen, Mette Voldby; Hasman, Henrik; Aarestrup, Frank Møller; Lund, Ole

2016-01-01

Recent advances in whole genome sequencing have made the technology available for routine use in microbiological laboratories. However, a major obstacle for using this technology is the availability of simple and automatic bioinformatics tools. Based on previously published and already available web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the individual services. The reported results enable a rapid overview of the major results, and comparing that to the previously found results showed that the platform is reliable and able to correctly predict the species and find most of the expected genes automatically. In conclusion, a combined bioinformatics platform was developed and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 and it is the intention that it will continue to be expanded with new features as these become available.

Molecular characterization of Giardia psittaci by multilocus sequence analysis.

PubMed

Abe, Niichiro; Makino, Ikuko; Kojima, Atsushi

2012-12-01

Multilocus sequence analyses targeting small subunit ribosomal DNA (SSU rDNA), elongation factor 1 alpha (ef1α), glutamate dehydrogenase (gdh), and beta giardin (β-giardin) were performed on Giardia psittaci isolates from three Budgerigars (Melopsittacus undulates) and four Barred parakeets (Bolborhynchus lineola) kept in individual households or imported from overseas. Nucleotide differences and phylogenetic analyses at four loci indicate the distinction of G. psittaci from the other known Giardia species: Giardia muris, Giardia microti, Giardia ardeae, and Giardia duodenalis assemblages. Furthermore, G. psittaci was related more closely to G. duodenalis than to the other known Giardia species, except for G. microti. Conflicting signals regarded as "double peaks" were found at the same nucleotide positions of the ef1α in all isolates. However, the sequences of the other three loci, including gdh and β-giardin, which are known to be highly variable, from all isolates were also mutually identical at every locus. They showed no double peaks. These results suggest that double peaks found in the ef1α sequences are caused not by mixed infection with genetically different G. psittaci isolates but by allelic sequence heterogeneity (ASH), which is observed in diplomonad lineages including G. duodenalis. No sequence difference was found in any G. psittaci isolates at the gdh and β-giardin, suggesting that G. psittaci is indeed not more diverse genetically than other Giardia species. This report is the first to provide evidence related to the genetic characteristics of G. psittaci obtained using multilocus sequence analysis. Copyright © 2012 Elsevier B.V. All rights reserved.

Multilocus sequence typing, biochemical and antibiotic resistance characterizations reveal diversity of North American strains of the honey bee pathogen Paenibacillus larvae.

PubMed

Krongdang, Sasiprapa; Evans, Jay D; Pettis, Jeffery S; Chantawannakul, Panuwan

2017-01-01

Paenibacillus larvae is a Gram positive bacterium and the causative agent of the most widespread fatal brood disease of honey bees, American foulbrood (AFB). A total of thirty-three independent Paenibacillus larvae isolates from various geographical origins in North America and five reference strains were investigated for genetic diversity using multilocus sequence typing (MLST). This technique is regarded to be a powerful tool for epidemiological studies of pathogenic bacteria and is widely used in genotyping assays. For MLST, seven housekeeping gene loci, ilvD (dihydroxy-acid dyhydrogenase), tri (triosephosphate isomerase), purH (phospharibosyl-aminoimidazolecarboxamide), recF (DNA replication and repair protein), pyrE (orotate phosphoribosyltransferase), sucC (succinyl coenzyme A synthetase β subunit) and glpF (glycerol uptake facilitator protein) were studied and applied for primer designs. Previously, ERIC type DNA fingerprinting was applied to these same isolates and the data showed that almost all represented the ERIC I type, whereas using BOX-PCR gave an indication of more diversity. All isolates were screened for resistance to four antibiotics used by U.S. beekeepers, showing extensive resistance to tetracycline and the first records of resistance to tylosin and lincomycin. Our data highlight the intraspecies relationships of P. larvae and the potential application of MLST methods in enhancing our understanding of epidemiological relationships among bacterial isolates of different origins.

Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence typing primers.

PubMed

Adamiak, Paul; Vanderkooi, Otto G; Kellner, James D; Schryvers, Anthony B; Bettinger, Julie A; Alcantara, Joenel

2014-06-03

Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate's sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles. The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years. The inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative primers, to research groups seeking to undertake S. pneumoniae MLST based studies.

Veterinary Fusarioses within the United States

PubMed Central

Sutton, Deanna A.; Wiederhold, Nathan; Robert, Vincent A. R. G.; Crous, Pedro W.; Geiser, David M.

2016-01-01

Multilocus DNA sequence data were used to assess the genetic diversity and evolutionary relationships of 67 Fusarium strains from veterinary sources, most of which were from the United States. Molecular phylogenetic analyses revealed that the strains comprised 23 phylogenetically distinct species, all but two of which were previously known to infect humans, distributed among eight species complexes. The majority of the veterinary isolates (47/67 = 70.1%) were nested within the Fusarium solani species complex (FSSC), and these included 8 phylospecies and 33 unique 3-locus sequence types (STs). Three of the FSSC species (Fusarium falciforme, Fusarium keratoplasticum, and Fusarium sp. FSSC 12) accounted for four-fifths of the veterinary strains (38/47) and STs (27/33) within this clade. Most of the F. falciforme strains (12/15) were recovered from equine keratitis infections; however, strains of F. keratoplasticum and Fusarium sp. FSSC 12 were mostly (25/27) isolated from marine vertebrates and invertebrates. Our sampling suggests that the Fusarium incarnatum-equiseti species complex (FIESC), with eight mycoses-associated species, may represent the second most important clade of veterinary relevance within Fusarium. Six of the multilocus STs within the FSSC (3+4-eee, 1-b, 12-a, 12-b, 12-f, and 12-h) and one each within the FIESC (1-a) and the Fusarium oxysporum species complex (ST-33) were widespread geographically, including three STs with transoceanic disjunctions. In conclusion, fusaria associated with veterinary mycoses are phylogenetically diverse and typically can only be identified to the species level using DNA sequence data from portions of one or more informative genes. PMID:27605713

Method for identifying mutagenic agents which induce large, multilocus deletions in DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradley, W.E.C.; Belouchi, A.; Dewyse, P.

1993-07-13

A method of identifying a mutagenic agent is described which includes a large, multilocus deletions in DNA in mammalian cells comprising: (i) exposing a class III heterozygous CHO cell line to a potential mutagenic agent under investigation, and allowing any mutation of the cell line to proceed, said cell line being characterized in that a restriction fragment length variation exists in on mutation it becomes resistant to 2,6-diaminopurine and in that the DNA sequence adjacent to the two alleles of the APRT gene such that the DNA sequence adjacent to one of the two alleles can be digested with themore » enzyme BclI but the DNA sequence variation adjacent to the other of the two alleles cannot be digested with BclI, (ii) isolating induced mutations of the cell line deficient in APRT function, (iii) isolating DNA from the induced mutants, (iv) digesting the isolated DNA with BclI enzyme to produce digested fragments including a 19 kb fragment and any 2 kb fragment, which fragments hybridize with the labeled probe derived from DNA fragment PDI, (v) separating any digested fragments, (vi) transferring the separated fragments of (v) to a solid support, (vii) hybridizing the supported separated fragments with a labeled probe derived from the clone DNA fragment PD 1, (viii) determining fragments having undergone loss of the 2 kb band identified by the probe, as an identification of parent mutants in which the loss occurred, and (ix) evaluating the mutating ability of the potential mutagenic agent.« less

Veterinary Fusarioses within the United States.

PubMed

O'Donnell, Kerry; Sutton, Deanna A; Wiederhold, Nathan; Robert, Vincent A R G; Crous, Pedro W; Geiser, David M

2016-11-01

Multilocus DNA sequence data were used to assess the genetic diversity and evolutionary relationships of 67 Fusarium strains from veterinary sources, most of which were from the United States. Molecular phylogenetic analyses revealed that the strains comprised 23 phylogenetically distinct species, all but two of which were previously known to infect humans, distributed among eight species complexes. The majority of the veterinary isolates (47/67 = 70.1%) were nested within the Fusarium solani species complex (FSSC), and these included 8 phylospecies and 33 unique 3-locus sequence types (STs). Three of the FSSC species (Fusarium falciforme, Fusarium keratoplasticum, and Fusarium sp. FSSC 12) accounted for four-fifths of the veterinary strains (38/47) and STs (27/33) within this clade. Most of the F. falciforme strains (12/15) were recovered from equine keratitis infections; however, strains of F. keratoplasticum and Fusarium sp. FSSC 12 were mostly (25/27) isolated from marine vertebrates and invertebrates. Our sampling suggests that the Fusarium incarnatum-equiseti species complex (FIESC), with eight mycoses-associated species, may represent the second most important clade of veterinary relevance within Fusarium Six of the multilocus STs within the FSSC (3+4-eee, 1-b, 12-a, 12-b, 12-f, and 12-h) and one each within the FIESC (1-a) and the Fusarium oxysporum species complex (ST-33) were widespread geographically, including three STs with transoceanic disjunctions. In conclusion, fusaria associated with veterinary mycoses are phylogenetically diverse and typically can only be identified to the species level using DNA sequence data from portions of one or more informative genes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Population genetics of Enterocytozoon bieneusi in captive giant pandas of China.

PubMed

Li, Wei; Song, Yuan; Zhong, Zhijun; Huang, Xiangming; Wang, Chengdong; Li, Caiwu; Yang, Haidi; Liu, Haifeng; Ren, Zhihua; Lan, Jingchao; Wu, Kongju; Peng, Guangneng

2017-10-18

Most studies on Enterocytozoon bieneusi are conducted based on the internal transcribed spacer (ITS) region of the rRNA gene, whereas some have examined E. bieneusi population structures. Currently, the population genetics of this pathogen in giant panda remains unknown. The objective of this study was to determine the E. bieneusi population in captive giant pandas in China. We examined 69 E. bieneusi-positive specimens from captive giant pandas in China using five loci (ITS, MS1, MS3, MS4 and MS7) to infer E. bieneusi population genetics. For multilocus genotype (MLG) analysis of E. bieneusi-positive isolates, the MS1, MS3, MS4, and MS7 microsatellite and minisatellite loci were amplified and sequenced in 48, 45, 50 and 47 specimens, respectively, generating ten, eight, nine and five types. We successfully amplified 36 specimens and sequenced all five loci, forming 24 MLGs. Multilocus sequence analysis revealed a strong and significant linkage disequilibrium (LD), indicating a clonal population. This result was further supported by measurements of pairwise intergenic LD and a standardized index of association (I S A ) from allelic profile data. The analysis in STRUCTURE suggested three subpopulations in E. bieneusi, further confirmed using right's fixation index (F ST ). Subpopulations 1 and 2 exhibited an epidemic structure, whereas subpopulation 3 had a clonal structure. Our results describe E. bieneusi population genetics in giant pandas for the first time, improving the current understanding E. bieneusi epidemiology in the studied region. These data also benefit future studies exploring potential transmission risks from pandas to other animals, including humans.

A web-based genomic sequence database for the Streptomycetaceae: a tool for systematics and genome mining

USDA-ARS?s Scientific Manuscript database

The ARS Microbial Genome Sequence Database (http://199.133.98.43), a web-based database server, was established utilizing the BIGSdb (Bacterial Isolate Genomics Sequence Database) software package, developed at Oxford University, as a tool to manage multi-locus sequence data for the family Streptomy...

Characterization of Multi-Drug Resistant Enterococcus faecalis Isolated from Cephalic Recording Chambers in Research Macaques (Macaca spp.).

PubMed

Woods, Stephanie E; Lieberman, Mia T; Lebreton, Francois; Trowel, Elise; de la Fuente-Núñez, César; Dzink-Fox, Joanne; Gilmore, Michael S; Fox, James G

2017-01-01

Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two E. faecalis sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included aac(6')-aph(2"), aph(3')-III, str, ant(6)-Ia, tetM, tetS, tetL, ermB, bcrABR, cat, and dfrG, and polymorphisms in parC (S80I) and gyrA (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (esp, agg, ace, SrtA, gelE, ebpABC), hyaluronidases (hylA, hylB), and other survival genes (ElrA, tpx). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-cas. This macaque colony represents a unique model for studying E. faecalis infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.

New Insights on Taxonomy, Phylogeny and Population Genetics of Leishmania (Viannia) Parasites Based on Multilocus Sequence Analysis

PubMed Central

Boité, Mariana C.; Mauricio, Isabel L.; Miles, Michael A.; Cupolillo, Elisa

2012-01-01

The Leishmania genus comprises up to 35 species, some with status still under discussion. The multilocus sequence typing (MLST)—extensively used for bacteria—has been proposed for pathogenic trypanosomatids. For Leishmania, however, a detailed analysis and revision on the taxonomy is still required. We have partially sequenced four housekeeping genes—glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), mannose phosphate isomerase (MPI) and isocitrate dehydrogenase (ICD)—from 96 Leishmania (Viannia) strains and assessed their discriminatory typing capacity. The fragments had different degrees of diversity, and are thus suitable to be used in combination for intra- and inter-specific inferences. Species-specific single nucleotide polymorphisms were detected, but not for all species; ambiguous sites indicating heterozygosis were observed, as well as the putative homozygous donor. A large number of haplotypes were detected for each marker; for 6PGD a possible ancestral allele for L. (Viannia) was found. Maximum parsimony-based haplotype networks were built. Strains of different species, as identified by multilocus enzyme electrophoresis (MLEE), formed separated clusters in each network, with exceptions. NeighborNet of concatenated sequences confirmed species-specific clusters, suggesting recombination occurring in L. braziliensis and L. guyanensis. Phylogenetic analysis indicates L. lainsoni and L. naiffi as the most divergent species and does not support L. shawi as a distinct species, placing it in the L. guyanensis cluster. BURST analysis resulted in six clonal complexes (CC), corresponding to distinct species. The L. braziliensis strains evaluated correspond to one widely geographically distributed CC and another restricted to one endemic area. This study demonstrates the value of systematic multilocus sequence analysis (MLSA) for determining intra- and inter-species relationships and presents an approach to validate the species status of some entities. Furthermore, it contributes to the phylogeny of L. (Viannia) and might be helpful for epidemiological and population genetics analysis based on haplotype/diplotype determinations and inferences. PMID:23133690

Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data.

PubMed

Tanabe, Akifumi S

2011-09-01

Proportional and separate models able to apply different combination of substitution rate matrix (SRM) and among-site rate variation model (ASRVM) to each locus are frequently used in phylogenetic studies of multilocus data. A proportional model assumes that branch lengths are proportional among partitions and a separate model assumes that each partition has an independent set of branch lengths. However, the selection from among nonpartitioned (i.e., a common combination of models is applied to all-loci concatenated sequences), proportional and separate models is usually based on the researcher's preference rather than on any information criteria. This study describes two programs, 'Kakusan4' (for DNA sequences) and 'Aminosan' (for amino-acid sequences), which allow the selection of evolutionary models based on several types of information criteria. The programs can handle both multilocus and single-locus data, in addition to providing an easy-to-use wizard interface and a noninteractive command line interface. In the case of multilocus data, SRMs and ASRVMs are compared at each locus and at all-loci concatenated sequences, after which nonpartitioned, proportional and separate models are compared based on information criteria. The programs also provide model configuration files for mrbayes, paup*, phyml, raxml and Treefinder to support further phylogenetic analysis using a selected model. When likelihoods are optimized by Treefinder, the best-fit models were found to differ depending on the data set. Furthermore, differences in the information criteria among nonpartitioned, proportional and separate models were much larger than those among the nonpartitioned models. These findings suggest that selecting from nonpartitioned, proportional and separate models results in a better phylogenetic tree. Kakusan4 and Aminosan are available at http://www.fifthdimension.jp/. They are licensed under gnugpl Ver.2, and are able to run on Windows, MacOS X and Linux. © 2011 Blackwell Publishing Ltd.

“Epidemic Clones” of Listeria monocytogenes Are Widespread and Ancient Clonal Groups

PubMed Central

Cantinelli, Thomas; Chenal-Francisque, Viviane; Diancourt, Laure; Frezal, Lise; Leclercq, Alexandre; Wirth, Thierry

2013-01-01

The food-borne pathogen Listeria monocytogenes is genetically heterogeneous. Although some clonal groups have been implicated in multiple outbreaks, there is currently no consensus on how “epidemic clones” should be defined. The objectives of this work were to compare the patterns of sequence diversity on two sets of genes that have been widely used to define L. monocytogenes clonal groups: multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MvLST). Further, we evaluated the diversity within clonal groups by pulsed-field gel electrophoresis (PFGE). Based on 125 isolates of diverse temporal, geographical, and source origins, MLST and MvLST genes (i) had similar patterns of sequence polymorphisms, recombination, and selection, (ii) provided concordant phylogenetic clustering, and (iii) had similar discriminatory power, which was not improved when we combined both data sets. Inclusion of representative strains of previous outbreaks demonstrated the correspondence of epidemic clones with previously recognized MLST clonal complexes. PFGE analysis demonstrated heterogeneity within major clones, most of which were isolated decades before their involvement in outbreaks. We conclude that the “epidemic clone” denominations represent a redundant but largely incomplete nomenclature system for MLST-defined clones, which must be regarded as successful genetic groups that are widely distributed across time and space. PMID:24006010

Pantoea allii sp. nov., isolated from onion plants and seed.

PubMed

Brady, Carrie L; Goszczynska, Teresa; Venter, Stephanus N; Cleenwerck, Ilse; De Vos, Paul; Gitaitis, Ronald D; Coutinho, Teresa A

2011-04-01

Eight yellow-pigmented, Gram-negative, rod-shaped, oxidase-negative, motile, facultatively anaerobic bacteria were isolated from onion seed in South Africa and from an onion plant exhibiting centre rot symptoms in the USA. The isolates were assigned to the genus Pantoea on the basis of phenotypic and biochemical tests. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), based on gyrB, rpoB, infB and atpD sequences, confirmed the allocation of the isolates to the genus Pantoea. MLSA further indicated that the isolates represented a novel species, which was phylogenetically most closely related to Pantoea ananatis and Pantoea stewartii. Amplified fragment length polymorphism analysis also placed the isolates into a cluster separate from P. ananatis and P. stewartii. Compared with type strains of species of the genus Pantoea that showed >97 % 16S rRNA gene sequence similarity with strain BD 390(T), the isolates exhibited 11-55 % whole-genome DNA-DNA relatedness, which confirmed the classification of the isolates in a novel species. The most useful phenotypic characteristics for the differentiation of the isolates from their closest phylogenetic neighbours are production of acid from amygdalin and utilization of adonitol and sorbitol. A novel species, Pantoea allii sp. nov., is proposed, with type strain BD 390(T) ( = LMG 24248(T)).

Pseudomonas aestus sp. nov., a plant growth-promoting bacterium isolated from mangrove sediments.

PubMed

Vasconcellos, Rafael L F; Santos, Suikinai Nobre; Zucchi, Tiago Domingues; Silva, Fábio Sérgio Paulino; Souza, Danilo Tosta; Melo, Itamar Soares

2017-10-01

Strain CMAA 1215 T , a Gram-reaction-negative, aerobic, catalase positive, polarly flagellated, motile, rod-shaped (0.5-0.8 × 1.3-1.9 µm) bacterium, was isolated from mangrove sediments, Cananéia Island, Brazil. Analysis of the 16S rRNA gene sequences showed that strain CMAA 1215 T forms a distinct phyletic line within the Pseudomonas putida subclade, being closely related to P. plecoglossicida ATCC 700383 T , P. monteilii NBRC 103158 T , and P. taiwanensis BCRC 17751 T of sequence similarity of 98.86, 98.73, and 98.71%, respectively. Genomic comparisons of the strain CMAA 1215 T with its closest phylogenetic type strains using average nucleotide index (ANI) and DNA:DNA relatedness approaches revealed 84.3-85.3% and 56.0-63.0%, respectively. A multilocus sequence analysis (MLSA) performed concatenating 16S rRNA, gyrB and rpoB gene sequences from the novel species was related with Pseudomonas putida subcluster and formed a new phylogenetic lineage. The phenotypic, physiological, biochemical, and genetic characteristics support the assignment of CMAA 1215 T to the genus Pseudomonas, representing a novel species. The name Pseudomonas aestus sp.nov. is proposed, with CMAA 1215 T (=NRRL B-653100 T  = CBMAI 1962 T ) as the type strain.

New Mutations of Penicillin-Binding Proteins in Streptococcus agalactiae Isolates from Cattle with Decreased Susceptibility to Penicillin.

PubMed

Hu, Yun; Kan, Yunchao; Zhang, Zhengtian; Lu, Zhanning; Li, Yanqiu; Leng, Chaoliang; Ji, Jun; Song, Shiyang; Shi, Hongfei

2018-02-23

Streptococcus agalactiae is a causal agent of bovine mastitis and is treated by β-lactam antibiotics (BLAs). Compared to penicillin-resistant S. agalactiae from humans, resistant strains in bovine are rarely reported. In this study, we aimed to investigate BLA resistance and mutations in penicillin-binding proteins (PBPs) of S. agalactiae in central and northeast China. The minimum inhibitory concentrations (MICs) of 129 penicillin-resistant S. agalactiae isolates from cows with mastitis were determined, and the related PBP genes were detected and sequenced. All strains were unsusceptible to penicillin G and mostly resistant to ampicillin, cefalexin, and ceftiofur sodium. One hundred twenty-nine strains were divided into 4 clonal groups and 8 sequence types by multilocus sequence typing analysis. We found a set of new substitutions in PBP1B, PBP2B, and PBP2X from most strains isolated from three provinces. The strains with high PBP mutations showed a broader unsusceptible spectrum and higher MICs than those with few or single mutation. Our research indicates unpredicted mutations in the PBP genes of S. agalactiae isolated from cows with mastitis treated by BLAs. This screening is the first of S. agalactiae from cattle.

Proposal of Vespertiliibacter pulmonis gen. nov., sp. nov. and two genomospecies as new members of the family Pasteurellaceae isolated from European bats.

PubMed

Mühldorfer, Kristin; Speck, Stephanie; Wibbelt, Gudrun

2014-07-01

Five bacterial strains isolated from bats of the family Vespertilionidae were characterized by phenotypic tests and multilocus sequence analysis (MLSA) using the 16S rRNA gene and four housekeeping genes (rpoA, rpoB, infB, recN). Phylogenetic analyses of individual and combined datasets indicated that the five strains represent a monophyletic cluster within the family Pasteurellaceae. Comparison of 16S rRNA gene sequences demonstrated a high degree of similarity (98.3-99.9%) among the group of bat-derived strains, while searches in nucleotide databases indicated less than 96% sequence similarity to known members of the Pasteurellaceae. The housekeeping genes rpoA, rpoB, infB and recN provided higher resolution compared with the 16S rRNA gene and subdivided the group according to the bat species from which the strains were isolated. Three strains derived from noctule bats shared 98.6-100% sequence similarity in all four genes investigated, whereas, based on rpoB, infB and recN gene sequences, 91.8-96% similarity was observed with and between the remaining two strains isolated from a serotine bat and a pipistrelle bat, respectively. Genome relatedness as deduced from recN gene sequences correlated well with the results of MLSA and indicated that the five strains represent a new genus. Based on these results, it is proposed to classify the five strains derived from bats within Vespertiliibacter pulmonis gen. nov., sp. nov. (the type species), Vespertiliibacter genomospecies 1 and Vespertiliibacter genomospecies 2. The genus can be distinguished phenotypically from recognized genera of the Pasteurellaceae by at least three characteristics. All strains are nutritionally fastidious and require a chemically defined supplement with NAD for growth. The DNA G+C content of strain E127/08(T) is 38.2 mol%. The type strain of Vespertiliibacter pulmonis gen. nov., sp. nov. is E127/08(T) ( = CCUG 64585(T) = DSM 27238(T)). The reference strains of Vespertiliibacter genomospecies 1 and 2 are E145/08 and E157/08, respectively. © 2014 IUMS.

High Prevalence of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae in Wild Fish from the Mediterranean Sea in Algeria.

PubMed

Brahmi, Soumia; Touati, Abdelaziz; Dunyach-Remy, Catherine; Sotto, Albert; Pantel, Alix; Lavigne, Jean-Philippe

2018-04-01

We investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae among wild fish from the coast of Bejaia (Algeria) in the Mediterranean Sea. From March 2012 to August 2013, gut and gill samples of wild fish were screened for the presence of ESBL-producing Enterobacteriaceae. Strains were characterized with regard to antibiotic resistance, β-lactamase content, plasmid-mediated quinolone resistance, aminoglycoside resistance genes, and clonality (repetitive sequence-based polymerase chain reaction profiles and multilocus sequence typing). Virulence traits were performed for Escherichia coli and Klebsiella pneumoniae isolates. Of the 300 fish studied, 64 (21.3%) isolates were screened as positive for ESBL producing by the double-disc method. The isolates corresponded to E. coli, K. pneumoniae, Enterobacter cloacae, Morganella morganii, Citrobacter freundii, and Proteus vulgaris. A predominance of bla CTX-M gene was observed with a prevalence of 60.5% (n = 46). Furthermore, our study describes the association of important coresistance and virulence factors in E. coli and K. pneumoniae. Twelve of the ESBL producers carried genes of the qnr family and oqxAB gene and six carried the aac(6')-Ib-cr gene. Our results highlight for the first time the diffusion of multidrug-resistant Enterobacteriaceae isolates carrying resistance and virulence genes in fish from the Mediterranean Sea in Algeria.

Unique core genomes of the bacterial family vibrionaceae: insights into niche adaptation and speciation.

PubMed

Kahlke, Tim; Goesmann, Alexander; Hjerde, Erik; Willassen, Nils Peder; Haugen, Peik

2012-05-10

The criteria for defining bacterial species and even the concept of bacterial species itself are under debate, and the discussion is apparently intensifying as more genome sequence data is becoming available. However, it is still unclear how the new advances in genomics should be used most efficiently to address this question. In this study we identify genes that are common to any group of genomes in our dataset, to determine whether genes specific to a particular taxon exist and to investigate their potential role in adaptation of bacteria to their specific niche. These genes were named unique core genes. Additionally, we investigate the existence and importance of unique core genes that are found in isolates of phylogenetically non-coherent groups. These groups of isolates, that share a genetic feature without sharing a closest common ancestor, are termed genophyletic groups. The bacterial family Vibrionaceae was used as the model, and we compiled and compared genome sequences of 64 different isolates. Using the software orthoMCL we determined clusters of homologous genes among the investigated genome sequences. We used multilocus sequence analysis to build a host phylogeny and mapped the numbers of unique core genes of all distinct groups of isolates onto the tree. The results show that unique core genes are more likely to be found in monophyletic groups of isolates. Genophyletic groups of isolates, in contrast, are less common especially for large groups of isolate. The subsequent annotation of unique core genes that are present in genophyletic groups indicate a high degree of horizontally transferred genes. Finally, the annotation of the unique core genes of Vibrio cholerae revealed genes involved in aerotaxis and biosynthesis of the iron-chelator vibriobactin. The presented work indicates that genes specific for any taxon inside the bacterial family Vibrionaceae exist. These unique core genes encode conserved metabolic functions that can shed light on the adaptation of a species to its ecological niche. Additionally, our study suggests that unique core genes can be used to aid classification of bacteria and contribute to a bacterial species definition on a genomic level. Furthermore, these genes may be of importance in clinical diagnostics and drug development.

A RESTful application programming interface for the PubMLST molecular typing and genome databases

PubMed Central

Bray, James E.; Maiden, Martin C. J.

2017-01-01

Abstract Molecular typing is used to differentiate microorganisms at the subspecies or strain level for epidemiological investigations, infection control, public health and environmental sampling. DNA sequence-based typing methods require authoritative databases that link sequence variants to nomenclature in order to facilitate communication and comparison of identified types in national or global settings. The PubMLST website (https://pubmlst.org/) fulfils this role for over a hundred microorganisms for which it hosts curated molecular sequence typing data, providing sequence and allelic profile definitions for multi-locus sequence typing (MLST) and single-gene typing approaches. In recent years, these have expanded to cover the whole genome with schemes such as core genome MLST (cgMLST) and whole genome MLST (wgMLST) which catalogue the allelic diversity found in hundreds to thousands of genes. These approaches provide a common nomenclature for high-resolution strain characterization and comparison. Molecular typing information is linked to isolate provenance, phenotype, and increasingly genome assemblies, providing a resource for outbreak investigation and research in to population structure, gene association, global epidemiology and vaccine coverage. A Representational State Transfer (REST) Application Programming Interface (API) has been developed for the PubMLST website to make these large quantities of structured molecular typing and whole genome sequence data available for programmatic access by any third party application. The API is an integral component of the Bacterial Isolate Genome Sequence Database (BIGSdb) platform that is used to host PubMLST resources, and exposes all public data within the site. In addition to data browsing, searching and download, the API supports authentication and submission of new data to curator queues. Database URL: http://rest.pubmlst.org/ PMID:29220452

Penicillin-resistant, ampicillin-susceptible Enterococcus faecalis of hospital origin: pbp4 gene polymorphism and genetic diversity.

PubMed

Conceição, Natália; da Silva, Lucas Emanuel Pinheiro; Darini, Ana Lúcia da Costa; Pitondo-Silva, André; de Oliveira, Adriana Gonçalves

2014-12-01

Despite the spread of penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates in diverse countries, the mechanisms leading to this unusual resistance phenotype have not yet been investigated. The aim of this study was to evaluate whether polymorphism in the pbp4 gene is associated with penicillin resistance in PRASEF isolates and to determine their genetic diversity. E. faecalis isolates were recovered from different clinical specimens of hospitalized patients from February 2006 to June 2010. The β-lactam minimal inhibitory concentrations (MICs) were determined by E-test®. The PCR-amplified pbp4 gene was sequenced with an automated sequencer. The genetic diversities of the isolates were established by PFGE (pulsed-field gel electrophoresis) and MLST (multilocus sequencing typing). Seventeen non-producing β-lactamase PRASEF and 10 penicillin-susceptible, ampicillin-susceptible E. faecalis (PSASEF) strains were analyzed. A single-amino-acid substitution (Asp-573→Glu) in the penicillin-binding domain was significantly found in all PRASEF isolates by sequencing of the pbp4 gene but not in the penicillin-susceptible isolates. In contrast to the PSASEF isolates, a majority of the PRASEFs had similar PFGE profiles. Six representative PRASEF isolates were resolved by MLST into ST9 and ST524 and belong to the globally dispersed clonal complex 9 (CC9). In conclusion, it appears quite likely that the amino acid alteration (Asp-573→Glu) found in the PBP4 of the Brazilian PRASEF isolates may account for their reduced susceptibility to penicillin, although other resistance mechanisms remain to be investigated. Copyright © 2014 Elsevier B.V. All rights reserved.

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

PubMed

Spring-Pearson, Senanu M; Stone, Joshua K; Doyle, Adina; Allender, Christopher J; Okinaka, Richard T; Mayo, Mark; Broomall, Stacey M; Hill, Jessica M; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; McNew, Lauren A; Rosenzweig, C Nicole; Gibbons, Henry S; Currie, Bart J; Wagner, David M; Keim, Paul; Tuanyok, Apichai

2015-01-01

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

PubMed Central

Sanz, Yolanda

2017-01-01

Abstract The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems. PMID:28605506

Molecular epidemiology of Pseudomonas aeruginosa clinical isolates from Korea producing β-lactamases with extended-spectrum activity.

PubMed

Bae, Il Kwon; Suh, Borum; Jeong, Seok Hoon; Wang, Kang-Kyun; Kim, Yong-Rok; Yong, Dongeun; Lee, Kyungwon

2014-07-01

This study was performed to investigate the prevalence and molecular epidemiology of Pseudomonas aeruginosa isolates from Korea that produce enzymes with extended-spectrum (ES) activity to β-lactams. A total of 205 non-duplicate P. aeruginosa clinical isolates were collected from 18 university hospitals in Korea. PCR and sequencing experiments were performed to identify genes encoding β-lactamases. PCR mapping and sequencing of the regions surrounding the β-lactamase genes were performed. Multilocus sequence typing experiments were performed. The most common sequence type (ST) was ST235 (n = 96), and 2 single-locus variants of ST235, ST1015 (n = 1) and ST1162 (n = 1), were also identified. These 3 STs were grouped as a clonal complex (CC), CC235. The remaining 107 isolates were identified as 59 different STs. Isolates belonging to CC235 showed higher rates of non-susceptibility to imipenem (85.4% versus 47.7%) and meropenem (92.7% versus 52.3%) compared to non-CC235 isolates. All the metallo-β-lactamase (MBL)-producing isolates were identified as CC235, except for 1 ST591. Genes encoding OXA-17 and OXA-142 were detected in 1 isolate and 4 isolates of CC235, respectively; while the bla(SHV-12) gene was detected in 4 non-CC235 isolates. Class A and D β-lactamases with ES activity play a role in acquiring ceftazidime resistance in P. aeruginosa in Korea. Production of IMP-6 and VIM-2 MBLs is the main mechanisms in acquiring resistance to ceftazidime and carbapenems in P. aeruginosa isolates in Korea. Clonal spread of P. aeruginosa CC235 may be an important conduit for the dissemination of MBL genes in Korea. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?

PubMed

Antonov, Valery A; Tkachenko, Galina A; Altukhova, Viktoriya V; Savchenko, Sergey S; Zinchenko, Olga V; Viktorov, Dmitry V; Zamaraev, Valery S; Ilyukhin, Vladimir I; Alekseev, Vladimir V

2008-12-01

Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains.

Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

PubMed Central

Unemo, Magnus; Dillon, Jo-Anne R.

2011-01-01

Summary: Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding. PMID:21734242

Streptococcal toxic shock syndrome by an iMLS resistant M type 77 Streptococcus pyogenes in the Netherlands.

PubMed

Gooskens, J; Neeling, A J De; Willems, R J; Wout, J W Van 't; Kuijper, E J

2005-01-01

An increasing number of group A streptococci (GAS) with constitutive or inducible resistance to macrolide-lincosamide-streptogramin B antibiotics (cMLS or iMLS phenotype) is observed in Europe, but MLS resistant GAS associated with streptococcal toxic shock syndrome (STSS) has not been reported. We describe a patient admitted with STSS caused by an iMLS resistant T28 M77 Streptococcus pyogenes carrying the ermA [subclass TR] gene. A 2-y retrospective analysis among 701 nationwide collected GAS strains revealed an incidence of 3.1% of this M type 77 GAS. Analysis of 17 available M77 strains (12 T28 and 5 T13) indicated that 2 (12%) were MLS resistant due to the ermA [TR] gene. Both MLS resistant strains were cultured from blood and belonged to T28 serotype. Multilocus sequence typing (MLST) showed that all M77 isolates belonged to sequence type 63. We conclude that 17 M77 GAS collected in the Netherlands in a 2-y period were associated with invasive disease and belonged to the same clonal complex. Since only 12% carried the ermA [TR] resistance gene, it is very likely that the gene has been acquired by horizontal transmission rather than from spread of a resistant circulating clone.

High Diversity of Antimicrobial Resistance Genes, Class 1 Integrons, and Genotypes of Multidrug-Resistant Escherichia coli in Beef Carcasses.

PubMed

Chen, Chih-Ming; Ke, Se-Chin; Li, Chia-Ru; Wu, Ying-Chen; Chen, Ter-Hsin; Lai, Chih-Ho; Wu, Xin-Xia; Wu, Lii-Tzu

2017-10-01

Multidrug-resistant Escherichia coli can contaminate food meat during processing and cause human infection. Phenotypic and genotypic characterization of the antimicrobial resistance were conducted for 45 multidrug-resistant E. coli isolates from 208 samples of beef carcasses. The mechanisms of resistance were evaluated using polymerase chain reaction and sequencing methods, and the clonal relationship among isolates was evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Different variants of bla, tet, flo, dfrA, and aadA genes were detected in most of the strains resistant to β-lactam, tetracycline, chloramphenicol, sulfonamides, and aminoglycosides, respectively. Extended-spectrum β-lactamase (ESBL)-producing E. coli was found in 42.2% of the 45 E. coli isolates and the most commonly detected ESBL genotypes were CTX-M group 1 and 9. Class 1 integrons with nine different arrangements of gene cassettes were present in 28 of 45 E. coli isolates. Twenty-nine PFGE groups and 24 MLST types were identified in their clonal structure. This study revealed that E. coli isolates from beef contained high diversity of antimicrobial resistance genes, integrons, and genotypes. These results highlighted the role of beef meat as a potential source for multidrug-resistant E. coli strains and the need for controlling beef safety.

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles

PubMed Central

Nandi, Tannistha; Holden, Matthew T.G.; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A.; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J.; Titball, Richard; Chen, Swaine L.; Parkhill, Julian

2015-01-01

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

Biogeography of sulfur-oxidizing Acidithiobacillus populations in extremely acidic cave biofilms

PubMed Central

Jones, Daniel S; Schaperdoth, Irene; Macalady, Jennifer L

2016-01-01

Extremely acidic (pH 0–1.5) Acidithiobacillus-dominated biofilms known as snottites are found in sulfide-rich caves around the world. Given the extreme geochemistry and subsurface location of the biofilms, we hypothesized that snottite Acidithiobacillus populations would be genetically isolated. We therefore investigated biogeographic relationships among snottite Acidithiobacillus spp. separated by geographic distances ranging from meters to 1000s of kilometers. We determined genetic relationships among the populations using techniques with three levels of resolution: (i) 16S rRNA gene sequencing, (ii) 16S–23S intergenic transcribed spacer (ITS) region sequencing and (iii) multi-locus sequencing typing (MLST). We also used metagenomics to compare functional gene characteristics of select populations. Based on 16S rRNA genes, snottites in Italy and Mexico are dominated by different sulfur-oxidizing Acidithiobacillus spp. Based on ITS sequences, Acidithiobacillus thiooxidans strains from different cave systems in Italy are genetically distinct. Based on MLST of isolates from Italy, genetic distance is positively correlated with geographic distance both among and within caves. However, metagenomics revealed that At. thiooxidans populations from different cave systems in Italy have different sulfur oxidation pathways and potentially other significant differences in metabolic capabilities. In light of those genomic differences, we argue that the observed correlation between genetic and geographic distance among snottite Acidithiobacillus populations is partially explained by an evolutionary model in which separate cave systems were stochastically colonized by different ancestral surface populations, which then continued to diverge and adapt in situ. PMID:27187796

Multilocus sequence typing of Streptococcus thermophilus from naturally fermented dairy foods in China and Mongolia.

PubMed

Yu, Jie; Sun, Zhihong; Liu, Wenjun; Xi, Xiaoxia; Song, Yuqin; Xu, Haiyan; Lv, Qiang; Bao, Qiuhua; Menghe, Bilige; Sun, Tiansong

2015-10-26

Streptococcus thermophilus is a major dairy starter used for manufacturing of dairy products. In the present study, we developed a multilocus sequence typing (MLST) scheme for this important food bacterium. Sequences of 10 housekeeping genes (carB, clpX, dnaA, murC, murE, pepN, pepX, pyrG, recA, and rpoB) were obtained for 239 S. thermophilus strains, which were isolated from home-made fermented dairy foods in 18 different regions of Mongolia and China. All 10 genes of S. thermophilus were sequenced, aligned, and defined sequence types (STs) using the BioNumerics Software. The nucleotide diversity was calculated by START v2.0. The population structure, phylogenetic relationships and the role of recombination were inferred using ClonalFrame v1.2, SplitsTree 4.0 and Structure v2.3. The 239 S. thermophilus isolates and 18 reference strains could be assigned into 119 different STs, which could be further separated into 16 clonal complexes (CCs) and 38 singletons. Among the 10 loci, a total of 132 polymorphic sites were detected. The standardized index of association (IAS=0.0916), split-decomposition and ρ/θ (relative frequency of occurrence of recombination and mutation) and r/m value (relative impact of recombination and mutation in the diversification) confirms that recombination may have occurred, but it occurred at a low frequency in these 10 loci. Phylogenetic trees indicated that there were five lineages in the S. thermophilus isolates used in our study. MSTree and ClonalFrame tree analyses suggest that the evolution of S. thermophilus isolates have little relationship with geographic locality, but revealed no association with the types of fermented dairy product. Phylogenetic analysis of 36 whole genome strains (18 S. thermophilus, 2 S. vestibularis and 16 S. salivarius strains) indicated that our MLST scheme could clearly separate three closely related species within the salivarius group and is suitable for analyzing the population structure of the other two species in the salivarius group. Our newly developed MLST scheme improved the understanding on the genetic diversity and population structure of the S. thermophilus, as well as provided useful information for further studies on the genotyping and evolutionary research for S. thermophilus strains with global diversity.

Extrahuman Epidemiology of Acinetobacter baumannii in Lebanon

PubMed Central

Rafei, Rayane; Hamze, Monzer; Pailhoriès, Hélène; Eveillard, Matthieu; Marsollier, Laurent; Joly-Guillou, Marie-Laure; Dabboussi, Fouad

2015-01-01

The presence of Acinetobacter baumannii outside hospitals is still a controversial issue. The objective of our study was to explore the extrahospital epidemiology of A. baumannii in Lebanon. From February 2012 to October 2013, a total of 73 water samples, 51 soil samples, 37 raw cow milk samples, 50 cow meat samples, 7 raw cheese samples, and 379 animal samples were analyzed by cultural methods for the presence of A. baumannii. Species identification was performed by rpoB gene sequencing. Antibiotic susceptibility was investigated, and the A. baumannii population was studied by two genotyping approaches: multilocus sequence typing (MLST) and blaOXA-51 sequence-based typing (SBT). A. baumannii was detected in 6.9% of water samples, 2.7% of milk samples, 8.0% of meat samples, 14.3% of cheese samples, and 7.7% of animal samples. All isolates showed a susceptible phenotype against most of the antibiotics tested and lacked carbapenemase-encoding genes, except one that harbored a blaOXA-143 gene. MLST analysis revealed the presence of 36 sequence types (STs), among which 24 were novel STs reported for the first time in this study. blaOXA-51 SBT showed the presence of 34 variants, among which 21 were novel and all were isolated from animal origins. Finally, 30 isolates had new partial rpoB sequences and were considered putative new Acinetobacter species. In conclusion, animals can be a potential reservoir for A. baumannii and the dissemination of new emerging carbapenemases. The roles of the novel animal clones identified in community-acquired infections should be investigated. PMID:25616788

[Isolation of a carbapenem-resistant K1 serotype Klebsiella pneumonia strain and the study of resistance mechanism].

PubMed

Zhang, Rong; Wang, Xuan; Lü, Jianxin

2014-12-16

To study the virulence and mechanism of carbapenem resistance of a clinical isolate of carbapenem-resistant K1 serotype Klebsiella pneumonia strain. Identification of isolate was carried out with VITEK-2 compact system. Antimicrobial susceptibility was determined by E-test; Metallo β-lactamases and carbapenemases screening were conducted by imipenem-EDTA double disc synergy test and modified Hodge test, respectively.Specific polymerehse chain reaction (PCR) and DNA sequencing were preformed to detect the virulence genes including K1, K2, K5, K20, K54, K57, magA, rmpA, wcaG and a series of β-lactamase resistence genes. Conjunction experiment was also performed. The plasmids of transconjugants were submitted to PCR-based replicon typing (PBRT) method. Molecular typing was performed by multilocus sequence typing (MLST). Antimicrobial susceptibility testing revealed that the Klebsiella pneumonia strain was resistant to most of the antibiotics used in clinic. Phynotype confirmary rest revealed the production of carbapanemases, while Metallo β-lactamases were negative; PCR and DNA sequencing confirmed the isolate was positive for blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-1 and virulence genes K1, magA, rmpA, wcaG simultaneously; blaKPC-2 was transferred from donor to Escherichia EC600 by conjunction experiment, while no virulence genes were found in the transconjugants. PBRT revealed that Frep plasmid was found in transconjugants. MLST analysis revealed that this strain belonged to ST23. K1 serotype Klebsiella pneumonia strain carries virulence genes and carbapenem resistance gene blaKPC-2, noteworthily the carbapenem resistance genes can be transferred through horizontal transmission on plasmids.

Multilocus approach to clarify species status and the divergence history of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex.

PubMed

Hsieh, Chia-Hung; Ko, Chiun-Cheng; Chung, Cheng-Han; Wang, Hurng-Yi

2014-07-01

The sweet potato whitefly, Bemisia tabaci, is a highly differentiated species complex. Despite consisting of several morphologically indistinguishable entities and frequent invasions on all continents with important associated economic losses, the phylogenetic relationships, species status, and evolutionary history of this species complex is still debated. We sequenced and analyzed one mitochondrial and three single-copy nuclear genes from 9 of the 12 genetic groups of B. tabaci and 5 closely related species. Bayesian species delimitation was applied to investigate the speciation events of B. tabaci. The species statuses of the different genetic groups were strongly supported under different prior settings and phylogenetic scenarios. Divergence histories were estimated by a multispecies coalescence approach implemented in (*)BEAST. Based on mitochondrial locus, B. tabaci was originated 6.47 million years ago (MYA). Nevertheless, the time was 1.25MYA based on nuclear loci. According to the method of approximate Bayesian computation, this difference is probably due to different degrees of migration among loci; i.e., although the mitochondrial locus had differentiated, gene flow at nuclear loci was still possible, a scenario similar to parapatric mode of speciation. This is the first study in whiteflies using multilocus data and incorporating Bayesian coalescence approaches, both of which provide a more biologically realistic framework for delimiting species status and delineating the divergence history of B. tabaci. Our study illustrates that gene flow during species divergence should not be overlooked and has a great impact on divergence time estimation. Copyright © 2014 Elsevier Inc. All rights reserved.

The human clone ST22 SCCmec IV methicillin-resistant Staphylococcus aureus isolated from swine herds and wild primates in Nepal: is man the common source?

PubMed

Roberts, Marilyn C; Joshi, Prabhu Raj; Greninger, Alexander L; Melendez, Daira; Paudel, Saroj; Acharya, Mahesh; Bimali, Nabin Kishor; Koju, Narayan P; No, David; Chalise, Mukesh; Kyes, Randall C

2018-05-01

Swine nasal samples [n = 282] were collected from 12 randomly selected farms around Kathmandu, Nepal, from healthy animals. In addition, wild monkey (Macaca mulatta) saliva samples [n = 59] were collected near temples areas in Kathmandu using a non-invasive sampling technique. All samples were processed for MRSA using standardized selective media and conventional biochemical tests. MRSA verification was done and isolates characterized by SCCmec, multilocus sequence typing, whole genome sequencing [WGS] and antibiotic susceptibilities. Six (2.1%) swine MRSA were isolated from five of the different swine herds tested, five were ST22 type IV and one ST88 type V. Four (6.8%) macaques MRSA were isolated, with three ST22 SCCmec type IV and one ST239 type III. WGS sequencing showed that the eight ciprofloxacin resistant ST22 isolates carried gyrA mutation [S84L]. Six isolates carried the erm(C) genes, five isolates carried aacC-aphD genes and four isolates carried blaZ genes. The swine linezolid resistant ST22 did not carry any known acquired linezolid resistance genes but had a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69], both previously associated with linezolid resistance. Multiple virulence factors were also identified. This is the first time MRSA ST22 SCCmec IV has been isolated from livestock or primates.

Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability.

PubMed

Joardar, Vinita; Abrams, Natalie F; Hostetler, Jessica; Paukstelis, Paul J; Pakala, Suchitra; Pakala, Suman B; Zafar, Nikhat; Abolude, Olukemi O; Payne, Gary; Andrianopoulos, Alex; Denning, David W; Nierman, William C

2012-12-12

The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25-36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing.

dCITE: Measuring Necessary Cladistic Information Can Help You Reduce Polytomy Artefacts in Trees.

PubMed

Wise, Michael J

2016-01-01

Biologists regularly create phylogenetic trees to better understand the evolutionary origins of their species of interest, and often use genomes as their data source. However, as more and more incomplete genomes are published, in many cases it may not be possible to compute genome-based phylogenetic trees due to large gaps in the assembled sequences. In addition, comparison of complete genomes may not even be desirable due to the presence of horizontally acquired and homologous genes. A decision must therefore be made about which gene, or gene combinations, should be used to compute a tree. Deflated Cladistic Information based on Total Entropy (dCITE) is proposed as an easily computed metric for measuring the cladistic information in multiple sequence alignments representing a range of taxa, without the need to first compute the corresponding trees. dCITE scores can be used to rank candidate genes or decide whether input sequences provide insufficient cladistic information, making artefactual polytomies more likely. The dCITE method can be applied to protein, nucleotide or encoded phenotypic data, so can be used to select which data-type is most appropriate, given the choice. In a series of experiments the dCITE method was compared with related measures. Then, as a practical demonstration, the ideas developed in the paper were applied to a dataset representing species from the order Campylobacterales; trees based on sequence combinations, selected on the basis of their dCITE scores, were compared with a tree constructed to mimic Multi-Locus Sequence Typing (MLST) combinations of fragments. We see that the greater the dCITE score the more likely it is that the computed phylogenetic tree will be free of artefactual polytomies. Secondly, cladistic information saturates, beyond which little additional cladistic information can be obtained by adding additional sequences. Finally, sequences with high cladistic information produce more consistent trees for the same taxa.

dCITE: Measuring Necessary Cladistic Information Can Help You Reduce Polytomy Artefacts in Trees

PubMed Central

2016-01-01

Biologists regularly create phylogenetic trees to better understand the evolutionary origins of their species of interest, and often use genomes as their data source. However, as more and more incomplete genomes are published, in many cases it may not be possible to compute genome-based phylogenetic trees due to large gaps in the assembled sequences. In addition, comparison of complete genomes may not even be desirable due to the presence of horizontally acquired and homologous genes. A decision must therefore be made about which gene, or gene combinations, should be used to compute a tree. Deflated Cladistic Information based on Total Entropy (dCITE) is proposed as an easily computed metric for measuring the cladistic information in multiple sequence alignments representing a range of taxa, without the need to first compute the corresponding trees. dCITE scores can be used to rank candidate genes or decide whether input sequences provide insufficient cladistic information, making artefactual polytomies more likely. The dCITE method can be applied to protein, nucleotide or encoded phenotypic data, so can be used to select which data-type is most appropriate, given the choice. In a series of experiments the dCITE method was compared with related measures. Then, as a practical demonstration, the ideas developed in the paper were applied to a dataset representing species from the order Campylobacterales; trees based on sequence combinations, selected on the basis of their dCITE scores, were compared with a tree constructed to mimic Multi-Locus Sequence Typing (MLST) combinations of fragments. We see that the greater the dCITE score the more likely it is that the computed phylogenetic tree will be free of artefactual polytomies. Secondly, cladistic information saturates, beyond which little additional cladistic information can be obtained by adding additional sequences. Finally, sequences with high cladistic information produce more consistent trees for the same taxa. PMID:27898695

First report on the occurrence of Theileria sp. OT3 in China.

PubMed

Tian, Zhancheng; Liu, Guangyuan; Yin, Hong; Xie, Junren; Wang, Suyan; Yuan, Xiaosong; Wang, Fangfang; Luo, Jin

2014-04-01

Theileria sp. OT3 was firstly detected and identified from clinically healthy sheep in Xinjiang Uygur Autonomous Region of China (XUAR) through comparing the complete 18S rDNA gene sequences available in GenBank database and the phylogenetic status based on the internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene by the methods of a partitioned multi-locus analysis in BEAST and Maximum likelihood analysis in PhyML. Moreover, the findings were confirmed by the species-specific PCR for Theileria sp. OT3 and the prevalence of Theileria sp. OT3 was 14.9% in the north of XUAR. This study is the first report on the occurrence of Theileria sp. OT3 in China. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Molecular Characterization of OXA-198 Carbapenemase-Producing Pseudomonas aeruginosa Clinical Isolates.

PubMed

Bonnin, Rémy A; Bogaerts, Pierre; Girlich, Delphine; Huang, Te-Din; Dortet, Laurent; Glupczynski, Youri; Naas, Thierry

2018-06-01

Carbapenemase-producing Pseudomonadaceae have increasingly been reported worldwide, with an ever-increasing heterogeneity of carbapenem resistance mechanisms, depending on the bacterial species and the geographical location. OXA-198 is a plasmid-encoded class D β-lactamase involved in carbapenem resistance in one Pseudomonas aeruginosa isolate from Belgium. In the setting of a multicenter survey of carbapenem resistance in P. aeruginosa strains in Belgian hospitals in 2013, three additional OXA-198-producing P. aeruginosa isolates originating from patients hospitalized in one hospital were detected. To reveal the molecular mechanism underlying the reduced susceptibility to carbapenems, MIC determinations, whole-genome sequencing, and PCR analyses to confirm the genetic organization were performed. The plasmid harboring the bla OXA-198 gene was characterized, along with the genetic relatedness of the four P. aeruginosa isolates. The bla OXA-198 gene was harbored on a class 1 integron carried by an ∼49-kb IncP-type plasmid proposed as IncP-11. The same plasmid was present in all four P. aeruginosa isolates. Multilocus sequence typing revealed that the isolates all belonged to sequence type 446, and single-nucleotide polymorphism analysis revealed only a few differences between the isolates. This report describes the structure of a 49-kb plasmid harboring the bla OXA-198 gene and presents the first description of OXA-198-producing P. aeruginosa isolates associated with a hospital-associated cluster episode. Copyright © 2018 American Society for Microbiology.

First Case of NDM-1-Producing Klebsiella pneumoniae in Annaba University Hospital, Algeria.

PubMed

Abderrahim, Amel; Djahmi, Nassima; Pujol, Charlotte; Nedjai, Sabina; Bentakouk, Mohamed Cherif; Kirane-Gacemi, Djamila; Dekhil, Mazouz; Sotto, Albert; Lavigne, Jean-Philippe; Pantel, Alix

2017-10-01

The aim of this study was to characterize two carbapenem-resistant Klebsiella pneumoniae isolates recovered from urine samples in a patient hospitalized at Annaba University hospital (Algeria) in 2014. Two K. pneumoniae isolates were studied because they proved resistant to almost all antibiotics tested with a high level resistance to ertapenem (minimum inhibitory concentration = 32 mg/L). The results of modified Hodge test and combined disk test (ROSCO Diagnostica, Taastrup, Denmark) were positive. The two isolates harbored the bla NDM-1 gene and one was also positive for bla CTX-M-15 . Screening of aminoglycoside-modifying enzymes and plasmid-mediated quinolone resistance contents detected aac(6')-Ib-cr, aac(3')-II, qnrB2, and oqxAB in both isolates. Multilocus sequence typing demonstrated that the two isolates belonged to sequence type 147. However, repetitive sequence-based PCR and pulsed-field gel electrophoresis showed that they were not clonally related. The bla NDM-1 gene and all other resistant genes were contained on an IncR plasmid of c.a. 85 kb. This study comprises the first identification of NDM-1-producing K. pneumoniae in Algeria. We thus confirm the concerning worldwide dissemination of this carbapenemase that involves the emergence of the IncR plasmid and the success of the ST147 clonal complex harboring it.

Detection of VIM-2-, IMP-1- and NDM-1-producing multidrug resistant Pseudomonas aeruginosa in Malaysia.

PubMed

Liew, Siew Mun; Rajasekaram, Ganeswrei; Puthucheary, Savithri D; Chua, Kek Heng

2018-02-09

The increasing incidence of carbapenem-resistant Pseudomonas aeruginosa along with the discovery of novel metallo-β-lactamases (MBLs) is of concern. In this study, the isolation of Malaysian MBL-producing P. aeruginosa clinical strains was investigated. Fifty-three P. aeruginosa clinical strains were isolated from different patients in Sultanah Aminah Hospital, Johor Bahru, Malaysia in 2015. Antimicrobial susceptibility test was conducted. Minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by Etest. The carbapenem-resistant strains were screened for MBL production by IMP-EDTA double disk synergy test (DDST), MBL imipenem/imipenem-inhibitor (IP/IPI) Etest and polymerase chain reaction (PCR). Genotyping was performed by multilocus sequence typing (MLST) analysis. Three (5.7%) clinical strains were identified as MBL producers. Multidrug resistance was observed in the three strains, and two were resistant to all the antimicrobials tested. Sequencing analysis confirmed the three strains to harbour carbapenemase genes: one with bla IMP-1 , one with bla VIM-2 and the other with bla NDM-1 genes. These multidrug resistant strains were identified as sequence type (ST) 235 and ST308. None of the bla IMP-1 and bla NDM-1 genes have been reported in Malaysian P. aeruginosa. The emergence of imipenemase 1 (IMP-1)- and New Delhi metallo-β-lactamase 1 (NDM-1)-producing P. aeruginosa in Malaysia maybe travel-associated. Copyright © 2018. Published by Elsevier Ltd.

Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

PubMed Central

Yang, Yongchun; Liu, Yinglong; Ding, Yunlei; Yi, Li; Ma, Zhe; Fan, Hongjie; Lu, Chengping

2013-01-01

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae. PMID:23874442

Detection and multilocus genotyping of Giardia duodenalis in dogs in Sichuan province, China.

PubMed

Zhang, Yue; Zhong, Zhijun; Deng, Lei; Wang, Maoqing; Li, Wei; Gong, Chao; Fu, Hualin; Cao, Suizhong; Shi, Xianpeng; Wu, Kongju; Peng, Guangneng

2017-01-01

Giardia duodenalis (also known as G. intestinalis) is a flagellated protozoan that parasitizes the small intestine and is a common causal agent of zoonotic infections in humans and animals. To assess the genetic diversity and zoonotic transmission potential of G. duodenalis in stray dogs, 159 fecal specimens were collected from dogs in Chengdu, Yaan, and Leshan in Sichuan province, China. Of the 159 fecal samples from stray dogs, 18 (11.3%) were G. duodenalis-positive based on nested PCR amplification of the beta giardin (bg) gene, and the occurrence varied from 1.8% to 35% in different cities. Dog-specific assemblages C (n = 9) and D (n = 9) were identified. The glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes of all bg-positive isolates were characterized. A total of 16 and 8 isolates were positive for the gdh and tpi genes, respectively. Two novel sequences of the bg locus were detected among genetic assemblage D isolates, and one novel gdh sequence and four novel tpi sequences were identified among genetic assemblage C isolates. Mixed infections of assemblages C and D were also detected. Assemblages A and B, which have high zoonotic potential, were not detected. Our results show that G. duodenalis is prevalent and a cause of diarrhea in dogs in Sichuan province, China. © Y. Zhang et al., published by EDP Sciences, 2017.

Highly virulent M1 Streptococcus pyogenes isolates resistant to clindamycin.

PubMed

Plainvert, C; Martin, C; Loubinoux, J; Touak, G; Dmytruk, N; Collobert, G; Fouet, A; Ploy, M-C; Poyart, C

2015-01-01

Emm1-type group A Streptococcus (GAS), or Streptococcus pyogenes, is mostly responsible for invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The recommended treatment of severe invasive GAS infections is a combination of clindamycin and penicillin. Until 2012, almost all emm1 isolates were susceptible to clindamycin. We aimed to identify the phenotypic and genotypic characteristics of emm1 GAS clone resistant to clindamycin. GAS strains were characterized by emm sequence typing, detection of genes encoding pyrogenic exotoxins or superantigens. Cluster analysis was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic susceptibility was assessed using disk diffusion and resistance genes were detected by PCR. A total of 1321 GAS invasive isolates were analyzed between January 2011 and December 2012. The overall number of invasive isolates resistant to clindamycin was 52 (3.9%); seven of them were emm1 isolates. All isolates had the same genomic markers: macrolide resistance due to the presence of the erm(B) gene, emm subtype 1.0, the same toxin or superantigen profile, PFGE pattern and sequence type. This is the first description of highly virulent GAS emm1 isolates resistant to clindamycin in France. This article strengthens the need for monitoring the epidemiology of invasive GAS strains as they could lead to changes in treatment guidelines. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Molecular Typing and Virulence Gene Profiles of Enterotoxin Gene Cluster (egc)-Positive Staphylococcus aureus Isolates Obtained from Various Food and Clinical Specimens.

PubMed

Song, Minghui; Shi, Chunlei; Xu, Xuebing; Shi, Xianming

2016-11-01

The enterotoxin gene cluster (egc) has been proposed to contribute to the Staphylococcus aureus colonization, which highlights the need to evaluate genetic diversity and virulence gene profiles of the egc-positive population. Here, a total of 43 egc-positive isolates (16.2%) were identified from 266 S. aureus isolates that were obtained from various food and clinical specimens in Shanghai. Seven different egc profiles were found based on the polymerase chain reaction (PCR) result for egc genes. Then, these 43 egc-positive isolates were further typed by multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number tandem-repeat analysis (MLVA), and accessory gene regulatory (agr) typing. It showed that the 43 egc-positive isolates displayed 17 sequence types, 28 PFGE patterns, 29 MLVA types, and 4 agr types, respectively. Among them, the dominant clonal lineage was CC5-agr II (48.84%). Thirty toxin and 20 adhesion-associated genes were detected by PCR in egc-positive isolates. Notably, invasive toxin genes showed a high prevalence, such as 76.7% for Panton-Valentine leukocidin encoding genes, 27.9% for sec, and 23.3% for tsst-1. Most of the examined adhesion-associated genes were found to be conserved (76.7-100%), whereas the fnbB gene was only found in 8 (18.6%) isolates. In addition, 33 toxin gene profiles and 13 adhesion gene profiles were identified, respectively. Our results imply that isolates belonging to the same clonal lineage harbored similar adhesion gene profiles but diverse toxin gene profiles. Overall, the high prevalence of invasive virulence genes increases the potential risk of egc-positive isolates in S. aureus infection.

Staphylococcal food poisoning caused by Staphylococcus argenteus harboring staphylococcal enterotoxin genes.

PubMed

Wakabayashi, Yuki; Umeda, Kaoru; Yonogi, Shinya; Nakamura, Hiromi; Yamamoto, Kaori; Kumeda, Yuko; Kawatsu, Kentaro

2018-01-16

Staphylococcal food poisoning (SFP) is caused by staphylococcal enterotoxins (SEs) preformed in food materials. SE genes are encoded on mobile genetic elements and are widely found across Staphylococcus species including S. argenteus, although most SFP cases are caused by S. aureus. S. argenteus, recently discriminated from S. aureus as a novel species, are non-pigmented staphylococci phenotypically related to S. aureus. In 2014 and 2015, two independent food poisoning cases occurred in Osaka, Japan, in which non-pigmented staphylococci were predominantly isolated. Several enterotoxin genes (seb, seg, sei, sem, sen, seo, and selu2) were found in their genome and the production of SEB was confirmed by reverse passive agglutination tests. The non-pigmented isolates from patients, food handlers, food, and cooking utensils all produced the same pulsed-field gel electrophoresis pattern. These non-pigmented isolates were coagulase-positive and biochemically identical to S. aureus. We performed further genetic analysis using nucA sequencing and multi-locus sequence typing, and identified these isolates as S. argenteus. We also found that seb was encoded on the Staphylococcus aureus pathogenicity island, while seg, sei, sem, sen, seo, and selu2 were encoded on the enterotoxin gene cluster. From these results, we concluded that the two food poisoning outbreaks were SFP cases caused by S. argenteus harboring SE genes. Copyright © 2017 Elsevier B.V. All rights reserved.

"Silent" dissemination of Klebsiella pneumoniae isolates bearing K. pneumoniae carbapenemase in a long-term care facility for children and young adults in Northeast Ohio.

PubMed

Viau, Roberto A; Hujer, Andrea M; Marshall, Steven H; Perez, Federico; Hujer, Kristine M; Briceño, David F; Dul, Michael; Jacobs, Michael R; Grossberg, Richard; Toltzis, Philip; Bonomo, Robert A

2012-05-01

Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (bla(KPC)) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection. Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed. The DNA microarray detected bla(KPC) in all 12 isolates, and bla(KPC-3) was identified by PCR amplification and sequencing of the amplicon. In addition, a bla(SHV-11) gene was detected in all isolates. Immunoblotting revealed "low-level" production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all bla(KPC-3)-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with bla(KPC) carriage. Plasmids from 3 representative isolates readily transferred the bla(KPC-3) to Escherichia coli J-53 recipients. Our findings reveal the "silent" dissemination of bla(KPC-3) as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing bla(KPC-3) in an LTCF caring for neurologically impaired children and young adults.

Characterization of Streptococcus pyogenes from Animal Clinical Specimens, Spain

PubMed Central

Vela, Ana Isabel; Villalón, Pilar; Sáez-Nieto, Juan Antonio; Chacón, Gema; Domínguez, Lucas

2017-01-01

Streptococcus pyogenes appears to be almost exclusively restricted to humans, with few reports on isolation from animals. We provide a detailed characterization (emm typing, pulsed-field gel electrophoresis [PFGE], and multilocus sequence typing [MLST]) of 15 S. pyogenes isolates from animals associated with different clinical backgrounds. We also investigated erythromycin resistance mechanisms and phenotypes and virulence genes. We observed 2 emm types: emm12 (11 isolates) and emm77 (4 isolates). Similarly, we observed 2 genetic linages, sequence type (ST) 26 and ST63. Most isolates exhibited the M macrolide resistance phenotype and the mefA/ermB genotype. Isolates were grouped into 2 clones on the basis of emm-MLST-PFGE-virulence gene profile combinations: clone 1, characterized by the combined genotype emm12-ST36-pulsotype A-speG; and clone 2, characterized by the genotype emm77-ST63-pulsotype B-speC. Our results do not show conclusively that animals may represent a new reservoir of S. pyogenes but indicate the ability of human-derived S. pyogenes isolates to colonize and infect animals. PMID:29148379

Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico.

PubMed

Guerrero, Abraham; Gómez Gil Rodríguez, Bruno; Wong-Chang, Irma; Lizárraga-Partida, Marcial Leonardo

2015-01-01

Vibrio vulnificus strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to vvhA, vcg genotype, PFGE, multilocus sequence typing (MLST), and rtxA1. Analyses included a comparison with rtxA1 reference sequences. Environmental (vcgE) and clinical (vcgC) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by vcgE or vcgC genotype. Select housekeeping genes for MLST and primers that were designed for rtxA1 domains divided the strains into two clusters according to the E or C genotype. Reference rtxA1 sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to vcg genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

PubMed

Clark, Clifford G; Berry, Chrystal; Walker, Matthew; Petkau, Aaron; Barker, Dillon O R; Guan, Cai; Reimer, Aleisha; Taboada, Eduardo N

2016-12-03

Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

PubMed

Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

2016-01-01

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Phylogenetic diversity, antimicrobial susceptibility and virulence gene profiles of Brachyspira hyodysenteriae isolates from pigs in Germany

PubMed Central

Joerling, Jessica; Barth, Stefanie A.; Schlez, Karen; Willems, Hermann

2018-01-01

Swine dysentery (SD) is an economically important diarrheal disease in pigs caused by different strongly hemolytic Brachyspira (B.) species, such as B. hyodysenteriae, B. suanatina and B. hampsonii. Possible associations of epidemiologic data, such as multilocus sequence types (STs) to virulence gene profiles and antimicrobial susceptibility are rather scarce, particularly for B. hyodysenteriae isolates from Germany. In this study, B. hyodysenteriae (n = 116) isolated from diarrheic pigs between 1990 and 2016 in Germany were investigated for their STs, susceptibility to the major drugs used for treatment of SD (tiamulin and valnemulin) and genes that were previously linked with virulence and encode for hemolysins (tlyA, tlyB, tlyC, hlyA, BHWA1_RS02885, BHWA1_RS09085, BHWA1_RS04705, and BHWA1_RS02195), outer membrane proteins (OMPs) (bhlp16, bhlp17.6, bhlp29.7, bhmp39f, and bhmp39h) as well as iron acquisition factors (ftnA and bitC). Multilocus sequence typing (MLST) revealed that 79.4% of the isolates belonged to only three STs, namely ST52 (41.4%), ST8 (12.1%), and ST112 (25.9%) which have been observed in other European countries before. Another 24 isolates belonged to twelve new STs (ST113-118, ST120-123, ST131, and ST193). The temporal distribution of STs revealed the presence of new STs as well as the regular presence of ST52 over three decades (1990s–2000s). The proportion of strains that showed resistance to both tiamulin und valnemulin (39.1%) varied considerably among the most frequent STs ranging from 0% (0/14 isolates resistant) in ST8 isolates to 46.7% (14/30), 52.1% (25/48), and 85.7% (6/7) in isolates belonging to ST112, ST52, and ST114, respectively. All hemolysin genes as well as the iron-related gene ftnA and the OMP gene bhlp29.7 were regularly present in the isolates, while the OMP genes bhlp17.6 and bhmp39h could not be detected. Sequence analysis of hemolysin genes of selected isolates revealed co-evolution of tlyB, BHWA1_RS02885, BHWA1_RS09085, and BHWA1_RS02195 with the core genome and suggested independent evolution of tlyA, tlyC, and hlyA. Our data indicate that in Germany, swine dysentery might be caused by a limited number of B. hyodysenteriae clonal groups. Major STs (ST8, ST52, and ST112) are shared with other countries in Europe suggesting a possible role of the European intra-Community trade of pigs in the dissemination of certain clones. The identification of several novel STs, some of which are single or double locus variants of ST52, may on the other hand hint towards an ongoing diversification of the pathogen in the studied area. The linkage of pleuromutilin susceptibility and sequence type of an isolate might reflect a clonal expansion of the underlying resistance mechanism, namely mutations in the ribosomal RNA genes. A linkage between single virulence-associated genes (VAGs) or even VAG patterns and the phylogenetic background of the isolates could not be established, since almost all VAGs were regularly present in the isolates. PMID:29324785

Molecular characterization of fluoroquinolone and/or cephalosporin resistance in Shigella sonnei isolates from yaks.

PubMed

Zhu, Zhen; Shi, Yuxiang; Zhou, Xuzheng; Li, Bing; Zhang, Jiyu

2018-06-07

Members of the genus Shigella are intestinal pathogens and a major cause of seasonal outbreaks of bacterial diarrhea worldwide. Although humans are the conventional hosts of Shigella species, expansion of the Shigella host range to certain animals was recently reported. To investigate the prevalence of Shigella sonnei (S. sonnei) in yaks and perform molecular characterization, we analyzed 1132 fresh yak diarrheal stool samples and collected a total of 44 S. sonnei isolates. We performed multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) with XbaI-digested DNA to study genetic relatedness among the 44 isolates, which were differentiated into 4 sequence types (STs) and 32 PFGE types (PTs). All isolates harbored virulence genes, and 87.36% tested positive for invasion plasmid antigen H (ipaH), invasion associated locus (ial) and the Shigella enterotoxin gene sen. According to the results of antimicrobial susceptibility tests, 45.45% (20/44) were resistant to fluoroquinolones and/or cephalosporin. By sequencing the quinolone resistance determining region (QRDR) genes, we identified double mutations in gyrA (Ser83-Leu and Asp87-Asn) and a single mutation in parC (Ser80-Ile). All 12 fluoroquinolone-resistant S. sonnei isolates tested positive for the aac(6')-Ib-cr gene but negative for qepA. Three isolates harbored qnr genes, including two with qnrS and one with qnrB. In addition, three types of β-lactamase genes, bla TEM-1 , bla OXA-1 and bla CTX-M-14/79 , were detected in cephalosporin-resistant isolates. The findings of this study have enriched our knowledge of fluoroquinolone- and/or cephalosporin-resistant S. sonnei isolates from yaks, which has important public health significance.

Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

PubMed

Dolatabadi, Somayeh; Kolecka, Anna; Versteeg, Matthijs; de Hoog, Sybren G; Boekhout, Teun

2015-07-01

This study addresses the usefulness of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS for reliable identification of the two most frequently occurring clinical species of Rhizopus, namely Rhizopus arrhizus with its two varieties, arrhizus and delemar, and Rhizopus microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by internal transcribed spacer (ITS) sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF ) showed two monophylic clades for Rhizopus arrhizus and Rhizopus microsporus (bootstrap values of 99 %). Cluster analysis confirmed the presence of two distinct clades within Rhizopus arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between Rhizopus arrhizus and Rhizopus microsporus and the presence of two varieties within the species Rhizopus arrhizus. An in-house database of 30 reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an in-house database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on principal component analysis, two MSP dendrograms were created and showed concordance with the multi-locus tree; thus, MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.

Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand.

PubMed

Chonsin, Kaknokrat; Matsuda, Shigeaki; Theethakaew, Chonchanok; Kodama, Toshio; Junjhon, Jiraphan; Suzuki, Yasuhiko; Suthienkul, Orasa; Iida, Tetsuya

2016-01-01

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A divergent spirochete strain isolated from a resident of the southeastern United States was identified by multilocus sequence typing as Borrelia bissettii.

PubMed

Golovchenko, Maryna; Vancová, Marie; Clark, Kerry; Oliver, James H; Grubhoffer, Libor; Rudenko, Nataliia

2016-02-04

Out of 20 spirochete species from Borrelia burgdorferi sensu lato (s.l.) complex recognized to date some are considered to have a limited distribution, while others are worldwide dispersed. Among those are Borrelia burgdorferi sensu stricto (s.s.) and Borrelia bissettii which are distributed both in North America and in Europe. While B. burgdorferi s.s. is recognized as a cause of Lyme borreliosis worldwide, involvement of B. bissettii in human Lyme disease was not so definite yet. Multilocus sequence typing of spirochete isolates originating from residents of Georgia and Florida, USA, revealed the presence of two Borrelia burgdorferi sensu stricto strains highly similar to those from endemic Lyme borreliosis regions of the northeastern United States, and an unusual strain that differed from any previously described in Europe or North America. Based on phylogenetic analysis of eight chromosomally located housekeeping genes divergent strain clustered between Borrelia bissettii and Borrelia carolinensis, two species from the B.burgdorferi s.l. complex, widely distributed among the multiple hosts and vector ticks in the southeastern United States. The genetic distance analysis showed a close relationship of the diverged strain to B. bissettii. Here, we present the analysis of the first North American human originated live spirochete strain that revealed close relatedness to B. bissettii. The potential of B. bissettii to cause human disease, even if it is infrequent, is of importance for clinicians due to the extensive range of its geographic distribution.

Multilocus Sequence Typing Reveals a New Cluster of Closely Related Candida tropicalis Genotypes in Italian Patients With Neurological Disorders.

PubMed

Scordino, Fabio; Giuffrè, Letterio; Barberi, Giuseppina; Marino Merlo, Francesca; Orlando, Maria Grazia; Giosa, Domenico; Romeo, Orazio

2018-01-01

Candida tropicalis is a pathogenic yeast that has emerged as an important cause of candidemia especially in elderly patients with hematological malignancies. Infections caused by this species are mainly reported from Latin America and Asian-Pacific countries although recent epidemiological data revealed that C. tropicalis accounts for 6-16.4% of the Candida bloodstream infections (BSIs) in Italy by representing a relevant issue especially for patients receiving long-term hospital care. The aim of this study was to describe the genetic diversity of C. tropicalis isolates contaminating the hands of healthcare workers (HCWs) and hospital environments and/or associated with BSIs occurring in patients with different neurological disorders and without hematological disease. A total of 28 C. tropicalis isolates were genotyped using multilocus sequence typing analysis of six housekeeping ( ICL1, MDR1, SAPT2, SAPT4, XYR1 , and ZWF1 ) genes and data revealed the presence of only eight diploid sequence types (DSTs) of which 6 (75%) were completely new. Four eBURST clonal complexes (CC2, CC10, CC11, and CC33) contained all DSTs found in this study and the CC33 resulted in an exclusive, well-defined, clonal cluster from Italy. In conclusion, C. tropicalis could represent an important cause of BSIs in long-term hospitalized patients with no underlying hematological disease. The findings of this study also suggest a potential horizontal transmission of a specific C. tropicalis clone through hands of HCWs and expand our understanding of the molecular epidemiology of this pathogen whose population structure is still far from being fully elucidated as its complexity increases as different categories of patients and geographic areas are examined.

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water

NASA Astrophysics Data System (ADS)

Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Kao, Po Min; Shen, Shu Min; Chou Chiu, Yi; Chen, Jung Sheng

2013-04-01

Salmonella is one of the most important pathogens of waterborne diseases with outbreaks from contaminated water reported worldwide. In addition, Salmonella spp. can survive for long periods in aquatic environments. To realize genotypes and serovars of Salmonella in aquatic environments, we isolated the Salmonella strains by selective culture plates to identify the serovars of Salmonella by serological assay, and identify the genotypes by Multilocus sequence typing (MLST) based on the sequence data from University College Cork (UCC), respectively. The results show that 36 stream water samples (30.1%) and 18 drinking water samples (23.3%) were confirmed the existence of Salmonella using culture method combined PCR specific invA gene amplification. In this study, 24 cultured isolates of Salmonella from water samples were classified to fifteen Salmonella enterica serovars. In addition, we construct phylogenetic analysis using phylogenetic tree and Minimum spanning tree (MST) method to analyze the relationship of clinical, environmental, and geographical data. Phylogenetic tree showed that four main clusters and our strains can be distributed in all. The genotypes of isolates from stream water are more biodiversity while comparing the Salmonella strains genotypes from drinking water sources. According to MST data, we can found the positive correlation between serovars and genotypes of Salmonella. Previous studies revealed that the result of Pulsed field gel electrophoresis (PFGE) method can predict the serovars of Salmonella strain. Hence, we used the MLST data combined phylogenetic analysis to identify the serovars of Salmonella strain and achieved effectiveness. While using the geographical data combined phylogenetic analysis, the result showed that the dominant strains were existed in whole stream area in rainy season. Keywords: Salmonella spp., MLST, phylogenetic analysis, PFGE

Human Infections Attributable to the d-Tartrate-Fermenting Variant of Salmonella enterica Serovar Paratyphi B in Germany Originate in Reptiles and, on Rare Occasions, Poultry

PubMed Central

Toboldt, Anne; Tietze, Erhard; Helmuth, Reiner; Fruth, Angelika; Junker, Ernst

2012-01-01

In this study, the population structure, incidence, and potential sources of human infection caused by the d-tartrate-fermenting variant of Salmonella enterica serovar Paratyphi B [S. Paratyphi B (dT+)] was investigated. In Germany, the serovar is frequently isolated from broilers. Therefore, a selection of 108 epidemiologically unrelated S. enterica serovar Paratyphi B (dT+) strains isolated in Germany between 2002 and 2010 especially from humans, poultry/poultry meat, and reptiles was investigated by phenotypic and genotypic methods. Strains isolated from poultry and products thereof were strongly associated with multilocus sequence type ST28 and showed antimicrobial multiresistance profiles. Pulsed-field gel electrophoresis XbaI profiles were highly homogeneous, with only a few minor XbaI profile variants. All strains isolated from reptiles, except one, were strongly associated with ST88, another distantly related type. Most of the strains were susceptible to antimicrobial agents, and XbaI profiles were heterogeneous. Strains isolated from humans yielded seven sequence types (STs) clustering in three distantly related lineages. The first lineage, comprising five STs, represented mainly strains belonging to ST43 and ST149. The other two lineages were represented only by one ST each, ST28 and ST88. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was mostly in agreement with the multilocus sequence type. Because ST28 was frequently isolated from poultry but rarely in humans over the 9-year period investigated, overall, this study indicates that in Germany S. enterica serovar Paratyphi B (dT+) poses a health risk preferentially by contact with reptiles and, to a less extent, by exposure to poultry or poultry meat. PMID:22885742

Serogroup 19 pneumococci containing both mef and erm macrolide resistance determinants in an American city.

PubMed

Toltzis, Philip; Dul, Michael; O'Riordan, Mary Ann; Jacobs, Michael R; Blumer, Jeffrey

2006-01-01

Asia has experienced a striking incidence of infection by highly resistant pneumococi containing both principal macrolide resistance determinants, namely, the mef efflux pump and the erm ribosomal methylase. mef/erm-containing pneumococci have not been identified in significant numbers in North America. Pneumococci were isolated as part of a larger study in Cleveland, OH examining colonization patterns among children randomized to 1 of 4 outpatient antibiotics for acute otitis media. Azithromycin-resistant organisms were tested for the presence of mef and erm sequences by polymerase chain reaction. The clonal relationship of pneumococci containing both genes was determined by pulsed field gel electrophoresis and multilocus sequence testing. Selected characteristics of children harboring mef/erm-containing organisms were compared with other participants of the larger study. Of 221 children colonized by pneumococci, 17 (7.7%) were colonized with an organism containing both determinants. All mef/erm-positive organisms demonstrated azithromycin minimum inhibitory concentrations > or =256 microg/mL and were coresistant to all other agents tested. The mef/erm-containing organisms were serotype 19A and 19F, all but 1 of which manifested similar pulsed field gel electrophoresis patterns. Multilocus sequence testing analysis indicated a relationship to the Taiwan-14 macrolide-resistant strain that has spread throughout Eastern Asia. More than one-third of children colonized by a mef/erm-containing organism had received > or =1 dose of conjugate pneumococcal vaccine, a significantly higher proportion than children carrying less resistant organisms (P< 0.01). No other characteristics distinguished children harboring a mef/erm-containing pneumococcus from other children enrolled in the larger study. Clonally related mef/erm-containing serogroup 19 pneumococci were prominent among otherwise healthy children in a North American metropolitan area. Our findings suggest that spread of these organisms may be poorly contained by immunization.

Selection and Validation of a Multilocus Variable-Number Tandem-Repeat Analysis Panel for Typing Shigella spp.▿ †

PubMed Central

Gorgé, Olivier; Lopez, Stéphanie; Hilaire, Valérie; Lisanti, Olivier; Ramisse, Vincent; Vergnaud, Gilles

2008-01-01

The Shigella genus has historically been separated into four species, based on biochemical assays. The classification within each species relies on serotyping. Recently, genome sequencing and DNA assays, in particular the multilocus sequence typing (MLST) approach, greatly improved the current knowledge of the origin and phylogenetic evolution of Shigella spp. The Shigella and Escherichia genera are now considered to belong to a unique genomospecies. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses of highly homogeneous bacterial pathogens. Here, we assess the capability of MLVA for Shigella typing. Thirty-two potentially polymorphic VNTRs were selected by analyzing in silico five Shigella genomic sequences and subsequently evaluated. Eventually, a panel of 15 VNTRs was selected (i.e., MLVA15 analysis). MLVA15 analysis of 78 strains or genome sequences of Shigella spp. and 11 strains or genome sequences of Escherichia coli distinguished 83 genotypes. Shigella population cluster analysis gave consistent results compared to MLST. MLVA15 analysis showed capabilities for E. coli typing, providing classification among pathogenic and nonpathogenic E. coli strains included in the study. The resulting data can be queried on our genotyping webpage (http://mlva.u-psud.fr). The MLVA15 assay is rapid, highly discriminatory, and reproducible for Shigella and Escherichia strains, suggesting that it could significantly contribute to epidemiological trace-back analysis of Shigella infections and pathogenic Escherichia outbreaks. Typing was performed on strains obtained mostly from collections. Further studies should include strains of much more diverse origins, including all pathogenic E. coli types. PMID:18216214

Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis

PubMed Central

Sun, Jiufeng; Huang, Yan; Huang, Huaiqiu; Liang, Pei; Wang, Xiaoyun; Mao, Qiang; Men, Jingtao; Chen, Wenjun; Deng, Chuanhuan; Zhou, Chenhui; Lv, Xiaoli; Zhou, Juanjuan; Zhang, Fan; Li, Ran; Tian, Yanli; Lei, Huali; Liang, Chi; Hu, Xuchu; Xu, Jin; Li, Xuerong; XinbingYu

2013-01-01

Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis. PMID:23825605

Genetic Diversity of Giardia duodenalis: Multilocus Genotyping Reveals Zoonotic Potential between Clinical and Environmental Sources in a Metropolitan Region of Brazil

PubMed Central

Durigan, Mauricio; Abreu, Aluana Gonçalves; Zucchi, Maria Imaculada; Franco, Regina Maura Bueno; de Souza, Anete Pereira

2014-01-01

Background Giardia duodenalis is a flagellate protozoan that parasitizes humans and several other mammals. Protozoan contamination has been regularly documented at important environmental sites, although most of these studies were performed at the species level. There is a lack of studies that correlate environmental contamination and clinical infections in the same region. The aim of this study is to evaluate the genetic diversity of a set of clinical and environmental samples and to use the obtained data to characterize the genetic profile of the distribution of G. duodenalis and the potential for zoonotic transmission in a metropolitan region of Brazil. Methodology/Principal Findings The genetic assemblages and subtypes of G. duodenalis isolates obtained from hospitals, a veterinary clinic, a day-care center and important environmental sites were determined via multilocus sequence-based genotyping using three unlinked gene loci. Cysts of Giardia were detected at all of the environmental sites. Mixed assemblages were detected in 25% of the total samples, and an elevated number of haplotypes was identified. The main haplotypes were shared among the groups, and new subtypes were identified at all loci. Ten multilocus genotypes were identified: 7 for assemblage A and 3 for assemblage B. Conclusions/Significance There is persistent G. duodenalis contamination at important environmental sites in the city. The identified mixed assemblages likely represent mixed infections, suggesting high endemicity of Giardia in these hosts. Most Giardia isolates obtained in this study displayed zoonotic potential. The high degree of genetic diversity in the isolates obtained from both clinical and environmental samples suggests that multiple sources of infection are likely responsible for the detected contamination events. The finding that many multilocus genotypes (MLGs) and haplotypes are shared by different groups suggests that these sources of infection may be related and indicates that there is a notable risk of human infection caused by Giardia in this region. PMID:25536055

Neisseria meningitidis; clones, carriage, and disease.

PubMed

Read, R C

2014-05-01

Neisseria meningitidis, the cause of meningococcal disease, has been the subject of sophisticated molecular epidemiological investigation as a consequence of the significant public health threat posed by this organism. The use of multilocus sequence typing and whole genome sequencing classifies the organism into clonal complexes. Extensive phenotypic, genotypic and epidemiological information is available on the PubMLST website. The human nasopharynx is the sole ecological niche of this species, and carrier isolates show extensive genetic diversity as compared with hyperinvasive lineages. Horizontal gene exchange and recombinant events within the meningococcal genome during residence in the human nasopharynx result in antigenic diversity even within clonal complexes, so that individual clones may express, for example, more than one capsular polysaccharide (serogroup). Successful clones are capable of wide global dissemination, and may be associated with explosive epidemics of invasive disease. © 2014 The Author Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Detection of Epistasis for Flowering Time Using Bayesian Multilocus Estimation in a Barley MAGIC Population

PubMed Central

Mathew, Boby; Léon, Jens; Sannemann, Wiebke; Sillanpää, Mikko J.

2018-01-01

Gene-by-gene interactions, also known as epistasis, regulate many complex traits in different species. With the availability of low-cost genotyping it is now possible to study epistasis on a genome-wide scale. However, identifying genome-wide epistasis is a high-dimensional multiple regression problem and needs the application of dimensionality reduction techniques. Flowering Time (FT) in crops is a complex trait that is known to be influenced by many interacting genes and pathways in various crops. In this study, we successfully apply Sure Independence Screening (SIS) for dimensionality reduction to identify two-way and three-way epistasis for the FT trait in a Multiparent Advanced Generation Inter-Cross (MAGIC) barley population using the Bayesian multilocus model. The MAGIC barley population was generated from intercrossing among eight parental lines and thus, offered greater genetic diversity to detect higher-order epistatic interactions. Our results suggest that SIS is an efficient dimensionality reduction approach to detect high-order interactions in a Bayesian multilocus model. We also observe that many of our findings (genomic regions with main or higher-order epistatic effects) overlap with known candidate genes that have been already reported in barley and closely related species for the FT trait. PMID:29254994

Ancestral European roots of Helicobacter pylori in India

PubMed Central

Devi, S Manjulata; Ahmed, Irshad; Francalacci, Paolo; Hussain, M Abid; Akhter, Yusuf; Alvi, Ayesha; Sechi, Leonardo A; Mégraud, Francis; Ahmed, Niyaz

2007-01-01

Background The human gastric pathogen Helicobacter pylori is co-evolved with its host and therefore, origins and expansion of multiple populations and sub populations of H. pylori mirror ancient human migrations. Ancestral origins of H. pylori in the vast Indian subcontinent are debatable. It is not clear how different waves of human migrations in South Asia shaped the population structure of H. pylori. We tried to address these issues through mapping genetic origins of present day H. pylori in India and their genomic comparison with hundreds of isolates from different geographic regions. Results We attempted to dissect genetic identity of strains by multilocus sequence typing (MLST) of the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) and phylogeographic analysis of haplotypes using MEGA and NETWORK software while incorporating DNA sequences and genotyping data of whole cag pathogenicity-islands (cagPAI). The distribution of cagPAI genes within these strains was analyzed by using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined by gene sequencing. All the isolates analyzed revealed European ancestry and belonged to H. pylori sub-population, hpEurope. The cagPAI harbored by Indian strains revealed European features upon PCR based analysis and whole PAI sequencing. Conclusion These observations suggest that H. pylori strains in India share ancestral origins with their European counterparts. Further, non-existence of other sub-populations such as hpAfrica and hpEastAsia, at least in our collection of isolates, suggest that the hpEurope strains enjoyed a special fitness advantage in Indian stomachs to out-compete any endogenous strains. These results also might support hypotheses related to gene flow in India through Indo-Aryans and arrival of Neolithic practices and languages from the Fertile Crescent. PMID:17584914

Enterobacter cloacae Complex Isolates Harboring blaNMC-A or blaIMI-Type Class A Carbapenemase Genes on Novel Chromosomal Integrative Elements and Plasmids

PubMed Central

Boyd, David A.; Mataseje, Laura F.; Davidson, Ross; Delport, Johannes A.; Fuller, Jeff; Hoang, Linda; Lefebvre, Brigitte; Levett, Paul N.; Roscoe, Diane L.; Willey, Barbara M.

2017-01-01

ABSTRACT Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A blaNMC-A or blaIMI-type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species; however, blaNMC-A was highly associated with Enterobacter ludwigii. Whole-genome sequencing and bioinformatics analysis revealed that all NMC-A (n = 10), IMI-1 (n = 5), and IMI-9 (n = 2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements (EcloIMEXs) located in the identical chromosomal locus. Two novel genes, blaIMI-5 and blaIMI-6, were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring blaNMC-A/IMI-type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential. PMID:28223374

Enterobacter cloacae Complex Isolates Harboring blaNMC-A or blaIMI-Type Class A Carbapenemase Genes on Novel Chromosomal Integrative Elements and Plasmids.

PubMed

Boyd, David A; Mataseje, Laura F; Davidson, Ross; Delport, Johannes A; Fuller, Jeff; Hoang, Linda; Lefebvre, Brigitte; Levett, Paul N; Roscoe, Diane L; Willey, Barbara M; Mulvey, Michael R

2017-05-01

Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A bla NMC-A or bla IMI -type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species; however, bla NMC-A was highly associated with Enterobacter ludwigii Whole-genome sequencing and bioinformatics analysis revealed that all NMC-A ( n = 10), IMI-1 ( n = 5), and IMI-9 ( n = 2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements (EcloIMEXs) located in the identical chromosomal locus. Two novel genes, bla IMI-5 and bla IMI-6 , were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring bla NMC-A/IMI -type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential. © Crown copyright 2017.

Internalin profiling and multilocus sequence typing suggest four Listeria innocua subgroups with different evolutionary distances from Listeria monocytogenes

PubMed Central

2010-01-01

Background Ecological, biochemical and genetic resemblance as well as clear differences of virulence between L. monocytogenes and L. innocua make this bacterial clade attractive as a model to examine evolution of pathogenicity. This study was attempted to examine the population structure of L. innocua and the microevolution in the L. innocua-L. monocytogenes clade via profiling of 37 internalin genes and multilocus sequence typing based on the sequences of 9 unlinked genes gyrB, sigB, dapE, hisJ, ribC, purM, gap, tuf and betL. Results L. innocua was genetically monophyletic compared to L. monocytogenes, and comprised four subgroups. Subgroups A and B correlated with internalin types 1 and 3 (except the strain 0063 belonging to subgroup C) and internalin types 2 and 4 respectively. The majority of L. innocua strains belonged to these two subgroups. Subgroup A harbored a whole set of L. monocytogenes-L. innocua common and L. innocua-specific internalin genes, and displayed higher recombination rates than those of subgroup B, including the relative frequency of occurrence of recombination versus mutation (ρ/θ) and the relative effect of recombination versus point mutation (r/m). Subgroup A also exhibited a significantly smaller exterior/interior branch length ratio than expected under the coalescent model, suggesting a recent expansion of its population size. The phylogram based on the analysis with correction for recombination revealed that the time to the most recent common ancestor (TMRCA) of L. innocua subgroups A and B were similar. Additionally, subgroup D, which correlated with internalin type 5, branched off from the other three subgroups. All L. innocua strains lacked seventeen virulence genes found in L. monocytogenes (except for the subgroup D strain L43 harboring inlJ and two subgroup B strains bearing bsh) and were nonpathogenic to mice. Conclusions L. innocua represents a young species descending from L. monocytogenes and comprises four subgroups: two major subgroups A and B, and one atypical subgroup D serving as a link between L. monocytogenes and L. innocua in the evolutionary chain. Although subgroups A and B appeared at approximately the same time, subgroup A seems to have experienced a recent expansion of the population size with higher recombination frequency and effect than those of subgroup B, and might represent the possible evolutionary direction towards adaptation to enviroments. The evolutionary history in the L. monocytogenes-L. innocua clade represents a rare example of evolution towards reduced virulence of pathogens. PMID:20356375

Characterization of Streptococcus pneumoniae isolates from Austrian companion animals and horses.

PubMed

Ginders, Maximilian; Leschnik, Michael; Künzel, Frank; Kampner, Doris; Mikula, Claudia; Steindl, Georg; Eichhorn, Inga; Feßler, Andrea T; Schwarz, Stefan; Spergser, Joachim; Loncaric, Igor

2017-11-14

The aim of the present study was to investigate the genetic relatedness and the antimicrobial resistance profiles of a collection of Austrian Streptococcus pneumoniae isolates from companion animals and horses. A total of 12 non-repetitive isolates presumptively identified as S. pneumoniae were obtained during routinely diagnostic activities between March 2009 and January 2017. Isolates were confirmed as S. pneumoniae by bile solubility and optochin susceptibility testing, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry and sequence analysis of a part recA and the 16S rRNA genes. Isolates were further characterized by pneumolysin polymerase chain reaction (PCR) and genotyped by multilocus sequence typing (MLST). Antimicrobial susceptibility testing was performed and resistance genes were detected by specific PCR assays. All isolates were serotyped. Four sequence types (ST) (ST36, ST3546, ST6934 and ST6937) and four serotypes (3, 19A, 19F and 23F) were detected. Two isolates from twelve displayed a multidrug-resistance pheno- and genotype. This study represents the first comprehensive investigation on characteristics of S. pneumoniae isolates recovered from Austrian companion animals and horses. The obtained results indicate that common human sero- (23F) and sequence type (ST36) implicated in causing invasive pneumococcal disease (IPD) may circulate in dogs. Isolates obtained from other examined animals seem to be host-adapted.

A New Perspective on Polyploid Fragaria (Strawberry) Genome Composition Based on Large-Scale, Multi-Locus Phylogenetic Analysis

PubMed Central

Yang, Yilong

2017-01-01

Abstract The subgenomic compositions of the octoploid (2n = 8× = 56) strawberry (Fragaria) species, including the economically important cultivated species Fragaria x ananassa, have been a topic of long-standing interest. Phylogenomic approaches utilizing next-generation sequencing technologies offer a new window into species relationships and the subgenomic compositions of polyploids. We have conducted a large-scale phylogenetic analysis of Fragaria (strawberry) species using the Fluidigm Access Array system and 454 sequencing platform. About 24 single-copy or low-copy nuclear genes distributed across the genome were amplified and sequenced from 96 genomic DNA samples representing 16 Fragaria species from diploid (2×) to decaploid (10×), including the most extensive sampling of octoploid taxa yet reported. Individual gene trees were constructed by different tree-building methods. Mosaic genomic structures of diploid Fragaria species consisting of sequences at different phylogenetic positions were observed. Our findings support the presence in octoploid species of genetic signatures from at least five diploid ancestors (F. vesca, F. iinumae, F. bucharica, F. viridis, and at least one additional allele contributor of unknown identity), and questions the extent to which distinct subgenomes are preserved over evolutionary time in the allopolyploid Fragaria species. In addition, our data support divergence between the two wild octoploid species, F. virginiana and F. chiloensis. PMID:29045639

[Multilocus Sequence Typing analysis of human Campylobacter coli in Granada (Spain)].

PubMed

Carrillo-Ávila, J A; Sorlózano-Puerto, A; Pérez-Ruiz, M; Gutiérrez-Fernández, J

2016-12-01

Different subtypes of Campylobacter spp. have been associated with diarrhoea and a Multilocus Sequence Typing (MLST) method has been performed for subtyping. In the present work, MLST was used to analyse the genetic diversity of eight strains of Campylobacter coli. Nineteen genetic markers were amplified for MLST analysis: AnsB, DmsA, ggt, Cj1585c, CJJ81176-1367/1371, Tlp7, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, cstIII. After comparing the obtained sequences with the Campylobacter MLST database, the allele numbers, sequence types (STs) and clonal complexes (CCs) were assigned. The 8 C. coli isolates yielded 4 different STs belonging to 2 CCs. Seven isolates belong to ST-828 clonal complex and only one isolate belong to ST-21. Two samples came from the same patient, but were isolated in two different periods of time. MLST can be useful for taxonomic characterization of C. coli isolates.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1.

PubMed

Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie; Maubon, Danièle

2017-08-01

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1

PubMed Central

Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie

2017-01-01

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus. PMID:28726611

Phylogeography and Sex-Biased Dispersal across Riverine Manatee Populations (Trichechus inunguis and Trichechus manatus) in South America

PubMed Central

Satizábal, Paula; Mignucci-Giannoni, Antonio A.; Duchêne, Sebastián; Caicedo-Herrera, Dalila; Perea-Sicchar, Carlos M.; García-Dávila, Carmen R.; Trujillo, Fernando; Caballero, Susana J.

2012-01-01

Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian (Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop (Control Region, Mitochondrial DNA) sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments. PMID:23285054

Whole-genome sequencing of the blue whale and other rorquals finds signatures for introgressive gene flow

PubMed Central

Árnason, Úlfur; Kumar, Vikas

2018-01-01

Reconstructing the evolution of baleen whales (Mysticeti) has been problematic because morphological and genetic analyses have produced different scenarios. This might be caused by genomic admixture that may have taken place among some rorquals. We present the genomes of six whales, including the blue whale (Balaenoptera musculus), to reconstruct a species tree of baleen whales and to identify phylogenetic conflicts. Evolutionary multilocus analyses of 34,192 genome fragments reveal a fast radiation of rorquals at 10.5 to 7.5 million years ago coinciding with oceanic circulation shifts. The evolutionarily enigmatic gray whale (Eschrichtius robustus) is placed among rorquals, and the blue whale genome shows a high degree of heterozygosity. The nearly equal frequency of conflicting gene trees suggests that speciation of rorqual evolution occurred under gene flow, which is best depicted by evolutionary networks. Especially in marine environments, sympatric speciation might be common; our results raise questions about how genetic divergence can be established. PMID:29632892

Piggy: a rapid, large-scale pan-genome analysis tool for intergenic regions in bacteria.

PubMed

Thorpe, Harry A; Bayliss, Sion C; Sheppard, Samuel K; Feil, Edward J

2018-04-01

The concept of the "pan-genome," which refers to the total complement of genes within a given sample or species, is well established in bacterial genomics. Rapid and scalable pipelines are available for managing and interpreting pan-genomes from large batches of annotated assemblies. However, despite overwhelming evidence that variation in intergenic regions in bacteria can directly influence phenotypes, most current approaches for analyzing pan-genomes focus exclusively on protein-coding sequences. To address this we present Piggy, a novel pipeline that emulates Roary except that it is based only on intergenic regions. A key utility provided by Piggy is the detection of highly divergent ("switched") intergenic regions (IGRs) upstream of genes. We demonstrate the use of Piggy on large datasets of clinically important lineages of Staphylococcus aureus and Escherichia coli. For S. aureus, we show that highly divergent (switched) IGRs are associated with differences in gene expression and we establish a multilocus reference database of IGR alleles (igMLST; implemented in BIGSdb).

Phylogeography and sex-biased dispersal across riverine manatee populations (Trichechus inunguis and Trichechus manatus) in South America.

PubMed

Satizábal, Paula; Mignucci-Giannoni, Antonio A; Duchêne, Sebastián; Caicedo-Herrera, Dalila; Perea-Sicchar, Carlos M; García-Dávila, Carmen R; Trujillo, Fernando; Caballero, Susana J

2012-01-01

Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian (Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop (Control Region, Mitochondrial DNA) sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments.

Sequences associated with human iris pigmentation.

PubMed Central

Frudakis, Tony; Thomas, Matthew; Gaskin, Zach; Venkateswarlu, K; Chandra, K Suresh; Ginjupalli, Siva; Gunturi, Sitaram; Natrajan, Sivamani; Ponnuswamy, Viswanathan K; Ponnuswamy, K N

2003-01-01

To determine whether and how common polymorphisms are associated with natural distributions of iris colors, we surveyed 851 individuals of mainly European descent at 335 SNP loci in 13 pigmentation genes and 419 other SNPs distributed throughout the genome and known or thought to be informative for certain elements of population structure. We identified numerous SNPs, haplotypes, and diplotypes (diploid pairs of haplotypes) within the OCA2, MYO5A, TYRP1, AIM, DCT, and TYR genes and the CYP1A2-15q22-ter, CYP1B1-2p21, CYP2C8-10q23, CYP2C9-10q24, and MAOA-Xp11.4 regions as significantly associated with iris colors. Half of the associated SNPs were located on chromosome 15, which corresponds with results that others have previously obtained from linkage analysis. We identified 5 additional genes (ASIP, MC1R, POMC, and SILV) and one additional region (GSTT2-22q11.23) with haplotype and/or diplotypes, but not individual SNP alleles associated with iris colors. For most of the genes, multilocus gene-wise genotype sequences were more strongly associated with iris colors than were haplotypes or SNP alleles. Diplotypes for these genes explain 15% of iris color variation. Apart from representing the first comprehensive candidate gene study for variable iris pigmentation and constituting a first step toward developing a classification model for the inference of iris color from DNA, our results suggest that cryptic population structure might serve as a leverage tool for complex trait gene mapping if genomes are screened with the appropriate ancestry informative markers. PMID:14704187

Genomic Sequencing of Bordetella pertussis for Epidemiology and Global Surveillance of Whooping Cough.

PubMed

Bouchez, Valérie; Guglielmini, Julien; Dazas, Mélody; Landier, Annie; Toubiana, Julie; Guillot, Sophie; Criscuolo, Alexis; Brisse, Sylvain

2018-06-01

Bordetella pertussis causes whooping cough, a highly contagious respiratory disease that is reemerging in many world regions. The spread of antigen-deficient strains may threaten acellular vaccine efficacy. Dynamics of strain transmission are poorly defined because of shortcomings in current strain genotyping methods. Our objective was to develop a whole-genome genotyping strategy with sufficient resolution for local epidemiologic questions and sufficient reproducibility to enable international comparisons of clinical isolates. We defined a core genome multilocus sequence typing scheme comprising 2,038 loci and demonstrated its congruence with whole-genome single-nucleotide polymorphism variation. Most cases of intrafamilial groups of isolates or of multiple isolates recovered from the same patient were distinguished from temporally and geographically cocirculating isolates. However, epidemiologically unrelated isolates were sometimes nearly undistinguishable. We set up a publicly accessible core genome multilocus sequence typing database to enable global comparisons of B. pertussis isolates, opening the way for internationally coordinated surveillance.

Cryptosporidium genotyping in Europe: The current status and processes for a harmonised multi-locus genotyping scheme.

PubMed

Chalmers, Rachel M; Pérez-Cordón, Gregorio; Cacció, Simone M; Klotz, Christian; Robertson, Lucy J

2018-06-13

Due to the occurrence of genetic recombination, a reliable and discriminatory method to genotype Cryptosporidium isolates at the intra-species level requires the analysis of multiple loci, but a standardised scheme is not currently available. A workshop was held at the Robert Koch Institute, Berlin in 2016 that gathered 23 scientists with appropriate expertise (in either Cryptosporidium genotyping and/or surveillance, epidemiology or outbreaks) to discuss the processes for the development of a robust, standardised, multi-locus genotyping (MLG) scheme and propose an approach. The background evidence and main conclusions were outlined in a previously published report; the objectives of this further report are to describe 1) the current use of Cryptosporidium genotyping, 2) the elicitation and synthesis of the participants' opinions, and 3) the agreed processes and criteria for the development, evaluation and validation of a standardised MLG scheme for Cryptosporidium surveillance and outbreak investigations. Cryptosporidium was characterised to the species level in 7/12 (58%) participating European countries, mostly for human outbreak investigations. Further genotyping was mostly by sequencing the gp60 gene. A ranking exercise of performance and convenience criteria found that portability, biological robustness, typeability, and discriminatory power were considered by participants as the most important attributes in developing a multilocus scheme. The major barrier to implementation was lack of funding. A structured process for marker identification, evaluation, validation, implementation, and maintenance was proposed and outlined for application to Cryptosporidium, with prioritisation of Cryptosporidium parvum to support investigation of transmission in Europe. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetic background of novel sequence types of CTX-M-8- and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae from public wastewater treatment plants in São Paulo, Brazil.

PubMed

Dropa, Milena; Lincopan, Nilton; Balsalobre, Livia C; Oliveira, Danielle E; Moura, Rodrigo A; Fernandes, Miriam Rodriguez; da Silva, Quézia Moura; Matté, Glavur R; Sato, Maria I Z; Matté, Maria H

2016-03-01

The release of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae to the environment is a public health issue worldwide. The aim of this study was to investigate the genetic background of genes encoding ESBLs in wastewater treatment plants (WWTPs) in São Paulo, southeastern Brazil. In 2009, during a local surveillance study, seven ESBL-producing Enterobacteriaceae strains were recovered from five WWTPs and screened for ESBL genes and mobile genetic elements. Multilocus sequence typing (MLST) was carried out, and wild plasmids were transformed into electrocompetent Escherichia coli. S1-PFGE technique was used to verify the presence of high molecular weight plasmids in wild-type strains and in bla ESBL-containing E. coli transformants. Strains harbored bla CTX-M-8, bla CTX-M-15, and/or bla SHV-28. Sequencing results showed that bla CTX-M-8 and bla CTX-M-15 genes were associated with IS26. MLST revealed new sequence types for E. coli (ST4401, ST4402, ST4403, and ST4445) and Klebsiella pneumoniae (ST1574), except for one K. pneumoniae from ST307 and Enterobacter cloacae from ST131. PCR and S1-PFGE results showed CTX-M-producing E. coli transformants carried heavy plasmids sizing 48.5-209 kb, which belonged to IncI1, IncF, and IncM1 incompatibility groups. This is the first report of CTX-M-8 and SHV-28 enzymes in environmental samples, and the present results demonstrate the plasmid-mediated spread of CTX-M-encoding genes through five WWTPs in São Paulo, Brazil, suggesting WWTPs are hotspots for the transfer of ESBL genes and confirming the urgent need to improve the management of sewage in order to minimize the dissemination of resistance genes to the environment.

Bioaccessible Antioxidants in Milk Fermented by Bifidobacterium longum subsp. longum Strains

PubMed Central

Gagnon, Mérilie; Savard, Patricia; Rivière, Audrey; LaPointe, Gisèle

2015-01-01

Bifidobacterium longum subsp. longum is among the dominant species of the human gastrointestinal microbiota and could thus have potential as probiotics. New targets such as antioxidant properties have interest for beneficial effects on health. The objective of this study was to evaluate the bioaccessibility of antioxidants in milk fermented by selected B. longum subsp. longum strains during in vitro dynamic digestion. The antioxidant capacity of cell extracts from 38 strains, of which 32 belong to B. longum subsp. longum, was evaluated with the ORAC (oxygen radical absorbance capacity) method. On the basis of screening and gene sequence typing by multilocus locus sequence analysis (MLSA), five strains were chosen for fermenting reconstituted skim milk. Antioxidant capacity varied among the strains tested (P = 0.0009). Two strains of B. longum subsp. longum (CUETM 172 and 171) showed significantly higher ORAC values than the other bifidobacteria strains. However, there does not appear to be a relationship between gene sequence types and antioxidant capacity. The milk fermented by each of the five strains selected (CUETM 268, 172, 245, 247, or PRO 16-10) did not have higher initial ORAC values compared to the nonfermented milk samples. However, higher bioaccessibility of antioxidants in fermented milk (175–358%) was observed during digestion. PMID:25802836

Delineation of Streptococcus dysgalactiae, Its Subspecies, and Its Clinical and Phylogenetic Relationship to Streptococcus pyogenes

PubMed Central

Jensen, Anders

2012-01-01

The taxonomic status and structure of Streptococcus dysgalactiae have been the object of much confusion. Bacteria belonging to this species are usually referred to as Lancefield group C or group G streptococci in clinical settings in spite of the fact that these terms lack precision and prevent recognition of the exact clinical relevance of these bacteria. The purpose of this study was to develop an improved basis for delineation and identification of the individual species of the pyogenic group of streptococci in the clinical microbiology laboratory, with a special focus on S. dysgalactiae. We critically reexamined the genetic relationships of the species S. dysgalactiae, Streptococcus pyogenes, Streptococcus canis, and Streptococcus equi, which may share Lancefield group antigens, by phylogenetic reconstruction based on multilocus sequence analysis (MLSA) and 16S rRNA gene sequences and by emm typing combined with phenotypic characterization. Analysis of concatenated sequences of seven genes previously used for examination of viridans streptococci distinguished robust and coherent clusters. S. dysgalactiae consists of two separate clusters consistent with the two recognized subspecies dysgalactiae and equisimilis. Both taxa share alleles with S. pyogenes in several housekeeping genes, which invalidates identification based on single-locus sequencing. S. dysgalactiae, S. canis, and S. pyogenes constitute a closely related branch within the genus Streptococcus indicative of recent descent from a common ancestor, while S. equi is highly divergent from other species of the pyogenic group streptococci. The results provide an improved basis for identification of clinically important pyogenic group streptococci and explain the overlapping spectrum of infections caused by the species associated with humans. PMID:22075580

Insight into the Diversity of Penicillin-Binding Protein 2x Alleles and Mutations in Viridans Streptococci

PubMed Central

van der Linden, Mark; Otten, Julia; Bergmann, Carina; Latorre, Cristina; Liñares, Josefina

2017-01-01

ABSTRACT The identification of commensal streptococci species is an everlasting problem due to their ability to genetically transform. A new challenge in this respect is the recent description of Streptococcus pseudopneumoniae as a new species, which was distinguished from closely related pathogenic S. pneumoniae and commensal S. mitis by a variety of physiological and molecular biological tests. Forty-one atypical S. pneumoniae isolates have been collected at the German National Reference Center for Streptococci (GNRCS). Multilocus sequence typing (MLST) confirmed 35 isolates as the species S. pseudopneumoniae. A comparison with the pbp2x sequences from 120 commensal streptococci isolated from different continents revealed that pbp2x is distinct among penicillin-susceptible S. pseudopneumoniae isolates. Four penicillin-binding protein x (PBPx) alleles of penicillin-sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pseudopneumoniae, S. pneumoniae, and S. mitis, and S. infantis and S. oralis sequences were found in S. pneumoniae from Japan. PBP2x genes of the family of mosaic genes related to pbp2x in the S. pneumoniae clone Spain23F-1 were observed in S. oralis and S. infantis as well, confirming its global distribution. Thirty-eight sites were altered within the PBP2x transpeptidase domains of penicillin-resistant strains, excluding another 37 sites present in the reference genes of sensitive strains. Specific mutational patterns were detected depending on the parental sequence blocks, in agreement with distinct mutational pathways during the development of beta-lactam resistance. The majority of the mutations clustered around the active site, whereas others are likely to affect stability or interactions with the C-terminal domain or partner proteins. PMID:28193649

Serratia aquatilis sp. nov., isolated from drinking water systems.

PubMed

Kämpfer, Peter; Glaeser, Stefanie P

2016-01-01

A cream-white-pigmented, oxidase-negative bacterium (strain 2015-2462-01T), isolated from a drinking water system, was investigated in detail to determine its taxonomic position. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of strain 2015-2462-01T with sequences of the type strains of closely related species of the genus Serratia revealed highest similarity to Serratia fonticola (98.4 %), Serratia proteamaculans (97.8 %), Serratia liquefaciens and Serratia grimesii (both 97.7 %). 16S rRNA gene sequence similarities to all other Serratia species were below 97.4 %. Multilocus sequence analysis (MLSA) on the basis of concatenated partial gyrB, rpoB, infB and atpD gene sequences showed a clear distinction of strain 2015-2462-01T from the type strains of the closest related Serratia species. The fatty acid profile of the strain consisted of C16 : 1 ω7c, C16 : 0; C14 : 0 and C14 : 0 3-OH/iso-C16 : 1 I as major components. DNA-DNA hybridizations between 2015-2462-01T and S. fonticola ATCC 29844T resulted in a relatedness value of 27 % (reciprocal 20 %). This DNA-DNA hybridization result in combination with the MLSA results and the differential biochemical properties indicated that strain 2015-2462-01T represents a novel species of the genus Serratia, for which the name Serratia aquatilis sp. nov. is proposed. The type strain is 2015-2462-01T ( = LMG 29119T = CCM 8626T).

Emergence of Sequence Type 779 Methicillin-Resistant Staphylococcus aureus Harboring a Novel Pseudo Staphylococcal Cassette Chromosome mec (SCCmec)-SCC-SCCCRISPR Composite Element in Irish Hospitals

PubMed Central

Kinnevey, Peter M.; Shore, Anna C.; Brennan, Grainne I.; Sullivan, Derek J.; Ehricht, Ralf; Monecke, Stefan; Slickers, Peter

2013-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa (t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec (SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmec element (16.3 kb) carrying mecA with a novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4 allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrC allele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureus and coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureus and CoNS and show how this contributes to the emergence of novel SCCmec elements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmec typing methods. PMID:23147725

ISOLATION AND CHARACTERIZATION OF A NOVEL MARINE BRUCELLA FROM A SOUTHERN SEA OTTER (ENHYDRA LUTRIS NEREIS), CALIFORNIA, USA.

PubMed

Miller, Melissa A; Burgess, Tristan L; Dodd, Erin M; Rhyan, Jack C; Jang, Spencer S; Byrne, Barbara A; Gulland, Frances M D; Murray, Michael J; Toy-Choutka, Sharon; Conrad, Patricia A; Field, Cara L; Sidor, Inga F; Smith, Woutrina A

2017-04-01

We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.

The molecular epidemiology of cholera in Latin America.

PubMed

Wachsmuth, I K; Evins, G M; Fields, P I; Olsvik, O; Popovic, T; Bopp, C A; Wells, J G; Carrillo, C; Blake, P A

1993-03-01

To explain the sudden appearance and rapid spread of cholera in Latin America in January 1991, molecular techniques were used to define Vibrio cholerae O1 isolates from around the world. Restriction fragment length polymorphisms of rRNA and ctxA genes, DNA sequence of cholera toxin B subunit gene ctxB, and multilocus enzyme electrophoresis data were used to characterize 197 isolates. Worldwide, there are at least four distinct toxigenic El Tor V. cholerae O1 clones: the seventh pandemic (Eastern Hemisphere), US Gulf Coast, Australian, and Latin American. Nontoxigenic V. cholerae O1 previously isolated in Brazil, Mexico, and Peru are unlike current toxigenic isolates. The Latin American clone probably represents an extension of the seventh pandemic into the Western Hemisphere, while the US Gulf Coast clone most likely evolved separately. These data will be useful in monitoring the spread of cholera, determining the origin of outbreaks in both hemispheres, and implicating specific vehicles of transmission.

Faecal carriage of extended-spectrum β-lactamase-producing and AmpC β-lactamase-producing bacteria among Danish army recruits.

PubMed

Hammerum, A M; Lester, C H; Jakobsen, L; Porsbo, L J

2011-04-01

During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing and AmpC β-lactamase-producing bacteria. Three ESBL-producing (CTX-M-14a) Escherichia coli isolates, two AmpC-producing (CMY-2) E. coli isolates and one AmpC-producing (CMY-34) Citrobacter freundii isolate were detected. Two of the CTX-M-14a E. coli isolates had similar pulsed-field gel electrophoresis and multilocus sequence typing profiles, indicating the same origin or transmission between the two army recruits. The bla(CTX-M-14a) genes were transferable to an E. coli recipient. These commensal bacteria therefore constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria in the intestine. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

High prevalence of the PER-1 gene among carbapenem-resistant Acinetobacter baumannii in Riyadh, Saudi Arabia.

PubMed

Aly, M M; Abu Alsoud, N M; Elrobh, M S; Al Johani, S M; Balkhy, H H

2016-11-01

The prevalence of carbapenem-resistant Acinetobacter baumannii in Saudi Arabia and their resistance genetic mechanisms are yet to be identified. We studied the prevalence and genetic diversity of extended-spectrum beta-lactamase genes, particularly the PER-1 gene, among carbapenem-resistant A. baumannii strains from patients at a tertiary care hospital in Riyadh, Saudi Arabia between 2006 and 2014. Fresh subcultured samples were tested for antimicrobial susceptibility minimum inhibitory concentration (MIC). Total genomic DNA was extracted from each isolate and further used for polymerase chain reaction (PCR) genotyping, sequence-based typing (SBT) of PER-1 and OXA-51-like gene, and multilocus sequence typing (MLST) of positive isolates. Randomly selected clinical isolates (n = 100) were subjected to MLST. A total of 503 isolates were characterized as multidrug-resistant (MDR) using the MIC. Isolates were further PCR tested for bla -TEM and bla -PER-1 resistance genes (n = 503). The genotyping results showed that 68/503 (14 %) isolates were positive to bla TEM. The genotyping results of PER-1-like genes showed that 384/503 (76.3 %) were positive among MDR Acinetobacter isolates. Based on SBT, the majority of these isolates were clustered into three main groups including isolates harboring PER-1: AB11 (bla -PER-1 ), isolate AB16 (bla -PER-1 ), and, finally, the plasmid pAB154 (bla -PER-7 ). Remarkably, many isolates were concealing the PER-1 gene and harboring the TEM resistance genes as well. MLST results for selected isolates (n = 100) identified four main sequence types (STs: 2, 19, 20, and 25) and four novel isolates (ST 486-489). We report 76.3 % prevalence of the PER-1 resistance gene among Acinetobacter clinical isolates from Riyadh, Saudi Arabia. Further work is needed to explore the clinical risks and patient outcome with such resistance related to healthcare-associated infections and investigate the genetic and molecular mechanisms that confer the MDR phenotype.

Phylogenetic relationships in the family Streptomycetaceae using multi-locus sequence analysis

USDA-ARS?s Scientific Manuscript database

The family Streptomycetaceae, notably species in the genus Streptomyces, have long been the subject of investigation due to their well-known ability to produce secondary metabolites. The emergence of drug resistant pathogens and the relative ease of producing genome sequences has renewed the importa...

Hypervariability generated by natural selection in an extracellular complement-inhibiting protein of serotype M1 strains of group A Streptococcus.

PubMed

Stockbauer, K E; Grigsby, D; Pan, X; Fu, Y X; Mejia, L M; Cravioto, A; Musser, J M

1998-03-17

In many countries, M1 strains of the human pathogenic bacterium group A Streptococcus are the most common serotype recovered from patients with invasive disease episodes. Strains of this serotype express an extracellular protein that inhibits complement [streptococcal inhibitor of complement (Sic)] and is therefore believed to be a virulence factor. Comparative sequence analysis of the 915-bp sic gene in 165 M1 organisms recovered from diverse localities and infection types identified 62 alleles. Inasmuch as multilocus enzyme electrophoresis and pulsed-field gel electrophoresis previously showed that most M1 organisms represent a distinct streptococcal clone, the extent of sic gene polymorphism was unexpected. The level of polymorphism greatly exceeds that recorded for all other genes examined in serotype M1 strains. All insertions and deletions are in frame, and virtually all nucleotide substitutions alter the amino acid sequence of the Sic protein. These molecular features indicate that structural change in Sic is mediated by natural selection. Study of 70 strains recovered from two temporally distinct epidemics of streptococcal infections in the former East Germany found little sharing of Sic variants among strains recovered in the different time periods. Taken together, the data indicate that sic is a uniquely variable gene and provide insight into a potential molecular mechanism contributing to fluctuations in streptococcal disease frequency and severity.

Analysis of Clonality and Antibiotic Resistance among Early Clinical Isolates of Enterococcus faecium in the United States

PubMed Central

Galloway-Peña, Jessica R.; Nallapareddy, Sreedhar R.; Arias, Cesar A.; Eliopoulos, George M.; Murray, Barbara E.

2009-01-01

Background The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. Methods Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994 we determined the multi-locus sequence type, the presence of 16 putative virulence genes (hylEfm, espEfm and fms genes), resistance to ampicillin (AMPR), vancomycin (VANR) and high-levels of gentamicin and streptomycin. Results Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the US. The earliest CC17 isolates were part of an outbreak in 1982 in Richmond, VA. Characteristics of CC17 isolates included increases in AMPR, the presence of hylEfm and espEfm, emergence of VANR and the presence of at least 13/14 fms genes. Eight out of forty-one of the early AMPR isolates, however, were not within CC17. Conclusions While not all early US AMPR isolates were clonally related, E. faecium CC17 isolates have been circulating in the US since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment. PMID:19821720

Genetic Attributes of E. coli Isolates from Chlorinated Drinking Water

PubMed Central

Blyton, Michaela D. J.; Gordon, David M.

2017-01-01

Escherichia coli, is intimately associated with both human health and water sanitation. E. coli isolates from water can either be (i) host associated commensals, indicating recent faecal contamination; (ii) diarrheal pathogens or (iii) extra-intestinal pathogens that pose a direct health risk; or (iv) free-living. In this study we genetically characterised 28 E. coli isolates obtained from treated drinking water in south eastern Australia to ascertain their likely source. We used full genome sequencing to assign the isolates to their phylogenetic group and multi-locus sequence type. The isolates were also screened in silico for several virulence genes and genes involved in acquired antibiotic resistance. The genetic characteristics of the isolates indicated that four isolates were likely human pathogens. However, these isolates were not detected in sufficient numbers to present a health risk to the public. An additional isolate was a human associated strain. Nine isolates were water associated free-living strains that were unlikely to pose a health risk. Only 14% of the isolates belonged to the host associated phylogenetic group (B2) and only a single isolate had any antibiotic resistance genes. This suggests that the primary source of the drinking water E. coli isolates may not have been recent human faecal contamination. PMID:28107364

Genetic Attributes of E. coli Isolates from Chlorinated Drinking Water.

PubMed

Blyton, Michaela D J; Gordon, David M

2017-01-01

Escherichia coli, is intimately associated with both human health and water sanitation. E. coli isolates from water can either be (i) host associated commensals, indicating recent faecal contamination; (ii) diarrheal pathogens or (iii) extra-intestinal pathogens that pose a direct health risk; or (iv) free-living. In this study we genetically characterised 28 E. coli isolates obtained from treated drinking water in south eastern Australia to ascertain their likely source. We used full genome sequencing to assign the isolates to their phylogenetic group and multi-locus sequence type. The isolates were also screened in silico for several virulence genes and genes involved in acquired antibiotic resistance. The genetic characteristics of the isolates indicated that four isolates were likely human pathogens. However, these isolates were not detected in sufficient numbers to present a health risk to the public. An additional isolate was a human associated strain. Nine isolates were water associated free-living strains that were unlikely to pose a health risk. Only 14% of the isolates belonged to the host associated phylogenetic group (B2) and only a single isolate had any antibiotic resistance genes. This suggests that the primary source of the drinking water E. coli isolates may not have been recent human faecal contamination.

The evolutionary history of Saccharomyces species inferred from completed mitochondrial genomes and revision in the ‘yeast mitochondrial genetic code’

PubMed Central

Szabóová, Dana; Bielik, Peter; Poláková, Silvia; Šoltys, Katarína; Jatzová, Katarína; Szemes, Tomáš

2017-01-01

Abstract The yeast Saccharomyces are widely used to test ecological and evolutionary hypotheses. A large number of nuclear genomic DNA sequences are available, but mitochondrial genomic data are insufficient. We completed mitochondrial DNA (mtDNA) sequencing from Illumina MiSeq reads for all Saccharomyces species. All are circularly mapped molecules decreasing in size with phylogenetic distance from Saccharomyces cerevisiae but with similar gene content including regulatory and selfish elements like origins of replication, introns, free-standing open reading frames or GC clusters. Their most profound feature is species-specific alteration in gene order. The genetic code slightly differs from well-established yeast mitochondrial code as GUG is used rarely as the translation start and CGA and CGC code for arginine. The multilocus phylogeny, inferred from mtDNA, does not correlate with the trees derived from nuclear genes. mtDNA data demonstrate that Saccharomyces cariocanus should be assigned as a separate species and Saccharomyces bayanus CBS 380T should not be considered as a distinct species due to mtDNA nearly identical to Saccharomyces uvarum mtDNA. Apparently, comparison of mtDNAs should not be neglected in genomic studies as it is an important tool to understand the origin and evolutionary history of some yeast species. PMID:28992063

Identification of Infantile Diarrhea Caused by Breast Milk-Transmitted Staphylococcus aureus Infection.

PubMed

Chen, Zhong; Pan, Wei-Guang; Xian, Wei-Yi; Cheng, Hang; Zheng, Jin-Xin; Hu, Qing-Hua; Yu, Zhi-Jian; Deng, Qi-Wen

2016-10-01

Staphylococcus aureus is a well-known organism which is responsible for a variety of human infectious diseases including skin infections, pneumonia, bacteremia, and endocarditis. Few of the microorganisms can be transmitted from mother to the newborn or infant by milk breastfeeding. This study aims to identify transmission of S. aureus from healthy, lactating mothers to their infants by breastfeeding. Stool specimens of diarrheal infants and breast milk of their mother (totally three pairs) were collected and six Staphylococcus aureus isolates were cultured positively. Homology and molecular characters of isolated strains were tested using pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing. Furthermore, toxin genes detection was also performed. Each pair of isolates has the same PFGE type and spa type. Four Sequence types (STs) were found among all the isolates; they are ST15, ST188, and ST59, respectively. Among the strains, seb, sec, and tst genes were found, and all were negative for pvl gene. The homology of the S. aureus strains isolated from the infants' stool and the mothers' milk was genetically demonstrated, which indicated that breastfeeding may be important in the transmission of S. aureus infection, and the character of S. aureus needed to be further evaluated.

Emergence of Klebsiella pneumoniae ST273 Carrying blaNDM-7 and ST656 Carrying blaNDM-1 in Manila, Philippines.

PubMed

Chou, Andrew; Roa, Marylette; Evangelista, Michael A; Sulit, Arielle Kae; Lagamayo, Evelina; Torres, Brian C; Klinzing, David C; Daroy, Maria Luisa G; Navoa-Ng, Josephine; Sucgang, Richard; Zechiedrich, Lynn

2016-10-01

We sought to determine the epidemiology of carbapenem-resistant Enterobacteriaceae and to investigate the emergence of carbapenem-resistant Klebsiella pneumoniae in two teaching hospitals in Manila, Philippines. We screened 364 Enterobacteriaceae for carbapenem resistance between 2012 and 2013 and detected four carbapenem-resistant K. pneumoniae isolates from three different patients. We used whole genome sequencing to determine the antibiotic resistance profiles and confirmed the presence of carbapenemase genes by multiplex PCR. We used multilocus sequence typing and PCR-based replicon typing to genetically characterize the carbapenem-resistant isolates. The carbapenemase gene bla NDM was detected in K. pneumoniae isolates from two patients. The first patient had ventilator-associated pneumonia and lumbar shunt infection from K. pneumoniae ST273 carrying bla NDM-7 . The second patient had asymptomatic genitourinary colonization with K. pneumoniae ST656 carrying bla NDM-1 . The third patient had a gluteal abscess with K. pneumoniae ST1 that did not carry a carbapenemase gene, but did carry bla DHA-1 , bla OXA-1 , and bla SHV-1 . In this study, we report the first cases of bla NDM -carrying pathogens in the Philippines and add to the growing evidence of the worldwide spread of ST273 and NDM-7, a more efficient carbapenem hydrolyzer than NDM-1.

Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity.

PubMed

Scally, Mark; Schuenzel, Erin L; Stouthamer, Richard; Nunney, Leonard

2005-12-01

Multilocus sequence typing (MLST) identifies and groups bacterial strains based on DNA sequence data from (typically) seven housekeeping genes. MLST has also been employed to estimate the relative contributions of recombination and point mutation to clonal divergence. We applied MLST to the plant pathogen Xylella fastidiosa using an initial set of sequences for 10 loci (9.3 kb) of 25 strains from five different host plants, grapevine (PD strains), oleander (OLS strains), oak (OAK strains), almond (ALS strains), and peach (PP strains). An eBURST analysis identified six clonal complexes using the grouping criterion that each member must be identical to at least one other member at 7 or more of the 10 loci. These clonal complexes corresponded to previously identified phylogenetic clades; clonal complex 1 (CC1) (all PD strains plus two ALS strains) and CC2 (OLS strains) defined the X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi clades, while CC3 (ALS strains), CC4 (OAK strains), and CC5 (PP strains) were subclades of X. fastidiosa subsp. multiplex. CC6 (ALS strains) identified an X. fastidiosa subsp. multiplex-like group characterized by a high frequency of intersubspecific recombination. Compared to the recombination rate in other bacterial species, the recombination rate in X. fastidiosa is relatively low. Recombination between different alleles was estimated to give rise to 76% of the nucleotide changes and 31% of the allelic changes observed. The housekeeping loci holC, nuoL, leuA, gltT, cysG, petC, and lacF were chosen to form the basis of a public database for typing X. fastidiosa (www.mlst.net). These loci identified the same six clonal complexes using the strain grouping criterion of identity at five or more loci with at least one other member.

Genetic Relatedness Among Shiga Toxin-Producing Escherichia coli Isolated Along the Animal Food Supply Chain and in Gastroenteritis Cases in Qatar Using Multilocus Sequence Typing.

PubMed

Palanisamy, Srikanth; Chang, YuChen; Scaria, Joy; Penha Filho, Rafael Antonio Casarin; Peters, Kenlyn E; Doiphode, Sanjay H; Sultan, Ali; Mohammed, Hussni O

2017-06-01

Pathogenic Escherichia coli has been listed among the most important bacteria associated with foodborne illnesses around the world. We investigated the genetic relatedness among Shiga toxin-producing E. coli (STEC) isolated along the animal food supply chain and from humans diagnosed with gastroenteritis in Qatar. Samples were collected from different sources along the food supply chain and from patients admitted to the hospital with complaints of gastroenteritis. All samples were screened for the presence of E. coli O157:H7 and non-O157 STEC using a combination of bacterial enrichment and molecular detection techniques. A proportional sampling approach was used to select positive samples from each source for further multilocus sequence typing (MLST) analysis. Seven housekeeping genes described for STEC were amplified by polymerase chain reaction, sequenced, and analyzed by MLST. Isolates were characterized by allele composition, sequence type (ST) and assessed for epidemiologic relationship within and among different sources. Nei's genetic distance was calculated at the allele level between sample pools in each site downstream. E. coli O157:H7 occurred at a higher rate in slaughterhouse and retail samples than at the farm or in humans in our sampling. The ST171, an ST common to enterotoxigenic E. coli and atypical enteropathogenic E. coli, was the most common ST (15%) in the food supply chain. None of the genetic distances among the different sources was statistically significant. Enterohemorrhagic E. coli pathogenic strains are present along the supply chain at different levels and with varying relatedness. Clinical isolates were the most diverse, as expected, considering the polyclonal diversity in the human microbiota. The high occurrence of these food adulterants among the farm products suggests that implementation of sanitary measures at that level might reduce the risk of human exposure.

Epidemiological study of erythromycin-resistant Streptococcus pyogenes from Korea and Japan by emm genotyping and multilocus sequence typing.

PubMed

Takahashi, Takashi; Arai, Kazuaki; Lee, Dong Hyun; Koh, Eun Ha; Yoshida, Haruno; Yano, Hisakazu; Kaku, Mitsuo; Kim, Sunjoo

2016-01-01

We determined the epidemiological characteristics of erythromycin (EM)-resistant Streptococcus pyogenes (group A streptococci, GAS) strains isolated from Korea and Japan, using emm genotyping and multilocus sequence typing (MLST). Clinical isolates of GAS had been collected from 1992 to 2012 in Korea and from 2004 to 2009 in Japan. EM resistance was determined by the microdilution method, and resistance genotypes were assessed by PCR. The emm genotyping and MLST were performed by DNA sequencing. The emm genotypes and sequence types (STs) were concordant in 143 (85.1%) of 168 EM-resistant GAS strains from Korea. ST36/emm12 (35.1%), ST52/emm28 (22.6%), and ST49/emm75 (16.1%) were the most common types. Most of the ST36 (93.9%) and ST52 (95.8%) strains harbored erm(B), whereas strains ST49, ST42, and ST15 contained mef(A). The concordance between emm genotypes and STs was 41 (93.2%) among 44 EM-resistant GAS strains from Japan. ST36/emm12 (34.1%), ST49/emm75 (18.2%), and ST28/emm1 (15.9%) were the major types. ST36 isolates harbored either erm(B) (56.3%) or mef(A) (37.5%), whereas isolates ST28, ST49, and ST38 carried only mef(A). The proportion of erm(B) and mef(A) was 66.1% and 33.3% in Korea and 22.7% and 68.2% in Japan, respectively. The common STs in Korea and Japan were ST36 and ST49, whereas ST52 was present only in Korea and ST28 only in Japan. Genotype erm(B) was predominant in Korea, whereas mef(A) was frequent in Japan. There were differences between Korea and Japan regarding the frequencies of emm genotypes, STs, and EM resistance genes among the EM-resistant GAS.

Does typing of Chlamydia trachomatis using housekeeping multilocus sequence typing reveal different sexual networks among heterosexuals and men who have sex with men?

PubMed

Versteeg, Bart; Bruisten, Sylvia M; van der Ende, Arie; Pannekoek, Yvonne

2016-04-18

Chlamydia trachomatis infections remain the most common bacterial sexually transmitted infection worldwide. To gain more insight into the epidemiology and transmission of C. trachomatis, several schemes of multilocus sequence typing (MLST) have been developed. We investigated the clustering of C. trachomatis strains derived from men who have sex with men (MSM) and heterosexuals using the MLST scheme based on 7 housekeeping genes (MLST-7) adapted for clinical specimens and a high-resolution MLST scheme based on 6 polymorphic genes, including ompA (hr-MLST-6). Specimens from 100 C. trachomatis infected men who have sex with men (MSM) and 100 heterosexual women were randomly selected from previous studies and sequenced. We adapted the MLST-7 scheme to a nested assay to be suitable for direct typing of clinical specimens. All selected specimens were typed using both the adapted MLST-7 scheme and the hr-MLST-6 scheme. Clustering of C. trachomatis strains derived from MSM and heterosexuals was assessed using minimum spanning tree analysis. Sufficient chlamydial DNA was present in 188 of the 200 (94 %) selected samples. Using the adapted MLST-7 scheme, full MLST profiles were obtained for 187 of 188 tested specimens resulting in a high success rate of 99.5 %. Of these 187 specimens, 91 (48.7 %) were from MSM and 96 (51.3 %) from heterosexuals. We detected 21 sequence types (STs) using the adapted MLST-7 and 79 STs using the hr-MLST-6 scheme. Minimum spanning tree analyses was used to examine the clustering of MLST-7 data, which showed no reflection of separate transmission in MSM and heterosexual hosts. Moreover, typing using the hr-MLST-6 scheme identified genetically related clusters within each of clusters that were identified by using the MLST-7 scheme. No distinct transmission of C. trachomatis could be observed in MSM and heterosexuals using the adapted MLST-7 scheme in contrast to using the hr-MLST-6. In addition, we compared clustering of both MLST schemes and demonstrated that typing using the hr-MLST-6 scheme is able to identify genetically related clusters of C. trachomatis strains within each of the clusters that were identified by using the MLST-7 scheme.

Prevalence and characterisation of Staphylococcus aureus causing community-acquired skin and soft tissue infections on Java and Bali, Indonesia.

PubMed

Santosaningsih, Dewi; Santoso, Sanarto; Setijowati, Nanik; Rasyid, Harun A; Budayanti, Nyoman S; Suata, Ketut; Widhyatmoko, Dicky B; Purwono, Priyo B; Kuntaman, Kuntaman; Damayanti, Damayanti; Prakoeswa, Cita R S; Laurens, Mitchell; van Nierop, Josephine W I; Nanninga, Geraldine L; Oudenes, Neline; de Regt, Michelle; Snijders, Susan V; Verbrugh, Henri A; Severin, Juliëtte A

2018-01-01

To define the role of Staphylococcus aureus in community settings among patients with skin and soft tissue infections (SSTI) in Indonesia. Staphylococcus aureus were cultured from anterior nares, throat and wounds of 567 ambulatory patients presenting with SSTI. The mecA gene and genes encoding Panton-Valentine leukocidin (PVL; lukF-PV and lukS-PV) and exfoliative toxin (ET; eta and etb) were determined by PCR. Clonal relatedness among methicillin-resistant S. aureus (MRSA) and PVL-positive S. aureus was analysed using multilocus variable-number tandem-repeat analysis (MLVA) typing, and multilocus sequence typing (MLST) for a subset of isolates. Staphylococcal cassette chromosome mec (SCCmec) was determined for all MRSA isolates. Moreover, determinants for S. aureus SSTI, and PVL/ET-positive vs PVL/ET-negative S. aureus were assessed. Staphylococcus aureus were isolated from SSTI wounds of 257 (45.3%) patients, eight (3.1%) of these were MRSA. Genes encoding PVL and ETs were detected in 21.8% and 17.5% of methicillin-susceptible S. aureus (MSSA), respectively. PVL-positive MRSA was not detected. Nasopharyngeal S. aureus carriage was an independent determinant for S. aureus SSTI (odds ratio [OR] 1.8). Primary skin infection (OR 5.4) and previous antibiotic therapy (OR 3.5) were associated with PVL-positive MSSA. Primary skin infection (OR 2.2) was the only factor associated with ET-positive MSSA. MLVA typing revealed two more prevalent MSSA clusters. One ST1-MRSA-SCCmec type IV isolate and a cluster of ST239-MRSA-SCCmec type III were found. Community-acquired SSTI in Indonesia was frequently caused by PVL-positive MSSA, and the hospital-associated ST239-MRSA may have spread from the hospital into the community. © 2017 John Wiley & Sons Ltd.

pLARmEB: integration of least angle regression with empirical Bayes for multilocus genome-wide association studies.

PubMed

Zhang, J; Feng, J-Y; Ni, Y-L; Wen, Y-J; Niu, Y; Tamba, C L; Yue, C; Song, Q; Zhang, Y-M

2017-06-01

Multilocus genome-wide association studies (GWAS) have become the state-of-the-art procedure to identify quantitative trait nucleotides (QTNs) associated with complex traits. However, implementation of multilocus model in GWAS is still difficult. In this study, we integrated least angle regression with empirical Bayes to perform multilocus GWAS under polygenic background control. We used an algorithm of model transformation that whitened the covariance matrix of the polygenic matrix K and environmental noise. Markers on one chromosome were included simultaneously in a multilocus model and least angle regression was used to select the most potentially associated single-nucleotide polymorphisms (SNPs), whereas the markers on the other chromosomes were used to calculate kinship matrix as polygenic background control. The selected SNPs in multilocus model were further detected for their association with the trait by empirical Bayes and likelihood ratio test. We herein refer to this method as the pLARmEB (polygenic-background-control-based least angle regression plus empirical Bayes). Results from simulation studies showed that pLARmEB was more powerful in QTN detection and more accurate in QTN effect estimation, had less false positive rate and required less computing time than Bayesian hierarchical generalized linear model, efficient mixed model association (EMMA) and least angle regression plus empirical Bayes. pLARmEB, multilocus random-SNP-effect mixed linear model and fast multilocus random-SNP-effect EMMA methods had almost equal power of QTN detection in simulation experiments. However, only pLARmEB identified 48 previously reported genes for 7 flowering time-related traits in Arabidopsis thaliana.

High Prevalence of ESBL-Producing Klebsiella pneumoniae Causing Community-Onset Infections in China

PubMed Central

Zhang, Jing; Zhou, Kai; Zheng, Beiwen; Zhao, Lina; Shen, Ping; Ji, Jinru; Wei, Zeqing; Li, Lanjuan; Zhou, Jianying; Xiao, Yonghong

2016-01-01

The aim of this work was to investigate the epidemiological and genetic characteristics of ESBL-producing Klebsiella pneumoniae (ESBL-Kp) causing community-onset infections. K. pneumoniae isolates were collected from 31 Chinese secondary hospitals between August 2010 and 2011. Genes encoding ESBL and AmpC beta-lactamases were detected by PCR. The isolates were assigned to sequence types (STs) using multi-locus sequence typing (MLST). Eleven ESBL-Kp strains were selected for whole-genome sequencing (WGS) for investigating the genetic environment and plasmids encoding ESBL genes. A total of 578 K. pneumoniae isolates were collected, and 184 (31.8%) carried ESBL genes. The prevalence of ESBL-Kp varied from different geographical areas of China (10.2–50.3%). The three most prevalent ESBL genes were blaCTX-M-14 (n = 74), blaCTX-M-15 (n = 60), and blaCTX-M-3 (n = 40). MLST assigned 127 CTX-M-14 and CTX-M-15 producers to 54 STs, and CC17 was the most prevalent population (12.6%). STs (23, 37, and 86) that were known frequently associated with hypervirulent K. pneumoniae (hvKP) account for 14.1% (18/127). Phylogenetic analysis by concatenating the seven loci of MLST revealed the existence of ESBL-producing K. quasipneumoniae (two strains) and K. varricola (one strain), which was further confirmed by WGS. This study highlights the challenge of community-onset infections caused by ESBL-Kp in China. The prevalence of STs frequently associating with hvKP should be of concern. Surveillance of ESBL-KP causing community-onset infections now appears imperative. PMID:27895637

Characterization of extensively drug-resistant Mycobacterium tuberculosis in Nepal.

PubMed

Poudel, Ajay; Maharjan, Bhagwan; Nakajima, Chie; Fukushima, Yukari; Pandey, Basu D; Beneke, Antje; Suzuki, Yasuhiko

2013-01-01

The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global control of TB. Although molecular characterization of drug resistance-associated mutations in multidrug-resistant isolates in Nepal has been made, mutations in XDR isolates and their genotypes have not been reported previously. In this study, we identified and characterized 13 XDR Mycobacterium tuberculosis isolates from clinical isolates in Nepal. The most prevalent mutations involved in rifampicin, isoniazid, ofloxacin, and kanamycin/capreomycin resistance were Ser531Leu in rpoB gene (92.3%), Ser315Thr in katG gene (92.3%), Asp94Gly in gyrA gene (53.9%) and A1400G in rrs gene (61.5%), respectively. Spoligotyping and multilocus sequence typing revealed that 69% belonged to Beijing family, especially modern types. Further typing with 26-loci variable number of tandem repeats suggested the current spread of XDR M. tuberculosis. Our result highlights the need to reinforce the TB policy in Nepal with regard to control and detection strategies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Linezolid-Resistant Staphylococcus aureus Strain 1128105, the First Known Clinical Isolate Possessing the cfr Multidrug Resistance Gene

PubMed Central

Zuill, Douglas E.; Scharn, Caitlyn R.; Deane, Jennifer; Sahm, Daniel F.; Denys, Gerald A.; Goering, Richard V.; Shaw, Karen J.

2014-01-01

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZDr clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination. PMID:25155597

Multilocus Variable-Number-Tandem-Repeats Analysis (MLVA) distinguishes a clonal complex of Clavibacter michiganensis subsp. michiganensis strains isolated from recent outbreaks of bacterial wilt and canker in Belgium

PubMed Central

2013-01-01

Background Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato. Cmm is present nearly in all European countries. During the last three years several local outbreaks were detected in Belgium. The lack of a convenient high-resolution strain-typing method has hampered the study of the routes of transmission of Cmm and epidemiology in tomato cultivation. In this study the genetic relatedness among a worldwide collection of Cmm strains and their relatives was approached by gyrB and dnaA gene sequencing. Further, we developed and applied a multilocus variable number of tandem repeats analysis (MLVA) scheme to discriminate among Cmm strains. Results A phylogenetic analysis of gyrB and dnaA gene sequences of 56 Cmm strains demonstrated that Belgian Cmm strains from recent outbreaks of 2010–2012 form a genetically uniform group within the Cmm clade, and Cmm is phylogenetically distinct from other Clavibacter subspecies and from non-pathogenic Clavibacter-like strains. MLVA conducted with eight minisatellite loci detected 25 haplotypes within Cmm. All strains from Belgian outbreaks, isolated between 2010 and 2012, together with two French strains from 2010 seem to form one monomorphic group. Regardless of the isolation year, location or tomato cultivar, Belgian strains from recent outbreaks belonged to the same haplotype. On the contrary, strains from diverse geographical locations or isolated over longer periods of time formed mostly singletons. Conclusions We hypothesise that the introduction might have originated from one lot of seeds or contaminated tomato seedlings that was the source of the outbreak in 2010 and that these Cmm strains persisted and induced infection in 2011 and 2012. Our results demonstrate that MLVA is a promising typing technique for a local surveillance and outbreaks investigation in epidemiological studies of Cmm. PMID:23738754

Multilocus variable-number-tandem-repeats analysis (MLVA) distinguishes a clonal complex of Clavibacter michiganensis subsp. michiganensis strains isolated from recent outbreaks of bacterial wilt and canker in Belgium.

PubMed

Zaluga, Joanna; Stragier, Pieter; Van Vaerenbergh, Johan; Maes, Martine; De Vos, Paul

2013-06-05

Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato. Cmm is present nearly in all European countries. During the last three years several local outbreaks were detected in Belgium. The lack of a convenient high-resolution strain-typing method has hampered the study of the routes of transmission of Cmm and epidemiology in tomato cultivation. In this study the genetic relatedness among a worldwide collection of Cmm strains and their relatives was approached by gyrB and dnaA gene sequencing. Further, we developed and applied a multilocus variable number of tandem repeats analysis (MLVA) scheme to discriminate among Cmm strains. A phylogenetic analysis of gyrB and dnaA gene sequences of 56 Cmm strains demonstrated that Belgian Cmm strains from recent outbreaks of 2010-2012 form a genetically uniform group within the Cmm clade, and Cmm is phylogenetically distinct from other Clavibacter subspecies and from non-pathogenic Clavibacter-like strains. MLVA conducted with eight minisatellite loci detected 25 haplotypes within Cmm. All strains from Belgian outbreaks, isolated between 2010 and 2012, together with two French strains from 2010 seem to form one monomorphic group. Regardless of the isolation year, location or tomato cultivar, Belgian strains from recent outbreaks belonged to the same haplotype. On the contrary, strains from diverse geographical locations or isolated over longer periods of time formed mostly singletons. We hypothesise that the introduction might have originated from one lot of seeds or contaminated tomato seedlings that was the source of the outbreak in 2010 and that these Cmm strains persisted and induced infection in 2011 and 2012. Our results demonstrate that MLVA is a promising typing technique for a local surveillance and outbreaks investigation in epidemiological studies of Cmm.

Molecular Phylogenetic Diversity of Dermatologic and Other Human Pathogenic Fusaria from Hospitals in Northern Italy

USDA-ARS?s Scientific Manuscript database

Fifty-eight fusaria isolated from 52 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusari...

Aspergillus section Versicolores: nine new species and multilocus DNA sequence based phylogeny

USDA-ARS?s Scientific Manuscript database

ß-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA, RNA polymerase, DNA replication licensing factor Mcm7, and pre-rRNA processing protein Tsr1 were amplified and sequenced from 62 A. versicolor clade isolates and analyzed phylogenetically using the concordance model to establis...

Aspergillus section Versicolores, nine new species and multilocus DNA sequence based phylogeny

USDA-ARS?s Scientific Manuscript database

ß-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA, RNA polymerase, DNA replication licensing factor Mcm7, and pre-rRNA processing protein Tsr1 were amplified and sequenced from 62 A. versicolor clade isolates and analyzed phylogenetically using the concordance model to establis...

Molecular evidence of Burkholderia pseudomallei genotypes based on geographical distribution.

PubMed

Zulkefli, Noorfatin Jihan; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Chong, Chun Wie; Thong, Kwai Lin; Ponnampalavanar, Sasheela; Vadivelu, Jamuna; Teh, Cindy Shuan Ju

2016-01-01

Background. Central intermediary metabolism (CIM) in bacteria is defined as a set of metabolic biochemical reactions within a cell, which is essential for the cell to survive in response to environmental perturbations. The genes associated with CIM are commonly found in both pathogenic and non-pathogenic strains. As these genes are involved in vital metabolic processes of bacteria, we explored the efficiency of the genes in genotypic characterization of Burkholderia pseudomallei isolates, compared with the established pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) schemes. Methods. Nine previously sequenced B. pseudomallei isolates from Malaysia were characterized by PFGE, MLST and CIM genes. The isolates were later compared to the other 39 B. pseudomallei strains, retrieved from GenBank using both MLST and sequence analysis of CIM genes. UniFrac and hierachical clustering analyses were performed using the results generated by both MLST and sequence analysis of CIM genes. Results. Genetic relatedness of nine Malaysian B. pseudomallei isolates and the other 39 strains was investigated. The nine Malaysian isolates were subtyped into six PFGE profiles, four MLST profiles and five sequence types based on CIM genes alignment. All methods demonstrated the clonality of OB and CB as well as CMS and THE. However, PFGE showed less than 70% similarity between a pair of morphology variants, OS and OB. In contrast, OS was identical to the soil isolate, MARAN. To have a better understanding of the genetic diversity of B. pseudomallei worldwide, we further aligned the sequences of genes used in MLST and genes associated with CIM for the nine Malaysian isolates and 39 B. pseudomallei strains from NCBI database. Overall, based on the CIM genes, the strains were subtyped into 33 profiles where majority of the strains from Asian countries were clustered together. On the other hand, MLST resolved the isolates into 31 profiles which formed three clusters. Hierarchical clustering using UniFrac distance suggested that the isolates from Australia were genetically distinct from the Asian isolates. Nevertheless, statistical significant differences were detected between isolates from Malaysia, Thailand and Australia. Discussion. Overall, PFGE showed higher discriminative power in clustering the nine Malaysian B. pseudomallei isolates and indicated its suitability for localized epidemiological study. Compared to MLST, CIM genes showed higher resolution in distinguishing those non-related strains and better clustering of strains from different geographical regions. A closer genetic relatedness of Malaysian isolates with all Asian strains in comparison to Australian strains was observed. This finding was supported by UniFrac analysis which resulted in geographical segregation between Australia and the Asian countries.

Multiple independent origins of mitochondrial control region duplications in the order Psittaciformes

PubMed Central

Schirtzinger, Erin E.; Tavares, Erika S.; Gonzales, Lauren A.; Eberhard, Jessica R.; Miyaki, Cristina Y.; Sanchez, Juan J.; Hernandez, Alexis; Müeller, Heinrich; Graves, Gary R.; Fleischer, Robert C.; Wright, Timothy F.

2012-01-01

Mitochondrial genomes are generally thought to be under selection for compactness, due to their small size, consistent gene content, and a lack of introns or intergenic spacers. As more animal mitochondrial genomes are fully sequenced, rearrangements and partial duplications are being identified with increasing frequency, particularly in birds (Class Aves). In this study, we investigate the evolutionary history of mitochondrial control region states within the avian order Psittaciformes (parrots and cockatoos). To this aim, we reconstructed a comprehensive multi-locus phylogeny of parrots, used PCR of three diagnostic fragments to classify the mitochondrial control region state as single or duplicated, and mapped these states onto the phylogeny. We further sequenced 44 selected species to validate these inferences of control region state. Ancestral state reconstruction using a range of weighting schemes identified six independent origins of mitochondrial control region duplications within Psittaciformes. Analysis of sequence data showed that varying levels of mitochondrial gene and tRNA homology and degradation were present within a given clade exhibiting duplications. Levels of divergence between control regions within an individual varied from 0–10.9% with the differences occurring mainly between 51 and 225 nucleotides 3′ of the goose hairpin in domain I. Further investigations into the fates of duplicated mitochondrial genes, the potential costs and benefits of having a second control region, and the complex relationship between evolutionary rates, selection, and time since duplication are needed to fully explain these patterns in the mitochondrial genome. PMID:22543055

Biodiversity of Vibrios

PubMed Central

Thompson, Fabiano L.; Iida, Tetsuya; Swings, Jean

2004-01-01

Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years. PMID:15353563

Clonal Transmission of Gram-Negative Bacteria with Carbapenemases NDM-1, VIM-1, and OXA-23/72 in a Bulgarian Hospital.

PubMed

Pfeifer, Yvonne; Trifonova, Angelina; Pietsch, Michael; Brunner, Magdalena; Todorova, Iva; Gergova, Ivanka; Wilharm, Gottfried; Werner, Guido; Savov, Encho

2017-04-01

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different β-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes bla OXA-23 or bla OXA-72 ; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Early detection of metallo-β-lactamase NDM-1- and OXA-23 carbapenemase-producing Acinetobacter baumannii in Libyan hospitals.

PubMed

Mathlouthi, Najla; El Salabi, Allaaeddin Ali; Ben Jomàa-Jemili, Mariem; Bakour, Sofiane; Al-Bayssari, Charbel; Zorgani, Abdulaziz A; Kraiema, Abdulmajeed; Elahmer, Omar; Okdah, Liliane; Rolain, Jean-Marc; Chouchani, Chedly

2016-07-01

Acinetobacter baumannii is an opportunistic pathogen causing various nosocomial infections. The aim of this study was to characterise the molecular support of carbapenem-resistant A. baumannii clinical isolates recovered from two Libyan hospitals. Bacterial isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antibiotic susceptibility testing was performed using disk diffusion and Etest methods, and carbapenem resistance determinants were studied by PCR amplification and sequencing. Multilocus sequence typing (MLST) was performed for typing of the isolates. All 36 imipenem-resistant isolates tested were identified as A. baumannii. The blaOXA-23 gene was detected in 29 strains (80.6%). The metallo-β-lactamase blaNDM-1 gene was detected in eight isolates (22.2%), showing dissemination of multidrug-resistant (MDR) A. baumannii in Tripoli Medical Center and Burn and Plastic Surgery Hospital in Libya, including one isolate that co-expressed the blaOXA-23 gene. MLST revealed several sequence types (STs). Imipenem-resistant A. baumannii ST2 was the predominant clone (16/36; 44.4%). This study shows that NDM-1 and OXA-23 contribute to antibiotic resistance in Libyan hospitals and represents the first incidence of the association of these two carbapenemases in an autochthonous MDR A. baumannii isolated from patients in Libya, indicating that there is a longstanding infection control problem in these hospitals. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Genomic Analysis of Bacillus sp. Strain B25, a Biocontrol Agent of Maize Pathogen Fusarium verticillioides.

PubMed

Douriet-Gámez, Nadia R; Maldonado-Mendoza, Ignacio E; Ibarra-Laclette, Enrique; Blom, Jochen; Calderón-Vázquez, Carlos L

2018-03-01

Bacillus sp. B25 is an effective biocontrol agent against the maize pathogenic fungus Fusarium verticillioides (Fv). Previous in vitro assays have shown that B25 has protease, glucanase, and chitinase activities and siderophores production; however, specific mechanisms by which B25 controls Fv are still unknown. To determine the genetic traits involved in biocontrol, B25 genome was sequenced and analyzed. B25 genome is composed of 5,113,413 bp and 5251 coding genes. A multilocus phylogenetic analysis (MLPA) suggests that B25 is closely related to the Bacillus cereus group and a high percentage (70-75%) of the genetic information is conserved between B25 and related strains, which include most of the genes associated to fungal antagonism. Some of these genes are shared with some biocontrol agents of the Bacillus genus and less with Pseudomonas and Serratia strains. We performed a genomic comparison between B25 and five Bacillus spp., Pseudomonas and Serratia strains. B25 contains genes involved in a wide variety of antagonistic mechanisms including chitinases, glycoside hydrolases, siderophores, antibiotics, and biofilm production that could be implicated in root colonization. Also, 24 genomic islands and 3 CRISPR sequences were identified in the B25 genome. This is the first comparative genome analysis between strains belonging to the B. cereus group and biocontrol agents of phytopathogenic fungi. These results are the starting point for further studies on B25 gene expression during its interaction with Fv.

Clonal structure of Streptococcus sanguinis strains isolated from endocarditis cases and the oral cavity.

PubMed

Do, T; Gilbert, S C; Klein, J; Warren, S; Wade, W G; Beighton, D

2011-10-01

A collection of Streptococcus sanguinis strains from patients with endocarditis (n = 21) and from the oral cavity (n = 34) was subjected to a multi-locus sequence typing analysis using seven housekeeping genes, carbamoyl-phosphate synthetase (carB), Co/Zn/Cd efflux system component (czcD), d-alanyl-d-alanine ligase (ddl), DNA polymerase III (dnaX), glucose-6-phosphate dehydrogenase (gdh), DNA-directed RNA polymerase, beta subunit (rpoB) and superoxide dismutase (sodA). The scheme was expanded by the inclusion of two the putative virulence genes, bacitracin-resistance protein (bacA) and saliva-binding protein (ssaB), to increase strain discrimination. Extensive intra-species recombination was apparent in all genes but inter-species recombination was also apparent with strains apparently harbouring gdh and ddl from unidentified sources and one isolate harboured a sodA allele apparently derived from Streptococcus oralis. The recombination/mutation ratio for the concatenated housekeeping gene sequences was 1.67 (95% confidence limits 1.25-2.72) and for the two virulence genes the r/m ratio was 3.99 (95% confidence limits 1.61-8.72); recombination was the major driver for genetic variation. All isolates were distinct and the endocarditis strains did not form distinct sub-clusters when the data were analysed using ClonalFrame. These data support the widely held opinion that infecting S. sanguinis strains are opportunistic human pathogens. © 2011 John Wiley & Sons A/S.

Multigene assessment of the species boundaries and sexual status of the basidiomycetous yeasts Cryptococcus flavescens and C. terrestris (Tremellales).

PubMed

Yurkov, Andrey; Guerreiro, Marco A; Sharma, Lav; Carvalho, Cláudia; Fonseca, Álvaro

2015-01-01

Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful.

Multigene Assessment of the Species Boundaries and Sexual Status of the Basidiomycetous Yeasts Cryptococcus flavescens and C. terrestris (Tremellales)

PubMed Central

Sharma, Lav; Carvalho, Cláudia; Fonseca, Álvaro

2015-01-01

Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful. PMID:25811603

Novel Plasmid-Encoded Ceftazidime-Hydrolyzing CTX-M-53 Extended-Spectrum β-Lactamase from Salmonella enterica Serotypes Westhampton and Senftenberg▿

PubMed Central

Doublet, Benoît; Granier, Sophie A.; Robin, Frédéric; Bonnet, Richard; Fabre, Laëtitia; Brisabois, Anne; Cloeckaert, Axel; Weill, François-Xavier

2009-01-01

We describe the characterization of a novel CTX-M β-lactamase from Salmonella enterica. Four S. enterica isolates (three of serotype Westhampton and one of serotype Senftenberg) resistant to extended-spectrum cephalosporins (cefotaxime and ceftazidime) were recovered in 2004 from living cockles in three supermarkets located in distant geographic areas in France, which got their supplies from the same fishery. The isolates were found to produce a novel extended-spectrum β-lactamase (ESBL) belonging to the CTX-M-1 phylogenetic group and named CTX-M-53. The CTX-M-53 β-lactamase harbored the substitution Asp240Gly, like the CTX-M-15 enzyme, which is specifically implicated in a higher catalytic efficiency against ceftazidime. The blaCTX-M-53 gene was located on a mobilizable 11-kb plasmid, pWES-1. The complete sequence of pWES-1 revealed the presence of a novel insertion sequence, ISSen2, and an IS26 element upstream and downstream of the blaCTX-M-53 gene, respectively; however, transposition assays of the blaCTX-M-53 gene were unsuccessful. IS26 elements may have contributed to the acquisition of the blaCTX-M-53 gene. Interestingly, the mobilization module of the pWES-1 plasmid was similar to that of quinolone resistance plasmids (carrying the qnrS2 gene) from aquatic sources. Although belonging to two serotypes differentiated on the basis of the O-antigen structure (E1 or E4 groups), the isolates were found to be genetically indistinguishable by pulsed-field gel electrophoresis. Multilocus sequence typing showed that the isolates of serotype Westhampton had a sequence type, ST14, common among isolates of serotype Senftenberg. This is the first characterization of the CTX-M-53 ESBL, which represents an additional ceftazidime-hydrolyzing CTX-M enzyme. PMID:19273683

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

PubMed

Tellapragada, Chaitanya; Kamthan, Aayushi; Shaw, Tushar; Ke, Vandana; Kumar, Subodh; Bhat, Vinod; Mukhopadhyay, Chiranjay

2016-01-01

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006-2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as "outliers" on the eBURST "Population snapshot", suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

Multilocus analyses indicate a mosaic distribution of hybrid populations in ground squirrels (genus Ictidomys)

PubMed Central

Thompson, Cody W; Anwarali Khan, Faisal Ali; Stangl, Frederick B; Baker, Robert J; Bradley, Robert D

2013-01-01

DNA sequence data from mitochondrial cytochrome-b (Cytb) and Y-linked structural maintenance of chromosomes (SmcY) genes were combined with 478 nuclear loci obtained from amplified fragment length polymorphisms (AFLP) to assess the extent of hybridization and genetic spatial structure of populations in two hybridizing species of ground squirrel (Ictidomys parvidens and Ictidomys tridecemlineatus). Based on AFLP analyses of 134 individuals from 28 populations, 10 populations were identified that possessed hybrid individuals. Overall estimates of FST values revealed strong support for population structure in the Cytb data set; however, analyses of the SmcY gene and the AFLP data indicated ongoing gene flow between species. Pairwise FST comparisons of populations were not significant for the SmcY gene; although they were significant for the Cytb gene, indicating that these populations were structured and that gene flow was minimal. Therefore, gene flow between I. parvidens and I. tridecemlineatus appeared to be restricted to populations that exhibited hybridization. In addition, the fragmented nature of the geographic landscape suggested limited gene flow between populations. As a result, the distributional pattern of interspersed parental and hybrid populations were compatible with a mosaic hybrid zone model. Because ground squirrels display female philopatry and male-biased dispersal, the ecology of these species is compatible with this hypothesis. PMID:24340186

Porphyromonas loveana sp. nov., isolated from the oral cavity of Australian marsupials.

PubMed

Bird, Philip S; Trott, Darren J; Mikkelsen, Deirdre; Milinovich, Gabriel J; Hillman, Kristine M; Burrell, Paul C; Blackall, Linda L

2016-10-01

An obligatory anaerobic, Gram-stain-negative coccobacillus with black-pigmented colonies was isolated from the oral cavity of selected Australian marsupial species. Phenotypic and molecular criteria showed that this bacterium was a distinct species within the genus Porphyromonas, and was closely related to Porphyromonas gingivalis and Porphyromonas gulae. This putative novel species and P. gulae could be differentiated from P. gingivalis by catalase activity. Further characterization by multi-locus enzyme electrophoresis of glutamate dehydrogenase and malate dehydrogenase enzyme mobility and matrix-assisted laser desorption ionization time-of-flight MS showed that this putative novel species could be differentiated phenotypically from P. gingivalis and P. gulae. Definitive identification by 16S rRNA gene sequencing showed that this bacterium belonged to a unique monophyletic lineage, phylogenetically distinct from P. gingivalis (94.9 % similarity) and P. gulae (95.5 %). This also was supported by 16S-23S rRNA intergenic spacer region and glutamate dehydrogenase gene sequencing. A new species epithet, Porphyromonas loveana sp. nov., is proposed for this bacterium, with DSM 28520T (=NCTC 13658T=UQD444T=MRK101T), isolated from a musky rat kangaroo, as the type strain.

Inter-hospital outbreak of Klebsiella pneumoniae producing KPC-2 carbapenemase in Ireland.

PubMed

Morris, Dearbháile; Boyle, Fiona; Morris, Carol; Condon, Iris; Delannoy-Vieillard, Anne-Sophie; Power, Lorraine; Khan, Aliya; Morris-Downes, Margaret; Finnegan, Cathriona; Powell, James; Monahan, Regina; Burns, Karen; O'Connell, Nuala; Boyle, Liz; O'Gorman, Alan; Humphreys, Hilary; Brisse, Sylvain; Turton, Jane; Woodford, Neil; Cormican, Martin

2012-10-01

To describe an outbreak of KPC-2-producing Klebsiella pneumoniae with inter-hospital spread and measures taken to control transmission. Between January and March 2011, 13 K. pneumoniae isolates were collected from nine patients at hospital A and two patients at hospital B. Meropenem, imipenem and ertapenem MICs were determined by Etest, carbapenemase production was confirmed by the modified Hodge method and by a disc synergy test, and confirmed carbapenemase producers were tested for the presence of carbapenemase-encoding genes by PCR. PFGE, plasmid analysis, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST) analysis were performed on all or a subset of isolates. Meropenem, imipenem and ertapenem MICs were 4 to >32, 8-32 and >16 mg/L, respectively. PCR and sequencing confirmed the presence of bla(KPC-2). PFGE identified four distinguishable (≥88%) pulsed-field profiles (PFPs). Isolates distinguishable by PFGE had identical MLVA profiles, and MLST analysis indicated all isolates belonged to the ST258 clone. Stringent infection prevention and control measures were implemented. Over a period of almost 8 months no further carbapenemase-producing Enterobacteriaceae (CPE) were isolated. However, KPC-2-producing K. pneumoniae was detected in two further patients in hospital A in August (PFP indistinguishable from previous isolates) and October 2011 (PFP similar to but distinguishable from previous isolates). Stringent infection prevention and control measures help contain CPE in the healthcare setting; however, in the case of hospital A, where CPE appears to be established in the population served, it may be virtually impossible to achieve eradication or avoid reintroduction into the hospital.

Fatal infection in three Grey Slender Lorises (Loris lydekkerianus nordicus) caused by clonally related Trueperella pyogenes.

PubMed

Nagib, Samy; Glaeser, Stefanie P; Eisenberg, Tobias; Sammra, Osama; Lämmler, Christoph; Kämpfer, Peter; Schauerte, Nicole; Geiger, Christina; Kaim, Ute; Prenger-Berninghoff, Ellen; Becker, André; Abdulmawjood, Amir

2017-08-29

Trueperella pyogenes is a worldwide known bacterium causing mastitis, abortion and various other pyogenic infections in domestic animals like ruminants and pigs. In this study we represent the first case report of three unusual fatal infections of Grey Slender Lorises caused by Trueperella pyogenes. Meanwhile, this study represents the first in-depth description of the multilocus sequence analysis (MLSA) on T. pyogenes species. Three Trueperella pyogenes were isolated from three different Grey Slender Lorises, which died within a period of two years at Frankfurt Zoo (Frankfurt am Main - Germany). The three Grey Slender Loris cases were suffering from severe sepsis and died from its complication. During the bacteriological investigation of the three cases, the T. pyogenes were isolated from different organisms in each case. The epidemiological relationship between the three isolates could be shown by four genomic DNA fingerprint methods (ERIC-PCR, BOX-PCR, (GTG) 5 -PCR, and RAPD-PCR) and by multilocus sequence analysis (MLSA) investigating four different housekeeping genes (fusA-tuf-metG-gyrA). In this study, we clearly showed by means of using three different rep-PCRs, by RAPD-PCR and by MLSA that the genomic fingerprinting of the investigated three T. pyogenes have the same clonal origin and are genetically identical. These results suggest that the same isolate contaminated the animal's facility and subsequently caused cross infection between the three different Grey Slender Lorises. To the best of our knowledge, this is the first epidemiological approach concentrating on T. pyogenes using MLSA.

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics.

PubMed

Couto, Natacha; Chlebowicz, Monika A; Raangs, Erwin C; Friedrich, Alex W; Rossen, John W

2018-04-05

The emergence of nosocomial infections by multidrug-resistant Staphylococcus haemolyticus isolates has been reported in several European countries. Here, we report the first two complete genome sequences of S. haemolyticus sequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics. Copyright © 2018 Couto et al.

Comparative Analysis of the Orphan CRISPR2 Locus in 242 Enterococcus faecalis Strains

PubMed Central

Hullahalli, Karthik; Rodrigues, Marinelle; Schmidt, Brendan D.; Li, Xiang; Bhardwaj, Pooja; Palmer, Kelli L.

2015-01-01

Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs. PMID:26398194

Members of a new subgroup of Streptococcus anginosus harbor virulence related genes previously observed in Streptococcus pyogenes.

PubMed

Babbar, Anshu; Kumar, Venkatesan Naveen; Bergmann, René; Barrantes, Israel; Pieper, Dietmar H; Itzek, Andreas; Nitsche-Schmitz, D Patric

2017-04-01

Conventionally categorized as commensals, the Streptococci of the species S. anginosus are facultative human pathogens that are difficult to diagnose and often overlooked. Furthermore, detailed investigation and diagnosis of S. anginosus infections is hampered by unexplored taxonomy and widely elusive molecular pathogenesis. To explore their pathogenic potential, S. anginosus isolates collected from patients of two geographical locations (Vellore, India and Leipzig, Germany) were subjected to multi-locus sequence analysis (MLSA). This analysis revealed the potential presence of a new distinct clade of the species S. anginosus, tentatively termed here as genomosubspecies vellorensis. A complementary PCR-based screening for S. pyogenes virulence factor as well as antibiotic resistance genes revealed not only the presence of superantigen- and extracellular DNase coding genes identical to corresponding genes of S. pyogenes, but also of erythromycin and tetracycline resistance genes in the genomes of the analyzed S. anginosus isolates, thus posing a matter of significant health concern. Identification of new pathogenic S. anginosus strains capable of causing difficult to treat infections may pose additional challenges to the diagnosis and treatment of Streptococcus based infections. Copyright © 2017 Elsevier GmbH. All rights reserved.

Characterization of Plasmid-Mediated Quinolone Resistance Determinants in High-Level Quinolone-Resistant Enterobacteriaceae Isolates from the Community: First Report of qnrD Gene in Algeria.

PubMed

Yanat, Betitera; Machuca, Jesús; Díaz-De-Alba, Paula; Mezhoud, Halima; Touati, Abdelaziz; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

2017-01-01

The objective was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR)-producing isolates in a collection of quinolone-resistant Enterobacteriaceae of community origin isolated in Bejaia, Algeria. A total of 141 nalidixic acid-resistant Enterobacteriaceae community isolates were collected in Bejaia (Northern Algeria) and screened for PMQR genes using polymerase chain reaction (PCR). For PMQR-positive strains, antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion. Mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected with a PCR-based method and sequencing. Southern blotting, conjugation and transformation assays and molecular typing by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing were also performed. The prevalence of PMQR-producing Enterobacteriaceae isolates was 13.5% (19/141); 11 of these isolates produced Aac(6')-Ib-cr and 8 were qnr-positive (4 qnrB1-like, 2 qnrS1-like, and 2 qnrD1-like), including the association with aac(6')-Ib-cr gene in three cases. PMQR gene transfer by conjugation was successful in 6 of 19 isolates tested. PFGE revealed that most of the PMQR-positive Escherichia coli isolates were unrelated, except for two groups comprising two and four isolates, respectively, including the virulent multidrug-resistant clone E. coli ST131 that were clonally related. Our findings indicate that PMQR determinants are prevalent in Enterobacteriaceae isolates from the community studied. We describe the first report of the qnrD gene in Algeria.

Multilocus family-based association analysis of seven candidate polymorphisms with essential hypertension in an african-derived semi-isolated brazilian population.

PubMed

Kimura, L; Angeli, C B; Auricchio, M T B M; Fernandes, G R; Pereira, A C; Vicente, J P; Pereira, T V; Mingroni-Netto, R C

2012-01-01

Background. It has been widely suggested that analyses considering multilocus effects would be crucial to characterize the relationship between gene variability and essential hypertension (EH). Objective. To test for the presence of multilocus effects between/among seven polymorphisms (six genes) on blood pressure-related traits in African-derived semi-isolated Brazilian populations (quilombos). Methods. Analyses were carried out using a family-based design in a sample of 652 participants (97 families). Seven variants were investigated: ACE (rs1799752), AGT (rs669), ADD2 (rs3755351), NOS3 (rs1799983), GNB3 (rs5441 and rs5443), and GRK4 (rs1801058). Sensitivity analyses were further performed under a case-control design with unrelated participants only. Results. None of the investigated variants were associated individually with both systolic and diastolic BP levels (SBP and DBP, respectively) or EH (as a binary outcome). Multifactor dimensionality reduction-based techniques revealed a marginal association of the combined effect of both GNB3 variants on DBP levels in a family-based design (P = 0.040), whereas a putative NOS3-GRK4 interaction also in relation to DBP levels was observed in the case-control design only (P = 0.004). Conclusion. Our results provide limited support for the hypothesis of multilocus effects between/among the studied variants on blood pressure in quilombos. Further larger studies are needed to validate our findings.

Comparison of Molecular Typing Methods Useful for Detecting Clusters of Campylobacter jejuni and C. coli Isolates through Routine Surveillance

PubMed Central

Taboada, Eduardo; Grant, Christopher C. R.; Blakeston, Connie; Pollari, Frank; Marshall, Barbara; Rahn, Kris; MacKinnon, Joanne; Daignault, Danielle; Pillai, Dylan; Ng, Lai-King

2012-01-01

Campylobacter spp. may be responsible for unreported outbreaks of food-borne disease. The detection of these outbreaks is made more difficult by the fact that appropriate methods for detecting clusters of Campylobacter have not been well defined. We have compared the characteristics of five molecular typing methods on Campylobacter jejuni and C. coli isolates obtained from human and nonhuman sources during sentinel site surveillance during a 3-year period. Comparative genomic fingerprinting (CGF) appears to be one of the optimal methods for the detection of clusters of cases, and it could be supplemented by the sequencing of the flaA gene short variable region (flaA SVR sequence typing), with or without subsequent multilocus sequence typing (MLST). Different methods may be optimal for uncovering different aspects of source attribution. Finally, the use of several different molecular typing or analysis methods for comparing individuals within a population reveals much more about that population than a single method. Similarly, comparing several different typing methods reveals a great deal about differences in how the methods group individuals within the population. PMID:22162562

Bradyrhizobium sacchari sp. nov., a legume nodulating bacterium isolated from sugarcane roots.

PubMed

de Matos, Gustavo Feitosa; Zilli, Jerri Edson; de Araújo, Jean Luiz Simões; Parma, Marcia Maria; Melo, Itamar Soares; Radl, Viviane; Baldani, José Ivo; Rouws, Luc Felicianus Marie

2017-11-01

Members of the genus Bradyrhizobium are well-known as nitrogen-fixing microsymbionts of a wide variety of leguminous species, but they have also been found in different environments, notably as endophytes in non-legumes such as sugarcane. This study presents a detailed polyphasic characterization of four Bradyrhizobium strains (type strain BR 10280 T ), previously isolated from roots of sugarcane in Brazil. 16S rRNA sequence analysis, multilocus sequence analysis (MLSA) and analysis of the 16S-23S rRNA internal transcribed spacer showed that these strains form a novel clade close to, but different from B. huanghuaihaiense strain CCBAU 23303 T . Average nucleotide identity (ANI) analyses confirmed that BR 10280 T represents a novel species. Phylogenetic analysis based on nodC gene sequences also placed the strains close to CCBAU 23303 T , but different from this latter strain, the sugarcane strains did not nodulate soybean, although they effectively nodulated Vigna unguiculata, Cajanus cajan and Macroptilium atropurpureum. Physiological traits are in agreement with the placement of the strains in the genus Bradyrhizobium as a novel species for which the name Bradyrhizobium sacchari sp. nov. is proposed.

Detection and characterization of Histoplasma capsulatum in a German badger (Meles meles) by ITS sequencing and multilocus sequencing analysis

USDA-ARS?s Scientific Manuscript database

A wild badger (Meles meles) with a severe nodular dermatitis was presented for post mortem examination. Numerous cutaneous granulomas with superficial ulceration were present especially on head, dorsum, and forearms were found at necropsy. Histopathological examination of the skin revealed a severe ...

Utility of Whole-Genome Sequencing of Escherichia coli O157 for Outbreak Detection and Epidemiological Surveillance.

PubMed

Holmes, Anne; Allison, Lesley; Ward, Melissa; Dallman, Timothy J; Clark, Richard; Fawkes, Angie; Murphy, Lee; Hanson, Mary

2015-11-01

Detailed laboratory characterization of Escherichia coli O157 is essential to inform epidemiological investigations. This study assessed the utility of whole-genome sequencing (WGS) for outbreak detection and epidemiological surveillance of E. coli O157, and the data were used to identify discernible associations between genotypes and clinical outcomes. One hundred five E. coli O157 strains isolated over a 5-year period from human fecal samples in Lothian, Scotland, were sequenced with the Ion Torrent Personal Genome Machine. A total of 8,721 variable sites in the core genome were identified among the 105 isolates; 47% of the single nucleotide polymorphisms (SNPs) were attributable to six "atypical" E. coli O157 strains and included recombinant regions. Phylogenetic analyses showed that WGS correlated well with the epidemiological data. Epidemiological links existed between cases whose isolates differed by three or fewer SNPs. WGS also correlated well with multilocus variable-number tandem repeat analysis (MLVA) typing data, with only three discordant results observed, all among isolates from cases not known to be epidemiologically related. WGS produced a better-supported, higher-resolution phylogeny than MLVA, confirming that the method is more suitable for epidemiological surveillance of E. coli O157. A combination of in silico analyses (VirulenceFinder, ResFinder, and local BLAST searches) were used to determine stx subtypes, multilocus sequence types (15 loci), and the presence of virulence and acquired antimicrobial resistance genes. There was a high level of correlation between the WGS data and our routine typing methods, although some discordant results were observed, mostly related to the limitation of short sequence read assembly. The data were used to identify sublineages and clades of E. coli O157, and when they were correlated with the clinical outcome data, they showed that one clade, Ic3, was significantly associated with severe disease. Together, the results show that WGS data can provide higher resolution of the relationships between E. coli O157 isolates than that provided by MLVA. The method has the potential to streamline the laboratory workflow and provide detailed information for the clinical management of patients and public health interventions. Copyright © 2015, Holmes et al.

Utility of Whole-Genome Sequencing of Escherichia coli O157 for Outbreak Detection and Epidemiological Surveillance

PubMed Central

Allison, Lesley; Ward, Melissa; Dallman, Timothy J.; Clark, Richard; Fawkes, Angie; Murphy, Lee; Hanson, Mary

2015-01-01

Detailed laboratory characterization of Escherichia coli O157 is essential to inform epidemiological investigations. This study assessed the utility of whole-genome sequencing (WGS) for outbreak detection and epidemiological surveillance of E. coli O157, and the data were used to identify discernible associations between genotypes and clinical outcomes. One hundred five E. coli O157 strains isolated over a 5-year period from human fecal samples in Lothian, Scotland, were sequenced with the Ion Torrent Personal Genome Machine. A total of 8,721 variable sites in the core genome were identified among the 105 isolates; 47% of the single nucleotide polymorphisms (SNPs) were attributable to six “atypical” E. coli O157 strains and included recombinant regions. Phylogenetic analyses showed that WGS correlated well with the epidemiological data. Epidemiological links existed between cases whose isolates differed by three or fewer SNPs. WGS also correlated well with multilocus variable-number tandem repeat analysis (MLVA) typing data, with only three discordant results observed, all among isolates from cases not known to be epidemiologically related. WGS produced a better-supported, higher-resolution phylogeny than MLVA, confirming that the method is more suitable for epidemiological surveillance of E. coli O157. A combination of in silico analyses (VirulenceFinder, ResFinder, and local BLAST searches) were used to determine stx subtypes, multilocus sequence types (15 loci), and the presence of virulence and acquired antimicrobial resistance genes. There was a high level of correlation between the WGS data and our routine typing methods, although some discordant results were observed, mostly related to the limitation of short sequence read assembly. The data were used to identify sublineages and clades of E. coli O157, and when they were correlated with the clinical outcome data, they showed that one clade, Ic3, was significantly associated with severe disease. Together, the results show that WGS data can provide higher resolution of the relationships between E. coli O157 isolates than that provided by MLVA. The method has the potential to streamline the laboratory workflow and provide detailed information for the clinical management of patients and public health interventions. PMID:26354815

Inference on the Strength of Balancing Selection for Epistatically Interacting Loci

PubMed Central

Buzbas, Erkan Ozge; Joyce, Paul; Rosenberg, Noah A.

2011-01-01

Existing inference methods for estimating the strength of balancing selection in multi-locus genotypes rely on the assumption that there are no epistatic interactions between loci. Complex systems in which balancing selection is prevalent, such as sets of human immune system genes, are known to contain components that interact epistatically. Therefore, current methods may not produce reliable inference on the strength of selection at these loci. In this paper, we address this problem by presenting statistical methods that can account for epistatic interactions in making inference about balancing selection. A theoretical result due to Fearnhead (2006) is used to build a multi-locus Wright-Fisher model of balancing selection, allowing for epistatic interactions among loci. Antagonistic and synergistic types of interactions are examined. The joint posterior distribution of the selection and mutation parameters is sampled by Markov chain Monte Carlo methods, and the plausibility of models is assessed via Bayes factors. As a component of the inference process, an algorithm to generate multi-locus allele frequencies under balancing selection models with epistasis is also presented. Recent evidence on interactions among a set of human immune system genes is introduced as a motivating biological system for the epistatic model, and data on these genes are used to demonstrate the methods. PMID:21277883

Prenatal maternal depression and child serotonin transporter linked polymorphic region (5-HTTLPR) and dopamine receptor D4 (DRD4) genotype predict negative emotionality from 3 to 36 months.

PubMed

Green, Cathryn Gordon; Babineau, Vanessa; Jolicoeur-Martineau, Alexia; Bouvette-Turcot, Andrée-Anne; Minde, Klaus; Sassi, Roberto; St-André, Martin; Carrey, Normand; Atkinson, Leslie; Kennedy, James L; Steiner, Meir; Lydon, John; Gaudreau, Helene; Burack, Jacob A; Levitan, Robert; Meaney, Michael J; Wazana, Ashley

2017-08-01

Prenatal maternal depression and a multilocus genetic profile of two susceptibility genes implicated in the stress response were examined in an interaction model predicting negative emotionality in the first 3 years. In 179 mother-infant dyads from the Maternal Adversity, Vulnerability, and Neurodevelopment cohort, prenatal depression (Center for Epidemiologic Studies Depressions Scale) was assessed at 24 to 36 weeks. The multilocus genetic profile score consisted of the number of susceptibility alleles from the serotonin transporter linked polymorphic region gene (5-HTTLPR): no long-rs25531(A) (LA: short/short, short/long-rs25531(G) [LG], or LG/LG] vs. any LA) and the dopamine receptor D4 gene (six to eight repeats vs. two to five repeats). Negative emotionality was extracted from the Infant Behaviour Questionnaire-Revised at 3 and 6 months and the Early Child Behavior Questionnaire at 18 and 36 months. Mixed and confirmatory regression analyses indicated that prenatal depression and the multilocus genetic profile interacted to predict negative emotionality from 3 to 36 months. The results were characterized by a differential susceptibility model at 3 and 6 months and by a diathesis-stress model at 36 months.

A New Perspective on Polyploid Fragaria (Strawberry) Genome Composition Based on Large-Scale, Multi-Locus Phylogenetic Analysis.

PubMed

Yang, Yilong; Davis, Thomas M

2017-12-01

The subgenomic compositions of the octoploid (2n = 8× = 56) strawberry (Fragaria) species, including the economically important cultivated species Fragaria x ananassa, have been a topic of long-standing interest. Phylogenomic approaches utilizing next-generation sequencing technologies offer a new window into species relationships and the subgenomic compositions of polyploids. We have conducted a large-scale phylogenetic analysis of Fragaria (strawberry) species using the Fluidigm Access Array system and 454 sequencing platform. About 24 single-copy or low-copy nuclear genes distributed across the genome were amplified and sequenced from 96 genomic DNA samples representing 16 Fragaria species from diploid (2×) to decaploid (10×), including the most extensive sampling of octoploid taxa yet reported. Individual gene trees were constructed by different tree-building methods. Mosaic genomic structures of diploid Fragaria species consisting of sequences at different phylogenetic positions were observed. Our findings support the presence in octoploid species of genetic signatures from at least five diploid ancestors (F. vesca, F. iinumae, F. bucharica, F. viridis, and at least one additional allele contributor of unknown identity), and questions the extent to which distinct subgenomes are preserved over evolutionary time in the allopolyploid Fragaria species. In addition, our data support divergence between the two wild octoploid species, F. virginiana and F. chiloensis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.

PubMed

Barony, Gustavo M; Tavares, Guilherme C; Pereira, Felipe L; Carvalho, Alex F; Dorella, Fernanda A; Leal, Carlos A G; Figueiredo, Henrique C P

2017-10-19

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Use of Whole-Genus Genome Sequence Data To Develop a Multilocus Sequence Typing Tool That Accurately Identifies Yersinia Isolates to the Species and Subspecies Levels

PubMed Central

Hall, Miquette; Chattaway, Marie A.; Reuter, Sandra; Savin, Cyril; Strauch, Eckhard; Carniel, Elisabeth; Connor, Thomas; Van Damme, Inge; Rajakaruna, Lakshani; Rajendram, Dunstan; Jenkins, Claire; Thomson, Nicholas R.

2014-01-01

The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica. PMID:25339391

Comparison of a newly developed binary typing with ribotyping and multilocus sequence typing methods for Clostridium difficile.

PubMed

Li, Zhirong; Liu, Xiaolei; Zhao, Jianhong; Xu, Kaiyue; Tian, Tiantian; Yang, Jing; Qiang, Cuixin; Shi, Dongyan; Wei, Honglian; Sun, Suju; Cui, Qingqing; Li, Ruxin; Niu, Yanan; Huang, Bixing

2018-04-01

Clostridium difficile is the causative pathogen for antibiotic-related nosocomial diarrhea. For epidemiological study and identification of virulent clones, a new binary typing method was developed for C. difficile in this study. The usefulness of this newly developed optimized 10-loci binary typing method was compared with two widely used methods ribotyping and multilocus sequence typing (MLST) in 189 C. difficile samples. The binary typing, ribotyping and MLST typed the samples into 53 binary types (BTs), 26 ribotypes (RTs), and 33 MLST sequence types (STs), respectively. The typing ability of the binary method was better than that of either ribotyping or MLST expressed in Simpson Index (SI) at 0.937, 0.892 and 0.859, respectively. The ease of testing, portability and cost-effectiveness of the new binary typing would make it a useful typing alternative for outbreak investigations within healthcare facilities and epidemiological research. Copyright © 2018 Elsevier B.V. All rights reserved.

Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China.

PubMed

Yang, Yong; Yu, Xiaofeng; Zhan, Li; Chen, Jiancai; Zhang, Yunyi; Zhang, Junyan; Chen, Honghu; Zhang, Zheng; Zhang, Yanjun; Lu, Yiyu; Mei, Lingling

2017-04-01

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multilocus Sequence Types of Campylobacter jejuni Isolates from Different Sources in Eastern China.

PubMed

Zhang, Gong; Zhang, Xiaoyan; Hu, Yuanqing; Jiao, Xin-An; Huang, Jinlin

2015-09-01

Campylobacter jejuni is a major food-borne pathogen that causes human gastroenteritis in many developed countries. In our study, we applied multilocus sequence typing (MLST) technology to 167 C. jejuni isolates from diverse sources in Eastern China to examine their genetic diversity. MLST defined 94 sequence types (STs) belonging to 18 clonal complexes (CCs). Forty-five STs from 60 isolates (36%) and 22 alleles have not been previously documented in an international database. One hundred and two isolates, accounting for 61.1% of all isolates, belonged to eight clonal complexes. The eight major CCs were also the most common complexes from different sources. The most common ST type of isolates from human and food was ST-353. The dominant ST type in chicken and foods was ST-354. Among 21 STs that contained two or more different sources isolates, 15 STs contained human isolates and isolates from other sources, suggesting that potentially pathogenic strains are not restricted to specific lineages.

Distinct Bacteriophages Encoding Panton-Valentine Leukocidin (PVL) among International Methicillin-Resistant Staphylococcus aureus Clones Harboring PVL▿

PubMed Central

Boakes, E.; Kearns, A. M.; Ganner, M.; Perry, C.; Hill, R. L.; Ellington, M. J.

2011-01-01

Genetically diverse community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) can harbor a bacteriophage encoding Panton-Valentine leukocidin (PVL) lysogenized into its chromosome (prophage). Six PVL phages (ΦPVL, Φ108PVL, ΦSLT, ΦSa2MW, ΦSa2USA, and ΦSa2958) are known, and single-nucleotide polymorphisms (SNPs) in the PVL genes have been reported. We sought to determine the distribution of lysogenized PVL phages among MRSA strains with PVL (PVL-MRSA strains), the PVL gene sequences, and the chromosomal phage insertion sites in 114 isolates comprising nine clones of PVL-MRSA that were selected for maximal underlying genetic diversity. The six PVL phages were identified by PCR; ΦSa2USA was present in the highest number of different lineages (multilocus sequence type clonal complex 1 [CC1], CC5, CC8, and sequence type 93 [ST93]) (n = 37 isolates). Analysis of 92 isolates confirmed that PVL phages inserted into the same chromosomal insertion locus in CC22, -30, and -80 but in a different locus in isolates of CC1, -5, -8, -59, and -88 and ST93 (and CC22 in two isolates). Within the two different loci, specific attachment motifs were found in all cases, although some limited inter- and intralineage sequence variation occurred. Overall, lineage-specific relationships between the PVL phage, the genes that encode the toxin, and the position at which the phage inserts into the host chromosome were identified. These analyses provide important insights into the microepidemiology of PVL-MRSA, will prove a valuable adjunct in outbreak investigation, and may help predict the emergence of new strains. PMID:21106787

Prevalence of Complement-Mediated Cell Lysis-like Gene (sicG) in Streptococcus dysgalactiae subsp. equisimilis Isolates From Japan (2014-2016).

PubMed

Takahashi, Takashi; Fujita, Tomohiro; Shibayama, Akiyoshi; Tsuyuki, Yuzo; Yoshida, Haruno

2017-07-01

Streptococcus dysgalactiae subsp. equisimilis (SDSE; a β-hemolytic streptococcus of human or animal origin) infections are emerging worldwide. We evaluated the clonal distribution of complement-mediated cell lysis-like gene (sicG) among SDSE isolates from three central prefectures of Japan. Group G/C β-hemolytic streptococci were collected from three institutions from April 2014 to March 2016. Fifty-five strains (52 from humans and three from animals) were identified as SDSE on the basis of 16S rRNA sequencing data.; they were obtained from 25 sterile (blood, joint fluid, and cerebrospinal fluid) and 30 non-sterile (skin-, respiratory tract-, and genitourinary tract-origin) samples. emm genotyping, multilocus sequence typing, sicG amplification/sequencing, and random amplified polymorphic DNA (RAPD) analysis of sicG-positive strains were performed. sicG was detected in 30.9% of the isolates (16 human and one canine) and the genes from the 16 human samples (blood, 10; open pus, 3; sputum, 2; throat swab, 1) and one canine sample (open pus) showed the same sequence pattern. All sicG-harboring isolates belonged to clonal complex (CC) 17, and the most prevalent emm type was stG6792 (82.4%). There was a significant association between sicG presence and the development of skin/soft tissue infections. CC17 isolates with sicG could be divided into three subtypes by RAPD analysis. CC17 SDSE harboring sicG might have spread into three closely-related prefectures in central Japan during 2014-2016. Clonal analysis of isolates from other areas might be needed to monitor potentially virulent strains in humans and animals. © The Korean Society for Laboratory Medicine

Multidrug Resistant Pseudomonas aeruginosa Causing Prosthetic Valve Endocarditis: A Genetic-Based Chronicle of Evolving Antibiotic Resistance.

PubMed

Domitrovic, T Nicholas; Hujer, Andrea M; Perez, Federico; Marshall, Steven H; Hujer, Kristine M; Woc-Colburn, Laila E; Parta, Mark; Bonomo, Robert A

2016-10-01

Background.  Successful treatment of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa is thwarted by the emergence of antibiotic resistance and biofilm formation on prosthetic devices. Our aims were to decipher the molecular basis of resistance in a unique case of prosthetic valve endocarditis (PVE) caused by MDR P. aeruginosa . Methods.  Five sequential MDR P. aeruginosa blood isolates collected during a 7-month period were recovered from a patient suffering from PVE previously exposed to β-lactam antibiotics. Minimum inhibitory concentrations (MICs) of several classes of antibiotics were used to indicate clinical resistance characteristics; relatedness of the isolates was determined using multilocus sequence typing and repetitive sequence-based polymerase chain reaction. Amplification and sequencing of regulatory and resistance genes was performed. Results.  All isolates belonged to ST 298, possessed bla PDC-16 , and were resistant to fluoroquinolones and carbapenems. In the course of therapy, we observed a >2-fold increase in cephalosporin resistance (4 µg/mL to >16 µg/mL). Sequencing of the AmpC regulator, amp R, revealed a D135N point mutation in cephalosporin-resistant isolates. Common carbapenemase genes were not identified. All isolates demonstrated a premature stop codon at amino acid 79 of the outer membrane protein OprD and mutations in the quinolone resistance-determining regions of gyr A and par C. Point mutations in nal C, an efflux pump regulator, were also observed. Conclusions.  In this analysis, we chart the molecular evolution of β-lactam resistance in a case of PVE. We show that mutations in regulatory genes controlling efflux and cephalosporinase production contributed to the MDR phenotype.

Lactobacillus delbrueckii subsp. jakobsenii subsp. nov., isolated from dolo wort, an alcoholic fermented beverage in Burkina Faso.

PubMed

Adimpong, David B; Nielsen, Dennis S; Sørensen, Kim I; Vogensen, Finn K; Sawadogo-Lingani, Hagrétou; Derkx, Patrick M F; Jespersen, Lene

2013-10-01

Lactobacillus delbrueckii is divided into five subspecies based on phenotypic and genotypic differences. A novel isolate, designated ZN7a-9(T), was isolated from malted sorghum wort used for making an alcoholic beverage (dolo) in Burkina Faso. The results of 16S rRNA gene sequencing, DNA-DNA hybridization and peptidoglycan cell-wall structure type analyses indicated that it belongs to the species L. delbrueckii. The genome sequence of isolate ZN7a-9(T) was determined by Illumina-based sequencing. Multilocus sequence typing (MLST) and split-decomposition analyses were performed on seven concatenated housekeeping genes obtained from the genome sequence of strain ZN7a-9(T) together with 41 additional L. delbrueckii strains. The results of the MLST and split-decomposition analyses could not establish the exact subspecies of L. delbrueckii represented by strain ZN7a-9(T) as it clustered with L. delbrueckii strains unassigned to any of the recognized subspecies of L. delbrueckii. Strain ZN7a-9(T) additionally differed from the recognized type strains of the subspecies of L. delbrueckii with respect to its carbohydrate fermentation profile. In conclusion, the cumulative results indicate that strain ZN7a-9(T) represents a novel subspecies of L. delbrueckii closely related to Lactobacillus delbrueckii subsp. lactis and Lactobacillus delbrueckii subsp. delbrueckii for which the name Lactobacillus delbrueckii subsp. jakobsenii subsp. nov. is proposed. The type strain is ZN7a-9(T) = DSM 26046(T) = LMG 27067(T).

In Silico identification of pathogenic strains of Cronobacter from Biochemical data reveals association of inositol fermentation with pathogenicity.

PubMed

Hamby, Stephen E; Joseph, Susan; Forsythe, Stephen J; Chuzhanova, Nadia

2011-09-20

Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known to cause neonatal meningitis, septicaemia and death. Current diagnostic tests for identification of Cronobacter do not differentiate between species, necessitating time consuming 16S rDNA gene sequencing or multilocus sequence typing (MLST). The organism is ubiquitous, being found in the environment and in a wide range of foods, although there is variation in pathogenicity between Cronobacter isolates and between species. Therefore to be able to differentiate between the pathogenic and non-pathogenic strains is of interest to the food industry and regulators. Here we report the use of Expectation Maximization clustering to categorise 98 strains of Cronobacter as pathogenic or non-pathogenic based on biochemical test results from standard diagnostic test kits. Pathogenicity of a strain was postulated on the basis of either pathogenic symptoms associated with strain source or corresponding MLST sequence types, allowing the clusters to be labelled as containing either pathogenic or non-pathogenic strains. The resulting clusters gave good differentiation of strains into pathogenic and non-pathogenic groups, corresponding well to isolate source and MLST sequence type. The results also revealed a potential association between pathogenicity and inositol fermentation. An investigation of the genomes of Cronobacter sakazakii and C. turicensis revealed the gene for inositol monophosphatase is associated with putative virulence factors in pathogenic strains of Cronobacter. We demonstrated a computational approach allowing existing diagnostic kits to be used to identify pathogenic strains of Cronobacter. The resulting clusters correlated well with MLST sequence types and revealed new information about the pathogenicity of Cronobacter species.

Discrimination of Anopheles species of the Arribalzagia series in Colombia using a multilocus approach.

PubMed

Álvarez, Natalí; Gómez, Giovan F; Naranjo-Díaz, Nelson; Correa, Margarita M

2018-06-18

The Arribalzagia Series of the Anopheles Subgenus comprises morphologically similar species or members of species complexes which makes correct species identification difficult. Therefore, the aim of this work was to discriminate the morphospecies of the Arribalzagia Series present in Colombia using a multilocus approach based on ITS2, COI and CAD sequences. Specimens of the Arribalzagia Series collected at 32 localities in nine departments were allocated to seven species. Individual and concatenated Bayesian analyses showed high support for each of the species and reinforced the previous report of the Apicimacula species Complex with distribution in the Pacific Coast and northwestern Colombia. In addition, a new molecular operational taxonomic unit-MOTU was identified, herein denominated near Anopheles peryassui, providing support for the existence of a Peryassui species Complex. Further, the CAD gene, just recently used for Anopheles taxonomy and phylogeny, demonstrated its power in resolving phylogenetic relationships among species of the Arribalzagia Series. The divergence times for these species correspond to the early Pliocene and the Miocene. Considering the epidemiological importance of some species of the Series and their co-occurrence in malaria endemic regions of Colombia, their discrimination constitutes an important step for vector incrimination and control in the country. Copyright © 2018. Published by Elsevier B.V.

Recurrent hybridization and recent origin obscure phylogenetic relationships within the ‘white-headed’ gull (Larus sp.) complex

USGS Publications Warehouse

Sonsthagen, Sarah A.; Wilson, Robert E.; Chesser, Terry; Pons, Jean-Marc; Crochet, Pierre-Andre; Driscoll, Amy; Dove, Carla

2016-01-01

Species complexes that have undergone recent radiations are often characterized by extensive allele sharing due to recent ancestry and (or) introgressive hybridization. This can result in discordant evolutionary histories of genes and heterogeneous genomes, making delineating species limits difficult. Here we examine the phylogenetic relationships among a complex group of birds, the white-headed gulls (Aves: Laridae), which offer a unique window into the speciation process due to their recent evolutionary history and propensity to hybridize. Relationships were examined among 17 species (61 populations) using a multilocus approach, including mitochondrial and nuclear intron DNA sequences and microsatellite genotype information. Analyses of microsatellite and intron data resulted in some species-based groupings, although most species were not represented by a single cluster. Considerable allele and haplotype sharing among white-headed gull species was observed; no locus contained a species-specific clade. Despite this, our multilocus approach provided better resolution among some species than previous studies. Interestingly, most clades appear to correspond to geographic locality: our BEAST analysis recovered strong support for a northern European/Icelandic clade, a southern European/Russian clade, and a western North American/canus clade, with weak evidence for a high latitude clade spanning North America and northwestern Europe. This geographical structuring is concordant with behavioral observations of pervasive hybridization in areas of secondary contact. The extent of allele and haplotype sharing indicates that ecological and sexual selection are likely not strong enough to complete reproductive isolation within several species in the white-headed gull complex. This suggests that just a few genes are driving the speciation process.

Multilocus sequence typing of total-genome-sequenced bacteria.

PubMed

Larsen, Mette V; Cosentino, Salvatore; Rasmussen, Simon; Friis, Carsten; Hasman, Henrik; Marvig, Rasmus Lykke; Jelsbak, Lars; Sicheritz-Pontén, Thomas; Ussery, David W; Aarestrup, Frank M; Lund, Ole

2012-04-01

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

“Silent” Dissemination of Klebsiella pneumoniae Isolates Bearing K. pneumoniae Carbapenemase in a Long-term Care Facility for Children and Young Adults in Northeast Ohio

PubMed Central

Viau, Roberto A.; Hujer, Andrea M.; Marshall, Steven H.; Perez, Federico; Hujer, Kristine M.; Briceño, David F.; Dul, Michael; Jacobs, Michael R.; Grossberg, Richard; Toltzis, Philip

2012-01-01

Background. Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (blaKPC) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection. Methods. Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed. Results. The DNA microarray detected blaKPC in all 12 isolates, and blaKPC-3 was identified by PCR amplification and sequencing of the amplicon. In addition, a blaSHV-11 gene was detected in all isolates. Immunoblotting revealed “low-level” production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all blaKPC-3-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with blaKPC carriage. Plasmids from 3 representative isolates readily transferred the blaKPC-3 to Escherichia coli J-53 recipients. Conclusions. Our findings reveal the “silent” dissemination of blaKPC-3 as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing blaKPC-3 in an LTCF caring for neurologically impaired children and young adults. PMID:22492318

Genotypes of Ciprofloxacin-Resistant Klebsiella pneumoniae in Korea and Their Characteristics According to the Genetic Lineages.

PubMed

Park, Dong Jin; Yu, Jin Kyung; Park, Kang Gyun; Park, Yeon-Joon

2015-12-01

We investigated the molecular genotypes of ciprofloxacin-resistant Klebsiella pneumoniae and their characteristics according to the genetic lineages. For 160 K. pneumoniae collected in 2013, ciprofloxacin minimum inhibitory concentrations (MICs) were determined by agar dilution method. The genotypes of ciprofloxacin-resistant K. pneumoniae isolates were determined by multilocus sequence typing (MLST) and wzi gene typing. The presence of plasmid-mediated resistance determinants [qnrA, qnrB, qnrS, aac(6')-Ib-cr, blaCTX-M, and blaSHV] was investigated. The gyrA and parC genes were sequenced. Fifty-seven isolates showed ciprofloxacin resistance. By MLST, four major sequence types (STs) or clonal complexes (CCs), that is, ST307, CC11, CC147, and ST15, were found and the two most prevalent STs were ST307 (14/57, 24.6%) and ST11 (12/57, 21.1%). By wzi gene sequencing, 46 of the 57 isolates could be differentiated. All the ST307 isolates had an identical wzi sequence and harbored qnrB. The majority of them harbored aac(6')-Ib-cr (85.7%) and CTX-M-15 (92.9%). In contrast, 12 ST11 isolates were divided into five sublineages by wzi sequence and qnrB, qnrS, and aac(6')-Ib-cr were carried by nine, seven, and three isolates, respectively. They harbored SHV-type extended-spectrum β-lactamase more frequently than CTX-M-15 (nine and four isolates, respectively). The prevalence of CTX-M-15, qnrB1, and aac(6')-Ib-cr was significantly higher in ST307 than in ST11 (p=0.003, p=0.000, and p=0.002, respectively). Both clones had identical amino acid substitution in gyrA (S83I) and parC (S80I). K. pneumoniae ST307 and ST11 were the two most common clones, and the ST307 isolates were highly homogeneous, suggesting their recent emergence.

Multilocus genetics to reconstruct aeromonad evolution

PubMed Central

2012-01-01

Background Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. Results The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. Conclusions Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or “disease specialized” while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters. PMID:22545815

Molecular Typing and Carbapenem Resistance Mechanisms of Pseudomonas aeruginosa Isolated From a Chinese Burn Center From 2011 to 2016

PubMed Central

Yin, Supeng; Chen, Ping; You, Bo; Zhang, Yulong; Jiang, Bei; Huang, Guangtao; Yang, Zichen; Chen, Yu; Chen, Jing; Yuan, Zhiqiang; Zhao, Yan; Li, Ming; Hu, Fuquan; Gong, Yali; Peng, Yizhi

2018-01-01

Pseudomonas aeruginosa is the leading cause of infection in burn patients. The increasing carbapenem resistance of P. aeruginosa has become a serious challenge to clinicians. The present study investigated the molecular typing and carbapenem resistance mechanisms of 196 P. aeruginosa isolates from the bloodstream and wound surface of patients in our burn center over a period of 6 years. By multilocus sequence typing (MLST), a total of 58 sequence types (STs) were identified. An outbreak of ST111, a type that poses a high international risk, occurred in 2014. The isolates from wound samples of patients without bacteremia were more diverse and more susceptible to antibiotics than strains collected from the bloodstream or the wound surface of patients with bacteremia. Importantly, a large proportion of the patients with multisite infection (46.51%) were simultaneously infected by different STs in the bloodstream and wound surface. Antimicrobial susceptibility testing of these isolates revealed high levels of resistance to carbapenems, with 35.71% susceptibility to imipenem and 32.14% to meropenem. To evaluate mechanisms associated with carbapenem resistance, experiments were conducted to determine the prevalence of carbapenemase genes, detect alterations of the oprD porin gene, and measure expression of the ampC β-lactamase gene and the mexB multidrug efflux gene. The main mechanism associated with carbapenem resistance was mutational inactivation of oprD (88.65%), accompanied by overexpression of ampC (68.09%). In some cases, oprD was inactivated by insertion sequence element IS1411, which has not been found previously in P. aeruginosa. These findings may help control nosocomial P. aeruginosa infections and improve clinical practice. PMID:29896186

Serotype distribution, antimicrobial resistance, and molecular characterization of invasive group B Streptococcus isolates recovered from Chinese neonates.

PubMed

Wang, Ping; Ma, Zhuoya; Tong, Jingjing; Zhao, Ruizhen; Shi, Wei; Yu, Sangjie; Yao, Kaihu; Zheng, Yuejie; Yang, Yonghong

2015-08-01

Group B Streptococcus (GBS) is an important neonatal pathogen associated with high morbidity and mortality in developed countries. However, data describing neonatal GBS disease in developing countries, particularly in Asia, are largely incomplete. The aim of this study was to determine the serotype distribution, antimicrobial resistance, and molecular characteristics of invasive GBS isolates recovered from Chinese neonates. From 2008 to 2013, 40 GBS isolates were recovered from infected neonates less than 3 months of age. All isolates were identified with the CAMP test and commercially available techniques. Serotyping was performed by latex agglutination. Antibiotic susceptibility was tested with Etest strips and the disk diffusion method. Multilocus sequence typing and erythromycin resistance gene detection (ermB and mefA) were performed by PCR. Four serotypes were identified. Serotype III (85%) was the most prevalent, followed by Ia (7.5%), Ib (5%), and V (2.5%). All isolates were sensitive to penicillin, ceftriaxone, and levofloxacin. However, resistance to erythromycin (92.5%), clindamycin (87.5%), and tetracycline (100%) was observed. Among erythromycin-resistant isolates, 73.0% carried the ermB gene alone, 5.4% carried the mefA gene alone, and 21.6% expressed both ermB and mefA genes. A total of seven sequence types (STs) were identified; the most prevalent was ST17, accounting for 80% of all isolates. Further, serotype III isolates contained ST17 (94.2%), ST19 (2.9%), and ST650 (2.9%). Serotype distribution, antimicrobial susceptibility, and sequence type characterization in Asia and in other global regions may contribute to improve the prevention and treatment of neonatal GBS infections. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Safety of the Surrogate Microorganism Enterococcus faecium NRRL B-2354 for Use in Thermal Process Validation

PubMed Central

Kopit, Lauren M.; Kim, Eun Bae; Siezen, Roland J.; Harris, Linda J.

2014-01-01

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products. PMID:24413604

Population genetics of Vibrio vulnificus: identification of two divisions and a distinct eel-pathogenic clone.

PubMed

Gutacker, Michaela; Conza, Nadine; Benagli, Cinzia; Pedroli, Ambra; Bernasconi, Marco Valerio; Permin, Lise; Aznar, Rosa; Piffaretti, Jean-Claude

2003-06-01

Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V. vulnificus population into two divisions (divisions I and II). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peter's fish (biotype 3). RAPD analysis of the 62 V. vulnificus isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V. vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the V. vulnificus population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions I and II) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.

The coding region of the UFGT gene is a source of diagnostic SNP markers that allow single-locus DNA genotyping for the assessment of cultivar identity and ancestry in grapevine (Vitis vinifera L.)

PubMed Central

2013-01-01

Background Vitis vinifera L. is one of society’s most important agricultural crops with a broad genetic variability. The difficulty in recognizing grapevine genotypes based on ampelographic traits and secondary metabolites prompted the development of molecular markers suitable for achieving variety genetic identification. Findings Here, we propose a comparison between a multi-locus barcoding approach based on six chloroplast markers and a single-copy nuclear gene sequencing method using five coding regions combined with a character-based system with the aim of reconstructing cultivar-specific haplotypes and genotypes to be exploited for the molecular characterization of 157 V. vinifera accessions. The analysis of the chloroplast target regions proved the inadequacy of the DNA barcoding approach at the subspecies level, and hence further DNA genotyping analyses were targeted on the sequences of five nuclear single-copy genes amplified across all of the accessions. The sequencing of the coding region of the UFGT nuclear gene (UDP-glucose: flavonoid 3-0-glucosyltransferase, the key enzyme for the accumulation of anthocyanins in berry skins) enabled the discovery of discriminant SNPs (1/34 bp) and the reconstruction of 130 V. vinifera distinct genotypes. Most of the genotypes proved to be cultivar-specific, and only few genotypes were shared by more, although strictly related, cultivars. Conclusion On the whole, this technique was successful for inferring SNP-based genotypes of grapevine accessions suitable for assessing the genetic identity and ancestry of international cultivars and also useful for corroborating some hypotheses regarding the origin of local varieties, suggesting several issues of misidentification (synonymy/homonymy). PMID:24298902

A multi-locus analysis of phylogenetic relationships within grass subfamily Pooideae (Poaceae) inferred from sequences of nuclear single copy gene regions compared with plastid DNA.

PubMed

Hochbach, Anne; Schneider, Julia; Röser, Martin

2015-06-01

To investigate phylogenetic relationships within the grass subfamily Pooideae we studied about 50 taxa covering all recognized tribes, using one plastid DNA (cpDNA) marker (matK gene-3'trnK exon) and for the first time four nuclear single copy gene loci. DNA sequence information from two parts of the nuclear genes topoisomerase 6 (Topo6) spanning the exons 8-13 and 17-19, the exons 9-13 encoding plastid acetyl-CoA-carboxylase (Acc1) and the partial exon 1 of phytochrome B (PhyB) were generated. Individual and nuclear combined data were evaluated using maximum parsimony, maximum likelihood and Bayesian methods. All of the phylogenetic results show Brachyelytrum and the tribe Nardeae as earliest diverging lineages within the subfamily. The 'core' Pooideae (Hordeeae and the Aveneae/Poeae tribe complex) are also strongly supported, as well as the monophyly of the tribes Brachypodieae, Meliceae and Stipeae (except PhyB). The beak grass tribe Diarrheneae and the tribe Duthieeae are not monophyletic in some of the analyses. However, the combined nuclear DNA (nDNA) tree yields the highest resolution and the best delimitation of the tribes, and provides the following evolutionary hypothesis for the tribes: Brachyelytrum, Nardeae, Duthieeae, Meliceae, Stipeae, Diarrheneae, Brachypodieae and the 'core' Pooideae. Within the individual datasets, the phylogenetic trees obtained from Topo6 exon 8-13 shows the most interesting results. The divergent positions of some clone sequences of Ampelodesmos mauritanicus and Trikeraia pappiformis, for instance, may indicate a hybrid origin of these stipoid taxa. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic diversity and symbiotic effectiveness of Bradyrhizobium strains nodulating selected annual grain legumes growing in Ethiopia.

PubMed

Degefu, Tulu; Wolde-Meskel, Endalkachew; Rasche, Frank

2018-01-01

Vigna unguiculata, Vigna radiata and Arachis hypogaea growing in Ethiopia are nodulated by a genetically diverse group of Bradyrhizobium strains. To determine the genetic identity and symbiotic effectiveness of these bacteria, a collection of 36 test strains originating from the root nodules of the three hosts was investigated using multilocus sequence analyses (MLSA) of core genes including 16S rRNA, recA, glnII, gyrB, atpD and dnaK. Sequence analysis of nodA and nifH genes along with tests for symbiotic effectiveness using δ 15 N analysis were also carried out. The phylogenetic trees derived from the MLSA grouped most test strains into four well-supported distinct positions designated as genospecies I-IV. The maximum likelihood (ML) tree that was constructed based on the nodA gene sequences separated the entire test strains into two lineages, where the majority of the test strains were clustered on one of a well-supported large branch that comprise Bradyrhizobium species from the tropics. This clearly suggested the monophyletic origin of the nodA genes within the bradyrhizobia of tropical origin. The δ 15 N-based symbiotic effectiveness test of seven selected strains revealed that strains GN100 (δ 15 N=0.73) and GN102 (δ 15 N=0.79) were highly effective nitrogen fixers when inoculated to cowpea, thus can be considered as inoculants in cowpea production. It was concluded that Ethiopian soils are a hotspot for rhizobial diversity. This calls for further research to unravel as yet unknown bradyrhizobia nodulating legume host species growing in the country. In this respect, prospective research should also address the mechanisms of symbiotic specificity that could lead to high nitrogen fixation in target legumes.

Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).

PubMed

Brzuszkiewicz, Elzbieta; Thürmer, Andrea; Schuldes, Jörg; Leimbach, Andreas; Liesegang, Heiko; Meyer, Frauke-Dorothee; Boelter, Jürgen; Petersen, Heiko; Gottschalk, Gerhard; Daniel, Rolf

2011-12-01

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo.

PubMed

Leekitcharoenphon, Pimlapas; Friis, Carsten; Zankari, Ea; Svendsen, Christina Aaby; Price, Lance B; Rahmani, Maral; Herrero-Fresno, Ana; Fashae, Kayode; Vandenberg, Olivier; Aarestrup, Frank M; Hendriksen, Rene S

2013-10-15

Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

Monomorphic genotypes within a generalist lineage of Campylobacter jejuni show signs of global dispersion

PubMed Central

Zhang, Ji; Vehkala, Minna; Välimäki, Niko; Hakkinen, Marjaana; Hänninen, Marja-Liisa; Roasto, Mati; Mäesaar, Mihkel; Taboada, Eduardo; Barker, Dillon; Garofolo, Giuliano; Cammà, Cesare; Di Giannatale, Elisabetta; Corander, Jukka; Rossi, Mirko

2016-01-01

The decreased costs of genome sequencing have increased the capability to apply whole-genome sequencing to epidemiological surveillance of zoonotic Campylobacter jejuni. However, knowledge of the genetic diversity of this bacteria is vital for inferring relatedness between epidemiologically linked isolates and a necessary prerequisite for correct application of this methodology. To address this issue in C. jejuni we investigated the spatial and temporal signals in the genomes of a major clonal complex and generalist lineage, ST-45 CC, by analysing the population structure and genealogy as well as applying genome-wide association analysis of 340 isolates from across Europe collected over a wide time range. The occurrence and strength of the geographical signal varied between sublineages and followed the clonal frame when present, while no evidence of a temporal signal was found. Certain sublineages of ST-45 formed discrete and genetically isolated clades containing isolates with extremely similar genomes regardless of time and location of sampling. Based on a separate data set, these monomorphic genotypes represent successful C. jejuni clones, possibly spread around the globe by rapid animal (migrating birds), food or human movement. In addition, we observed an incongruence between the genealogy of the strains and multilocus sequence typing (MLST), challenging the existing clonal complex definition and the use of whole-genome gene-by-gene hierarchical nomenclature schemes for C. jejuni. PMID:28348829

Sequencing of the Litchi Downy Blight Pathogen Reveals It Is a Phytophthora Species With Downy Mildew-Like Characteristics.

PubMed

Ye, Wenwu; Wang, Yang; Shen, Danyu; Li, Delong; Pu, Tianhuizi; Jiang, Zide; Zhang, Zhengguang; Zheng, Xiaobo; Tyler, Brett M; Wang, Yuanchao

2016-07-01

On the basis of its downy mildew-like morphology, the litchi downy blight pathogen was previously named Peronophythora litchii. Recently, however, it was proposed to transfer this pathogen to Phytophthora clade 4. To better characterize this unusual oomycete species and important fruit pathogen, we obtained the genome sequence of Phytophthora litchii and compared it to those from other oomycete species. P. litchii has a small genome with tightly spaced genes. On the basis of a multilocus phylogenetic analysis, the placement of P. litchii in the genus Phytophthora is strongly supported. Effector proteins predicted included 245 RxLR, 30 necrosis-and-ethylene-inducing protein-like, and 14 crinkler proteins. The typical motifs, phylogenies, and activities of these effectors were typical for a Phytophthora species. However, like the genome features of the analyzed downy mildews, P. litchii exhibited a streamlined genome with a relatively small number of genes in both core and species-specific protein families. The low GC content and slight codon preferences of P. litchii sequences were similar to those of the analyzed downy mildews and a subset of Phytophthora species. Taken together, these observations suggest that P. litchii is a Phytophthora pathogen that is in the process of acquiring downy mildew-like genomic and morphological features. Thus P. litchii may provide a novel model for investigating morphological development and genomic adaptation in oomycete pathogens.

Arthrobacter ruber sp. nov., isolated from glacier ice.

PubMed

Liu, Qing; Xin, Yu-Hua; Chen, Xiu-Ling; Liu, Hong-Can; Zhou, Yu-Guang; Chen, Wen-Xin

2018-05-01

A Gram-stain-positive strain designated MDB1-42 T was isolated from ice collected from Midui glacier in Tibet, PR China. Strain MDB1-42 T was catalase-positive, oxidase-negative and grew optimally at 25-28 °C and pH 7.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that MDB1-42 T represented a member of the genus Arthrobacter. The highest level of 16S rRNA gene sequence similarity (99.86 %) was found with Arthrobacter agilis NBRC 15319 T . Multilocus sequence analysis revealed low similarity of 91.93 % between MDB1-42 T and Arthrobacter agilis NBRC 15319 T . Average nucleotide identity and digital DNA-DNA hybridization values between MDB1-42 T and the most closely related strain, Arthrobacter agilis DSM 20550 T , were 81.36 and 24.5 %, respectively. The genomic DNA G+C content was 69.0 mol%. The major cellular fatty acids of MDB1-42 T were anteiso-C15 : 0 and anteiso-C17:0. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, one unidentified glycolipid and one unidentified lipid. The predominant menaquinone was MK-9(H2). On the basis of results obtained using a polyphasic approach, a novel species Arthrobacter ruber sp. nov. is proposed, with MDB1-42 T (=CGMCC 1.9772 T =NBRC 113088 T ) as the type strain.

Characterisation of extended-spectrum β-lactamase and AmpC β-lactamase-producing Enterobacteriaceae isolated from companion animals in New Zealand.

PubMed

Karkaba, A; Grinberg, A; Benschop, J; Pleydell, E

2017-03-01

To assess the occurrence of, and characterise, extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC)-producing Enterobacteriaceae isolated by veterinary diagnostic laboratories from infection sites in companion animals in New Zealand. Selected Enterobacteriaceae isolates were submitted by seven New Zealand veterinary diagnostic laboratories. They were isolated from infection sites in companion animals between June 2012 and June 2013, and were resistant to amoxicillin-clavulanic acid, fluoroquinolones, or any combination of two or more antimicrobials. Based on disk diffusion test results, the isolates were phenotypically categorised according to production of ESBL and AmpC. Genes for ESBL and AmpC production were amplified by PCR and sequenced. Escherichia coli isolates were also typed by multilocus sequence typing. A total of 115 isolates matching the inclusion criteria were obtained from the participating laboratories, of which 74 (64%) originated from dogs and 29 (25%) from cats. Seven bacterial species were identified, of which E. coli was the most common (87/115, 76%). Of the 115 isolates, 10 (9%) expressed the ESBL phenotype, 43 (37%) the AmpC phenotype, and seven (6%) both ESBL and AmpC phenotypes. Of the 60 ESBL and AmpC-producing isolates, 36 (60%) were E. coli. Amongst these isolates, 27/60 (45%) were classified as multidrug resistant, compared with 15/55 (27%) non-ESBL or AmpC-producing isolates (p<0.01). Ninety five isolates were resistant to amoxicillin-clavulanic acid and 58 (61%) of these were ESBL or AmpC-producing. The predominant ESBL genes were bla CTX-M-14 and bla CTX-M-15 , and the dominant plasmid-encoded AmpC gene was bla CMY-2 . Thirty-eight E. coli multilocus sequence types (ST) were identified, and the most prevalent were ST12 (12/89, 13%), ST131 (6/89, 7%) and ST648 (6/89, 7%). ESBL and AmpC-producing isolates accounted for 35/1,082 (3.2%) of the Enterobacteriaceae isolated by one laboratory network over the study period. ESBL and AmpC-producing Enterobacteriaceae were associated with clinical infections in companion animals in New Zealand, and were often multidrug resistant. In this study, these organisms accounted for <5% of all Enterobacteriaceae isolated from infection sites by one laboratory network, but their prevalence among isolates resistant to amoxicillin-clavulanic acid was 61%. Therefore routine secondary testing for ESBL and AmpC production by Enterobacteriaceae that are resistant to amoxicillin-clavulanic acid in primary testing could improve the accuracy of definitive antimicrobial therapy in companion animals in New Zealand.

Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips

DTIC Science & Technology

2010-05-12

Sequence Types. The evolutionary history of all the B. cereus MLST concatenated Sequence Types (545 taxa, 2,394 nucleotide positions) was inferred using...the Neighbor-Joining method [28]. The bootstrap consensus tree inferred from 100 replicates was taken to represent the evolutionary history of the... Chlamydia (manuscript in preparation) and performed pilot studies on Staphylococcus aureus and Streptoccus pneumoniae (Data S4 and Text S2). Another potential

Genetic characterization and antimicrobial resistance of Staphylococcus aureus isolated from bovine milk in Tunisia.

PubMed

Ben Said, M; Abbassi, M S; Bianchini, V; Sghaier, S; Cremonesi, P; Romanò, A; Gualdi, V; Hassen, A; Luini, M V

2016-12-01

Staphylococcus aureus is a major agent of bovine mastitis in dairy herds, causing economic losses in dairy industry worldwide. In addition, milk and milk-products contaminated by Staph. aureus can cause harmful human diseases. The aim of this study was to characterize Staph. aureus strains isolated from dairy farms in Tunisia. Bulk tank milk (n = 32) and individual cow milk (n = 130) samples were collected during the period of 2013-2014. Forty-three Staph. aureus isolates were recovered and typed by spa typing, 16S-23S rRNA intergenic spacer (RS-PCR) and multiplex PCRs for 22 virulence genes. Antimicrobial resistance was also investigated with a disc diffusion test. A selected subsample of 22 strains was additionally genotyped by multilocus sequence typing. Seventeen spa types were recovered, and t2421 (n = 10), t521 (n = 6) and t2112 (n = 5) were the most common. Fourteen different RS-PCR genotypes grouped into 11 clusters were detected in our study, with predominance of the R VI genotype (n = 24). Eight sequence types were identified and Clonal Complex 97, corresponding to RS-PCR cluster R, was the most common (n = 10), followed by CC1 (n = 4), CC15 (n = 3) and other four accounting for one or two strains. Different combinations of virulence genes were reported, and enterotoxin genes were present in few strains (seh, n = 4; sea, n = 2; sea and seh, n = 2; sec and sel, n = 2). The majority of strains were resistant only to penicillin; only one strain was found to be multiresistant and no methicillin-resistant Staph. aureus was demonstrated. Our study reported the isolation of CC97 from bovine milk in Tunisia for the first time and confirmed the relevance of this lineage in intramammary infection in cows. This paper describes the characteristics of Staphylococcus aureus isolated from bulk tank and individual cow milk in Tunisia. All strains were genotyped by spa typing and RS-PCR, a method based on the amplification of the 16S-23S rRNA intergenic spacer region, and multiplex PCRs for 22 virulence genes. A selected subsample of strains was also genotyped by multilocus sequence typing. All strains were tested for antimicrobial resistance. Our study evidences a predominance of strains belonging to Clonal Complex 97. Methicillin-resistant strains were not detected, and overall low level of antimicrobial resistance was reported. © 2016 The Society for Applied Microbiology.

Virulence gene typing of methicillin-resistant Staphylococcus aureus as a complement in epidemiological typing.

PubMed

Nowrouzian, Forough L; Karami, Nahid; Welinder-Olsson, Christina; Ahrén, Christina

2013-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) has widely spread to all parts of the world. For surveillance and effective infection control molecular typing is required. We have evaluated the utility of virulence gene determination as a complementary tool for epidemiological typing of MRSA in relation to spa-typing and pulsed-field gel electrophoresis (PFGE). We assessed 63 community-acquired MRSA (CA-MRSA) isolates detected in the West part of Sweden for 30 virulence factor genes (VF) and agr allele variations by serial polymerase chain reaction (PCR) assays. These isolates belonged to sequence types (ST) 8, 80, 45 and 30 as classified by multilocus sequence typing. The isolates in each spa-type and PFGE-type were examined over an extended time-period and constituted a varying number of PFGE-subtypes (5-14) and spa-types (3-11) within four major PFGE types. Each ST had a unique VF profile. For isolates within a major PFGE type showing high diversity both in PFGE subtypes and spa the VF profile varied as well in contrast to those with low diversity where no alterations were seen. Thus, the accuracy of each typing method does not only vary by the method per se but is rather dependent on the genetic repertoire of the typed strains and genes evaluated. For strains demonstrating high diversity VF typing may be a useful complement in the epidemiological investigations, and may highlight the accurate discriminatory power of spa or PFGE typing. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of Haemophilus influenzae clones associated with invasive disease a decade after introduction of H. influenzae serotype b vaccination in Italy.

PubMed

Giufrè, Maria; Cardines, Rita; Accogli, Marisa; Pardini, Manuela; Cerquetti, Marina

2013-08-01

The introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines has changed the epidemiology of invasive H. influenzae disease, with a shift in the predominant serotype from Hib to nonencapsulated H. influenzae (ncHi). The objective of this study was to identify the genotypes/clones associated with invasive H. influenzae disease in Italy. Eighty-seven H. influenzae strains isolated in the years 2009 to 2011 within the National Surveillance of Invasive Bacterial Disease program were analyzed. Strains were characterized by serotyping, antimicrobial susceptibility testing, and multilocus sequence typing (MLST). Genetic polymorphisms in the bla(TEM) gene promoter region as well as the occurrence of both adhesin genes (hmwA and hia) and the IgA1 protease-encoding gene (igaB) were also investigated. Of 87 strains, 67 were ncHi and 20 were encapsulated. Eleven strains were β-lactamase positive, harboring the bla(TEM) gene. Most bla(TEM) genes (10/11) were associated with a Pdel promoter region exhibiting a 135-bp deletion; the remaining strain possessed the Pa/Pb overlapping promoter. MLST analysis showed that encapsulated isolates were clonal, with each serotype sharing a few related sequence types (STs). Forty-six different STs were identified among the 67 ncHi strains. Despite this heterogeneity, a group of closely related STs (ST103, ST139, and ST145) encompassed almost 25% of all ncHi strains and 45.5% of the β-lactamase producers carrying the Pdel promoter. These major ST clones were found to be associated with the hmwA gene but not with the igaB gene. To conclude, although the heterogeneity of the ncHi population was confirmed, diffusion of major successful ST clones was documented.

Molecular sequence typing reveals genotypic diversity among Escherichia coli isolates recovered from a cantaloupe packinghouse in Northwestern Mexico

USDA-ARS?s Scientific Manuscript database

The increase in the consumption of fresh produce in the United States has correlated with a rise in the number of reported foodborne illnesses. To identify potential risk factors associated with post-harvest practices, the present study employed multilocus sequence typing (MLST) for the genotypic c...

Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers.

PubMed

Depoorter, Eliza; Bull, Matt J; Peeters, Charlotte; Coenye, Tom; Vandamme, Peter; Mahenthiralingam, Eshwar

2016-06-01

Burkholderia is an incredibly diverse and versatile Gram-negative genus, within which over 80 species have been formally named and multiple other genotypic groups likely represent new species. Phylogenetic analysis based on the 16S rRNA gene sequence and core genome ribosomal multilocus sequence typing analysis indicates the presence of at least three major clades within the genus. Biotechnologically, Burkholderia are well-known for their bioremediation and biopesticidal properties. Within this review, we explore the ability of Burkholderia to synthesise a wide range of antimicrobial compounds ranging from historically characterised antifungals to recently described antibacterial antibiotics with activity against multiresistant clinical pathogens. The production of multiple Burkholderia antibiotics is controlled by quorum sensing and examples of quorum sensing pathways found across the genus are discussed. The capacity for antibiotic biosynthesis and secondary metabolism encoded within Burkholderia genomes is also evaluated. Overall, Burkholderia demonstrate significant biotechnological potential as a source of novel antibiotics and bioactive secondary metabolites.

Population structure of Streptococcus oralis

PubMed Central

Do, Thuy; Jolley, Keith A.; Maiden, Martin C. J.; Gilbert, Steven C.; Clark, Douglas; Wade, William G.; Beighton, David

2009-01-01

Streptococcus oralis is a member of the normal human oral microbiota, capable of opportunistic pathogenicity; like related oral streptococci, it exhibits appreciable phenotypic and genetic variation. A multilocus sequence typing (MLST) scheme for S. oralis was developed and the resultant data analysed to examine the population structure of the species. Analysis of 113 isolates, confirmed as belonging to the S. oralis/mitis group by 16S rRNA gene sequencing, characterized the population as highly diverse and undergoing inter- and intra-species recombination with a probable clonal complex structure. ClonalFrame analysis of these S. oralis isolates along with examples of Streptococcus pneumoniae, Streptococcus mitis and Streptococcus pseudopneumoniae grouped the named species into distinct, coherent populations and did not support the clustering of S. pseudopneumoniae with S. mitis as reported previously using distance-based methods. Analysis of the individual loci suggested that this discrepancy was due to the possible hybrid nature of S. pseudopneumoniae. The data are available on the public MLST website (http://pubmlst.org/soralis/). PMID:19423627

Description of a new species of crested newt, previously subsumed in Triturus ivanbureschi (Amphibia: Caudata: Salamandridae).

PubMed

Wielstra, B; Arntzen, J W

2016-05-05

Multilocus molecular data play a pivotal role in diagnosing cryptic species (i.e. genetically distinct but morphologically similar species). A multilocus phylogeographic survey has provided compelling evidence that Triturus ivanbureschi sensu lato comprises two distinct gene pools with restricted gene flow. We conclude that this taxon had better be treated as two distinct (albeit morphologically cryptic) species. The name T. ivanbureschi should be restricted to the western species, which is distributed in western Asiatic Turkey plus the south-eastern Balkan Peninsula. No name is as yet available for the eastern species, which is distributed in northern Asiatic Turkey. We propose the name T. anatolicus sp. nov. for the eastern species and provide a formal species description.

Emergence of Klebsiella pneumoniae ST273 Carrying blaNDM-7 and ST656 Carrying blaNDM-1 in Manila, Philippines

PubMed Central

Chou, Andrew; Roa, Marylette; Evangelista, Michael A.; Sulit, Arielle Kae; Lagamayo, Evelina; Torres, Brian C.; Klinzing, David C.; Daroy, Maria Luisa G.; Navoa-Ng, Josephine; Sucgang, Richard

2016-01-01

We sought to determine the epidemiology of carbapenem-resistant Enterobacteriaceae and to investigate the emergence of carbapenem-resistant Klebsiella pneumoniae in two teaching hospitals in Manila, Philippines. We screened 364 Enterobacteriaceae for carbapenem resistance between 2012 and 2013 and detected four carbapenem-resistant K. pneumoniae isolates from three different patients. We used whole genome sequencing to determine the antibiotic resistance profiles and confirmed the presence of carbapenemase genes by multiplex PCR. We used multilocus sequence typing and PCR-based replicon typing to genetically characterize the carbapenem-resistant isolates. The carbapenemase gene blaNDM was detected in K. pneumoniae isolates from two patients. The first patient had ventilator-associated pneumonia and lumbar shunt infection from K. pneumoniae ST273 carrying blaNDM-7. The second patient had asymptomatic genitourinary colonization with K. pneumoniae ST656 carrying blaNDM-1. The third patient had a gluteal abscess with K. pneumoniae ST1 that did not carry a carbapenemase gene, but did carry blaDHA-1, blaOXA-1, and blaSHV-1. In this study, we report the first cases of blaNDM-carrying pathogens in the Philippines and add to the growing evidence of the worldwide spread of ST273 and NDM-7, a more efficient carbapenem hydrolyzer than NDM-1. PMID:27032000

Evidence for rare capsular switching in Streptococcus agalactiae.

PubMed

Martins, Elisabete Raquel; Melo-Cristino, José; Ramirez, Mário

2010-03-01

The polysaccharide capsule is a major antigenic factor in Streptococcus agalactiae (Lancefield group B streptococcus [GBS]). Previous observations suggest that exchange of capsular loci is likely to occur rather frequently in GBS, even though GBS is not known to be naturally transformable. We sought to identify and characterize putative capsular switching events, by means of a combination of phenotypic and genotypic methods, including pulsed-field gel electrophoretic profiling, multilocus sequence typing, and surface protein and pilus gene profiling. We show that capsular switching by horizontal gene transfer is not as frequent as previously suggested. Serotyping errors may be the main reason behind the overestimation of capsule switching, since phenotypic techniques are prone to errors of interpretation. The identified putative capsular transformants involved the acquisition of the entire capsular locus and were not restricted to the serotype-specific central genes, the previously suggested main mechanism underlying capsular switching. Our data, while questioning the frequency of capsular switching, provide clear evidence for in vivo capsular transformation in S. agalactiae, which may be of critical importance in planning future vaccination strategies against this pathogen.

Evidence for Rare Capsular Switching in Streptococcus agalactiae▿

PubMed Central

Martins, Elisabete Raquel; Melo-Cristino, José; Ramirez, Mário

2010-01-01

The polysaccharide capsule is a major antigenic factor in Streptococcus agalactiae (Lancefield group B streptococcus [GBS]). Previous observations suggest that exchange of capsular loci is likely to occur rather frequently in GBS, even though GBS is not known to be naturally transformable. We sought to identify and characterize putative capsular switching events, by means of a combination of phenotypic and genotypic methods, including pulsed-field gel electrophoretic profiling, multilocus sequence typing, and surface protein and pilus gene profiling. We show that capsular switching by horizontal gene transfer is not as frequent as previously suggested. Serotyping errors may be the main reason behind the overestimation of capsule switching, since phenotypic techniques are prone to errors of interpretation. The identified putative capsular transformants involved the acquisition of the entire capsular locus and were not restricted to the serotype-specific central genes, the previously suggested main mechanism underlying capsular switching. Our data, while questioning the frequency of capsular switching, provide clear evidence for in vivo capsular transformation in S. agalactiae, which may be of critical importance in planning future vaccination strategies against this pathogen. PMID:20023016

Analysis of the genetic diversity of ovine herpesvirus 2 in samples from livestock with malignant catarrhal fever.

PubMed

Russell, George C; Scholes, Sandra F; Twomey, David F; Courtenay, Ann E; Grant, Dawn M; Lamond, Bruce; Norris, David; Willoughby, Kim; Haig, David M; Stewart, James P

2014-08-06

In order to define better virus isolates from animals with malignant catarrhal fever (MCF), segments of three genes of ovine herpesvirus-2 were amplified from diagnostic samples representing MCF cases with a range of clinical presentations in cattle, including head and eye, alimentary and neurological. The variation within each gene segment was estimated by DNA sequencing, which confirmed that the newly-annotated Ov9.5 gene was significantly more polymorphic than either of the other loci tested (segments of ORF50 and ORF75), with alleles that differed at over 60% of nucleotide positions. Despite this, the nine Ov9.5 alleles characterised had identical predicted splicing patterns and could be translated into Ov9.5 polypeptides with at least 49% amino acid identity. This multi-locus approach has potential for use in epidemiological studies and in charactering chains of infection. However there was no association between specific variants of OvHV-2 and the clinical/pathological presentation of MCF in the cattle analysed. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of Brachyspira hyodysenteriae Isolates Recovered from Pigs in Apparently Healthy Multiplier Herds with Isolates from Herds with Swine Dysentery

PubMed Central

La, Tom; Rohde, Judith; Phillips, Nyree Dale; Hampson, David J.

2016-01-01

Swine dysentery (SD) is a mucohaemorrhagic colitis of grower/finisher pigs classically resulting from infection by the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. This study aimed to determine whether B. hyodysenteriae isolates from pigs in three healthy German multiplier herds supplying gilts to other farms differed from isolates from nine German production herds with SD. Isolates were subjected to whole genomic sequencing, and in silico multilocus sequence typing showed that those from the three multiplier herds were of previously undescribed sequence types (ST132, ST133 and ST134), with all isolates from the same herd having the same ST. All isolates were examined for the presence of 332 genes encoding predicted virulence or virulence lifestyle associated factors, and these were well conserved. Isolates from one multiplier herd were atypical in being weakly haemolytic: they had 10 amino acid substitutions in the haemolysin III protein and five in the haemolysin activation protein compared to reference strain WA1, and had a disruption in the promoter site of the hlyA gene. These changes likely contribute to the weakly haemolytic phenotype and putative lack of virulence. These same isolates also had nine base pair insertions in the iron metabolism genes bitB and bitC and lacked five of six plasmid genes that previously have been associated with colonisation. Other overall differences between isolates from the different herds were in genes from three of five outer membrane proteins, which were not found in all the isolates, and in members of a block of six plasmid genes. Isolates from three herds with SD had all six plasmid genes, while isolates lacking some of these genes were found in the three healthy herds—but also in isolates from six herds with SD. Other differences in genes of unknown function or in gene expression may contribute to variation in virulence; alternatively, superior husbandry and better general health may have made pigs in the two multiplier herds colonised by “typical” strongly haemolytic isolates less susceptible to disease expression. PMID:27489956

Comparison of Brachyspira hyodysenteriae Isolates Recovered from Pigs in Apparently Healthy Multiplier Herds with Isolates from Herds with Swine Dysentery.

PubMed

La, Tom; Rohde, Judith; Phillips, Nyree Dale; Hampson, David J

2016-01-01

Swine dysentery (SD) is a mucohaemorrhagic colitis of grower/finisher pigs classically resulting from infection by the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. This study aimed to determine whether B. hyodysenteriae isolates from pigs in three healthy German multiplier herds supplying gilts to other farms differed from isolates from nine German production herds with SD. Isolates were subjected to whole genomic sequencing, and in silico multilocus sequence typing showed that those from the three multiplier herds were of previously undescribed sequence types (ST132, ST133 and ST134), with all isolates from the same herd having the same ST. All isolates were examined for the presence of 332 genes encoding predicted virulence or virulence lifestyle associated factors, and these were well conserved. Isolates from one multiplier herd were atypical in being weakly haemolytic: they had 10 amino acid substitutions in the haemolysin III protein and five in the haemolysin activation protein compared to reference strain WA1, and had a disruption in the promoter site of the hlyA gene. These changes likely contribute to the weakly haemolytic phenotype and putative lack of virulence. These same isolates also had nine base pair insertions in the iron metabolism genes bitB and bitC and lacked five of six plasmid genes that previously have been associated with colonisation. Other overall differences between isolates from the different herds were in genes from three of five outer membrane proteins, which were not found in all the isolates, and in members of a block of six plasmid genes. Isolates from three herds with SD had all six plasmid genes, while isolates lacking some of these genes were found in the three healthy herds-but also in isolates from six herds with SD. Other differences in genes of unknown function or in gene expression may contribute to variation in virulence; alternatively, superior husbandry and better general health may have made pigs in the two multiplier herds colonised by "typical" strongly haemolytic isolates less susceptible to disease expression.

Multi-locus sequence typing of Salmonella enterica serovar Typhimurium isolates from wild birds in northern England suggests host-adapted strain.

PubMed

Hughes, L A; Wigley, P; Bennett, M; Chantrey, J; Williams, N

2010-10-01

Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi-locus sequence typing (MLST)to investigate evolutionary relationships between them. Multi-locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19. Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted. Host-pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird-feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird-feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host-adapted strain with increased virulence.

Comparative genomic analysis of a new tellurite-resistant Psychrobacter strain isolated from the Antarctic Peninsula.

PubMed

Muñoz-Villagrán, Claudia Melissa; Mendez, Katterinne N; Cornejo, Fabian; Figueroa, Maximiliano; Undabarrena, Agustina; Morales, Eduardo Hugo; Arenas-Salinas, Mauricio; Arenas, Felipe Alejandro; Castro-Nallar, Eduardo; Vásquez, Claudio Christian

2018-01-01

The Psychrobacter genus is a cosmopolitan and diverse group of aerobic, cold-adapted, Gram-negative bacteria exhibiting biotechnological potential for low-temperature applications including bioremediation. Here, we present the draft genome sequence of a bacterium from the Psychrobacter genus isolated from a sediment sample from King George Island, Antarctica (3,490,622 bp; 18 scaffolds; G + C = 42.76%). Using phylogenetic analysis, biochemical properties and scanning electron microscopy the bacterium was identified as Psychrobacter glacincola BNF20, making it the first genome sequence reported for this species. P. glacincola BNF20 showed high tellurite (MIC 2.3 mM) and chromate (MIC 6.0 mM) resistance, respectively. Genome-wide nucleotide identity comparisons revealed that P. glacincola BNF20 is highly similar (>90%) to other uncharacterized Psychrobacter spp. such as JCM18903, JCM18902, and P11F6. Bayesian multi-locus phylogenetic analysis showed that P. glacincola BNF20 belongs to a polyphyletic clade with other bacteria isolated from polar regions. A high number of genes related to metal(loid) resistance were found, including tellurite resistance genetic determinants located in two contigs: Contig LIQB01000002.1 exhibited five ter genes, each showing putative promoter sequences (terACDEZ), whereas contig LIQB1000003.2 showed a variant of the terZ gene. Finally, investigating the presence and taxonomic distribution of ter genes in the NCBI's RefSeq bacterial database (5,398 genomes, as January 2017), revealed that 2,623 (48.59%) genomes showed at least one ter gene. At the family level, most (68.7%) genomes harbored one ter gene and 15.6% exhibited five (including P. glacincola BNF20). Overall, our results highlight the diverse nature (genetic and geographic diversity) of the Psychrobacter genus, provide insights into potential mechanisms of metal resistance, and exemplify the benefits of sampling remote locations for prospecting new molecular determinants.

Genetic diversity of Streptococcus suis isolated from three pig farms of China obtained by acquiring antibiotic resistance genes.

PubMed

Huang, Jinhu; Shang, Kexin; Kashif, Jam; Wang, Liping

2015-05-01

Acquiring antibiotic resistance genes may change an organism's genetic characteristics and the effect of antibiotics, resulting in a rapid transmission of microbial pathogens. The objectives of this experiment were to identify the features of Streptococcus suis (S. suis) isolated from three pig farms in China which are geographically isolated. Among the isolates, 56.52% were sequence type 7 (ST7), followed by ST1 (26.09%), indicating that ST7 prevails in China, as revealed by multi-locus sequence typing (MLST). Statistical analysis indicated an association between geography, sequence types and antibiotic resistance genotypes. 66.67% of the isolates in Sichuan province presented a (ermB(-) + mefA(-) + tetO(-) + tetM(-)) + ST7 type. The tetM(+) +ST7 type was the most prevalent in Jiangsu province, whereas the strains from Hebei province had a phenotype ermB(+) +tetO(+) +ST1 (63.64%). Pulsed-field gel electrophoresis (PGFE) pattern A2 with 100% similarity reflected the clonal dissemination between Sichuan and Jiangsu provinces. Strains carrying or not carrying antibiotic resistance genes presented different PFGE patterns in Hebei province. ST7 is widespread in many regions of China and a clonal dissemination occurred between Sichuan and Jiangsu provinces in diseased pigs. However, ST1 strains with macrolide and tetracycline resistance (ermB(+) +tetO(+) +ST1) isolated from a farm in Hebei province demonstrated that the genetic diversity was contributed by horizontal acquiring of ermB and tetO carrying elements. © 2014 Society of Chemical Industry.

Dominance of serotype Ia among group B Streptococci causing invasive infections in nonpregnant adults in Portugal.

PubMed

Martins, E R; Melo-Cristino, J; Ramirez, M

2012-04-01

The population of group B streptococci (GBS) associated with invasive infections in nonpregnant adults from 2001 to 2008 was analyzed in isolates submitted from 24 hospital laboratories in Portugal (n = 225). The isolates were characterized by antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and surface protein gene profiling. GBS invasive cases were found more frequently among men in all age groups. In addition, serotype Ia was the most frequent in our collection, whereas serotype V is dominant elsewhere. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and surface protein gene eps and by ST24 and bca, similarly to neonatal invasive infections in Portugal, indicating that the same genetic lineages can be responsible for both vaginal colonization and invasive disease in all age groups. In contrast, the hypervirulent serotype III/ST17 neonatal lineage was responsible for a minority of infections. Serotype V isolates were distributed into two genetic lineages, one defined by ST1 and surface protein gene alp3 and macrolide resistant, and another presenting with ST2 and eps and fully susceptible to all antimicrobials tested. The erm(TR) gene was the most frequently found among erythromycin-resistant isolates, while the bovine-associated tet(O) gene was found in a minority of tetracycline-resistant isolates. Our data emphasize the importance of local identification of the genetic lineages responsible for GBS invasive infections in nonpregnant adults. The dominance of serotype Ia in invasive disease in Portugal highlights the importance of this serotype in GBS pathogenesis.

Characterization of Genetic and Phenotypic Diversity of Invasive Nontypeable Haemophilus influenzae

PubMed Central

Erwin, Alice L.; Nelson, Kevin L.; Mhlanga-Mutangadura, Tendai; Bonthuis, Paul J.; Geelhood, Jennifer L.; Morlin, Gregory; Unrath, William C. T.; Campos, Jose; Crook, Derrick W.; Farley, Monica M.; Henderson, Frederick W.; Jacobs, Richard F.; Mühlemann, Kathrin; Satola, Sarah W.; van Alphen, Loek; Golomb, Miriam; Smith, Arnold L.

2005-01-01

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains. PMID:16113304

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk.

PubMed

Jørgensen, H J; Mørk, T; Caugant, D A; Kearns, A; Rørvik, L M

2005-12-01

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.

Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk

PubMed Central

Jørgensen, H. J.; Mørk, T.; Caugant, D. A.; Kearns, A.; Rørvik, L. M.

2005-01-01

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex. PMID:16332822

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

PubMed

Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Characterization of carbapenem resistance mechanisms and integrons in Pseudomonas aeruginosa strains from blood samples in a French hospital.

PubMed

Rojo-Bezares, Beatriz; Cavalié, Laurent; Dubois, Damien; Oswald, Eric; Torres, Carmen; Sáenz, Yolanda

2016-04-01

Metallo-β-lactamases (MBLs), porin OprD, integrons, virulence factors and the clonal relationships were characterized in imipenem-resistant Pseudomonas aeruginosa (IRPA) isolates. Fifty-six IRPA strains were recovered from blood samples of different patients at a Toulouse teaching hospital from 2011 to 2013. Susceptibility testing of 14 antibiotics was performed by the disc diffusion method. Detection and characterization of MBLs, the oprD gene and integrons were studied by PCR and sequencing. Thirteen genes involved in the virulence of P. aeruginosa were analysed. Molecular typing of IRPA strains was performed by PFGE and multilocus sequence typing. In this study, 61 % of the IRPA isolates showed a multi-resistance phenotype. The MBL phenotype, detected in three isolates (5.4 %), was linked to the blaVIM-2 gene. The oprD gene was amplified in 55 (98.2 %) IRPA strains, and variations were observed in 54 of them. Insertion sequences (IS) truncating oprD were detected in eight IRPA strains, with the novel ISPa56 identified in two strains. Class 1 integrons were detected in 24 (42.9 %) IRPA strains. The blaVIM-2 gene was found inside the class 1 integron arrangements. The new integrons In1054 (intI1-aacA56-qacEΔ1-sul1) and In1160 (intI1-aacA4-aacC1d-ISKpn4-gcuE-qacEΔ1-sul1) have been described for the first time, to the best of our knowledge, in this study. A high clonal diversity was found in our strains. Among the variety of sequence types (STs) found, ST175, ST233, ST235, ST244 and ST654 were noteworthy as epidemic clones. In conclusion, 5.4 % of IRPA strains showed an MBL phenotype linked to the blaVIM-2 gene. The identified oprD high polymorphism could be implicated in the variable resistance to carbapenems in IRPA strains. The dissemination of high-risk clones is a cause of concern.

Bradyrhizobium tropiciagri sp. nov. and Bradyrhizobium embrapense sp. nov., nitrogen-fixing symbionts of tropical forage legumes.

PubMed

Delamuta, Jakeline Renata Marçon; Ribeiro, Renan Augusto; Ormeño-Orrillo, Ernesto; Parma, Marcia Maria; Melo, Itamar Soares; Martínez-Romero, Esperanza; Hungria, Mariangela

2015-12-01

Biological nitrogen fixation is a key process for agricultural production and environmental sustainability, but there are comparatively few studies of symbionts of tropical pasture legumes, as well as few described species of the genus Bradyrhizobium, although it is the predominant rhizobial genus in the tropics. A detailed polyphasic study was conducted with two strains of the genus Bradyrhizobium used in commercial inoculants for tropical pastures in Brazil, CNPSo 1112T, isolated from perennial soybean (Neonotonia wightii), and CNPSo 2833T, from desmodium (Desmodium heterocarpon). Based on 16S-rRNA gene phylogeny, both strains were grouped in the Bradyrhizobium elkanii superclade, but were not clearly clustered with any known species. Multilocus sequence analysis of three (glnII, gyrB and recA) and five (plus atpD and dnaK) housekeeping genes confirmed that the strains are positioned in two distinct clades. Comparison with intergenic transcribed spacer sequences of type strains of described species of the genus Bradyrhizobium showed similarity lower than 93.1 %, and differences were confirmed by BOX-PCR analysis. Nucleotide identity of three housekeeping genes with type strains of described species ranged from 88.1 to 96.2 %. Average nucleotide identity of genome sequences showed values below the threshold for distinct species of the genus Bradyrhizobium ( < 90.6 %), and the value between the two strains was also below this threshold (91.2 %). Analysis of nifH and nodC gene sequences positioned the two strains in a clade distinct from other species of the genus Bradyrhizobium. Morphophysiological, genotypic and genomic data supported the description of two novel species in the genus Bradyrhizobium, Bradyrhizobium tropiciagri sp. nov. (type strain CNPSo 1112T = SMS 303T = BR 1009T = SEMIA 6148T = LMG 28867T) and Bradyrhizobium embrapense sp. nov. (type strain CNPSo 2833T = CIAT 2372T = BR 2212T = SEMIA 6208T = U674T = LMG 2987).

Multilocus phylogeny and phylogenomics of Eriochrysis P. Beauv. (Poaceae-Andropogoneae): Taxonomic implications and evidence of interspecific hybridization.

PubMed

Welker, Cassiano A D; Souza-Chies, Tatiana T; Longhi-Wagner, Hilda M; Peichoto, Myriam Carolina; McKain, Michael R; Kellogg, Elizabeth A

2016-06-01

Species delimitation is a vital issue concerning evolutionary biology and conservation of biodiversity. However, it is a challenging task for several reasons, including the low interspecies variability of markers currently used in phylogenetic reconstructions and the occurrence of reticulate evolution and polyploidy in many lineages of flowering plants. The first phylogeny of the grass genus Eriochrysis is presented here, focusing on the New World species, in order to examine its relationships to other genera of the subtribe Saccharinae/tribe Andropogoneae and to define the circumscriptions of its taxonomically complicated species. Molecular cloning and sequencing of five regions of four low-copy nuclear genes (apo1, d8, ep2-ex7 and ep2-ex8, kn1) were performed, as well as complete plastome sequencing. Trees were reconstructed using maximum parsimony, maximum likelihood, and Bayesian inference analyses. The present phylogenetic analyses indicate that Eriochrysis is monophyletic and the Old World E. pallida is sister to the New World species. Subtribe Saccharinae is polyphyletic, as is the genus Eulalia. Based on nuclear and plastome sequences plus morphology, we define the circumscriptions of the New World species of Eriochrysis: E. laxa is distinct from E. warmingiana, and E. villosa is distinct from E. cayennensis. Natural hybrids occur between E. laxa and E. villosa. The hybrids are probably tetraploids, based on the number of paralogues in the nuclear gene trees. This is the first record of a polyploid taxon in the genus Eriochrysis. Some incongruities between nuclear genes and plastome analyses were detected and are potentially caused by incomplete lineage sorting and/or ancient hybridization. The set of low-copy nuclear genes used in this study seems to be sufficient to resolve phylogenetic relationships and define the circumscriptions of other species complexes in the grass family and relatives, even in the presence of polyploidy and reticulate evolution. Complete plastome sequencing is also a promising tool for phylogenetic inference. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular analysis of Acinetobacter baumannii strains isolated in Lebanon using four different typing methods.

PubMed

Rafei, Rayane; Dabboussi, Fouad; Hamze, Monzer; Eveillard, Matthieu; Lemarié, Carole; Gaultier, Marie-Pierre; Mallat, Hassan; Moghnieh, Rima; Husni-Samaha, Rola; Joly-Guillou, Marie-Laure; Kempf, Marie

2014-01-01

This study analyzed 42 Acinetobacter baumannii strains collected between 2009-2012 from different hospitals in Beyrouth and North Lebanon to better understand the epidemiology and carbapenem resistance mechanisms in our collection and to compare the robustness of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), repetitive sequence-based PCR (rep-PCR) and blaOXA-51 sequence-based typing (SBT). Among 31 carbapenem resistant strains, we have detected three carbapenem resistance genes: 28 carried the blaOXA-23 gene, 1 the blaOXA-24 gene and 2 strains the blaOXA-58 gene. This is the first detection of blaOXA-23 and blaOXA-24 in Lebanon. PFGE identified 11 types and was the most discriminating technique followed by rep-PCR (9 types), blaOXA-51 SBT (8 types) and MLST (7 types). The PFGE type A'/ST2 was the dominant genotype in our collection present in Beyrouth and North Lebanon. The clustering agreement between all techniques was measured by adjust Wallace coefficient. An overall agreement has been demonstrated. High values of adjust Wallace coefficient were found with followed combinations: PFGE to predict MLST types  = 100%, PFGE to predict blaOXA-51 SBT = 100%, blaOXA-51 SBT to predict MLST = 100%, MLST to predict blaOXA-51 SBT = 84.7%, rep-PCR to predict MLST = 81.5%, PFGE to predict rep-PCR = 69% and rep-PCR to predict blaOXA-51 SBT = 67.2%. PFGE and MLST are gold standard methods for outbreaks investigation and population structure studies respectively. Otherwise, these two techniques are technically, time and cost demanding. We recommend the use of blaOXA-51 SBT as first typing method to screen isolates and assign them to their corresponding clonal lineages. Repetitive sequence-based PCR is a rapid tool to access outbreaks but careful interpretation of results must be always performed.

Resistant mechanisms and molecular epidemiology of imipenem-resistant Acinetobacter baumannii.

PubMed

Xiao, Shu-Zhen; Chu, Hai-Qing; Han, Li-Zhong; Zhang, Zhe-Min; Li, Bing; Zhao, Lan; Xu, Liyun

2016-09-01

The aim of the study was to investigate the resistant mechanisms and homology of imipenem-resistant Acinetobacter baumannii (A. baumannii). A total of 46 non-duplicate imipenem‑resistant A. baumannii clinical isolates were collected from three tertiary hospitals between July, 2011 and June, 2012. The minimal inhibitory concentrations (MICs) of antimicrobial agents were determined using the agar dilution method. Phenylalanine‑arginine β-naphthylamide was used to detect the presence of the efflux pump-mediated resistant mechanism. Polymerase chain reaction was employed to amplify genes associated with drug resistance, including β‑lactamase genes, efflux pump genes and outer membrane protein gene CarO. A few amplicons were randomly selected and sequenced. Multilocus sequence analysis (MLST) was employed in typing A. baumanni. A. baumannii was resistant to imipenem, simultaneously showing resistance to several other antimicrobials. In addtition, 13 A. baumannii were found to mediate drug resistance through operation of the efflux pump. Of the various drug resistance genes tested, blaOXA‑51 was present in 46 isolates, blaOXA‑23 gene was present in 44 isolates and blaNDM gene was found in only one strain. Other drug resistant‑associated genes, including blaKPC, blaIMP, blaOXA-24, blaOXA‑58, blaSHV, blaGIM and blaVIM were not detected. Mutation of adeS and outer membrane protein gene CarO were found in a few of the imipenem‑resistant isolates. The MLST analysis revealed that all 46 clinical isolates were clustered into 11 genotypes and the most frequent genotype was ST208. In conclusion, β‑lactamase genes, genes involved in efflux pump and mutation of outer membrane protein encoding gene may be important in mediating imipenem resistance in A. baumannii. Of the 11 different genotypes, ST11 was shared by the majority of A. baumannii, which may be due to horizontal transfer of patients from hospitals.

Occidiofungin is an important component responsible for the antifungal activity of Burkholderia pyrrocinia strain Lyc2.

PubMed

Wang, X Q; Liu, A X; Guerrero, A; Liu, J; Yu, X Q; Deng, P; Ma, L; Baird, S M; Smith, L; Li, X D; Lu, S E

2016-03-01

To identify the taxonomy of tobacco rhizosphere-isolated strain Lyc2 and investigate the mechanisms of the antifungal activities, focusing on antimicrobials gene clusters identification and function analysis. Multilocus sequence typing and 16S rRNA analyses indicated that strain Lyc2 belongs to Burkholderia pyrrocinia. Bioassay results indicated strain Lyc2 showed significant antifungal activities against a broad range of plant and animal fungal pathogens and control efficacy on seedling damping off disease of cotton. A 55·2-kb gene cluster which was homologous to ocf gene clusters in Burkholderia contaminans MS14 was confirmed to be responsible for antifungal activities by random mutagenesis; HPLC was used to verify the production of antifungal compounds. Multiple antibiotic and secondary metabolized biosynthesis gene clusters predicated by antiSMASH revealed the broad spectrum of antimicrobials activities of the strain. Our results revealed the mechanisms of antifungal activities of strain Lyc2 and expand our knowledge about production of occidiofungin in the bacteria Burkholderia. Understanding the mechanisms of antifungal activities of strain Lyc2 has contributed to discovery of new antibiotics and expand our knowledge of production of occidiofungin in the bacteria Burkholderia. © 2015 The Society for Applied Microbiology.

Wing patterning genes and coevolution of Müllerian mimicry in Heliconius butterflies: Support from phylogeography, cophylogeny, and divergence times.

PubMed

Hoyal Cuthill, Jennifer F; Charleston, Michael

2015-12-01

Examples of long-term coevolution are rare among free-living organisms. Müllerian mimicry in Heliconius butterflies had been suggested as a key example of coevolution by early genetic studies. However, research over the last two decades has been dominated by the idea that the best-studied comimics, H. erato and H. melpomene, did not coevolve at all. Recently sequenced genes associated with wing color pattern phenotype offer a new opportunity to resolve this controversy. Here, we test the hypothesis of coevolution between H. erato and H. melpomene using Bayesian multilocus analysis of five color pattern genes and five neutral genetic markers. We first explore the extent of phylogenetic agreement versus conflict between the different genes. Coevolution is then tested against three aspects of the mimicry diversifications: phylogenetic branching patterns, divergence times, and, for the first time, phylogeographic histories. We show that all three lines of evidence are compatible with strict coevolution of the diverse mimicry wing patterns, contrary to some recent suggestions. Instead, these findings tally with a coevolutionary diversification driven primarily by the ecological force of Müllerian mimicry. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

Additive Genetic Risk from Five Serotonin System Polymorphisms Interacts with Interpersonal Stress to Predict Depression

PubMed Central

Vrshek-Schallhorn, Suzanne; Stroud, Catherine B.; Mineka, Susan; Zinbarg, Richard E.; Adam, Emma K.; Redei, Eva E.; Hammen, Constance; Craske, Michelle G.

2016-01-01

Behavioral genetic research supports polygenic models of depression in which many genetic variations each contribute a small amount of risk, and prevailing diathesis-stress models suggest gene-environment interactions (GxE). Multilocus profile scores of additive risk offer an approach that is consistent with polygenic models of depression risk. In a first demonstration of this approach in a GxE predicting depression, we created an additive multilocus profile score from five serotonin system polymorphisms (one each in the genes HTR1A, HTR2A, HTR2C, and two in TPH2). Analyses focused on two forms of interpersonal stress as environmental risk factors. Using five years of longitudinal diagnostic and life stress interviews from 387 emerging young adults in the Youth Emotion Project, survival analyses show that this multilocus profile score interacts with major interpersonal stressful life events to predict major depressive episode onsets (HR = 1.815, p = .007). Simultaneously, there was a significant protective effect of the profile score without a recent event (HR = 0.83, p = .030). The GxE effect with interpersonal chronic stress was not significant (HR = 1.15, p = .165). Finally, effect sizes for genetic factors examined ignoring stress suggested such an approach could lead to overlooking or misinterpreting genetic effects. Both the GxE effect and the protective simple main effect were replicated in a sample of early adolescent girls (N = 105). We discuss potential benefits of the multilocus genetic profile score approach and caveats for future research. PMID:26595467

Application of DNA barcoding in biodiversity studies of shallow-water octocorals: molecular proxies agree with morphological estimates of species richness in Palau

NASA Astrophysics Data System (ADS)

McFadden, C. S.; Brown, A. S.; Brayton, C.; Hunt, C. B.; van Ofwegen, L. P.

2014-06-01

The application of DNA barcoding to anthozoan cnidarians has been hindered by their slow rates of mitochondrial gene evolution and the failure to identify alternative molecular markers that distinguish species reliably. Among octocorals, however, multilocus barcodes can distinguish up to 70 % of morphospecies, thereby facilitating the identification of species that are ecologically important but still very poorly known taxonomically. We tested the ability of these imperfect DNA barcodes to estimate species richness in a biodiversity survey of the shallow-water octocoral fauna of Palau using multilocus ( COI, mtMutS, 28S rDNA) sequences obtained from 305 specimens representing 38 genera of octocorals. Numbers and identities of species were estimated independently (1) by a taxonomic expert using morphological criteria and (2) by assigning sequences to molecular operational taxonomic units (MOTUs) using predefined genetic distance thresholds. Estimated numbers of MOTUs ranged from 73 to 128 depending on the barcode and distance threshold applied, bracketing the estimated number of 118 morphospecies. Concordance between morphospecies identifications and MOTUs ranged from 71 to 75 % and differed little among barcodes. For the speciose and ecologically dominant genus Sinularia, however, we were able to identify 95 % of specimens correctly simply by comparing mtMutS sequences and in situ photographs of colonies to an existing vouchered database. Because we lack a clear understanding of species boundaries in most of these taxa, numbers of morphospecies and MOTUs are both estimates of the true species diversity, and we cannot currently determine which is more accurate. Our results suggest, however, that the two methods provide comparable estimates of species richness for shallow-water Indo-Pacific octocorals. Use of molecular barcodes in biodiversity surveys will facilitate comparisons of species richness and composition among localities and over time, data that do not currently exist for any octocoral community.

Is the extremely rare Iberian endemic plant species Castrilanthemum debeauxii (Compositae, Anthemideae) a 'living fossil'? Evidence from a multi-locus species tree reconstruction.

PubMed

Tomasello, Salvatore; Álvarez, Inés; Vargas, Pablo; Oberprieler, Christoph

2015-01-01

The present study provides results of multi-species coalescent species tree analyses of DNA sequences sampled from multiple nuclear and plastid regions to infer the phylogenetic relationships among the members of the subtribe Leucanthemopsidinae (Compositae, Anthemideae), to which besides the annual Castrilanthemum debeauxii (Degen, Hervier & É.Rev.) Vogt & Oberp., one of the rarest flowering plant species of the Iberian Peninsula, two other unispecific genera (Hymenostemma, Prolongoa), and the polyploidy complex of the genus Leucanthemopsis belong. Based on sequence information from two single- to low-copy nuclear regions (C16, D35, characterised by Chapman et al. (2007)), the multi-copy region of the nrDNA internal transcribed spacer regions ITS1 and ITS2, and two intergenic spacer regions of the cpDNA gene trees were reconstructed using Bayesian inference methods. For the reconstruction of a multi-locus species tree we applied three different methods: (a) analysis of concatenated sequences using Bayesian inference (MrBayes), (b) a tree reconciliation approach by minimizing the number of deep coalescences (PhyloNet), and (c) a coalescent-based species-tree method in a Bayesian framework ((∗)BEAST). All three species tree reconstruction methods unequivocally support the close relationship of the subtribe with the hitherto unclassified genus Phalacrocarpum, the sister-group relationship of Castrilanthemum with the three remaining genera of the subtribe, and the further sister-group relationship of the clade of Hymenostemma+Prolongoa with a monophyletic genus Leucanthemopsis. Dating of the (∗)BEAST phylogeny supports the long-lasting (Early Miocene, 15-22Ma) taxonomical independence and the switch from the plesiomorphic perennial to the apomorphic annual life-form assumed for the Castrilanthemum lineage that may have occurred not earlier than in the Pliocene (3Ma) when the establishment of a Mediterranean climate with summer droughts triggered evolution towards annuality. Copyright © 2014 Elsevier Inc. All rights reserved.

Ancestral Polymorphisms and Sex-Biased Migration Shaped the Demographic History of Brown Bears and Polar Bears

PubMed Central

Nakagome, Shigeki; Mano, Shuhei; Hasegawa, Masami

2013-01-01

Recent studies have reported discordant gene trees in the evolution of brown bears and polar bears. Genealogical histories are different among independent nuclear loci and between biparentally inherited autosomal DNA (aDNA) and matrilineal mitochondrial DNA (mtDNA). Based on multi-locus genomic sequences from aDNA and mtDNA, we inferred the population demography of brown and polar bears and found that brown bears have 6 times (aDNA) or more than 14 times (mtDNA) larger population sizes than polar bears and that polar bear lineage is derived from within brown bear diversity. In brown bears, the effective population size ratio of mtDNA to aDNA was at least 0.62, which deviated from the expected value of 0.25, suggesting matriarchal population due to female philopatry and male-biased migration. These results emphasize that ancestral polymorphisms and sex-biased migration may have contributed to conflicting branching patterns in brown and polar bears across aDNA genes and mtDNA. PMID:24236053

Ancestral polymorphisms and sex-biased migration shaped the demographic history of brown bears and polar bears.

PubMed

Nakagome, Shigeki; Mano, Shuhei; Hasegawa, Masami

2013-01-01

Recent studies have reported discordant gene trees in the evolution of brown bears and polar bears. Genealogical histories are different among independent nuclear loci and between biparentally inherited autosomal DNA (aDNA) and matrilineal mitochondrial DNA (mtDNA). Based on multi-locus genomic sequences from aDNA and mtDNA, we inferred the population demography of brown and polar bears and found that brown bears have 6 times (aDNA) or more than 14 times (mtDNA) larger population sizes than polar bears and that polar bear lineage is derived from within brown bear diversity. In brown bears, the effective population size ratio of mtDNA to aDNA was at least 0.62, which deviated from the expected value of 0.25, suggesting matriarchal population due to female philopatry and male-biased migration. These results emphasize that ancestral polymorphisms and sex-biased migration may have contributed to conflicting branching patterns in brown and polar bears across aDNA genes and mtDNA.

Human Staphylococcus aureus lineages among Zoological Park residents in Greece

PubMed Central

Drougka, E.; Foka, A.; Posantzis, D.; Giormezis, N.; Anastassiou, E.D.; Petinaki, E.; Spiliopoulou, I.

2015-01-01

Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL) and tst (toxic shock syndrome toxin-1) were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST), spa type and Pulsed-Field Gel Electrophoresis (PFGE). Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred. PMID:26623381

Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

PubMed

Kubo, Yuji; Rooney, Alejandro P; Tsukakoshi, Yoshiki; Nakagawa, Rikio; Hasegawa, Hiromasa; Kimura, Keitarou

2011-09-01

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.

Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production ▿

PubMed Central

Kubo, Yuji; Rooney, Alejandro P.; Tsukakoshi, Yoshiki; Nakagawa, Rikio; Hasegawa, Hiromasa; Kimura, Keitarou

2011-01-01

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group. PMID:21764950

Genetic relatedness, antimicrobial and biocide susceptibility comparative analysis of methicillin-resistant and -susceptible Staphylococcus pseudintermedius from Portugal.

PubMed

Couto, Natacha; Belas, Adriana; Couto, Isabel; Perreten, Vincent; Pomba, Constança

2014-08-01

Forty methicillin-resistant and -susceptible Staphylococcus pseudintermedius (MRSP and MSSP, respectively) from colonization and infection in dogs and cats were characterized for clonality, antimicrobial, and biocide susceptibility. MSSP were genetically more diverse than MRSP by multi-locus sequence typing and pulsed-field gel electrophoresis. Three different spa types (t06, t02, t05) and two SCCmec types (II-III and V) were detected in the MRSP isolates. All MRSP and two MSSP strains were multidrug-resistant. Several antibiotic resistance genes (mecA, blaZ, tet(M), tet(K), aac(6')-Ie-aph(2')-Ia, aph(3')-III, ant(6)-Ia, sat4, erm(B), lnu(A), dfr(G), and catp(C221)) were identified by microarray and double mutations in the gyrA and grlA genes and a single mutation in the rpoB gene were detected by sequence analysis. No differences were detected between MSSP and MRSP in the chlorhexidine acetate (CHA) minimum inhibitory concentrations (MICs). However, two MSSP had elevated MIC to triclosan (TCL) and one to benzalkonium chloride and ethidium bromide. One MSSP isolate harboured a qacA gene, while in another a qacB gene was detected. None of the isolates harboured the sh-fabI gene. Three of the biocide products studied had high bactericidal activity (Otodine(®), Clorexyderm Spot Gel(®), Dermocanis Piocure-M(®)), while Skingel(®) failed to achieve a five log reduction in the bacterial counting. S. pseudintermedius have become a serious therapeutic challenge in particular if methicillin- resistance and/or multidrug-resistance are involved. Biocides, like CHA and TCL, seem to be clinically effective and safe topical therapeutic options.

Whole-Genome Sequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods.

PubMed

Kamada, Mayumi; Hase, Sumitaka; Fujii, Kazushi; Miyake, Masato; Sato, Kengo; Kimura, Keitarou; Sakakibara, Yasubumi

2015-01-01

Bacillus subtilis is the main component in the fermentation of soybeans. To investigate the genetics of the soybean-fermenting B. subtilis strains and its relationship with the productivity of extracellular poly-γ-glutamic acid (γPGA), we sequenced the whole genome of eight B. subtilis stains isolated from non-salted fermented soybean foods in Southeast Asia. Assembled nucleotide sequences were compared with those of a natto (fermented soybean food) starter strain B. subtilis BEST195 and the laboratory standard strain B. subtilis 168 that is incapable of γPGA production. Detected variants were investigated in terms of insertion sequences, biotin synthesis, production of subtilisin NAT, and regulatory genes for γPGA synthesis, which were related to fermentation process. Comparing genome sequences, we found that the strains that produce γPGA have a deletion in a protein that constitutes the flagellar basal body, and this deletion was not found in the non-producing strains. We further identified diversity in variants of the bio operon, which is responsible for the biotin auxotrophism of the natto starter strains. Phylogenetic analysis using multilocus sequencing typing revealed that the B. subtilis strains isolated from the non-salted fermented soybeans were not clustered together, while the natto-fermenting strains were tightly clustered; this analysis also suggested that the strain isolated from "Tua Nao" of Thailand traces a different evolutionary process from other strains.

Two Atypical Cases of Kingella kingae Invasive Infection with Concomitant Human Rhinovirus Infection

PubMed Central

Basmaci, Romain; Ilharreborde, Brice; Doit, Catherine; Presedo, Ana; Lorrot, Mathie; Alison, Marianne; Mazda, Keyvan; Bidet, Philippe

2013-01-01

We describe two atypical cases of Kingella kingae infection in children diagnosed by PCR, one case involving a soft tissue abscess and one case a femoral Brodie abscess. Both patients had concomitant human rhinovirus infection. K. kingae strains, isolated from an oropharyngeal swab, were characterized by multilocus sequence typing and rtxA sequencing. PMID:23784119

Significant spread of extensively drug-resistant Acinetobacter baumannii genotypes of clonal complex 92 among intensive care unit patients in a university hospital in southern Iran.

PubMed

Saffari, Fereshteh; Monsen, Tor; Karmostaji, Afsaneh; Azimabad, Fahimeh Bahadori; Widerström, Micael

2017-11-01

Infections associated with Acinetobacter baumannii represent an increasing threat in healthcare settings. Therefore, we investigated the epidemiological relationship between clinical isolates of A. baumannii obtained from patients in a university hospital in Bandar Abbas in southern Iran. Sixty-four consecutive non-duplicate clinical isolates collected during 2014-2015 were subjected to susceptibility testing, clonal relationship analysis using PFGE, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST), and examined for the presence of carbapenemases and integrons. Almost all A. baumannii isolates were extensively drug-resistant (XDR; 98 %) and carried an OXA carbapenemase gene (blaOXA-23-like; 98 %) and class 1 integrons (48 %). PFGE and MLST analysis identified three major genotypes, all belonging to clonal complex 92 (CC92): sequence type 848 (ST848) (n=23), ST451 (n=16) and ST195 (n=8). CC92 has previously been documented in the hospital setting in northern Iran, and ST195 has been reported in Arab States of the Persian Gulf. These data suggest national and global transmission of A. baumannii CC92. This report demonstrates the occurrence and potential spread of closely related XDR genotypes of A. baumannii CC92 within a university hospital in southern Iran. These genotypes were found in the majority of the investigated isolates, showed high prevalence of blaOXA-23 and integron class 1, and were associated with stay in the intensive care unit. Very few treatment options remain for healthcare-adapted XDR A. baumannii, and hence effective measures are desperately needed to reduce the spread of these strains and resultant infections in the healthcare setting.

Systematic characterization of Bacillus Genetic Stock Center Bacillus thuringiensis strains using Multi-Locus Sequence Typing.

PubMed

Wang, Kui; Shu, Changlong; Soberón, Mario; Bravo, Alejandra; Zhang, Jie

2018-04-30

The goal of this work was to perform a systematic characterization of Bacillus thuringiensis (Bt) strains from the Bacillus Genetic Stock Center (BGSC) collection using Multi-Locus Sequence Typing (MLST). Different genetic markers of 158 Bacillus thuringiensis (Bt) strains from 73 different serovars stored in the BGSC, that represented 92% of the different Bt serovars of the BGSC were analyzed, the 8% that were not analyzed were not available. In addition, we analyzed 72 Bt strains from 18 serovars available at the pubMLST bcereus database, and Bt strains G03, HBF18 and Bt185, with no H serovars provided by our laboratory. We performed a systematic MLST analysis using seven housekeeping genes (glpF, gmK, ilvD, pta, pur, pycA and tpi) and analyzed correlation of the results of this analysis with strain serovars. The 233 Bt strains analyzed were assigned to 119 STs from which 19 STs were new. Genetic relationships were established by phylogenetic analysis and showed that STs could be grouped in two major Clusters containing 21 sub-groups. We found that a significant number of STs (101 in total) correlated with specific serovars, such as ST13 that corresponded to nine Bt isolates from B. thuringiensis serovar kenyae. However, other serovars showed high genetic variability and correlated with multiple STs; for example, B. thuringiensis serovar morrisoni correlated with 11 different STs. In addition, we found that 16 different STs correlated with multiple serovars (2-4 different serovars); for example, ST12 correlated with B. thuringiensis serovar alesti, dakota, palmanyolensis and sotto/dendrolimus. These data indicated that only partial correspondence between MLST and serotyping can be established. Copyright © 2018 Elsevier Inc. All rights reserved.

Multilocus Sequence Analysis of Streptococcus canis Confirms the Zoonotic Origin of Human Infections and Reveals Genetic Exchange with Streptococcus dysgalactiae subsp. equisimilis

PubMed Central

Pinho, M. D.; Matos, S. C.; Pomba, C.; Lübke-Becker, A.; Wieler, L. H.; Preziuso, S.; Melo-Cristino, J.

2013-01-01

Streptococcus canis is an animal pathogen that occasionally causes human infections. Isolates recovered from infections of animals (n = 78, recovered from 2000 to 2010 in three European countries, mainly from house pets) and humans (n = 7, recovered from 2006 to 2010 in Portugal) were identified by phenotypic and genotypic methods and characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and emm typing. S. canis isolates presented considerable variability in biochemical profiles and 16S rRNA. Resistance to antimicrobial agents was low, with the most significant being tet(M)- and tet(O)-mediated tetracycline resistance. MLST analysis revealed a polyclonal structure of the S. canis population causing infections, where the same genetic lineages were found infecting house pets and humans and were disseminated in distinct geographic locations. Phylogenetic analysis indicated that S. canis was a divergent taxon of the sister species Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis and found evidence of acquisition of genetic material by S. canis from S. dysgalactiae subsp. equisimilis. PFGE confirmed the MLST findings, further strengthening the similarity between animal and human isolates. The presence of emm-like genes was restricted to a few isolates and correlated with some MLST-based genetic lineages, but none of the human isolates could be emm typed. Our data show that S. canis isolates recovered from house pets and humans constitute a single population and demonstrate that isolates belonging to the main genetic lineages identified have the ability to infect the human host, providing strong evidence for the zoonotic nature of S. canis infection. PMID:23345291

Multilocus sequence analysis of Streptococcus canis confirms the zoonotic origin of human infections and reveals genetic exchange with Streptococcus dysgalactiae subsp. equisimilis.

PubMed

Pinho, M D; Matos, S C; Pomba, C; Lübke-Becker, A; Wieler, L H; Preziuso, S; Melo-Cristino, J; Ramirez, M

2013-04-01

Streptococcus canis is an animal pathogen that occasionally causes human infections. Isolates recovered from infections of animals (n = 78, recovered from 2000 to 2010 in three European countries, mainly from house pets) and humans (n = 7, recovered from 2006 to 2010 in Portugal) were identified by phenotypic and genotypic methods and characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and emm typing. S. canis isolates presented considerable variability in biochemical profiles and 16S rRNA. Resistance to antimicrobial agents was low, with the most significant being tet(M)- and tet(O)-mediated tetracycline resistance. MLST analysis revealed a polyclonal structure of the S. canis population causing infections, where the same genetic lineages were found infecting house pets and humans and were disseminated in distinct geographic locations. Phylogenetic analysis indicated that S. canis was a divergent taxon of the sister species Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis and found evidence of acquisition of genetic material by S. canis from S. dysgalactiae subsp. equisimilis. PFGE confirmed the MLST findings, further strengthening the similarity between animal and human isolates. The presence of emm-like genes was restricted to a few isolates and correlated with some MLST-based genetic lineages, but none of the human isolates could be emm typed. Our data show that S. canis isolates recovered from house pets and humans constitute a single population and demonstrate that isolates belonging to the main genetic lineages identified have the ability to infect the human host, providing strong evidence for the zoonotic nature of S. canis infection.

Multilocus Sequence Typing of Bartonella henselae in the United Kingdom Indicates that Only a Few, Uncommon Sequence Types Are Associated with Zoonotic Disease▿†

PubMed Central

Chaloner, Gemma L.; Harrison, Timothy G.; Coyne, Karen P.; Aanensen, David M.; Birtles, Richard J.

2011-01-01

Bartonella henselae is one of the most common zoonotic agents acquired from companion animals (cats) in industrialized countries. Nonetheless, although the prevalence of infections in cats is high, the number of human cases reported is relatively low. One hypothesis for this discrepancy is that B. henselae strains vary in their zoonotic potential. To test this hypothesis, we employed structured sampling to explore the population structure of B. henselae in the United Kingdom and to determine the distribution of strains associated with zoonotic disease within this structure. A total of 118 B. henselae strains were delineated into 12 sequence types (STs) using multilocus sequence typing. We observed that most (85%) of the zoonosis-associated strains belonged to only three genotypes, i.e., ST2, ST5, and ST8. Conversely, most (74%) of the feline isolates belonged to ST4, ST6, and ST7. The difference in host association of ST2, ST5, and ST8 (zoonosis associated) and ST6 (feline) was statistically significant (P < 0.05), indicating that a few, uncommon STs were responsible for the majority of symptomatic human infections. PMID:21471345

The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa.

PubMed

Nunney, L; Elfekih, S; Stouthamer, R

2012-05-01

Microbial identification methods have evolved rapidly over the last few decades. One such method is multilocus sequence typing (MLST). MLST is a powerful tool for understanding the evolutionary dynamics of pathogens and to gain insight into their genetic diversity. We illustrate the importance of accurate typing by reporting on three problems that have arisen in the study of a single bacterial species, the plant pathogen Xylella fastidiosa. Two of these were particularly serious since they concerned contamination of important research material that has had detrimental consequences for Xylella research: the contamination of DNA used in the sequencing of an X. fastidiosa genome (Ann-1) with DNA from another X. fastidiosa strain, and the unrecognized mislabeling of a strain (Temecula1) distributed from a culture collection (ATCC). We advocate the routine use of MLST to define strains maintained in culture collections and emphasize the importance of confirming the purity of DNA submitted for sequencing. We also present a third example that illustrates the value of MLST in guiding the choice of taxonomic types. Beyond these situations, there is a strong case for MLST whenever an isolate is used experimentally, especially where genotypic differences are suspected to influence the outcome.

The importance of molecular analyses for understanding the genetic diversity of Histoplasma capsulatum: an overview.

PubMed

Vite-Garín, Tania; Estrada-Bárcenas, Daniel Alfonso; Cifuentes, Joaquín; Taylor, Maria Lucia

2014-01-01

Advances in the classification of the human pathogen Histoplasma capsulatum (H. capsulatum) (ascomycete) are sustained by the results of several genetic analyses that support the high diversity of this dimorphic fungus. The present mini-review highlights the great genetic plasticity of H. capsulatum. Important records with different molecular tools, mainly single- or multi-locus sequence analyses developed with this fungus, are discussed. Recent phylogenetic data with a multi-locus sequence analysis using 5 polymorphic loci support a new clade and/or phylogenetic species of H. capsulatum for the Americas, which was associated with fungal isolates obtained from the migratory bat Tadarida brasiliensis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Life on the rocks: Multilocus phylogeography of rock hyrax (Procavia capensis) from southern Africa.

PubMed

Maswanganye, K Amanda; Cunningham, Michael J; Bennett, Nigel C; Chimimba, Christian T; Bloomer, Paulette

2017-09-01

Understanding the role of geography and climatic cycles in determining patterns of biodiversity is important in comparative and evolutionary biology and conservation. We studied the phylogeographic pattern and historical demography of a rock-dwelling small mammal species from southern Africa, the rock hyrax Procavia capensis capensis. Using a multilocus coalescent approach, we assessed the influence of strong habitat dependence and fluctuating regional climates on genetic diversity. We sequenced a mitochondrial gene (cytochrome b) and two nuclear introns (AP5, PRKC1) supplemented with microsatellite genotyping, in order to assess evolutionary processes over multiple temporal scales. In addition, distribution modelling was used to investigate the current and predicted distribution of the species under different climatic scenarios. Collectively, the data reveal a complex history of isolation followed by secondary contact shaping the current intraspecific diversity. The cyt b sequences confirmed the presence of two previously proposed geographically and genetically distinct lineages distributed across the southern African Great Escarpment and north-western mountain ranges. Molecular dating suggests Miocene divergence of the lineages, yet there are no discernible extrinsic barriers to gene flow. The nuclear markers reveal incomplete lineage sorting or ongoing mixing of the two lineages. Although the microsatellite data lend some support to the presence of two subpopulations, there is weak structuring within and between lineages. These data indicate the presence of gene flow from the northern into the southern parts of the southern African sub-region likely following the secondary contact. The distribution modelling predictably reveal the species' preference for rocky areas, with stable refugia through time in the northern mountain ranges, the Great Escarpment, as well as restricted areas of the Northern Cape Province and the Cape Fold Mountains of South Africa. Different microclimatic variables appear to determine the distributional range of the species. Despite strong habitat preference, the micro-habitat offered by rocky crevices and unique life history traits likely promoted the adaptability of P. capensis, resulting in the widespread distribution and persistence of the species over a long evolutionary period. Spatio-temporal comparison of the evolutionary histories of other co-distributed species across the rocky landscapes of southern Africa will improve our understanding of the regional patterns of biodiversity and local endemism. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA Microarray Profiling of a Diverse Collection of Nosocomial Methicillin-Resistant Staphylococcus aureus Isolates Assigns the Majority to the Correct Sequence Type and Staphylococcal Cassette Chromosome mec (SCCmec) Type and Results in the Subsequent Identification and Characterization of Novel SCCmec-SCCM1 Composite Islands

PubMed Central

Brennan, Orla M.; Deasy, Emily C.; Rossney, Angela S.; Kinnevey, Peter M.; Ehricht, Ralf; Monecke, Stefan; Coleman, David C.

2012-01-01

One hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus (MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were characterized by spa typing (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmec types and subtypes and 35 spa types. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100/107) were assigned an ST, with 98% (98/100) correlating with MLST. The array assigned all isolates to the correct SCCmec type, but subtyping of only some SCCmec elements was possible. Additional SCCmec/SCC genes or DNA sequence variation not detected by SCCmec typing was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50/fusC. Novel SCCmec/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmec IIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCCM1 from ST8/t024-MRSA, SCCmec VIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100/107) and immune evasion cluster (91%; 97/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs/STs and SCCmec types and provided further evidence of the diversity of SCCmec/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate. PMID:22869569

Multilocus resolution of Mugilidae phylogeny (Teleostei: Mugiliformes): Implications for the family's taxonomy.

PubMed

Xia, Rong; Durand, Jean-Dominique; Fu, Cuizhang

2016-03-01

The interrelationships among mugilids (Mugiliformes: Mugilidae) remain highly debated. Using a mitochondrial gene-based phylogeny as criterion, a revised classification with 25 genera in the Mugilidae has recently been proposed. However, phylogenetic relationships of major mitochondrial lineages remain unresolved and to gain a general acceptance the classification requires confirmation based on multilocus evidence and diagnostic morphological characters. Here, we construct a species-tree using twelve nuclear and three mitochondrial loci and infer the evolution of 71 morphological characters. Our multilocus phylogeny does not agree with previous morphology-based hypotheses for the relationships within Mugilidae, confirms the revised classification with 25 genera and further resolves their phylogenetic relationships. Using the well-resolved multilocus phylogeny as the criterion, we reclassify Mugilidae genera into three new subfamilies (Myxinae, Rhinomugilinae, and Cheloninae) and one new, recombined, subfamily (Mugilinae). The Rhinomugilinae subfamily is further divided into four tribes. The revised classification of Mugilidae is supported by morpho-anatomical synapomorphies or a combination of characters. These characters are used to erect a key to the subfamilies and genera. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of Assays Using Hexokinase and Phosphoglucomutase Gene Sequences That Distinguish Strains of Leishmania tropica from Different Zymodemes and Microsatellite Clusters and Their Application to Palestinian Foci of Cutaneous Leishmaniasis

PubMed Central

Azmi, Kifaya; Schonian, Gabriele; Schnur, Lionel F.; Nasereddin, Abedelmajeed; Ereqat, Suheir; Abdeen, Ziad

2013-01-01

Background/Objectives Palestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307. Methodology/Principle Findings Assays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system ‘correctly’ identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter. Conclusions/Significance Both assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE. PMID:24086789

Multilocus Sex Determination Revealed in Two Populations of Gynodioecious Wild Strawberry, Fragaria vesca subsp. bracteata

PubMed Central

Ashman, Tia-Lynn; Tennessen, Jacob A.; Dalton, Rebecca M.; Govindarajulu, Rajanikanth; Koski, Matthew H.; Liston, Aaron

2015-01-01

Gynodioecy, the coexistence of females and hermaphrodites, occurs in 20% of angiosperm families and often enables transitions between hermaphroditism and dioecy. Clarifying mechanisms of sex determination in gynodioecious species can thus illuminate sexual system evolution. Genetic determination of gynodioecy, however, can be complex and is not fully characterized in any wild species. We used targeted sequence capture to genetically map a novel nuclear contributor to male sterility in a self-pollinated hermaphrodite of Fragaria vesca subsp. bracteata from the southern portion of its range. To understand its interaction with another identified locus and possibly additional loci, we performed crosses within and between two populations separated by 2000 km, phenotyped the progeny and sequenced candidate markers at both sex-determining loci. The newly mapped locus contains a high density of pentatricopeptide repeat genes, a class commonly involved in restoration of fertility caused by cytoplasmic male sterility. Examination of all crosses revealed three unlinked epistatically interacting loci that determine sexual phenotype and vary in frequency between populations. Fragaria vesca subsp. bracteata represents the first wild gynodioecious species with genomic evidence of both cytoplasmic and nuclear genes in sex determination. We propose a model for the interactions between these loci and new hypotheses for the evolution of sex determining chromosomes in the subdioecious and dioecious Fragaria. PMID:26483011

New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP131-18.

PubMed

Paulus, Constanze; Rebets, Yuriy; Tokovenko, Bogdan; Nadmid, Suvd; Terekhova, Larisa P; Myronovskyi, Maksym; Zotchev, Sergey B; Rückert, Christian; Braig, Simone; Zahler, Stefan; Kalinowski, Jörn; Luzhetskyy, Andriy

2017-02-10

Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family - lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites.

New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP131-18

PubMed Central

Paulus, Constanze; Rebets, Yuriy; Tokovenko, Bogdan; Nadmid, Suvd; Terekhova, Larisa P.; Myronovskyi, Maksym; Zotchev, Sergey B.; Rückert, Christian; Braig, Simone; Zahler, Stefan; Kalinowski, Jörn; Luzhetskyy, Andriy

2017-01-01

Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family – lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites. PMID:28186197

Highly diverse recombining populations of Vibrio cholerae and Vibrio parahaemolyticus in French Mediterranean coastal lagoons

PubMed Central

Esteves, Kévin; Mosser, Thomas; Aujoulat, Fabien; Hervio-Heath, Dominique; Monfort, Patrick; Jumas-Bilak, Estelle

2015-01-01

Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species found in Mediterranean coastal systems can induce infections in humans. Environmental isolates of V. cholerae (n = 109) and V. parahaemolyticus (n = 89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus, and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity condition for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity condition. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk. PMID:26236294