The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution

PubMed Central

McDowell, Andrew; Nagy, István; Magyari, Márta; Barnard, Emma; Patrick, Sheila

2013-01-01

We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST4) that correctly predicted the phylogroup (IA1, IA2, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http://pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST4 method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination; although the latter still plays an important role in the bacterium's evolution. The secreted/cell-associated ‘virulence’ factors tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P. acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and the effect such treatments may have on commensal lineages. PMID:24058439

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.

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.

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.

Insights into the Emergent Bacterial Pathogen Cronobacter spp., Generated by Multilocus Sequence Typing and Analysis

PubMed Central

Joseph, Susan; Forsythe, Stephen J.

2012-01-01

Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognized route of infection being the consumption of contaminated infant formula. As a recently recognized bacterial pathogen of considerable importance and regulatory control, appropriate detection, and identification schemes are required. The application of multilocus sequence typing (MLST) and analysis (MLSA) of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB, and ppsA (concatenated length 3036 base pairs) has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognized genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti) and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health. PMID:23189075

The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus.

PubMed

Blanchard, Adam M; Jolley, Keith A; Maiden, Martin C J; Coffey, Tracey J; Maboni, Grazieli; Staley, Ceri E; Bollard, Nicola J; Warry, Andrew; Emes, Richard D; Davies, Peers L; Tötemeyer, Sabine

2018-01-01

Dichelobacter nodosus ( D. nodosus ) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

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

Whole genome sequencing analyses of Listeria monocytogenes that persisted in a milkshake machine for a year and caused illnesses in Washington State.

PubMed

Li, Zhen; Pérez-Osorio, Ailyn; Wang, Yu; Eckmann, Kaye; Glover, William A; Allard, Marc W; Brown, Eric W; Chen, Yi

2017-06-15

In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates. The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

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.

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.

Multilocus sequence typing and pulsed-field gel electrophoresis analysis of Oenococcus oeni from different wine-producing regions of China.

PubMed

Wang, Tao; Li, Hua; Wang, Hua; Su, Jing

2015-04-16

The present study established a typing method with NotI-based pulsed-field gel electrophoresis (PFGE) and stress response gene schemed multilocus sequence typing (MLST) for 55 Oenococcus oeni strains isolated from six individual regions in China and two model strains PSU-1 (CP000411) and ATCC BAA-1163 (AAUV00000000). Seven stress response genes, cfa, clpL, clpP, ctsR, mleA, mleP and omrA, were selected for MLST testing, and positive selective pressure was detected for these genes. Furthermore, both methods separated the strains into two clusters. The PFGE clusters are correlated with the region, whereas the sequence types (STs) formed by the MLST confirm the two clusters identified by PFGE. In addition, the population structure was a mixture of evolutionary pathways, and the strains exhibited both clonal and panmictic characteristics. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Study of Streptococcus thermophilus population on a world-wide and historical collection by a new MLST scheme.

PubMed

Delorme, Christine; Legravet, Nicolas; Jamet, Emmanuel; Hoarau, Caroline; Alexandre, Bolotin; El-Sharoud, Walid M; Darwish, Mohamed S; Renault, Pierre

2017-02-02

We analyzed 178 Streptococcus thermophilus strains isolated from diverse products, from around the world, over a 60-year period with a new multilocus sequence typing (MLST) scheme. This collection included isolates from two traditional cheese-making sites with different starter-use practices, in sampling campaigns carried out over a three years period. The nucleotide diversity of the S. thermophilus population was limited, but 116 sequence types (ST) were identified. Phylogenetic analysis of the concatenated sequences of the six housekeeping genes revealed the existence of groups confirmed by eBURST analysis. Deeper analyses performed on 25 strains by CRISPR and whole-genome analysis showed that phylogenies obtained by MLST and whole-genome analysis were in agreement but differed from that inferred by CRISPR analysis. Strains isolated from traditional products could cluster in specific groups indicating their origin, but also be mixed in groups containing industrial starter strains. In the traditional cheese-making sites, we found that S. thermophilus persisted on dairy equipment, but that occasionally added starter strains may become dominant. It underlined the impact of starter use that may reshape S. thermophilus populations including in traditional products. This new MLST scheme thus provides a framework for analyses of S. thermophilus populations and the management of its biodiversity. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

Phylogenetic, epidemiological and functional analyses of the Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme.

PubMed

Jans, Christoph; de Wouters, Tomas; Bonfoh, Bassirou; Lacroix, Christophe; Kaindi, Dasel Wambua Mulwa; Anderegg, Janine; Böck, Désirée; Vitali, Sabrina; Schmid, Thomas; Isenring, Julia; Kurt, Fabienne; Kogi-Makau, Wambui; Meile, Leo

2016-06-21

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises seven (sub)species classified as human and animal commensals, emerging opportunistic pathogens and food fermentative organisms. Changing taxonomy, shared habitats, natural competence and evidence for horizontal gene transfer pose difficulties for determining their phylogeny, epidemiology and virulence mechanisms. Thus, novel phylogenetic and functional classifications are required. An SBSEC overarching multi locus sequence type (MLST) scheme targeting 10 housekeeping genes was developed, validated and combined with host-related properties of adhesion to extracellular matrix proteins (ECM), activation of the immune responses via NF-KB and survival in simulated gastric juice (SGJ). Commensal and pathogenic SBSEC strains (n = 74) of human, animal and food origin from Europe, Asia, America and Africa were used in the MLST scheme yielding 66 sequence types and 10 clonal complexes differentiated into distinct habitat-associated and mixed lineages. Adhesion to ECMs collagen I and mucin type II was a common characteristic (23 % of strains) followed by adhesion to fibronectin and fibrinogen (19.7 %). High adhesion abilities were found for East African dairy and human blood isolate branches whereas commensal fecal SBSEC displayed low adhesion. NF-KB activation was observed for a limited number of dairy and blood isolates suggesting the potential of some pathogenic strains for reduced immune activation. Strains from dairy MLST clades displayed the highest relative survival to SGJ independently of dairy adaptation markers lacS/lacZ. Combining phylogenetic and functional analyses via SBSEC MLST enabled the clear delineation of strain clades to unravel the complexity of this bacterial group. High adhesion values shared between certain dairy and blood strains as well as the behavior of NF-KB activation are concerning for specific lineages. They highlighted the health risk among shared lineages and establish the basis to elucidate (zoonotic-) transmission, host specificity, virulence mechanisms and enhanced risk assessment as pathobionts in an overarching One Health approach.

Multilocus sequence typing (MLST) and M13 PCR fingerprinting revealed heterogeneity amongst Cryptococcus species obtained from Italian veterinary isolates.

PubMed

Danesi, Patrizia; Firacative, Carolina; Cogliati, Massimo; Otranto, Domenico; Capelli, Gioia; Meyer, Wieland

2014-09-01

Cryptococcosis represents a fungal disease acquired from the environment with animals serving as host sentinels for human exposure. The aim of this study was to investigate the genetic characteristics of Cryptococcus isolates from veterinary sources (cats, dogs and birds) to understand their epidemiology and the genetic variability of the casual isolates. Mating-type PCR in connection with MLST analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex was used to genotype 17 C. neoformans isolates. In the absence of an MLST typing scheme Cryptococcus adeliensis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus and C. uniguttulatus strains were typed using M13 PCR fingerprinting. All C. neoformans isolates were MATα mating type, but hybrids possessed αADa and aADα mating and serotypes. Two C. neoformans molecular types VNI, VNIV and VNIII and VNII/VNIV hybrids were identified. Amongst the 66 non-C. neoformans strains investigated 55 M13 PCR fingerprinting types were identified. The wide variety of MLST types of C. neoformans and the occurrence of αADa and aADα hybrids in our study supports the notion of genetic recombination in the area studied. The heterogeneity of the non-C. neoformans isolates remains open to further investigations and should be taken into consideration when identifying emergent pathogens. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Tracking the blue: a MLST approach to characterise the Pseudomonas fluorescens group.

PubMed

Andreani, N A; Martino, M E; Fasolato, L; Carraro, L; Montemurro, F; Mioni, R; Bordin, P; Cardazzo, B

2014-05-01

The Pseudomonas fluorescens group comprises several closely related species that are involved in food contamination and spoilage. Specifically, the interest in P. fluorescens as a spoiler of dairy products increased after the cases of "blue mozzarella" that occurred in Italy in 2010. A Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise 136 isolates (reference strains and food borne isolates) at strain level, to reveal the genetic relationships among them and to disclose any possible genetic clustering of phenotypic markers involved in food spoilage (protease, lipase, lecithinase activities and pigmented or fluorescent molecule production). The production of dark blue diffusible pigment was evaluated on several bacterial culture media and directly on mozzarella cheese. The MLST scheme provided precise genotyping at the strain level, and the population analyses of the concatenated sequences allowed major taxa to be defined. This approach was revealed to be suitable for tracking the strains according to their origin, such as dairy plants or food matrices. The genetic analysis revealed the presence of a connection between the blue pigment production and a specific phylogenetic cluster. The development of the online database specific to the P. fluorescens group (http://pubmlst.org/pfluorescens) will facilitate the application of the scheme and the sharing of the data. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reprint of 'Tracking the blue: a MLST approach to characterise the Pseudomonas fluorescens group'.

PubMed

Andreani, N A; Martino, M E; Fasolato, L; Carraro, L; Montemurro, F; Mioni, R; Bordin, P; Cardazzo, B

2015-02-01

The Pseudomonas fluorescens group comprises several closely related species that are involved in food contamination and spoilage. Specifically, the interest in P. fluorescens as a spoiler of dairy products increased after the cases of "blue mozzarella" that occurred in Italy in 2010. A Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise 136 isolates (reference strains and food borne isolates) at strain level, to reveal the genetic relationships among them and to disclose any possible genetic clustering of phenotypic markers involved in food spoilage (protease, lipase, lecithinase activities and pigmented or fluorescent molecule production). The production of dark blue diffusible pigment was evaluated on several bacterial culture media and directly on mozzarella cheese. The MLST scheme provided precise genotyping at the strain level, and the population analyses of the concatenated sequences allowed major taxa to be defined. This approach was revealed to be suitable for tracking the strains according to their origin, such as dairy plants or food matrices. The genetic analysis revealed the presence of a connection between the blue pigment production and a specific phylogenetic cluster. The development of the online database specific to the P. fluorescens group (http://pubmlst.org/pfluorescens) will facilitate the application of the scheme and the sharing of the data. Copyright © 2014. Published by Elsevier Ltd.

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.

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.

Leptospira species molecular epidemiology in the genomic era.

PubMed

Caimi, K; Repetto, S A; Varni, V; Ruybal, P

2017-10-01

Leptospirosis is a zoonotic disease which global burden is increasing often related to climatic change. Hundreds of whole genome sequences from worldwide isolates of Leptospira spp. are available nowadays, together with online tools that permit to assign MLST sequence types (STs) directly from raw sequence data. In this work we have applied R7L-MLST to near 500 genomes and strains collection globally distributed. All 10 pathogenic species as well as intermediate were typed using this MLST scheme. The correlation observed between STs and serogroups in our previous work, is still satisfied with this higher dataset sustaining the implementation of MLST to assist serological classification as a complementary approach. Bayesian phylogenetic analysis of concatenated sequences from R7-MLST loci allowed us to resolve taxonomic inconsistencies but also showed that events such as recombination, gene conversion or lateral gene transfer played an important role in the evolution of Leptospira genus. Whole genome sequencing allows us to contribute with suitable epidemiologic information useful to apply in the design of control strategies and also in diagnostic methods for this illness. Copyright © 2017 Elsevier B.V. All rights reserved.

A genomic overview of the population structure of Salmonella.

PubMed

Alikhan, Nabil-Fareed; Zhou, Zhemin; Sergeant, Martin J; Achtman, Mark

2018-04-01

For many decades, Salmonella enterica has been subdivided by serological properties into serovars or further subdivided for epidemiological tracing by a variety of diagnostic tests with higher resolution. Recently, it has been proposed that so-called eBurst groups (eBGs) based on the alleles of seven housekeeping genes (legacy multilocus sequence typing [MLST]) corresponded to natural populations and could replace serotyping. However, this approach lacks the resolution needed for epidemiological tracing and the existence of natural populations had not been independently validated by independent criteria. Here, we describe EnteroBase, a web-based platform that assembles draft genomes from Illumina short reads in the public domain or that are uploaded by users. EnteroBase implements legacy MLST as well as ribosomal gene MLST (rMLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST) and currently contains over 100,000 assembled genomes from Salmonella. It also provides graphical tools for visual interrogation of these genotypes and those based on core single nucleotide polymorphisms (SNPs). eBGs based on legacy MLST are largely consistent with eBGs based on rMLST, thus demonstrating that these correspond to natural populations. rMLST also facilitated the selection of representative genotypes for SNP analyses of the entire breadth of diversity within Salmonella. In contrast, cgMLST provides the resolution needed for epidemiological investigations. These observations show that genomic genotyping, with the assistance of EnteroBase, can be applied at all levels of diversity within the Salmonella genus.

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

Multiple mitochondrial introgression events and heteroplasmy in trypanosoma cruzi revealed by maxicircle MLST and next generation sequencing.

PubMed

Messenger, Louisa A; Llewellyn, Martin S; Bhattacharyya, Tapan; Franzén, Oscar; Lewis, Michael D; Ramírez, Juan David; Carrasco, Hernan J; Andersson, Björn; Miles, Michael A

2012-01-01

Mitochondrial DNA is a valuable taxonomic marker due to its relatively fast rate of evolution. In Trypanosoma cruzi, the causative agent of Chagas disease, the mitochondrial genome has a unique structural organization consisting of 20-50 maxicircles (∼20 kb) and thousands of minicircles (0.5-10 kb). T. cruzi is an early diverging protist displaying remarkable genetic heterogeneity and is recognized as a complex of six discrete typing units (DTUs). The majority of infected humans are asymptomatic for life while 30-35% develop potentially fatal cardiac and/or digestive syndromes. However, the relationship between specific clinical outcomes and T. cruzi genotype remains elusive. The availability of whole genome sequences has driven advances in high resolution genotyping techniques and re-invigorated interest in exploring the diversity present within the various DTUs. To describe intra-DTU diversity, we developed a highly resolutive maxicircle multilocus sequence typing (mtMLST) scheme based on ten gene fragments. A panel of 32 TcI isolates was genotyped using the mtMLST scheme, GPI, mini-exon and 25 microsatellite loci. Comparison of nuclear and mitochondrial data revealed clearly incongruent phylogenetic histories among different geographical populations as well as major DTUs. In parallel, we exploited read depth data, generated by Illumina sequencing of the maxicircle genome from the TcI reference strain Sylvio X10/1, to provide the first evidence of mitochondrial heteroplasmy (heterogeneous mitochondrial genomes in an individual cell) in T. cruzi. mtMLST provides a powerful approach to genotyping at the sub-DTU level. This strategy will facilitate attempts to resolve phenotypic variation in T. cruzi and to address epidemiologically important hypotheses in conjunction with intensive spatio-temporal sampling. The observations of both general and specific incidences of nuclear-mitochondrial phylogenetic incongruence indicate that genetic recombination is geographically widespread and continues to influence the natural population structure of TcI, a conclusion which challenges the traditional paradigm of clonality in T. cruzi.

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.

Whole-genome-based Mycobacterium tuberculosis surveillance: a standardized, portable, and expandable approach.

PubMed

Kohl, Thomas A; Diel, Roland; Harmsen, Dag; Rothgänger, Jörg; Walter, Karen Meywald; Merker, Matthias; Weniger, Thomas; Niemann, Stefan

2014-07-01

Whole-genome sequencing (WGS) allows for effective tracing of Mycobacterium tuberculosis complex (MTBC) (tuberculosis pathogens) transmission. However, it is difficult to standardize and, therefore, is not yet employed for interlaboratory prospective surveillance. To allow its widespread application, solutions for data standardization and storage in an easily expandable database are urgently needed. To address this question, we developed a core genome multilocus sequence typing (cgMLST) scheme for clinical MTBC isolates using the Ridom SeqSphere(+) software, which transfers the genome-wide single nucleotide polymorphism (SNP) diversity into an allele numbering system that is standardized, portable, and not computationally intensive. To test its performance, we performed WGS analysis of 26 isolates with identical IS6110 DNA fingerprints and spoligotyping patterns from a longitudinal outbreak in the federal state of Hamburg, Germany (notified between 2001 and 2010). The cgMLST approach (3,041 genes) discriminated the 26 strains with a resolution comparable to that of SNP-based WGS typing (one major cluster of 22 identical or closely related and four outlier isolates with at least 97 distinct SNPs or 63 allelic variants). Resulting tree topologies are highly congruent and grouped the isolates in both cases analogously. Our data show that SNP- and cgMLST-based WGS analyses facilitate high-resolution discrimination of longitudinal MTBC outbreaks. cgMLST allows for a meaningful epidemiological interpretation of the WGS genotyping data. It enables standardized WGS genotyping for epidemiological investigations, e.g., on the regional public health office level, and the creation of web-accessible databases for global TB surveillance with an integrated early warning system. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

MLST-Based Population Genetic Analysis in a Global Context Reveals Clonality amongst Cryptococcus neoformans var. grubii VNI Isolates from HIV Patients in Southeastern Brazil

PubMed Central

Ferreira-Paim, Kennio; Andrade-Silva, Leonardo; Fonseca, Fernanda M.; Ferreira, Thatiana B.; Mora, Delio J.; Andrade-Silva, Juliana; Khan, Aziza; Dao, Aiken; Reis, Eduardo C.; Almeida, Margarete T. G.; Maltos, Andre; Junior, Virmondes R.; Trilles, Luciana; Rickerts, Volker; Chindamporn, Ariya; Sykes, Jane E.; Cogliati, Massimo; Nielsen, Kirsten; Boekhout, Teun; Fisher, Matthew; Kwon-Chung, June; Engelthaler, David M.; Lazéra, Marcia; Meyer, Wieland; Silva-Vergara, Mario L.

2017-01-01

Cryptococcosis is an important fungal infection in immunocompromised individuals, especially those infected with HIV. In Brazil, despite the free availability of antiretroviral therapy (ART) in the public health system, the mortality rate due to Cryptococcus neoformans meningitis is still high. To obtain a more detailed picture of the population genetic structure of this species in southeast Brazil, we studied 108 clinical isolates from 101 patients and 35 environmental isolates. Among the patients, 59% had a fatal outcome mainly in HIV-positive male patients. All the isolates were found to be C. neoformans var. grubii major molecular type VNI and mating type locus alpha. Twelve were identified as diploid by flow cytometry, being homozygous (AαAα) for the mating type and by PCR screening of the STE20, GPA1, and PAK1 genes. Using the ISHAM consensus multilocus sequence typing (MLST) scheme, 13 sequence types (ST) were identified, with one being newly described. ST93 was identified from 81 (75%) of the clinical isolates, while ST77 and ST93 were identified from 19 (54%) and 10 (29%) environmental isolates, respectively. The southeastern Brazilian isolates had an overwhelming clonal population structure. When compared with populations from different continents based on data extracted from the ISHAM-MLST database (mlst.mycologylab.org) they showed less genetic variability. Two main clusters within C. neoformans var. grubii VNI were identified that diverged from VNB around 0.58 to 4.8 million years ago. PMID:28099434

Analysis of nosocomial Staphylococcus haemolyticus by MLST and MALDI-TOF mass spectrometry.

PubMed

Kornienko, Maria; Ilina, Elena; Lubasovskaya, Ludmila; Priputnevich, Tatiana; Falova, Oksana; Sukhikh, Gennadiy; Govorun, Vadim

2016-04-01

Coagulase-negative staphylococci (CoNS) are a major component of normal human skin and mucosae flora. However, some species of CoNS can lead to infections in immunocompromised patients and premature newborns. The choice of a rapid and reliable typing method is one of the major problems in the epidemiological monitoring of CoNS, especially Staphylococcushaemolyticus isolates. In this study, we have tested 71 isolates of S. haemolyticus obtained from newborns using the multilocus sequence typing (MLST) and the direct bacterial MALDI-TOF mass spectrometry profiling approaches. To date, there is no standard MLST scheme for investigating the diversity of S. haemolyticus strains. The novel variant of MLST scheme including the tpiA, pta, sh1200, rphE, tphK, mvaK1, and arc loki was tested. The discriminatory power was estimated by the Hunter-Gaston discriminatory index (D) as 0.95. The Composition Correlation Index Matrix (CCI matrix) was calculated to typing the isolates through the analysis of mass spectra; also, the values of the correlation index for different groups of isolates were evaluated. Closely related isolates obtained from the same hospital are characterized by increased values of correlation indices in comparison with these values of isolates collected from various hospitals. The data obtained by both methods allow to describe a clonal structure of S. haemolyticus population and to designate the presence of endemic clones of S. haemolyticus. Copyright © 2015 Elsevier B.V. All rights reserved.

Diversity and evolution of Lactobacillus casei group isolated from fermented dairy products in Tibet.

PubMed

Feng, Jing; Jiang, Yujun; Li, Mingyu; Zhao, Siyu; Zhang, Yanming; Li, Xuesong; Wang, Hui; Lin, Guangen; Wang, Hao; Li, Tiejing; Man, Chaoxin

2018-05-25

Bacteria in Lactobacillus casei group, including Lactobacillus casei (L. casei), Lactobacillus paracasei (L. paracasei), and Lactobacillus rhamnosus (L. rhamnosus) are important lactic acid bacteria in the production of fermented dairy products and are faced with the controversial nomenclatural status due to their close phylogenetic similarity. To probe the evolution and phylogeny of L. casei group, 100 isolates of lactic acid bacteria originated from naturally fermented dairy products in Tibet of China were subjected to multilocus sequence typing (MLST). The MLST scheme, based on analysis of the housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA and recG, revealed that all the isolates belonged to a group containing the L. paracasei reference strains and were clearly different from the strains of L. casei and L. rhamnosus. Although nucleotide diversity (π) was low for the seven genes (ranging from 0.00341 for fusA to 0.01307 for recG), high genetic diversity represented by 83 sequence types (STs) with a discriminatory index of 0.98 was detected. A network-like structure based on split decomposition analysis, and the high values of the relative effect of recombination and mutation in the diversification of the lineages (r/m = 4.76) and the relative frequency of occurrence of recombination and mutation (ρ/θ = 2.62) indicated that intra-species recombination occurred frequently and homologous recombination played a key role in generating genotypic diversity amongst L. paracasei strains in Tibet. The discovery of 51 new STs and the results of STRUCTURE analysis suggested that the L. casei group in Tibet had an individual and particular population structure in comparison to European isolates. Overall, this research might be the first report about genetic diversity and population structure of Lactobacillus populations isolated from naturally fermented dairy products in Tibet based on MLST scheme.

A CRISPR-based MLST Scheme for Understanding the Population Biology and Epidemiology of Salmonella Enterica

DTIC Science & Technology

2015-05-26

in other systems , or whether it has alternative functions. Here, we report that CRISPR can be used to subtype Salmonella enterica serovariants...protects the bacteria against foreign DNA as described in other systems , or whether it has alternative functions. Here, we report that CRISPR can be...N. Shariat, R. E. Timme, J. B. Pettengill, R. Barrangou, E. G. Dudley. Characterization and evolution of Salmonella CRISPR-Cas systems

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.

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.

Clonal Dissemination of Extended-Spectrum β-Lactamase (ESBL)-Producing Klebsiella pneumoniae Isolates in a Korean Hospital

PubMed Central

Ko, Kwan Soo; Yeom, Joon-Sup; Lee, Mi Young; Peck, Kyong Ran

2008-01-01

In this study, we investigated the molecular characteristics of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates that were recovered from an outbreak in a Korean hospital. A new multilocus sequence typing (MLST) scheme for K. pneumoniae based on five housekeeping genes was developed and was evaluated for 43 ESBL-producing isolates from an outbreak as well as 38 surveillance isolates from Korea and also a reference strain. Overall, a total of 37 sequence types (STs) and six clonal complexes (CCs) were identified among the 82 K. pneumoniae isolates. The result of MLST analysis was concordant with that of pulsedfield gel electrophoresis. Most of the outbreak isolates belonged to a certain clone (ST2), and they produced SHV-1 and CTX-M14 enzymes, which was a different feature from that of the K. pneumoniae isolates from other Korean hospitals (ST20 and SHV-12). We also found a different distribution of CCs between ESBL-producing and -nonproducing K. pneumoniae isolates. The MLST method we developed in this study could provide unambiguous and well-resolved data for the epidemiologic study of K. pneumoniae. The outbreak isolates showed different molecular characteristics from the other K. pneumoniae isolates from other Korean hospitals. PMID:18303199

Isolation and clinical sample typing of human leptospirosis cases in Argentina.

PubMed

Chiani, Yosena; Jacob, Paulina; Varni, Vanina; Landolt, Noelia; Schmeling, María Fernanda; Pujato, Nazarena; Caimi, Karina; Vanasco, Bibiana

2016-01-01

Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients. Copyright © 2015 Elsevier B.V. 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.

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

Population structure and genetic diversity of the parasite Trichomonas vaginalis in Bristol, UK.

PubMed

Hawksworth, Joseph; Levy, Max; Smale, Chloe; Cheung, Dean; Whittle, Alice; Longhurst, Denise; Muir, Peter; Gibson, Wendy

2015-08-01

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, an extremely common, but non-life-threatening, sexually-transmitted disease throughout the world. Recent population genetics studies of T. vaginalis have detected high genetic diversity and revealed a two-type population structure, associated with phenotypic differences in sensitivity to metronidazole, the drug commonly used for treatment, and presence of T. vaginalis virus. There is currently a lack of data on UK isolates; most isolates examined to date are from the US. Here we used a recently described system for multilocus sequence typing (MLST) of T. vaginalis to study diversity of clinical isolates from Bristol, UK. We used MLST to characterise 23 clinical isolates of T. vaginalis collected from female patients during 2013. Seven housekeeping genes were PCR-amplified for each isolate and sequenced. The concatenated sequences were then compared with data from other MLST-characterised isolates available from http://tvaginalis.mlst.net/ to analyse the population structure and construct phylogenetic trees. Among the 23 isolates from the Bristol population of T. vaginalis, we found 23 polymorphic nucleotide sites, 25 different alleles and 19 sequence types (genotypes). Most isolates had a unique genotype, in agreement with the high levels of heterogeneity observed elsewhere in the world. A two-type population structure was evident from population genetic analysis and phylogenetic reconstruction split the isolates into two major clades. Tests for recombination in the Bristol population of T. vaginalis gave conflicting results, suggesting overall a clonal pattern of reproduction. We conclude that the Bristol population of T. vaginalis parasites conforms to the two-type population structure found in most other regions of the world. We found the MLST scheme to be an efficient genotyping method. The online MLST database provides a useful repository and resource that will prove invaluable in future studies linking the genetics of T. vaginalis with the clinical manifestation of trichomoniasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Long-Term, Low-Frequency Cluster of a German-Imipenemase-1-Producing Enterobacter hormaechei ssp. steigerwaltii ST89 in a Tertiary Care Hospital in Germany.

PubMed

Wendel, Andreas F; Meyer, Sebastian; Deenen, René; Köhrer, Karl; Kolbe-Busch, Susanne; Pfeffer, Klaus; Willmann, Matthias; Kaasch, Achim J; MacKenzie, Colin R

2018-05-11

Enterobacter cloacae complex is a common cause of hospital outbreaks. A retrospective and prospective molecular analysis of carbapenem-resistant clinical isolates in a tertiary care center demonstrated an outbreak of a German-imipenemase-1 (GIM-1) metallo-beta-lactamase-producing Enterobacter hormaechei ssp. steigerwaltii affecting 23 patients between 2009 and 2016. Thirty-three isolates were sequence type 89 by conventional multilocus sequence typing (MLST) and displayed a maximum difference of 49 out of 3,643 targets in the ad-hoc core-genome MLST (cgMLST) scheme (SeqSphere+ software; Ridom, Münster, Germany). The relatedness of all isolates was confirmed by further maximum-likelihood phylogeny. One clonal complex of highly related isolates (≤15 allele difference in cgMLST) contained 17 patients, but epidemiological data only suggested five transmission events. The bla GIM-1 -gene was embedded in a class-1-integron (In770) and the Tn21-subgroup transposon Tn6216 (KC511628) on a 25-kb plasmid. Environmental screening detected one colonized sink trap in a service room. The outbreak was self-limited as no further bla GIM-1 -positive E. hormaechei has been isolated since 2016. Routine molecular screening of carbapenem-nonsusceptible gram-negative isolates detected a long-term, low-frequency outbreak of a GIM-1-producing E. hormaechei ssp. steigerwaltii clone. This highlights the necessity of molecular surveillance.

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.

Emergence in Taiwan of novel imipenem-resistant Acinetobacter baumannii ST455 causing bloodstream infection in critical patients.

PubMed

Lee, Hao-Yuan; Huang, Chih-Wei; Chen, Chyi-Liang; Wang, Yi-Hsin; Chang, Chee-Jen; Chiu, Cheng-Hsun

2015-12-01

Acinetobacter baumannii is one of the most important nosocomial pathogens worldwide. This study aimed to use multilocus sequence typing (MLST) for the epidemiological surveillance of A. baumannii isolates in Taiwan and analyze the clinical presentations and patients' outcome. MLST according to both Bartual's PubMLST and Pasteur's MLST schemes was applied to characterize bloodstream imipenem-resistant A. baumannii (IRAB) infection in intensive care units in a medical center. A total of 39 clinical IRAB bloodstream isolates in 2010 were enrolled. We also collected 13 imipenem-susceptible A. baumannii (ISAB) bloodstream isolates and 30 clinical sputum isolates (24 IRAB and 6 ISAB) for comparison. Clinical presentations and outcome of the patients were analyzed. We found that infection by ST455(B)/ST2(P) and inappropriate initial therapy were statistically significant risk factors for mortality. More than one-third of the IRAB isolates belonged to ST455(B)/ST2(P). Most ST455(B)/ST2(P) (80%) carried ISAba1-blaOXA-23, including 10 (66.7%) with Tn2006 (ISAba1-blaOXA-23-ISAba1) in an AbaR4-type resistance island. ST455(B)/ST2(P) appears to evolve from ST208(B)/ST2(P) of clonal complex (CC) 92(B)/CC2(P). In this hospital-based study, A. baumannii ST455 accounted for 38.5% of IRAB bacteremia, with a high mortality of 86.7%. Approximately 85% of ST455(B)/ST2(P)bacteremia had a primary source of ventilation-associated pneumonia. We report the emergence in Taiwan of IRAB ST455(B)/ST2(P), which is the current predominant clone of IRAB in our hospital and has been causing bacteremia with high mortality in critical patients. Copyright © 2015. Published by Elsevier B.V.

Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

PubMed

Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

2017-05-01

Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Minim typing--a rapid and low cost MLST based typing tool for Klebsiella pneumoniae.

PubMed

Andersson, Patiyan; Tong, Steven Y C; Bell, Jan M; Turnidge, John D; Giffard, Philip M

2012-01-01

Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.

Minim Typing – A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae

PubMed Central

Andersson, Patiyan; Tong, Steven Y. C.; Bell, Jan M.; Turnidge, John D.; Giffard, Philip M.

2012-01-01

Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit. PMID:22428067

Molecular and Epidemiological Review of Toxigenic Diphtheria Infections in England between 2007 and 2013

PubMed Central

Both, Leonard; Collins, Sarah; de Zoysa, Aruni; White, Joanne; Mandal, Sema

2014-01-01

Human infections caused by toxigenic corynebacteria occur sporadically across Europe. In this report, we undertook the epidemiological and molecular characterization of all toxigenic corynebacterium strains isolated in England between January 2007 and December 2013. Epidemiological aspects include case demographics, risk factors, clinical presentation, treatment, and outcome. Molecular characterization was performed using multilocus sequence typing (MLST) alongside traditional phenotypic methods. In total, there were 20 cases of toxigenic corynebacteria; 12 (60.0%) were caused by Corynebacterium ulcerans, where animal contact was the predominant risk factor. The remaining eight (40.0%) were caused by Corynebacterium diphtheriae strains; six were biovar mitis, which were associated with recent travel abroad. Adults 45 years and older were particularly affected (55.0%; 11/20), and typical symptoms included sore throat and fever. Respiratory diphtheria with the absence of a pharyngeal membrane was the most common presentation (50.0%; 10/20). None of the eight C. diphtheriae cases were fully immunized. Diphtheria antitoxin was issued in two (9.5%) cases; both survived. Two (9.5%) cases died, one due to a C. diphtheriae infection and one due to C. ulcerans. MLST demonstrated that the majority (87.5%; 7/8) of C. diphtheriae strains represented new sequence types (STs). By adapting several primer sequences, the MLST genes in C. ulcerans were also amplified, thereby providing the basis for extension of the MLST scheme, which is currently restricted to C. diphtheriae. Despite high population immunity, occasional toxigenic corynebacterium strains are identified in England and continued surveillance is required. PMID:25502525

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.

A typing scheme for the honeybee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants.

PubMed

Haynes, Edward; Helgason, Thorunn; Young, J Peter W; Thwaites, Richard; Budge, Giles E

2013-08-01

Melissococcus plutonius is the bacterial pathogen that causes European Foulbrood of honeybees, a globally important honeybee brood disease. We have used next-generation sequencing to identify highly polymorphic regions in an otherwise genetically homogenous organism, and used these loci to create a modified MLST scheme. This synthesis of a proven typing scheme format with next-generation sequencing combines reliability and low costs with insights only available from high-throughput sequencing technologies. Using this scheme we show that the global distribution of M.plutonius variants is not uniform. We use the scheme in epidemiological studies to trace movements of infective material around England, insights that would have been impossible to confirm without the typing scheme. We also demonstrate the persistence of local variants over time. © 2013 Crown copyright. Reproduced with the permission of the Controller of Her Majesty's Stationary Office/Queen’s Printer for Scotland and Food and Environment Research Agency.

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.

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.

Morchella MLST database

USDA-ARS?s Scientific Manuscript database

Welcome to the Morchella MLST database. This dedicated database was set up at the CBS-KNAW Biodiversity Center by Vincent Robert in February 2012, using BioloMICS software (Robert et al., 2011), to facilitate DNA sequence-based identifications of Morchella species via the Internet. The current datab...

Molecular genotyping of Trypanosoma cruzi for lineage assignment and population genetics.

PubMed

Messenger, Louisa A; Yeo, Matthew; Lewis, Michael D; Llewellyn, Martin S; Miles, Michael A

2015-01-01

Trypanosoma cruzi, the etiological agent of Chagas disease, remains a major public health problem in Latin America. Infection with T. cruzi is lifelong and can lead to a spectrum of pathological sequelae ranging from subclinical to lethal cardiac and/or gastrointestinal complications. Isolates of T. cruzi can be assigned to six genetic lineages or discrete typing units (DTUs), which are broadly associated with disparate ecologies, transmission cycles, and geographical distributions. This extensive genetic diversity is also believed to contribute to the clinical variation observed among chagasic patients. Unravelling the population structure of T. cruzi is fundamental to understanding Chagas disease epidemiology, developing control strategies, and resolving the relationship between parasite genotype and clinical prognosis. To date, no single, widely validated, genetic target allows unequivocal resolution to DTU-level. In this chapter we present standardized methods for strain DTU assignment using PCR-restriction fragment length polymorphism analysis (PCR-RFLP) and nuclear multilocus sequence typing (MLST). PCR-RFLPs have the advantages of simplicity and reproducibility, requiring limited expertise and few laboratory consumables. MLST data are more laborious to generate but more informative; DNA sequences are readily transferable between research groups and amenable to recombination detection and intra-lineage analyses. We also recommend a mitochondrial (maxicircle) MLST scheme and a panel of 28 microsatellite loci for higher resolution population genetics studies. Due to the scarcity of T. cruzi in blood and tissue, all of these genotyping techniques have limited sensitivity when applied directly to clinical or biological specimens, particularly when targets are single (MLST) or low copy number (PCR-RFLPs). We therefore describe essential protocols to isolate parasites, derive biological clones, and extract T. cruzi genomic DNA from field and clinical samples.

The Characteristics of Ubiquitous and Unique Leptospira Strains from the Collection of Russian Centre for Leptospirosis

PubMed Central

Voronina, Olga L.; Kunda, Marina S.; Aksenova, Ekaterina I.; Ryzhova, Natalia N.; Semenov, Andrey N.; Petrov, Evgeny M.; Didenko, Lubov V.; Lunin, Vladimir G.; Ananyina, Yuliya V.; Gintsburg, Alexandr L.

2014-01-01

Background and Aim. Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. Methods. The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). Results. The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. Conclusions. Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions. PMID:25276806

Population genetic and evolution analysis of controversial genus Edwardsiella by multilocus sequence typing.

PubMed

Buján, Noemí; Balboa, Sabela; L Romalde, Jesús; E Toranzo, Alicia; Magariños, Beatriz

2018-05-08

At present, the genus Edwardsiella compiles five species: E. tarda, E. hoshinae, E. ictaluri, E. piscicida and E. anguillarum. Some species of this genus such us E. ictaluri and E. piscicida are important pathogens of numerous fish species. With the description of the two latter species, the phylogeny of Edwardsiella became more complicated. With the aim to clarify the relationships among all species in the genus, a multilocus sequence typing (MLST) approach was developed and applied to characterize 56 isolates and 6 reference strains belonging to the five Edwardsiella species. Moreover, several analyses based on the MLST scheme were performed to investigate the evolution within the genus, as well as the influence of recombination and mutation in the speciation. Edwardsiella isolates presented a high genetic variability reflected in the fourteen sequence types (ST) represented by a single isolates out of eighteen total ST. Mutation events were considerably more frequent than recombination, although both approximately equal influenced the genetic diversification. However, the speciation among species occurred mostly by recombination. Edwardsiella genus displays a non-clonal population structure with some degree of geographical isolation followed by a population expansion of E. piscicida. A database from this study was created and hosted on pubmlst.org (http://pubmlst.org/edwardsiella/). Copyright © 2018 Elsevier Inc. All rights reserved.

A Comparative Analysis of the Lyve-SET Phylogenomics Pipeline for Genomic Epidemiology of Foodborne Pathogens

PubMed Central

Katz, Lee S.; Griswold, Taylor; Williams-Newkirk, Amanda J.; Wagner, Darlene; Petkau, Aaron; Sieffert, Cameron; Van Domselaar, Gary; Deng, Xiangyu; Carleton, Heather A.

2017-01-01

Modern epidemiology of foodborne bacterial pathogens in industrialized countries relies increasingly on whole genome sequencing (WGS) techniques. As opposed to profiling techniques such as pulsed-field gel electrophoresis, WGS requires a variety of computational methods. Since 2013, United States agencies responsible for food safety including the CDC, FDA, and USDA, have been performing whole-genome sequencing (WGS) on all Listeria monocytogenes found in clinical, food, and environmental samples. Each year, more genomes of other foodborne pathogens such as Escherichia coli, Campylobacter jejuni, and Salmonella enterica are being sequenced. Comparing thousands of genomes across an entire species requires a fast method with coarse resolution; however, capturing the fine details of highly related isolates requires a computationally heavy and sophisticated algorithm. Most L. monocytogenes investigations employing WGS depend on being able to identify an outbreak clade whose inter-genomic distances are less than an empirically determined threshold. When the difference between a few single nucleotide polymorphisms (SNPs) can help distinguish between genomes that are likely outbreak-associated and those that are less likely to be associated, we require a fine-resolution method. To achieve this level of resolution, we have developed Lyve-SET, a high-quality SNP pipeline. We evaluated Lyve-SET by retrospectively investigating 12 outbreak data sets along with four other SNP pipelines that have been used in outbreak investigation or similar scenarios. To compare these pipelines, several distance and phylogeny-based comparison methods were applied, which collectively showed that multiple pipelines were able to identify most outbreak clusters and strains. Currently in the US PulseNet system, whole genome multi-locus sequence typing (wgMLST) is the preferred primary method for foodborne WGS cluster detection and outbreak investigation due to its ability to name standardized genomic profiles, its central database, and its ability to be run in a graphical user interface. However, creating a functional wgMLST scheme requires extended up-front development and subject-matter expertise. When a scheme does not exist or when the highest resolution is needed, SNP analysis is used. Using three Listeria outbreak data sets, we demonstrated the concordance between Lyve-SET SNP typing and wgMLST. Availability: Lyve-SET can be found at https://github.com/lskatz/Lyve-SET. PMID:28348549

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

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.

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

Characterization of Salmonella enterica isolates causing bacteremia in Lima, Peru, using multiple typing methods

PubMed Central

Betancor, Laura; García, Coralith; Astocondor, Lizeth; Hinostroza, Noemí; Bisio, Julieta; Rivera, Javier; Perezgasga, Lucía; Pérez Escanda, Victoria; Yim, Lucía; Jacobs, Jan; García-del Portillo, Francisco; Chabalgoity, José A.; Puente, José L.

2017-01-01

In this study, different molecular typing tools were applied to characterize 95 Salmonella enterica blood isolates collected between 2008 and 2013 from patients at nine public hospitals in Lima, Peru. Combined results of multiplex PCR serotyping, two- and seven-loci multilocus sequence typing (MLST) schemes, serotyping, IS200 amplification and RAPD fingerprints, showed that these infections were caused by eight different serovars: Enteritidis, Typhimurium, Typhi, Choleraesuis, Dublin, Paratyphi A, Paratyphi B and Infantis. Among these, Enteritidis, Typhimurium and Typhi were the most prevalent, representing 45, 36 and 11% of the isolates, respectively. Most isolates (74%) were not resistant to ten primarily used antimicrobial drugs; however, 37% of the strains showed intermediate susceptibility to ciprofloxacin (ISC). Antimicrobial resistance integrons were carried by one Dublin (dfra1 and aadA1) and two Infantis (aadA1) isolates. The two Infantis isolates were multidrug resistant and harbored a large megaplasmid. Amplification of spvC and spvRA regions showed that all Enteritidis (n = 42), Typhimurium (n = 34), Choleraesuis (n = 3) and Dublin (n = 1) isolates carried the Salmonella virulence plasmid (pSV). We conclude that the classic serotyping method can be substituted by the multiplex PCR and, when necessary, sequencing of only one or two loci of the MLST scheme is a valuable tool to confirm the results. The effectiveness and feasibility of different typing tools is discussed. PMID:29267322

The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies.

PubMed

Yoshida, Catherine E; Kruczkiewicz, Peter; Laing, Chad R; Lingohr, Erika J; Gannon, Victor P J; Nash, John H E; Taboada, Eduardo N

2016-01-01

For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub-typing allows for continuity with historical serotyping data as we transition towards the increasing adoption of genomic analyses in epidemiology. The SISTR platform is freely available on the web at https://lfz.corefacility.ca/sistr-app/.

Characterization and Expression of Drug Resistance Genes in MDROs Originating from Combat Wound Infections

DTIC Science & Technology

2016-09-01

assigned a classification. MLST analysis MLST was determined using an in-house automated pipeline that first searches for homologs of each gene of...and virulence mechanism contributing to their success as pathogens in the wound environment. A novel bioinformatics pipeline was used to incorporate...monitored in two ways: read-based genome QC and assembly based metrics. The JCVI Genome QC pipeline samples sequence reads and performs BLAST

Global MLST of Salmonella Typhi Revisited in Post-genomic Era: Genetic Conservation, Population Structure, and Comparative Genomics of Rare Sequence Types.

PubMed

Yap, Kien-Pong; Ho, Wing S; Gan, Han M; Chai, Lay C; Thong, Kwai L

2016-01-01

Typhoid fever, caused by Salmonella enterica serovar Typhi, remains an important public health burden in Southeast Asia and other endemic countries. Various genotyping methods have been applied to study the genetic variations of this human-restricted pathogen. Multilocus sequence typing (MLST) is one of the widely accepted methods, and recently, there is a growing interest in the re-application of MLST in the post-genomic era. In this study, we provide the global MLST distribution of S. Typhi utilizing both publicly available 1,826 S. Typhi genome sequences in addition to performing conventional MLST on S. Typhi strains isolated from various endemic regions spanning over a century. Our global MLST analysis confirms the predominance of two sequence types (ST1 and ST2) co-existing in the endemic regions. Interestingly, S. Typhi strains with ST8 are currently confined within the African continent. Comparative genomic analyses of ST8 and other rare STs with genomes of ST1/ST2 revealed unique mutations in important virulence genes such as flhB, sipC, and tviD that may explain the variations that differentiate between seemingly successful (widespread) and unsuccessful (poor dissemination) S. Typhi populations. Large scale whole-genome phylogeny demonstrated evidence of phylogeographical structuring and showed that ST8 may have diverged from the earlier ancestral population of ST1 and ST2, which later lost some of its fitness advantages, leading to poor worldwide dissemination. In response to the unprecedented increase in genomic data, this study demonstrates and highlights the utility of large-scale genome-based MLST as a quick and effective approach to narrow the scope of in-depth comparative genomic analysis and consequently provide new insights into the fine scale of pathogen evolution and population structure.

Clonal Relatedness of Enterotoxigenic Escherichia coli (ETEC) Strains Expressing LT and CS17 Isolated from Children with Diarrhoea in La Paz, Bolivia

PubMed Central

Rodas, Claudia; Klena, John D.; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Åsa

2011-01-01

Background Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. Methodology/Principal Findings In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNPbol in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNPbol) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. Conclusion/Significance The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors. PMID:22140423

Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia.

PubMed

Rodas, Claudia; Klena, John D; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Asa

2011-01-01

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.

Real-Time Pathogen Detection in the Era of Whole-Genome Sequencing and Big Data: Comparison of k-mer and Site-Based Methods for Inferring the Genetic Distances among Tens of Thousands of Salmonella Samples.

PubMed

Pettengill, James B; Pightling, Arthur W; Baugher, Joseph D; Rand, Hugh; Strain, Errol

2016-01-01

The adoption of whole-genome sequencing within the public health realm for molecular characterization of bacterial pathogens has been followed by an increased emphasis on real-time detection of emerging outbreaks (e.g., food-borne Salmonellosis). In turn, large databases of whole-genome sequence data are being populated. These databases currently contain tens of thousands of samples and are expected to grow to hundreds of thousands within a few years. For these databases to be of optimal use one must be able to quickly interrogate them to accurately determine the genetic distances among a set of samples. Being able to do so is challenging due to both biological (evolutionary diverse samples) and computational (petabytes of sequence data) issues. We evaluated seven measures of genetic distance, which were estimated from either k-mer profiles (Jaccard, Euclidean, Manhattan, Mash Jaccard, and Mash distances) or nucleotide sites (NUCmer and an extended multi-locus sequence typing (MLST) scheme). When analyzing empirical data (whole-genome sequence data from 18,997 Salmonella isolates) there are features (e.g., genomic, assembly, and contamination) that cause distances inferred from k-mer profiles, which treat absent data as informative, to fail to accurately capture the distance between samples when compared to distances inferred from differences in nucleotide sites. Thus, site-based distances, like NUCmer and extended MLST, are superior in performance, but accessing the computing resources necessary to perform them may be challenging when analyzing large databases.

Real-Time Pathogen Detection in the Era of Whole-Genome Sequencing and Big Data: Comparison of k-mer and Site-Based Methods for Inferring the Genetic Distances among Tens of Thousands of Salmonella Samples

DOE PAGES

Pettengill, James B.; Pightling, Arthur W.; Baugher, Joseph D.; ...

2016-11-10

The adoption of whole-genome sequencing within the public health realm for molecular characterization of bacterial pathogens has been followed by an increased emphasis on real-time detection of emerging outbreaks (e.g., food-borne Salmonellosis). In turn, large databases of whole-genome sequence data are being populated. These databases currently contain tens of thousands of samples and are expected to grow to hundreds of thousands within a few years. For these databases to be of optimal use one must be able to quickly interrogate them to accurately determine the genetic distances among a set of samples. Being able to do so is challenging duemore » to both biological (evolutionary diverse samples) and computational (petabytes of sequence data) issues. We evaluated seven measures of genetic distance, which were estimated from either k-mer profiles (Jaccard, Euclidean, Manhattan, Mash Jaccard, and Mash distances) or nucleotide sites (NUCmer and an extended multi-locus sequence typing (MLST) scheme). Finally, when analyzing empirical data (wholegenome sequence data from 18,997 Salmonella isolates) there are features (e.g., genomic, assembly, and contamination) that cause distances inferred from k-mer profiles, which treat absent data as informative, to fail to accurately capture the distance between samples when compared to distances inferred from differences in nucleotide sites. Thus, site-based distances, like NUCmer and extended MLST, are superior in performance, but accessing the computing resources necessary to perform them may be challenging when analyzing large databases.« less

Real-Time Pathogen Detection in the Era of Whole-Genome Sequencing and Big Data: Comparison of k-mer and Site-Based Methods for Inferring the Genetic Distances among Tens of Thousands of Salmonella Samples

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

Pettengill, James B.; Pightling, Arthur W.; Baugher, Joseph D.

The adoption of whole-genome sequencing within the public health realm for molecular characterization of bacterial pathogens has been followed by an increased emphasis on real-time detection of emerging outbreaks (e.g., food-borne Salmonellosis). In turn, large databases of whole-genome sequence data are being populated. These databases currently contain tens of thousands of samples and are expected to grow to hundreds of thousands within a few years. For these databases to be of optimal use one must be able to quickly interrogate them to accurately determine the genetic distances among a set of samples. Being able to do so is challenging duemore » to both biological (evolutionary diverse samples) and computational (petabytes of sequence data) issues. We evaluated seven measures of genetic distance, which were estimated from either k-mer profiles (Jaccard, Euclidean, Manhattan, Mash Jaccard, and Mash distances) or nucleotide sites (NUCmer and an extended multi-locus sequence typing (MLST) scheme). Finally, when analyzing empirical data (wholegenome sequence data from 18,997 Salmonella isolates) there are features (e.g., genomic, assembly, and contamination) that cause distances inferred from k-mer profiles, which treat absent data as informative, to fail to accurately capture the distance between samples when compared to distances inferred from differences in nucleotide sites. Thus, site-based distances, like NUCmer and extended MLST, are superior in performance, but accessing the computing resources necessary to perform them may be challenging when analyzing large databases.« less

Determination of Wolbachia Diversity in Butterflies from Western Ghats, India, by a Multigene Approach

PubMed Central

Salunke, Bipinchandra K.; Salunkhe, Rahul C.; Dhotre, Dhiraj P.; Walujkar, Sandeep A.; Khandagale, Avinash B.; Chaudhari, Rahul; Chandode, Rakesh K.; Ghate, Hemant V.; Patole, Milind S.; Werren, John H.

2012-01-01

Members of the genus Wolbachia are intracellular bacteria that are widespread in arthropods and establish diverse symbiotic associations with their hosts, ranging from mutualism to parasitism. Here we present the first detailed analyses of Wolbachia in butterflies from India with screening of 56 species. Twenty-nine species (52%) representing five families were positive for Wolbachia. This is the first report of Wolbachia infection in 27 of the 29 species; the other two were reported previously. This study also provides the first evidence of infection in the family Papilionidae. A striking diversity was observed among Wolbachia strains in butterfly hosts based on five multilocus sequence typing (MLST) genes, with 15 different sequence types (STs). Thirteen STs are new to the MLST database, whereas ST41 and ST125 were reported earlier. Some of the same host species from this study carried distinctly different Wolbachia strains, whereas the same or different butterfly hosts also harbored closely related Wolbachia strains. Butterfly-associated STs in the Indian sample originated by recombination and point mutation, further supporting the role of both processes in generating Wolbachia diversity. Recombination was detected only among the STs in this study and not in those from the MLST database. Most of the strains were remarkably similar in their wsp genotype, despite divergence in MLST. Only two wsp alleles were found among 25 individuals with complete hypervariable region (HVR) peptide profiles. Although both wsp and MLST show variability, MLST gives better separation between the strains. Completely different STs were characterized for the individuals sharing the same wsp alleles. PMID:22504801

Optimization of analytical parameters for inferring relationships among Escherichia coli isolates from repetitive-element PCR by maximizing correspondence with multilocus sequence typing data.

PubMed

Goldberg, Tony L; Gillespie, Thomas R; Singer, Randall S

2006-09-01

Repetitive-element PCR (rep-PCR) is a method for genotyping bacteria based on the selective amplification of repetitive genetic elements dispersed throughout bacterial chromosomes. The method has great potential for large-scale epidemiological studies because of its speed and simplicity; however, objective guidelines for inferring relationships among bacterial isolates from rep-PCR data are lacking. We used multilocus sequence typing (MLST) as a "gold standard" to optimize the analytical parameters for inferring relationships among Escherichia coli isolates from rep-PCR data. We chose 12 isolates from a large database to represent a wide range of pairwise genetic distances, based on the initial evaluation of their rep-PCR fingerprints. We conducted MLST with these same isolates and systematically varied the analytical parameters to maximize the correspondence between the relationships inferred from rep-PCR and those inferred from MLST. Methods that compared the shapes of densitometric profiles ("curve-based" methods) yielded consistently higher correspondence values between data types than did methods that calculated indices of similarity based on shared and different bands (maximum correspondences of 84.5% and 80.3%, respectively). Curve-based methods were also markedly more robust in accommodating variations in user-specified analytical parameter values than were "band-sharing coefficient" methods, and they enhanced the reproducibility of rep-PCR. Phylogenetic analyses of rep-PCR data yielded trees with high topological correspondence to trees based on MLST and high statistical support for major clades. These results indicate that rep-PCR yields accurate information for inferring relationships among E. coli isolates and that accuracy can be enhanced with the use of analytical methods that consider the shapes of densitometric profiles.

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.

Emergence of endemic MLST non-typeable vancomycin-resistant Enterococcus faecium.

PubMed

Carter, Glen P; Buultjens, Andrew H; Ballard, Susan A; Baines, Sarah L; Tomita, Takehiro; Strachan, Janet; Johnson, Paul D R; Ferguson, John K; Seemann, Torsten; Stinear, Timothy P; Howden, Benjamin P

2016-12-01

Enterococcus faecium is a major nosocomial pathogen causing significant morbidity and mortality worldwide. Assessment of E. faecium using MLST to understand the spread of this organism is an important component of hospital infection control measures. Recent studies, however, suggest that MLST might be inadequate for E. faecium surveillance. To use WGS to characterize recently identified vancomycin-resistant E. faecium (VREfm) isolates non-typeable by MLST that appear to be causing a multi-jurisdictional outbreak in Australia. Illumina NextSeq and Pacific Biosciences SMRT sequencing platforms were used to determine the genome sequences of 66 non-typeable E. faecium (NTEfm) isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools. Sixty-six E. faecium isolates were identified by WGS from multiple health jurisdictions in Australia that could not be typed by MLST due to a missing pstS allele. SMRT sequencing and complete genome assembly revealed a large chromosomal rearrangement in representative strain DMG1500801, which likely facilitated the deletion of the pstS region. Phylogenomic analysis of this population suggests that deletion of pstS within E. faecium has arisen independently on at least three occasions. Importantly, the majority of these isolates displayed a vancomycin-resistant genotype. We have identified NTEfm isolates that appear to be causing a multi-jurisdictional outbreak in Australia. Identification of these isolates has important implications for MLST-based typing activities designed to monitor the spread of VREfm and provides further evidence supporting the use of WGS for hospital surveillance of E. faecium. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Multilocus Sequence Typing of Cronobacter Strains Isolated from Retail Foods and Environmental Samples.

PubMed

Killer, Jiří; Skřivanová, Eva; Hochel, Igor; Marounek, Milan

2015-06-01

Cronobacter spp. are bacterial pathogens that affect children and immunocompromised adults. In this study, we used multilocus sequence typing (MLST) to determine sequence types (STs) in 11 Cronobacter spp. strains isolated from retail foods, 29 strains from dust samples obtained from vacuum cleaners, and 4 clinical isolates. Using biochemical tests, species-specific polymerase chain reaction, and MLST analysis, 36 strains were identified as Cronobacter sakazakii, and 6 were identified as Cronobacter malonaticus. In addition, one strain that originated from retail food and one from a dust sample from a vacuum cleaner were identified on the basis of MLST analysis as Cronobacter dublinensis and Cronobacter turicensis, respectively. Cronobacter spp. strains isolated from the retail foods were assigned to eight different MLST sequence types, seven of which were newly identified. The strains isolated from the dust samples were assigned to 7 known STs and 14 unknown STs. Three clinical isolates and one household dust isolate were assigned to ST4, which is the predominant ST associated with neonatal meningitis. One clinical isolate was classified based on MLST analysis as Cronobacter malonaticus and belonged to an as-yet-unknown ST. Three strains isolated from the household dust samples were assigned to ST1, which is another clinically significant ST. It can be concluded that Cronobacter spp. strains of different origin are genetically quite variable. The recovery of C. sakazakii strains belonging to ST1 and ST4 from the dust samples suggests the possibility that contamination could occur during food preparation. All of the novel STs and alleles for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis determined in this study were deposited in the Cronobacter MLST database available online ( http://pubmlst.org/cronobacter/).

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for Molecular Subtyping of Campylobacter jejuni by Using Capillary Electrophoresis

PubMed Central

Techaruvichit, Punnida; Vesaratchavest, Mongkol; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

2015-01-01

Campylobacter jejuni is a common cause of the frequently reported food-borne diseases in developed and developing nations. This study describes the development of multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) using capillary electrophoresis as a novel typing method for microbial source tracking and epidemiological investigation of C. jejuni. Among 36 tandem repeat loci detected by the Tandem Repeat Finder program, 7 VNTR loci were selected and used for characterizing 60 isolates recovered from chicken meat samples from retail shops, samples from chicken meat processing factory, and stool samples. The discrimination ability of MLVA was compared with that of multilocus sequence typing (MLST). MLVA (diversity index of 0.97 with 31 MLVA types) provided slightly higher discrimination than MLST (diversity index of 0.95 with 25 MLST types). The overall concordance between MLVA and MLST was estimated at 63% by adjusted Rand coefficient. MLVA predicted MLST type better than MLST predicted MLVA type, as reflected by Wallace coefficient (Wallace coefficient for MLVA to MLST versus MLST to MLVA, 86% versus 51%). MLVA is a useful tool and can be used for effective monitoring of C. jejuni and investigation of epidemics caused by C. jejuni. PMID:26025899

Whole-Genome Sequencing of Recent Listeria monocytogenes Isolates from Germany Reveals Population Structure and Disease Clusters.

PubMed

Halbedel, Sven; Prager, Rita; Fuchs, Stephan; Trost, Eva; Werner, Guido; Flieger, Antje

2018-06-01

Listeria monocytogenes causes foodborne outbreaks with high mortality. For improvement of outbreak cluster detection, the German consiliary laboratory for listeriosis implemented whole-genome sequencing (WGS) in 2015. A total of 424 human L. monocytogenes isolates collected in 2007 to 2017 were subjected to WGS and core-genome multilocus sequence typing (cgMLST). cgMLST grouped the isolates into 38 complexes, reflecting 4 known and 34 unknown disease clusters. Most of these complexes were confirmed by single nucleotide polymorphism (SNP) calling, but some were further differentiated. Interestingly, several cgMLST cluster types were further subtyped by pulsed-field gel electrophoresis, partly due to phage insertions in the accessory genome. Our results highlight the usefulness of cgMLST for routine cluster detection but also show that cgMLST complexes require validation by methods providing higher typing resolution. Twelve cgMLST clusters included recent cases, suggesting activity of the source. Therefore, the cgMLST nomenclature data presented here may support future public health actions. Copyright © 2018 American Society for Microbiology.

Persistence of Mycoplasma hyopneumoniae sequence types in spite of a control program for enzootic pneumonia in pigs.

PubMed

Overesch, Gudrun; Kuhnert, Peter

2017-09-15

Enzootic pneumonia (EP) in pigs caused by Mycoplasma (M.) hyopneumoniae has successfully been combatted in Switzerland. A control program was fully implemented in 2004 which is based on total depopulation strategies of affected fattening farms as well as partial depopulation on breeding farms. Thereby, the number of cases has dropped drastically from more than 200 in 2003 to two cases in 2013. Currently monitoring is done based on clinical observation and subsequent diagnostic of coughing pigs. Moreover, in case of more than 10% gross pathological lesions per slaughter batch laboratory confirmation for EP is compulsory. Despite these strict measures it was not possible to eliminate M. hyopneumoniae from Swiss pig production. In fact, during the last few years the number of EP cases has slightly increased. Therefore, genotyping of the involved M. hyopneumoniae strains was conducted in order to elucidate possible sources and routes of infection. All available and typeable samples from totally 22 cases during the period 2014-2016 were investigated by extended multilocus sequence typing (MLST). A total of 16 cases, including eight from 2014, five from 2015 and three from 2016 could thereby be included in the study. MLST revealed that the majority of cases in 2014/2015 were due to two major spread scenarios, i.e. two M. hyopneumoniae sequence types, each scenario involving six individual production farms in five to six different Cantons (states), respectively. Moreover, by comparison of archived sequences some sequence types were observed over ten years demonstrating their persistence over a long time and the possible partial failure of elimination measures in Switzerland. Insufficient sanitation on affected farms and subsequent animal transport of symptomless infected pigs could lead to recurrent cases. Wild boar harbor identical strains found with EP but solid data are missing to assign a role as reservoir to this wild animal. Implementing a monitoring scheme for M. hyopneumoniae in wild boar in combination with genotyping of all available samples from domestic pigs could direct responsible authorities to possible gaps and deficiencies of control measures taken for combating enzootic pneumonia. With the newly installed PubMLST database sequence types for M. hyopneumoniae are now available and allow tracing back strains on the international level. Copyright © 2017 Elsevier B.V. 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.

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China.

PubMed

Wang, Liyan; Ma, Lina; Liu, Yongan; Gao, Pengcheng; Li, Youquan; Li, Xuerui; Liu, Yongsheng

2016-10-01

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein-Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated 'nontypeable' strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei.

PubMed

Godoy, Daniel; Randle, Gaynor; Simpson, Andrew J; Aanensen, David M; Pitt, Tyrone L; Kinoshita, Reimi; Spratt, Brian G

2003-05-01

A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.

Molecular epidemiology of Staphylococcus aureus bacteremia in a single large Minnesota medical center in 2015 as assessed using MLST, core genome MLST and spa typing.

PubMed

Park, Kyung-Hwa; Greenwood-Quaintance, Kerryl E; Uhl, James R; Cunningham, Scott A; Chia, Nicholas; Jeraldo, Patricio R; Sampathkumar, Priya; Nelson, Heidi; Patel, Robin

2017-01-01

Staphylococcus aureus is a leading cause of bacteremia in hospitalized patients. Whether or not S. aureus bacteremia (SAB) is associated with clonality, implicating potential nosocomial transmission, has not, however, been investigated. Herein, we examined the epidemiology of SAB using whole genome sequencing (WGS). 152 SAB isolates collected over the course of 2015 at a single large Minnesota medical center were studied. Staphylococcus protein A (spa) typing was performed by PCR/Sanger sequencing; multilocus sequence typing (MLST) and core genome MLST (cgMLST) were determined by WGS. Forty-eight isolates (32%) were methicillin-resistant S. aureus (MRSA). The isolates encompassed 66 spa types, clustered into 11 spa clonal complexes (CCs) and 10 singleton types. 88% of 48 MRSA isolates belonged to spa CC-002 or -008. Methicillin-susceptible S. aureus (MSSA) isolates were more genotypically diverse, with 61% distributed across four spa CCs (CC-002, CC-012, CC-008 and CC-084). By MLST, there was 31 sequence types (STs), including 18 divided into 6 CCs and 13 singleton STs. Amongst MSSA isolates, the common MLST clones were CC5 (23%), CC30 (19%), CC8 (15%) and CC15 (11%). Common MRSA clones were CC5 (67%) and CC8 (25%); there were no MRSA isolates in CC45 or CC30. By cgMLST analysis, there were 9 allelic differences between two isolates, with the remaining 150 isolates differing from each other by over 40 alleles. The two isolates were retroactively epidemiologically linked by medical record review. Overall, cgMLST analysis resulted in higher resolution epidemiological typing than did multilocus sequence or spa typing.

A new scheme for strain typing of methicillin-resistant Staphylococcus aureus on the basis of matrix-assisted laser desorption ionization time-of-flight mass spectrometry by using machine learning approach.

PubMed

Wang, Hsin-Yao; Lee, Tzong-Yi; Tseng, Yi-Ju; Liu, Tsui-Ping; Huang, Kai-Yao; Chang, Yung-Ta; Chen, Chun-Hsien; Lu, Jang-Jih

2018-01-01

Methicillin-resistant Staphylococcus aureus (MRSA), one of the most important clinical pathogens, conducts an increasing number of morbidity and mortality in the world. Rapid and accurate strain typing of bacteria would facilitate epidemiological investigation and infection control in near real time. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid and cost-effective tool for presumptive strain typing. To develop robust method for strain typing based on MALDI-TOF spectrum, machine learning (ML) is a promising algorithm for the construction of predictive model. In this study, a strategy of building templates of specific types was used to facilitate generating predictive models of methicillin-resistant Staphylococcus aureus (MRSA) strain typing through various ML methods. The strain types of the isolates were determined through multilocus sequence typing (MLST). The area under the receiver operating characteristic curve (AUC) and the predictive accuracy of the models were compared. ST5, ST59, and ST239 were the major MLST types, and ST45 was the minor type. For binary classification, the AUC values of various ML methods ranged from 0.76 to 0.99 for ST5, ST59, and ST239 types. In multiclass classification, the predictive accuracy of all generated models was more than 0.83. This study has demonstrated that ML methods can serve as a cost-effective and promising tool that provides preliminary strain typing information about major MRSA lineages on the basis of MALDI-TOF spectra.

Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India.

PubMed

Panda, Sasmita; Jena, Smrutiti; Sharma, Savitri; Dhawan, Benu; Nath, Gopal; Singh, Durg Vijai

2016-01-01

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates.

Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India

PubMed Central

Panda, Sasmita; Jena, Smrutiti; Sharma, Savitri; Dhawan, Benu; Nath, Gopal

2016-01-01

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates. PMID:27824930

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.

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.

“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

Genetic variation of 'Candidatus Liberibacter solanacearum' haplotype C and identification of a novel haplotype from Trioza urticae and stinging nettle.

PubMed

Haapalainen, Minna L; Wang, Jinhui; Latvala, Satu; Lehtonen, Mikko T; Pirhonen, Minna; Nissinen, Anne I

2018-03-30

'Candidatus Liberibacter solanacearum' (CLso) haplotype C is associated with disease in carrots and transmitted by the carrot psyllid Trioza apicalis. To identify possible other sources and vectors of this pathogen in Finland, samples were taken of wild plants within and near the carrot fields, the psyllids feeding on these plants, parsnips growing next to carrots, and carrot seeds. For analyzing the genotype of the CLso positive samples, a multi-locus sequence typing (MLST) scheme was developed. CLso haplotype C was detected in 11% of the Trioza anthrisci samples, in 35% of the Anthriscus sylvestris plants with discoloration, and in parsnips showing leaf discoloration. MLST revealed that the CLso in T. anthrisci and most A. sylvestris plants represent different strains than the bacteria found in T. apicalis and the cultivated plants. CLso haplotype D was detected in two of the 34 carrot seed lots tested, but was not detected in the plants grown from these seeds. Phylogenetic analysis by UPGMA clustering suggested that the haplotype D is more closely related to the haplotype A than to C. A novel, sixth haplotype of CLso, most closely related to A and D, was found in the psyllid Trioza urticae and stinging nettle (Urtica dioica, Urticaceae), and named as haplotype U.

A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia.

PubMed

Bleidorn, Christoph; Gerth, Michael

2018-01-01

Wolbachia (Alphaproteobacteria, Rickettsiales) is the most common, and arguably one of the most important inherited symbionts. Molecular differentiation of Wolbachia strains is routinely performed with a set of five multilocus sequence typing (MLST) markers. However, since its inception in 2006, the performance of MLST in Wolbachia strain typing has not been assessed objectively. Here, we evaluate the properties of Wolbachia MLST markers and compare it to 252 other single copy loci present in the genome of most Wolbachia strains. Specifically, we investigated how well MLST performs at strain differentiation, at reflecting genetic diversity of strains, and as phylogenetic marker. We find that MLST loci are outperformed by other loci at all tasks they are currently employed for, and thus that they do not reflect the properties of a Wolbachia strain very well. We argue that whole genome typing approaches should be used for Wolbachia typing in the future. Alternatively, if few loci approaches are necessary, we provide a characterisation of 252 single copy loci for a number a criteria, which may assist in designing specific typing systems or phylogenetic studies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

BrucellaBase: Genome information resource.

PubMed

Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Khader, L K M Abdul; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2016-09-01

Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic diversity and population structure of food-borne Staphylococcus carnosus strains.

PubMed

Bückle Née Müller, Anne; Kranz, Markus; Schmidt, Herbert; Weiss, Agnes

2017-01-01

The species Staphylococcus carnosus is a non-pathogenic representative of the coagulase negative staphylococci. Specific strains are applied as meat starter cultures. The species consists of two subspecies, S. carnosus ssp. carnosus and S. carnosus ssp. utilis. In order to place S. carnosus strains, characterized in former studies, in a genetic background that allows a typing of candidates for starter cultures, a Multilocus Sequence Typing (MLST) scheme was developed. Internal fragments of the genes tpiA, xprT, dat, gmk, glpK, narG, cstA, encoding triosephosphate isomerase, xanthine phosphoribosyltransferase, d-amino acid aminotransferase, guanylate kinase, glycerol kinase, the α-chain of the respiratory nitrate reductase, and a carbon starvation protein where chosen. Genes were selected based on their equal distribution in the genome, taxonomic value in subspecies differentiation and metabolic function. This MLST was applied to 44 S. carnosus strains, most of them previously analyzed for their suitability as starter cultures. The number of alleles varied between zero (tpiA) and five (cstA) and allowed the definition of nine sequence types (ST). ST1 was most abundant (18 strains), followed by ST2 (8) and ST4 (6). The nine STs confirmed a close relationship of all strains despite their isolation source and year, but lacked correlation with physiological activities relevant for starter cultures. The low amount of STs in the strain set lets us suggest that recombination between strains is rare. Thus, it is hypothesized that evolutionary events seem to be due to single point mutations rather than intrachromosomal recombination, and that the species possesses a clonal population structure. Copyright © 2016 Elsevier GmbH. All rights reserved.

Molecular characterization of resistance, virulence and clonality in vancomycin-resistant Enterococcus faecium and Enterococcus faecalis: A hospital-based study in Beijing, China.

PubMed

Yang, Jing-xian; Li, Tong; Ning, Yong-zhong; Shao, Dong-hua; Liu, Jing; Wang, Shu-qin; Liang, Guo-wei

2015-07-01

The incidence of vancomycin-resistant enterococcus (VRE) in China is increasing, the molecular epidemiology of VRE in China is only partly known. This study was conducted to assess the molecular characterization of resistance, virulence and clonality of 69 vancomycin-resistant Enterococcus faecium (VREfm) and seven vancomycin-resistant Enterococcus faecalis (VREfs) isolates obtained from a Chinese hospital between July 2011 and July 2013. The glycopeptide resistance genes (VanA and VanB) were screened by multiplex PCR. The presence of five putative virulence genes (esp, gelE, asa1, hyl and cylA) were evaluated by another multiplex PCR. Multilocus sequence typing (MLST) scheme was used to assess the clonality. All 76 VRE isolates exhibited VanA phenotype and harbored VanA gene. Esp was the only gene detected both in VREfm and VREfs strains, accounting for 89.9% and 42.9%, respectively. The hyl gene was merely positive in 27.5% of VREfm strains. MLST analysis demonstrated three STs (ST6, ST4 and ST470) in VREfs and twelve STs (ST78, ST571, ST17, ST564, ST389, ST18, ST547, ST341, ST414, ST343, ST262 and ST203) in VREfm, which were all designated as CC17 by eBURST algorithm. An outbreak of VREfm belonging to ST571 was found to happen within the neurology ward in this hospital. To our knowledge, this is the first report of ST6 (CC2) VREfs strains in China and the first outbreak report of VREfm strains belonging to ST571 around the world. Our data could offer important information for understanding the molecular features of VRE in China. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

PubMed

Gurtler, Volker; Grando, Danilla; Mayall, Barrie C; Wang, Jenny; Ghaly-Derias, Shahbano

2012-09-01

In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with predictable instability based on recombination and concerted evolution of the rrn operon that will complement existing typing methods. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Vertical transmission of highly similar blaCTX-M-1-harboring IncI1 plasmids in Escherichia coli with different MLST types in the poultry production pyramid

PubMed Central

Zurfluh, Katrin; Wang, Juan; Klumpp, Jochen; Nüesch-Inderbinen, Magdalena; Fanning, Séamus; Stephan, Roger

2014-01-01

Objectives: The purpose of this study was to characterize sets of extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae collected longitudinally from different flocks of broiler breeders, meconium of 1-day-old broilers from theses breeder flocks, as well as from these broiler flocks before slaughter. Methods: Five sets of ESBL-producing Escherichia coli were studied by multi-locus sequence typing (MLST), phylogenetic grouping, PCR-based replicon typing and resistance profiling. The blaCTX-M-1-harboring plasmids of one set (pHV295.1, pHV114.1, and pHV292.1) were fully sequenced and subjected to comparative analysis. Results: Eleven different MLST sequence types (ST) were identified with ST1056 the predominant one, isolated in all five sets either on the broiler breeder or meconium level. Plasmid sequencing revealed that blaCTX-M-1 was carried by highly similar IncI1/ST3 plasmids that were 105 076 bp, 110 997 bp, and 117 269 bp in size, respectively. Conclusions: The fact that genetically similar IncI1/ST3 plasmids were found in ESBL-producing E. coli of different MLST types isolated at the different levels in the broiler production pyramid provides strong evidence for a vertical transmission of these plasmids from a common source (nucleus poultry flocks). PMID:25324838

Vertical transmission of highly similar bla CTX-M-1-harboring IncI1 plasmids in Escherichia coli with different MLST types in the poultry production pyramid.

PubMed

Zurfluh, Katrin; Wang, Juan; Klumpp, Jochen; Nüesch-Inderbinen, Magdalena; Fanning, Séamus; Stephan, Roger

2014-01-01

The purpose of this study was to characterize sets of extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae collected longitudinally from different flocks of broiler breeders, meconium of 1-day-old broilers from theses breeder flocks, as well as from these broiler flocks before slaughter. Five sets of ESBL-producing Escherichia coli were studied by multi-locus sequence typing (MLST), phylogenetic grouping, PCR-based replicon typing and resistance profiling. The bla CTX-M-1-harboring plasmids of one set (pHV295.1, pHV114.1, and pHV292.1) were fully sequenced and subjected to comparative analysis. Eleven different MLST sequence types (ST) were identified with ST1056 the predominant one, isolated in all five sets either on the broiler breeder or meconium level. Plasmid sequencing revealed that bla CTX-M-1 was carried by highly similar IncI1/ST3 plasmids that were 105 076 bp, 110 997 bp, and 117 269 bp in size, respectively. The fact that genetically similar IncI1/ST3 plasmids were found in ESBL-producing E. coli of different MLST types isolated at the different levels in the broiler production pyramid provides strong evidence for a vertical transmission of these plasmids from a common source (nucleus poultry flocks).

[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.

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.

Population analysis of Vibrio parahaemolyticus originating from different geographical regions demonstrates a high genetic diversity.

PubMed

Urmersbach, Sara; Alter, Thomas; Koralage, Madura Sanjeevani Gonsal; Sperling, Lisa; Gerdts, Gunnar; Messelhäusser, Ute; Huehn, Stephan

2014-03-08

Vibrio parahaemolyticus is frequently isolated from environmental and seafood samples and associated with gastroenteritis outbreakes in American, European, Asian and African countries. To distinguish between different lineages of V. parahaemolyticus various genotyping techniques have been used, incl. multilocus sequence typing (MLST). Even though some studies have already applied MLST analysis to characterize V. parahaemolyticus strain sets, these studies have been restricted to specific geographical areas (e.g. U.S. coast, Thailand and Peru), have focused exclusively on pandemic or non-pandemic pathogenic isolates or have been based on a limited strain number. To generate a global picture of V. parahaemolyticus genotype distribution, a collection of 130 environmental and seafood related V. parahaemolyticus isolates of different geographical origins (Sri Lanka, Ecuador, North Sea and Baltic Sea as well as German retail) was subjected to MLST analysis after modification of gyrB and recA PCRs. The V. parahaemolyticus population was composed of 82 unique Sequence Types (STs), of which 68 (82.9%) were new to the pubMLST database. After translating the in-frame nucleotide sequences into amino acid sequences, less diversity was detectable: a total of 31 different peptide Sequence Types (pSTs) with 19 (61.3%) new pSTs were generated from the analyzed isolates. Most STs did not show a global dissemination, but some were supra-regionally distributed and clusters of STs were dependent on geographical origin. On peptide level no general clustering of strains from specific geographical regions was observed, thereby the most common pSTs were found on all continents (Asia, South America and Europe) and rare pSTs were restricted to distinct countries or even geographical regions. One lineage of pSTs associated only with strains from North and Baltic Sea strains was identified. Our study reveals a high genetic diversity in the analyzed V. parahaemolyticus strain set as well as for geographical strain subsets, with a high proportion of newly discovered alleles and STs. Differences between the subsets were identified. Our data support the postulated population structure of V. parahaemolyticus which follows the 'epidemic' model of clonal expansion. Application of peptide based AA-MLST allowed the identification of reliable relationships between strains.

Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

PubMed

Do, Thuy; Gilbert, Steven C; Clark, Douglas; Ali, Farida; Fatturi Parolo, Clarissa C; Maltz, Marisa; Russell, Roy R; Holbrook, Peter; Wade, William G; Beighton, David

2010-02-05

Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However pi, nucleotide diversity per site, was low for all loci being in the range 0.019-0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3-1.15] compared to 8.3 [95% confidence interval 5.0-14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.

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.

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 CAD, a multifunctional protein involved in pyrimidine synthesis, with mLST8, a component of the mTOR complexes

PubMed Central

2013-01-01

Background mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. Results CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. Conclusion The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8. PMID:23594158

MLST genotypes of Campylobacter jejuni isolated from broiler products, dairy cattle and human campylobacteriosis cases in Lithuania.

PubMed

Ramonaite, Sigita; Tamuleviciene, Egle; Alter, Thomas; Kasnauskyte, Neringa; Malakauskas, Mindaugas

2017-06-15

Campylobacter (C.) jejuni is the leading cause of human campylobacteriosis worldwide. We performed a molecular epidemiological study to investigate the genetic relationship among C. jejuni strains isolated from human diarrhoeal patients, broiler products and dairy cattle in Lithuania. The C. jejuni isolates from human clinical cases, dairy cattle and broiler products were genotyped using multilocus sequence typing (MLST). Allele numbers for each housekeeping gene, sequence type (ST), and clonal complex (CC) were assigned by submitting the DNA sequences to the C. jejuni MLST database ( http://pubmlst.org/campylobacter ). Based on the obtained sequence data of the housekeeping genes a phylogenetic analysis of the strains was performed and a minimum spanning tree (MST) was calculated. Among the 262 C. jejuni strains (consisting of 43 strains isolated from dairy cattle, 102 strains isolated from broiler products and 117 clinical human C. jejuni strains), 82 different MLST sequence types and 22 clonal complexes were identified. Clonal complexes CC21 and CC353 predominated among the C. jejuni strains. On ST-level, five sequence types (ST-5, ST-21, ST-50, ST-464 and ST-6410) were dominating and these five STs accounted for 35.9% (n = 94) of our isolates. In addition, 51 (19.5%) C. jejuni strains representing 27 (32.9%) STs were reported for the first time in the PubMLST database ( http://pubmlst.org/campylobacter ). The highest Czekanowski index or proportional similarity index (PSI) was calculated for C. jejuni strains isolated from human campylobacteriosis cases and broiler products (PSI = 0.32) suggesting a strong link between broiler strains and human cases. The PSI of dairy cattle and human samples was lower (PSI = 0.11), suggesting a weaker link between bovine strains and human cases. The calculated Simpson's index of all C. jejuni isolates showed a high genetic diversity (D = 0.96). Our results suggest that broiler products are the most important source of human campylobacteriosis in Lithuania. The study provides information on MLST type distribution and genetic relatedness of C. jejuni strains from humans, broiler products and dairy cattle in Lithuania for the first time, enabling a better understanding of the transmission pathways of C. jejuni in this country.

Intrafamilial, Preferentially Mother-to-Child and Intraspousal, Helicobacter pylori Infection in Japan Determined by Mutilocus Sequence Typing and Random Amplified Polymorphic DNA Fingerprinting.

PubMed

Yokota, Shin-ichi; Konno, Mutsuko; Fujiwara, Shin-ichi; Toita, Nariaki; Takahashi, Michiko; Yamamoto, Soh; Ogasawara, Noriko; Shiraishi, Tsukasa

2015-10-01

The infection route of Helicobacter pylori has been recognized to be mainly intrafamilial, preferentially mother-to-child, especially in developed countries. To determine the transmission route, we examined whether multilocus sequence typing (MLST) was useful for analysis of intrafamilial infection. The possibility of intraspousal infection was also evaluated. Clonal relationships between strains derived from 35 index Japanese pediatric patients, and their family members were analyzed by two genetic typing procedures, MLST and random amplified polymorphic DNA (RAPD) fingerprinting. Mostly coincident results were obtained by MLST and RAPD. By MLST, the allele of loci in the isolates mostly matched between the index child and both the father and mother for 9 (25.7%) of the 35 patients, between the index child and the mother for 25 (60.0%) of the 35 patients. MLST is useful for analyzing the infection route of H. pylori as a highly reproducible method. Intrafamilial, especially mother-to-children and sibling, infection is the dominant transmission route. Intraspousal infection is also thought to occur in about a quarter in the Japanese families. © 2015 John Wiley & Sons Ltd.

Multilocus Sequence Typing Compared to Pulsed-Field Gel Electrophoresis for Molecular Typing of Pseudomonas aeruginosa▿

PubMed Central

Johnson, Jennifer K.; Arduino, Sonia M.; Stine, O. Colin; Johnson, Judith A.; Harris, Anthony D.

2007-01-01

For hospital epidemiologists, determining a system of typing that is discriminatory is essential for measuring the effectiveness of infection control measures. In situations in which the incidence of resistant Pseudomonas aeruginosa is increasing, the ability to discern whether it is due to patient-to-patient transmission versus an increase in patient endogenous strains is often made on the basis of molecular typing. The present study compared the discriminatory abilities of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for 90 P. aeruginosa isolates obtained from cultures of perirectal surveillance swabs from patients in an intensive care unit. PFGE identified 85 distinct types and 76 distinct groups when similarity cutoffs of 100% and 87%, respectively, were used. By comparison, MLST identified 60 sequence types that could be clustered into 11 clonal complexes and 32 singletons. By using the Simpson index of diversity (D), PFGE had a greater discriminatory ability than MLST for P. aeruginosa isolates (D values, 0.999 versus 0.975, respectively). Thus, while MLST was better for detecting genetic relatedness, we determined that PFGE was more discriminatory than MLST for determining genetic differences in P. aeruginosa. PMID:17881548

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.

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.

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.

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.

Fusarium MLST database

USDA-ARS?s Scientific Manuscript database

The CBS-KNAW Fungal Biodiversity Centre’s Fusarium MLST website (http://www.cbs.knaw.nl/Fusarium), and the corresponding Fusarium-ID site hosted at the Pennsylvania State University (http://isolate.fusariumdb.org; Geiser et al. 2004, Park et al. 2010) were constructed to facilitate identification of...

Using whole genome sequencing to study American foulbrood epidemiology in honeybees

PubMed Central

Ågren, Joakim; Schäfer, Marc Oliver

2017-01-01

American foulbrood (AFB), caused by Paenibacillus larvae, is a devastating disease in honeybees. In most countries, the disease is controlled through compulsory burning of symptomatic colonies causing major economic losses in apiculture. The pathogen is endemic to honeybees world-wide and is readily transmitted via the movement of hive equipment or bees. Molecular epidemiology of AFB currently largely relies on placing isolates in one of four ERIC-genotypes. However, a more powerful alternative is multi-locus sequence typing (MLST) using whole-genome sequencing (WGS), which allows for high-resolution studies of disease outbreaks. To evaluate WGS as a tool for AFB-epidemiology, we applied core genome MLST (cgMLST) on isolates from a recent outbreak of AFB in Sweden. The high resolution of the cgMLST allowed different bacterial clones involved in the disease outbreak to be identified and to trace the source of infection. The source was found to be a beekeeper who had sold bees to two other beekeepers, proving the epidemiological link between them. No such conclusion could have been made using conventional MLST or ERIC-typing. This is the first time that WGS has been used to study the epidemiology of AFB. The results show that the technique is very powerful for high-resolution tracing of AFB-outbreaks. PMID:29140998

Discriminatory Indices of Typing Methods for Epidemiologic Analysis of Contemporary Staphylococcus aureus Strains.

PubMed

Rodriguez, Marcela; Hogan, Patrick G; Satola, Sarah W; Crispell, Emily; Wylie, Todd; Gao, Hongyu; Sodergren, Erica; Weinstock, George M; Burnham, Carey-Ann D; Fritz, Stephanie A

2015-09-01

Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D = 0.88), followed by spa typing (D = 0.87), MLST (D = 0.84), PFGE (D = 0.76), and SCCmec typing (D = 0.60). The method with the highest D among MRSA isolates was repPCR (D = 0.64) followed by spa typing (D = 0.45) and MLST (D = 0.44). The method with the highest D among MSSA isolates was spa typing (D = 0.98), followed by MLST (D = 0.93), repPCR (D = 0.92), and PFGE (D = 0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D = 0.63). We identified 45 MRSA isolates which were classified as identical by PFGE, MLST, spa typing, and SCCmec typing (USA300, ST8, t008, SCCmec IV, respectively); within this collection, there were 5 distinct strain types identified by repPCR. The typing methods yielded comparable discriminatory power for S. aureus characterization overall; when discriminating among USA300 isolates, repPCR retained the highest discriminatory power. This property is advantageous for investigations conducted in the era of contemporary S. aureus infections.

Discriminatory Indices of Typing Methods for Epidemiologic Analysis of Contemporary Staphylococcus aureus Strains

PubMed Central

Rodriguez, Marcela; Hogan, Patrick G.; Satola, Sarah W.; Crispell, Emily; Wylie, Todd; Gao, Hongyu; Sodergren, Erica; Weinstock, George M.; Burnham, Carey-Ann D.; Fritz, Stephanie A.

2015-01-01

Abstract Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D = 0.88), followed by spa typing (D = 0.87), MLST (D = 0.84), PFGE (D = 0.76), and SCCmec typing (D = 0.60). The method with the highest D among MRSA isolates was repPCR (D = 0.64) followed by spa typing (D = 0.45) and MLST (D = 0.44). The method with the highest D among MSSA isolates was spa typing (D = 0.98), followed by MLST (D = 0.93), repPCR (D = 0.92), and PFGE (D = 0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D = 0.63). We identified 45 MRSA isolates which were classified as identical by PFGE, MLST, spa typing, and SCCmec typing (USA300, ST8, t008, SCCmec IV, respectively); within this collection, there were 5 distinct strain types identified by repPCR. The typing methods yielded comparable discriminatory power for S. aureus characterization overall; when discriminating among USA300 isolates, repPCR retained the highest discriminatory power. This property is advantageous for investigations conducted in the era of contemporary S. aureus infections. PMID:26376402

Analysis of β-Lactamase Resistance Determinants in Enterobacteriaceae from Chicago Children: a Multicenter Survey

PubMed Central

Hujer, Andrea M.; Marshall, Steven H.; Domitrovic, T. Nicholas; Rudin, Susan D.; Zheng, Xiaotian; Qureshi, Nadia K.; Hayden, Mary K.; Scaggs, Felicia A.; Karadkhele, Anand; Bonomo, Robert A.

2016-01-01

Multidrug-resistant (MDR) Enterobacteriaceae infections are increasing in U.S. children; however, there is a paucity of multicentered analyses of antibiotic resistance genes responsible for MDR phenotypes among pediatric Enterobacteriaceae isolates. In this study, 225 isolates phenotypically identified as extended-spectrum β-lactamase (ESBL) or carbapenemase producers, recovered from children ages 0 to 18 years hospitalized between January 2011 and April 2015 at three Chicago area hospitals, were analyzed. We used DNA microarray platforms to detect ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase type β-lactamase (bla) genes. Repetitive-sequence-based PCR and multilocus sequence typing (MLST) were performed to assess isolate similarity. Plasmid replicon typing was conducted to classify plasmids. The median patient age was 4.2 years, 56% were female, and 44% presented in the outpatient setting. The majority (60.9%) of isolates were Escherichia coli and from urinary sources (69.8%). Of 225 isolates exhibiting ESBL- or carbapenemase-producing phenotypes, 90.7% contained a bla gene. The most common genotype was the blaCTX-M-1 group (49.8%); 1.8% were carbapenem-resistant Enterobacteriaceae (three blaKPC and one blaIMP). Overall, pAmpC (blaACT/MIR and blaCMY) were present in 14.2%. The predominant E. coli phylogenetic group was the virulent B2 group (67.6%) associated with ST43/ST131 (Pasteur/Achtman MLST scheme) containing the blaCTX-M-1 group (84%), and plasmid replicon types FIA, FII, and FIB. K. pneumoniae harboring blaKPC were non-ST258 with replicon types I1 and A/C. Enterobacter spp. carrying blaACT/MIR contained plasmid replicon FIIA. We found that β-lactam resistance in children is diverse and that certain resistance mechanisms differ from known circulating genotypes in adults in an endemic area. The potential impact of complex molecular types and the silent dissemination of MDR Enterobacteriaceae in a vulnerable population needs to be studied further. PMID:27021322

The complete genome sequences of 65 Campylobacter jejuni and C. coli strains

USDA-ARS?s Scientific Manuscript database

Campylobacter jejuni (Cj) and C. coli (Cc) are genetically highly diverse based on various molecular methods including MLST, microarray-based comparisons and the whole genome sequences of a few strains. Cj and Cc diversity is also exhibited by variable capsular polysaccharides (CPS) that are the maj...

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.

A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China.

PubMed

Peng, Zhong; Wang, Haonan; Liang, Wan; Chen, Yibao; Tang, Xibiao; Chen, Huanchun; Wu, Bin

2018-01-01

Pasteurella multocida is a leading cause of respiratory disease in pigs worldwide. In this study, we determined the genetic characteristics of 115 P. multocida isolates from the lungs of pigs with respiratory disease in China in 2015 using capsular typing, lipopolysaccharide (LPS) genotyping, and virulence genotyping based on the detection of virulence-associated genes. The results showed that the isolates belonged to three capsular types: A (49.6%), D (46.1%), and nontypable (4.3%); and two LPS genotypes: L3 (22.6%) and L6 (77.4%). When combining the capsular types with the LPS genotypes, a genotype group D: L6 (46.1%) was the most prevalent among the strains. Among the 23 virulence-associated genes detected in this study, a small number of them displayed a certain level of "genotype-preference". We found that pfhA, hgbA, and hgbB had a close association with P. multocida LPS genotypes, while tadD was more associated with P. multocida capsular types. In addition, multilocus sequence typing (MLST) on 40 P. multocida isolates identified four sequence types: ST3, ST10, ST11, and ST16, and the distribution of ST11 was significantly higher than the other MLST genotypes. Interestingly, all of the ST11 isolates detected in this study were genotype D: L6 strains and they were 100% positive for hgbB. Our data suggest that a capsule/LPS/MLST genotype D/L6/ST11 is likely to be strongly associated with respiratory clinical manifestation of the disease in pigs.

DNA-Sequence Based Typing of the Cronobacter Genus Using MLST, CRISPR-cas Array and Capsular Profiling

PubMed Central

Ogrodzki, Pauline; Forsythe, Stephen J.

2017-01-01

The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST), capsular profiling of the K-antigen and colanic acid (CA) biosynthesis regions, and CRISPR-cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC) 1 and 4, sequence types (ST) 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis, C. dublinensis and C. universalis. The majority of CRISPR-cas types across the genus was the I-E (Ecoli) type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo) type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors. PMID:29033918

Genetic Characterization of Cronobacter sakazakii Recovered from the Environmental Surveillance Samples During a Sporadic Case Investigation of Foodborne Illness.

PubMed

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

2016-08-01

Cronobacter sakazakii is an opportunistic human-pathogenic bacterium known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. This human-pathogenic microorganism has been isolated from a variety of food and environmental samples, and has been also linked to foodborne outbreaks associated with powdered infant formula (PIF). The U.S. Food and Drug Administration have a policy of zero tolerance of these organisms in PIF. Thus, this agency utilizes the presence of these microorganisms as one of the criteria in implementing regulatory actions and assessing adulteration of food products of public health importance. In this study, we recovered two isolates of Cronobacter from the 91 environmental swab samples during an investigation of sporadic case of foodborne illness following conventional microbiological protocols. The isolated typical colonies were identified using VITEK2 and real-time PCR protocols. The recovered Cronobacter isolates were then characterized for species identification by sequencing the 16S rRNA locus. Further, multilocus sequence typing (MLST) was accomplished characterizing seven known C. sakazakii-specific MLST loci (atpD, fusA, glnS, gltB, gyrB, infB, and pps). Results of this study confirmed all of the recovered Cronobacter isolates from the environmental swab samples to be C. sakazakii. The MLST profile matched with the published profile of the complex 31 of C. sakazakii. Thus, rRNA and 7-loci MLST-based sequencing protocols are robust techniques for rapid detection and differentiation of Cronobacter species, and these molecular diagnostic tools can be used in implementing successful surveillance program and in the control and prevention of foodborne illness.

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 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.

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.

Use of Variable-Number Tandem Repeats To Examine Genetic Diversity of Neisseria meningitidis

PubMed Central

Yazdankhah, Siamak P.; Lindstedt, Bjørn-Arne; Caugant, Dominique A.

2005-01-01

Repetitive DNA motifs with potential variable-number tandem repeats (VNTR) were identified in the genome of Neisseria meningitidis and used to develop a typing method. A total of 146 meningococcal isolates recovered from carriers and patients were studied. These included 82 of the 107 N. meningitidis isolates previously used in the development of multilocus sequence typing (MLST), 45 isolates recovered from different counties in Norway in connection with local outbreaks, and 19 serogroup W135 isolates of sequence type 11 (ST-11), which were recovered in several parts of the world. The latter group comprised isolates related to the Hajj outbreak of 2000 and isolates recovered from outbreaks in Burkina Faso in 2001 and 2002. All isolates had been characterized previously by MLST or multilocus enzyme electrophoresis (MLEE). VNTR analysis showed that meningococcal isolates with similar MLST or MLEE types recovered from epidemiologically linked cases in a defined geographical area often presented similar VNTR patterns while isolates of the same MLST or MLEE types without an obvious epidemiological link showed variable VNTR patterns. Thus, VNTR analysis may be used for fine typing of meningococcal isolates after MLST or MLEE typing. The method might be especially valuable for differentiating among ST-11 strains, as shown by the VNTR analyses of serogroup W135 ST-11 meningococcal isolates recovered since the mid-1990s. PMID:15814988

Genomic Diversity of Lactobacillus salivarius▿ †

PubMed Central

Raftis, Emma J.; Salvetti, Elisa; Torriani, Sandra; Felis, Giovanna E.; O'Toole, Paul W.

2011-01-01

Strains of Lactobacillus salivarius are increasingly employed as probiotic agents for humans or animals. Despite the diversity of environmental sources from which they have been isolated, the genomic diversity of L. salivarius has been poorly characterized, and the implications of this diversity for strain selection have not been examined. To tackle this, we applied comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) to 33 strains derived from humans, animals, or food. The CGH, based on total genome content, including small plasmids, identified 18 major regions of genomic variation, or hot spots for variation. Three major divisions were thus identified, with only a subset of the human isolates constituting an ecologically discernible group. Omission of the small plasmids from the CGH or analysis by MLST provided broadly concordant fine divisions and separated human-derived and animal-derived strains more clearly. The two gene clusters for exopolysaccharide (EPS) biosynthesis corresponded to regions of significant genomic diversity. The CGH-based groupings of these regions did not correlate with levels of production of bound or released EPS. Furthermore, EPS production was significantly modulated by available carbohydrate. In addition to proving difficult to predict from the gene content, EPS production levels correlated inversely with production of biofilms, a trait considered desirable in probiotic commensals. L. salivarius displays a high level of genomic diversity, and while selection of L. salivarius strains for probiotic use can be informed by CGH or MLST, it also requires pragmatic experimental validation of desired phenotypic traits. PMID:21131523

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 ...

Analysis of co-evolving genes in campylobacter jejuni and C. coli

USDA-ARS?s Scientific Manuscript database

Background: The population structure of Campylobacter has been frequently studied by MLST, for which fragments of housekeeping genes are compared. We wished to determine if the used MLST genes are representative of the complete genome. Methods: A set of 1029 core gene families (CGF) was identifie...

Chlamydia trachomatis Strain Types Have Diversified Regionally and Globally with Evidence for Recombination across Geographic Divides

PubMed Central

Smelov, Vitaly; Vrbanac, Alison; van Ess, Eleanne F.; Noz, Marlies P.; Wan, Raymond; Eklund, Carina; Morgan, Tyler; Shrier, Lydia A.; Sanders, Blake; Dillner, Joakim; de Vries, Henry J. C.; Morre, Servaas A.; Dean, Deborah

2017-01-01

Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted diseases worldwide. The Ct Multi Locus Sequence Typing (MLST) scheme is effective in differentiating strain types (ST), deciphering transmission patterns and treatment failure, and identifying recombinant strains. Here, we analyzed 323 reference and clinical samples, including 58 samples from Russia, an area that has not previously been represented in Ct typing schemes, to expand our knowledge of the global diversification of Ct STs. The 323 samples resolved into 84 unique STs, a 3.23 higher typing resolution compared to the gold standard single locus ompA genotyping. Our MLST scheme showed a high discriminatory index, D, of 0.98 (95% CI 0.97–0.99) confirming the validity of this method for typing. Phylogenetic analyses revealed distinct branches for the phenotypic diseases of lymphogranuloma venereum, urethritis and cervicitis, and a sub-branch for ocular trachoma. Consistent with these findings, single nucleotide polymorphisms were identified that significantly correlated with each phenotype. While the overall number of unique STs per region was comparable across geographies, the number of STs was greater for Russia with a significantly higher ST/sample ratio of 0.45 (95% CI: 0.35–0.53) compared to Europe or the Americas (p < 0.009), which may reflect a higher level of sexual mixing with the introduction of STs from other regions and/or reassortment of alleles. Four STs were found to be significantly associated with a particular geographic region. ST23 [p = 0.032 (95% CI: 1–23)], ST34 [p = 0.019 (95% CI: 1.1–25)]; and ST19 [p = 0.001 (95% CI: 1.7–34.7)] were significantly associated with Netherlands compared to Russia or the Americas, while ST 30 [p = 0.031 (95% CI: 1.1–17.8)] was significantly associated with the Americas. ST19 was significantly associated with Netherlands and Russia compared with the Americans [p = 0.001 (95% CI: 1.7–34.7) and p = 0.006 (95% CI: 1.5–34.6), respectively]. Additionally, recombinant strains were ubiquitous in the data set [106 (32.8%)], although Europe had a significantly higher number than Russia or the Americas (p < 0.04), the majority of which were from Amsterdam [43 (87.8%) of 49)]. The higher number of recombinants in Europe indicates selective pressure and/or adaptive diversification that will require additional studies to elucidate. PMID:29180986

Discriminative power of Campylobacter phenotypic and genotypic typing methods.

PubMed

Duarte, Alexandra; Seliwiorstow, Tomasz; Miller, William G; De Zutter, Lieven; Uyttendaele, Mieke; Dierick, Katelijne; Botteldoorn, Nadine

2016-06-01

The aim of this study was to compare different typing methods, individually and combined, for use in the monitoring of Campylobacter in food. Campylobacter jejuni (n=94) and Campylobacter coli (n=52) isolated from different broiler meat carcasses were characterized using multilocus sequence typing (MLST), flagellin gene A restriction fragment length polymorphism typing (flaA-RFLP), antimicrobial resistance profiling (AMRp), the presence/absence of 5 putative virulence genes; and, exclusively for C. jejuni, the determination of lipooligosaccharide (LOS) class. Discriminatory power was calculated by the Simpson's index of diversity (SID) and the congruence was measured by the adjusted Rand index and adjusted Wallace coefficient. MLST was individually the most discriminative typing method for both C. jejuni (SID=0.981) and C. coli (SID=0.957). The most discriminative combination with a SID of 0.992 for both C. jejuni and C. coli was obtained by combining MLST with flaA-RFLP. The combination of MLST with flaA-RFLP is an easy and feasible typing method for short-term monitoring of Campylobacter in broiler meat carcass. Copyright © 2016 Elsevier B.V. All rights reserved.

Understanding the molecular epidemiology and global relationships of Brachyspira hyodysenteriae from swine herds in the United States: a multi-locus sequence typing approach.

PubMed

Mirajkar, Nandita S; Gebhart, Connie J

2014-01-01

Outbreaks of mucohemorrhagic diarrhea in pigs caused by Brachyspira hyodysenteriae in the late 2000s indicated the re-emergence of Swine Dysentery (SD) in the U.S. Although the clinical disease was absent in the U.S. since the early 1990s, it continued to cause significant economic losses to other swine rearing countries worldwide. This study aims to fill the gap in knowledge pertaining to the re-emergence and epidemiology of B. hyodysenteriae in the U.S. and its global relationships using a multi-locus sequence typing (MLST) approach. Fifty-nine post re-emergent isolates originating from a variety of sources in the U.S. were characterized by MLST, analyzed for epidemiological relationships (within and between multiple sites of swine systems), and were compared with pre re-emergent isolates from the U.S. Information for an additional 272 global isolates from the MLST database was utilized for international comparisons. Thirteen nucleotide sequence types (STs) including a predominant genotype (ST93) were identified in the post re-emergent U.S. isolates; some of which showed genetic similarity to the pre re-emergent STs thereby suggesting its likely role in the re-emergence of SD. In the U.S., in general, no more than one ST was found on a site; multiple sites of a common system shared a ST; and STs found in the U.S. were distinct from those identified globally. Of the 110 STs characterized from ten countries, only two were found in more than one country. The U.S. and global populations, identified as clonal and heterogeneous based on STs, showed close relatedness based on amino acid types (AATs). One predicted founder type (AAT9) and multiple predicted subgroup founder types identified for both the U.S. and the global population indicate the potential microevolution of this pathogen. This study elucidates the strain diversity and microevolution of B. hyodysenteriae, and highlights the utility of MLST for epidemiological and surveillance studies.

Listeria monocytogenes Circulating in Rabbit Meat Products and Slaughterhouses in Italy: Prevalence Data and Comparison Among Typing Results.

PubMed

De Cesare, Alessandra; Parisi, Antonio; Mioni, Renzo; Comin, Damiano; Lucchi, Alex; Manfreda, Gerardo

2017-03-01

Rabbit meat has outstanding dietetic and nutritional properties. However, few data on microbiological hazards associated with rabbit productions are available. In this study, the presence of Listeria monocytogenes was determined in 430 rabbit carcasses, 256 rabbit meat cuts and products, and 599 environmental sponges collected from four Italian rabbit slaughterhouses over a period of 1 year. Prevalence of L. monocytogenes among the 1285 rabbit meat and environmental samples was 11%, with statistically significant differences between slaughterhouses. The highest prevalence (33.6%) was observed in rabbit meat cuts and products; the majority of positive environmental samples were collected from conveyor belts. Overall, 27.9% and 14.3% of rabbit cuts and carcasses, respectively, had L. monocytogenes counts higher than 1 colony-forming unit (CFU)/10 g. A selection of 123 isolates from positive samples was genotyped and serotyped to determine genetic profiles and diversity among L. monocytogenes isolates contaminating different slaughterhouses and classes of products investigated. Discriminatory power and concordance among the results obtained using multilocus variable-number tandem-repeat analysis (MLVA), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), automated EcoRI ribotyping, and serotyping were assessed. The isolates selected for typing were classified into serotypes 1/2a (52.8%), 1/2c (32.5%), and 1/2b (14.6%). The majority of the isolates were classified as ST14 (34.1%), ST9 (35.5%), ST121 (17.9%), and ST224 (14.6%). The greatest discriminatory power was observed with the MLVA typing, followed by MLST, PFGE, and ribotyping. The best bidirectional concordance was achieved between PFGE and MLST. There was 100% correlation between both MLST and MLVA with serotype. Moreover, a high unidirectional correspondence was observed between MLVA and both MLST and PFGE, as well as between PFGE and both MLST and serotyping. The results of this study show for the first time in Italy prevalence and genetic profiles of L. monocytogenes isolated in rabbit products and slaughterhouses.

Expression of Sme efflux pumps and multilocus sequence typing in clinical isolates of Stenotrophomonas maltophilia.

PubMed

Cho, Hye Hyun; Sung, Ji Youn; Kwon, Kye Chul; Koo, Sun Hoe

2012-01-01

Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of smeABC and smeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia. In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMérieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S. maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays. The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed smeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs) and 23 allelic profiles were observed. The smeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, smeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.

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

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.

Dissemination of VIM-2 producing Pseudomonas aeruginosa ST233 at tertiary care hospitals in Egypt.

PubMed

Zafer, Mai Mahmoud; Al-Agamy, Mohamed Hamed; El-Mahallawy, Hadir Ahmed; Amin, Magdy Aly; El Din Ashour, Seif

2015-03-12

Pseudomonas aeruginosa is an important nosocomial pathogen, commonly causing infections in immunocompromised patients. The aim of this study was to examine the genetic relatedness of metallo-beta-lactamase (MBL) producing carbapenem resistant Pseudomonas aeruginosa clinical isolates collected from 2 tertiary hospitals in Cairo, Egypt using Multi Locus sequence typing (MLST). Phenotypic and genotypic detection of metallo-beta-lactamase for forty eight non-duplicate carbapenem resistant P. aeruginosa isolates were carried out. DNA sequencing and MLST were done. The bla VIM-2 gene was highly prevalent (28/33 strains, 85%) among 33 MBL-positive P.aeruginosa isolates. MLST revealed eleven distinct Sequence Types (STs). A unique ST233 clone producing VIM-2 was documented by MLST in P.aeruginosa strains isolated from Cairo university hospitals. The high prevalence of VIM-2 producers was not due to the spread of a single clone. The findings of the present study clearly demonstrate that clones of VIM-2 positive in our hospitals are different from those reported from European studies. Prevalence of VIM-2 producers of the same clone was detected from surgical specimens whereas oncology related specimens were showing diverse clones.

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.

The role of Surinamese migrants in the transmission of Chlamydia trachomatis between Paramaribo, Suriname and Amsterdam, The Netherlands.

PubMed

Bom, Reinier J M; van der Helm, Jannie J; Bruisten, Sylvia M; Grünberg, Antoon W; Sabajo, Leslie O A; Schim van der Loeff, Maarten F; de Vries, Henry J C

2013-01-01

The large Surinamese migrant population in the Netherlands is a major risk group for urogenital Chlamydia trachomatis infection. Suriname, a former Dutch colony, also has a high prevalence of C. trachomatis. Surinamese migrants travel extensively between the Netherlands and Suriname. Our objective was to assess whether the Surinamese migrants in the Netherlands form a bridge population facilitating transmission of C. trachomatis between Suriname and the Netherlands. If so, joint prevention campaigns involving both countries might be required. Between March 2008 and July 2010, participants were recruited at clinics in Paramaribo, Suriname and in Amsterdam, the Netherlands. Participants were grouped as native Surinamese, native Dutch, Surinamese migrant, Dutch migrant, or Other, based on country of residence and country of birth of the participant and of their parents. Risk behavior, such as sexual mixing between ethnic groups, was recorded and C. trachomatis positive samples were typed through multilocus sequence typing (MLST). A minimum spanning tree of samples from 426 participants showed four MLST clusters. The MLST strain distribution of Surinamese migrants differed significantly from both the native Surinamese and Dutch populations, but was not an intermediate state between these two populations. Sexual mixing between the Surinamese migrants and the Dutch and Surinamese natives occurred frequently. Yet, the MLST cluster distribution did not differ significantly between participants who mixed and those who did not. Sexual mixing occurred between Surinamese migrants in Amsterdam and the native populations of Suriname and the Netherlands. These migrants, however, did not seem to form an effective bridge population for C. trachomatis transmission between the native populations. Although our data do not seem to justify the need for joint campaigns to reduce the transmission of C. trachomatis strains between both countries, intensified preventive campaigns to decrease the C. trachomatis burden are required, both in Suriname and in the Netherlands.

Multilocus Sequence Typing Has Better Discriminatory Ability for Typing Vibrio cholerae than Does Pulsed-Field Gel Electrophoresis and Provides a Measure of Phylogenetic Relatedness

PubMed Central

Kotetishvili, Mamuka; Stine, O. Colin; Chen, Yuansha; Kreger, Arnold; Sulakvelidze, Alexander; Sozhamannan, Shanmuga; Morris, Jr., J. Glenn

2003-01-01

Twenty-two Vibrio cholerae isolates, including some from “epidemic” (O1 and O139) and “nonepidemic” serogroups, were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) by using three housekeeping genes, gyrB, pgm, and recA; sequence data were also obtained for the virulence-associated genes tcpA, ctxA, and ctxB. Even with the small number of loci used, MLST had better discriminatory ability than did PFGE. On MLST analysis, there was clear clustering of epidemic serogroups; much greater diversity was seen among tcpA- and ctxAB-positive V. cholerae strains from other, nonepidemic serogroups, with a number of tcpA and ctxAB alleles identified. PMID:12734277

Genotypic Diversity and Virulence Characteristics of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region

PubMed Central

Bier, Nadja; Bechlars, Silke; Diescher, Susanne; Klein, Florian; Hauk, Gerhard; Duty, Oliver; Strauch, Eckhard

2013-01-01

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region. PMID:23542621

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

Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus.

PubMed

Kim, Byoung-Jun; Kim, Ga-Na; Kim, Bo-Ram; Shim, Tae-Sun; Kook, Yoon-Hoh; Kim, Bum-Joon

2017-01-01

Recent multi locus sequence typing (MLST) and genome based studies indicate that lateral gene transfer (LGT) events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas), we applied rpoB typing (711 bp) to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp). The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60) genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46), showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41) PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated.

Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus

PubMed Central

Kim, Byoung-Jun; Kim, Ga-Na; Kim, Bo-Ram; Shim, Tae-Sun; Kook, Yoon-Hoh

2017-01-01

Recent multi locus sequence typing (MLST) and genome based studies indicate that lateral gene transfer (LGT) events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas), we applied rpoB typing (711 bp) to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp). The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60) genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46), showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41) PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated. PMID:28604829

Identification of Escherichia coli and Shigella Species from Whole-Genome Sequences.

PubMed

Chattaway, Marie A; Schaefer, Ulf; Tewolde, Rediat; Dallman, Timothy J; Jenkins, Claire

2017-02-01

Escherichia coli and Shigella species are closely related and genetically constitute the same species. Differentiating between these two pathogens and accurately identifying the four species of Shigella are therefore challenging. The organism-specific bioinformatics whole-genome sequencing (WGS) typing pipelines at Public Health England are dependent on the initial identification of the bacterial species by use of a kmer-based approach. Of the 1,982 Escherichia coli and Shigella sp. isolates analyzed in this study, 1,957 (98.4%) had concordant results by both traditional biochemistry and serology (TB&S) and the kmer identification (ID) derived from the WGS data. Of the 25 mismatches identified, 10 were enteroinvasive E. coli isolates that were misidentified as Shigella flexneri or S. boydii by the kmer ID, and 8 were S. flexneri isolates misidentified by TB&S as S. boydii due to nonfunctional S. flexneri O antigen biosynthesis genes. Analysis of the population structure based on multilocus sequence typing (MLST) data derived from the WGS data showed that the remaining discrepant results belonged to clonal complex 288 (CC288), comprising both S. boydii and S. dysenteriae strains. Mismatches between the TB&S and kmer ID results were explained by the close phylogenetic relationship between the two species and were resolved with reference to the MLST data. Shigella can be differentiated from E. coli and accurately identified to the species level by use of kmer comparisons and MLST. Analysis of the WGS data provided explanations for the discordant results between TB&S and WGS data, revealed the true phylogenetic relationships between different species of Shigella, and identified emerging pathoadapted lineages. © Crown copyright 2017.

Clonality and distribution of clinical Ureaplasma isolates recovered from male patients and infertile couples in China

PubMed Central

Ruan, Zhi; Yang, Ting; Shi, Xinyan; Kong, Yingying; Xie, Xinyou

2017-01-01

Ureaplasma spp. have gained increasing recognition as pathogens in both adult and neonatal patients with multiple clinical presentations. However, the clonality of this organism in the male population and infertile couples in China is largely unknown. In this study, 96 (53 U. parvum and 43 U. urealyticum) of 103 Ureaplasma spp. strains recovered from genital specimens from male patients and 15 pairs of infertile couples were analyzed using multilocus sequence typing (MLST)/expanded multilocus sequence typing (eMLST) schemes. A total of 39 sequence types (STs) and 53 expanded sequence types (eSTs) were identified, with three predominant STs (ST1, ST9 and ST22) and eSTs (eST16, eST41 and eST82). Moreover, phylogenetic analysis revealed two distinct clusters that were highly congruent with the taxonomic differences between the two Ureaplasma species. We found significant differences in the distributions of both clusters and sub-groups between the male and female patients (P < 0.001). Moreover, 66.7% and 40.0% of the male and female partners of the infertile couples tested positive for Ureaplasma spp. The present study also attained excellent agreement of the identification of both Ureaplasma species between paired urine and semen specimens from the male partners (k > 0.80). However, this concordance was observed only for the detection of U. urealyticum within the infertile couples. In conclusion, the distributions of the clusters and sub-groups significantly differed between the male and female patients. U. urealyticum is more likely to transmit between infertile couples and be associated with clinical manifestations by the specific epidemic clonal lineages. PMID:28859153

Clonality and distribution of clinical Ureaplasma isolates recovered from male patients and infertile couples in China.

PubMed

Ruan, Zhi; Yang, Ting; Shi, Xinyan; Kong, Yingying; Xie, Xinyou; Zhang, Jun

2017-01-01

Ureaplasma spp. have gained increasing recognition as pathogens in both adult and neonatal patients with multiple clinical presentations. However, the clonality of this organism in the male population and infertile couples in China is largely unknown. In this study, 96 (53 U. parvum and 43 U. urealyticum) of 103 Ureaplasma spp. strains recovered from genital specimens from male patients and 15 pairs of infertile couples were analyzed using multilocus sequence typing (MLST)/expanded multilocus sequence typing (eMLST) schemes. A total of 39 sequence types (STs) and 53 expanded sequence types (eSTs) were identified, with three predominant STs (ST1, ST9 and ST22) and eSTs (eST16, eST41 and eST82). Moreover, phylogenetic analysis revealed two distinct clusters that were highly congruent with the taxonomic differences between the two Ureaplasma species. We found significant differences in the distributions of both clusters and sub-groups between the male and female patients (P < 0.001). Moreover, 66.7% and 40.0% of the male and female partners of the infertile couples tested positive for Ureaplasma spp. The present study also attained excellent agreement of the identification of both Ureaplasma species between paired urine and semen specimens from the male partners (k > 0.80). However, this concordance was observed only for the detection of U. urealyticum within the infertile couples. In conclusion, the distributions of the clusters and sub-groups significantly differed between the male and female patients. U. urealyticum is more likely to transmit between infertile couples and be associated with clinical manifestations by the specific epidemic clonal lineages.

Population Genetics of Lactobacillus sakei Reveals Three Lineages with Distinct Evolutionary Histories

PubMed Central

Chaillou, Stéphane; Lucquin, Isabelle; Najjari, Afef; Zagorec, Monique; Champomier-Vergès, Marie-Christine

2013-01-01

Lactobacillus sakei plays a major role in meat fermentation and in the preservation of fresh meat. The large diversity of L. sakei strains represents a valuable and exploitable asset in the development of a variety of industrial applications; however, an efficient method to identify and classify these strains has yet to be developed. In this study, we used multilocus sequence typing (MLST) to analyze the polymorphism and allelic distribution of eight loci within an L. sakei population of 232 strains collected worldwide. Within this population, we identified 116 unique sequence types with an average pairwise nucleotide diversity per site (π) of 0.13%. Results from Structure, goeBurst, and ClonalFrame software analyses demonstrated that the L. sakei population analyzed here is derived from three ancestral lineages, each of which shows evidence of a unique evolutionary history influenced by independent selection scenarios. However, the signature of selective events in the contemporary population of isolates was somewhat masked by the pervasive phenomenon of homologous recombination. Our results demonstrate that lineage 1 is a completely panmictic subpopulation in which alleles have been continually redistributed through the process of intra-lineage recombination. In contrast, lineage 2 was characterized by a high degree of clonality. Lineage 3, the earliest-diverging branch in the genealogy, showed evidence of both clonality and recombination. These evolutionary histories strongly indicate that the three lineages may correspond to distinct ecotypes, likely linked or specialized to different environmental reservoirs. The MLST scheme developed in this study represents an easy and straightforward tool that can be used to further analyze the population dynamics of L. sakei strains in food products. PMID:24069179

Population genetics of Lactobacillus sakei reveals three lineages with distinct evolutionary histories.

PubMed

Chaillou, Stéphane; Lucquin, Isabelle; Najjari, Afef; Zagorec, Monique; Champomier-Vergès, Marie-Christine

2013-01-01

Lactobacillus sakei plays a major role in meat fermentation and in the preservation of fresh meat. The large diversity of L. sakei strains represents a valuable and exploitable asset in the development of a variety of industrial applications; however, an efficient method to identify and classify these strains has yet to be developed. In this study, we used multilocus sequence typing (MLST) to analyze the polymorphism and allelic distribution of eight loci within an L. sakei population of 232 strains collected worldwide. Within this population, we identified 116 unique sequence types with an average pairwise nucleotide diversity per site (π) of 0.13%. Results from Structure, goeBurst, and ClonalFrame software analyses demonstrated that the L. sakei population analyzed here is derived from three ancestral lineages, each of which shows evidence of a unique evolutionary history influenced by independent selection scenarios. However, the signature of selective events in the contemporary population of isolates was somewhat masked by the pervasive phenomenon of homologous recombination. Our results demonstrate that lineage 1 is a completely panmictic subpopulation in which alleles have been continually redistributed through the process of intra-lineage recombination. In contrast, lineage 2 was characterized by a high degree of clonality. Lineage 3, the earliest-diverging branch in the genealogy, showed evidence of both clonality and recombination. These evolutionary histories strongly indicate that the three lineages may correspond to distinct ecotypes, likely linked or specialized to different environmental reservoirs. The MLST scheme developed in this study represents an easy and straightforward tool that can be used to further analyze the population dynamics of L. sakei strains in food products.

Characterization of Listeria monocytogenes isolates in import food products of China from 8 provinces between 2005 and 2007.

PubMed

Wang, Ping; Yang, Hairong; Hu, Yue; Yuan, Fei; Zhao, Guiming; Zhao, Yongsheng; Chen, Ying

2012-04-01

A total of 48 Listeria monocytogenes isolates of different import food products from 8 provinces between 2005 and 2008 were characterized. The serotype and virulence were confirmed for each strain and molecular subtyping were analyzed by multilocus sequence typing (MLST). Twenty five strains were assigned to serotype 1/2a, and 11 isolates to serotype 1/2b, serotype 4b were found in 7 isolate, and the remaining 5 strains were grouped into serotypes 1/2c, 4a, and 4e. Molecular subtyping schemes found thirty two sequence types (STs) among these isolates and the majority of L. monocytogenes strains belonged to lineage II (56%), followed by lineage I (38%), lineage III (6%). Two molecular subtype clusters, cluster A included all isolates of lineage II, while cluster B contained the isolates of lineages I and lineages III. Two L. monocytogenes strains were not grouped in either of the two clusters. Fifty three isolates were as virulent as L. monocytogenes reference strain EGD in mouse virulence assay, while the isolates 22213 and 22265 had low pathogenicity. These results provide the first molecular insight into the L. monocytogenes strains isolated from import food products of 8 provinces in China and indicate the potential risk to cause human disease if intake by contaminated foods. MLST could be used as a routine subtyping method of L. monocytogenes isolates. In China, inspection and quarantine strategies of imported foods should be strengthened. There is a potential risk of listeriosis in China and routine subtyping of L. monocytogenes isolates is important. It is necessary for food hygiene management to strengthen the supervision of imported foods. © 2012 Institute of Food Technologists®

Molecular Epidemiology of Novel Pathogen "Brachyspira hampsonii" Reveals Relationships between Diverse Genetic Groups, Regions, Host Species, and Other Pathogenic and Commensal Brachyspira Species.

PubMed

Mirajkar, Nandita S; Bekele, Aschalew Z; Chander, Yogesh Y; Gebhart, Connie J

2015-09-01

Outbreaks of bloody diarrhea in swine herds in the late 2000s signaled the reemergence of an economically significant disease, swine dysentery, in the United States. Investigations confirmed the emergence of a novel spirochete in swine, provisionally designated "Brachyspira hampsonii," with two genetically distinct clades. Although it has since been detected in swine and migratory birds in Europe and North America, little is known about its genetic diversity or its relationships with other Brachyspira species. This study characterizes B. hampsonii using a newly developed multilocus sequence typing (MLST) approach and elucidates the diversity, distribution, population structure, and genetic relationships of this pathogen from diverse epidemiological sources globally. Genetic characterization of 81 B. hampsonii isolates, originating from six countries, with our newly established MLST scheme identified a total of 20 sequence types (STs) belonging to three clonal complexes (CCs). B. hampsonii showed a heterogeneous population structure with evidence of microevolution locally in swine production systems, while its clustering patterns showed associations with its epidemiological origins (country, swine production system, and host species). The close genetic relatedness of B. hampsonii isolates from different countries and host species highlights the importance of strict biosecurity control measures. A comparative analysis of 430 isolates representing seven Brachyspira species (pathogens and commensals) from 19 countries and 10 host species depicted clustering by microbial species. It revealed the close genetic relatedness of B. hampsonii with commensal Brachyspira species and also provided support for the two clades of B. hampsonii to be considered a single species. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing

PubMed Central

Momeni, Stephanie S.; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A.; Childers, Noel K.

2015-01-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African American children was examined using MLST. Serotype and presence of collagen-binding proteins (CBP) cnm/cbm were also assessed. One hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using START2 and MEGA. Thirty-four sequence types (ST) were identified of which 27 were unique to this population. Seventy-five percent of the isolates clustered into 16 clonal groups. Serotypes observed were c (n=84), e (n=3), and k (n=11). The prevalence of S. mutans isolates serotype k was notably high at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized populations studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study is higher than reported in most populations and is the first report of S. mutans serotype k in a US population. PMID:26443288

An Improved MLVF Method and Its Comparison with Traditional MLVF, spa Typing, MLST/SCCmec and PFGE for the Typing of Methicillin-Resistant Staphylococcus aureus

PubMed Central

Du, Xue-Fei; Xiao, Meng; Liang, Hong-Yan; Sun, Zhe; Jiang, Yue-Hong; Chen, Guo-Yu; Meng, Xiao-Yu; Zou, Gui-Ling; Zhang, Li; Liu, Ya-Li; Zhang, Hui; Sun, Hong-Li; Jiang, Xiao-Feng; Xu, Ying-Chun

2014-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen, causing considerable morbidity and mortality. During the last 20 years, a variety of genotyping methods have been introduced for screening the prevalence of MRSA. In this study, we developed and evaluated an improved approach capillary gel electrophoresis based multilocus variable-number tandem-repeat fingerprinting (CGE/MLVF) for rapid MRSA typing. A total of 42 well-characterized strains and 116 non-repetitive clinical MRSA isolates collected from six hospitals in northeast China between 2009 and 2010 were tested. The results obtained by CGE/MLVF against clinical isolates were compared with traditional MLVF, spa typing, Multilocus sequence typing/staphylococcal cassette chromosome mec (MLST/SCCmec) and pulse field gel electrophoresis (PFGE). The discriminatory power estimated by Simpson’s index of diversity was 0.855 (28 types), 0.855 (28 patterns), 0.623 (11 types), 0.517 (8 types) and 0.854 (28 patterns) for CGE/MLVF, traditional MLVF, spa typing, MLST/SCCmec and PFGE, respectively. All methods tested showed a satisfied concordance in clonal complex level calculated by adjusted Rand’s coefficient. CGE/MLVF showed better reproducibility and accuracy than traditional MLVF and PFGE methods. In addition, the CGE/MLVF has potential to produce portable results. In conclusion, CGE/MLVF is a rapid and easy to use MRSA typing method with lower cost, good reproducibility and high discriminatory power for monitoring the outbreak and clonal spread of MRSA isolates. PMID:24406728

A Nonautochthonous U.S. Strain of Vibrio parahaemolyticus Isolated from Chesapeake Bay Oysters Caused the Outbreak in Maryland in 2010

PubMed Central

Haendiges, Julie; Jones, Jessica; Myers, Robert A.; Mitchell, Clifford S.; Butler, Erin

2016-01-01

ABSTRACT In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to the consumption of oysters. Strains isolated from both stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). However, the oysters contained other potentially pathogenic V. parahaemolyticus strains exhibiting different PFGE patterns. In order to assess the identity, genetic makeup, relatedness, and potential pathogenicity of the V. parahaemolyticus strains, we sequenced 11 such strains (2 clinical strains and 9 oyster strains). We analyzed these genomes by in silico multilocus sequence typing (MLST) and determined their phylogeny using a whole-genome MLST (wgMLST) analysis. Our in silico MLST analysis identified six different sequence types (STs) (ST8, ST676, ST810, ST811, ST34, and ST768), with both of the clinical and four of the oyster strains being identified as belonging to ST8. Using wgMLST, we showed that the ST8 strains from clinical and oyster samples were nearly indistinguishable and belonged to the same outbreak, confirming that local oysters were the source of the infections. The remaining oyster strains were genetically diverse, differing in >3,000 loci from the Maryland ST8 strains. eBURST analysis comparing these strains with strains of other STs available at the V. parahaemolyticus MLST website showed that the Maryland ST8 strains belonged to a clonal complex endemic to Asia. This indicates that the ST8 isolates from clinical and oyster sources were likely not endemic to Maryland. Finally, this study demonstrates the utility of whole-genome sequencing (WGS) and associated analyses for source-tracking investigations. IMPORTANCE Vibrio parahaemolyticus is an important foodborne pathogen and the leading cause of bacterial infections in the United States associated with the consumption of seafood. In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to oyster consumption. Strains isolated from stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). The oysters also contained other potentially pathogenic V. parahaemolyticus strains with different PFGE patterns. Since their identity, genetic makeup, relatedness, and potential pathogenicity were unknown, their genomes were determined by using next-generation sequencing. Whole-genome sequencing (WGS) analysis by whole-genome multilocus sequence typing (wgMLST) allowed (i) identification of clinical and oyster strains with matching PFGE profiles as belonging to ST8, (ii) determination of oyster strain diversity, and (iii) identification of the clinical strains as belonging to a clonal complex (CC) described only in Asia. Finally, WGS and associated analyses demonstrated their utility for trace-back investigations. PMID:26994080

A Nonautochthonous U.S. Strain of Vibrio parahaemolyticus Isolated from Chesapeake Bay Oysters Caused the Outbreak in Maryland in 2010.

PubMed

Haendiges, Julie; Jones, Jessica; Myers, Robert A; Mitchell, Clifford S; Butler, Erin; Toro, Magaly; Gonzalez-Escalona, Narjol

2016-06-01

In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to the consumption of oysters. Strains isolated from both stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). However, the oysters contained other potentially pathogenic V. parahaemolyticus strains exhibiting different PFGE patterns. In order to assess the identity, genetic makeup, relatedness, and potential pathogenicity of the V. parahaemolyticus strains, we sequenced 11 such strains (2 clinical strains and 9 oyster strains). We analyzed these genomes by in silico multilocus sequence typing (MLST) and determined their phylogeny using a whole-genome MLST (wgMLST) analysis. Our in silico MLST analysis identified six different sequence types (STs) (ST8, ST676, ST810, ST811, ST34, and ST768), with both of the clinical and four of the oyster strains being identified as belonging to ST8. Using wgMLST, we showed that the ST8 strains from clinical and oyster samples were nearly indistinguishable and belonged to the same outbreak, confirming that local oysters were the source of the infections. The remaining oyster strains were genetically diverse, differing in >3,000 loci from the Maryland ST8 strains. eBURST analysis comparing these strains with strains of other STs available at the V. parahaemolyticus MLST website showed that the Maryland ST8 strains belonged to a clonal complex endemic to Asia. This indicates that the ST8 isolates from clinical and oyster sources were likely not endemic to Maryland. Finally, this study demonstrates the utility of whole-genome sequencing (WGS) and associated analyses for source-tracking investigations. Vibrio parahaemolyticus is an important foodborne pathogen and the leading cause of bacterial infections in the United States associated with the consumption of seafood. In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to oyster consumption. Strains isolated from stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). The oysters also contained other potentially pathogenic V. parahaemolyticus strains with different PFGE patterns. Since their identity, genetic makeup, relatedness, and potential pathogenicity were unknown, their genomes were determined by using next-generation sequencing. Whole-genome sequencing (WGS) analysis by whole-genome multilocus sequence typing (wgMLST) allowed (i) identification of clinical and oyster strains with matching PFGE profiles as belonging to ST8, (ii) determination of oyster strain diversity, and (iii) identification of the clinical strains as belonging to a clonal complex (CC) described only in Asia. Finally, WGS and associated analyses demonstrated their utility for trace-back investigations. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing.

PubMed

Momeni, Stephanie S; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A; Childers, Noel K

2015-12-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African-American children was examined using MLST. Serotype and the presence of collagen-binding proteins (CBPs) encoded by cnm/cbm were also assessed. One-hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start2 and mega. Thirty-four sequence types were identified, of which 27 were unique to this population. Seventy-five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population. © 2015 Eur J Oral Sci.

Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing.

PubMed

Magnússon, S H; Guðmundsdóttir, S; Reynisson, E; Rúnarsson, A R; Harðardóttir, H; Gunnarson, E; Georgsson, F; Reiersen, J; Marteinsson, V Th

2011-10-01

Campylobacter jejuni isolates from various sources in Iceland were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and campylobacter transmission routes to humans. A collection of 584 Campylobacter isolates were collected from clinical cases, food, animals and environment in Iceland in 1999-2002, during a period of national Campylobacter epidemic in Iceland. All isolates were characterized by pulse field gel electrophoresis (PFGE), and selected subset of 52 isolates representing the diversity of the identified PFGE types was further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. The results show a substantial diversity within the Icelandic Campylobacter population. Majority of the human Campylobacter infections originated from domestic chicken and cattle isolates. MLST showed the isolates to be distributed among previously reported and common sequence type complexes in the MLST database. The genotyping of Campylobacter from various sources has not previously been reported from Iceland, and the results of the study gave a valuable insight into the population structure of Camp. jejuni in Iceland, source distribution and transmission routes to humans. The geographical isolation of Iceland in the north Atlantic provides new information on Campylobacter population dynamics on a global scale. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology No claim to Icelandic Government works.

Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

PubMed Central

de Gier, Camilla; Kirkham, Lea-Ann S.

2015-01-01

Nonhemolytic variants of Haemophilus haemolyticus are difficult to differentiate from Haemophilus influenzae despite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the “fuzzy species” strains were identified as H. influenzae that had undergone complete deletion of the fucose operon. Such strains, which are untypeable by the H. influenzae multilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch of H. influenzae MLSA phylogenetic group II. We also found evidence of interspecies recombination between H. influenzae and H. haemolyticus within the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification of H. influenzae is important for disease surveillance and treatment. PMID:26378279

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA.

PubMed

Siarkou, Victoria I; Vorimore, Fabien; Vicari, Nadia; Magnino, Simone; Rodolakis, Annie; Pannekoek, Yvonne; Sachse, Konrad; Longbottom, David; Laroucau, Karine

2015-01-01

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.

Emergence of vanA Enterococcus faecium in Denmark, 2005-15.

PubMed

Hammerum, Anette M; Baig, Sharmin; Kamel, Yasmin; Roer, Louise; Pinholt, Mette; Gumpert, Heidi; Holzknecht, Barbara; Røder, Bent; Justesen, Ulrik S; Samulioniené, Jurgita; Kjærsgaard, Mona; Østergaard, Claus; Holm, Anette; Dzajic, Esad; Søndergaard, Turid Snekloth; Gaini, Shahin; Edquist, Petra; Alm, Erik; Lilje, Berit; Westh, Henrik; Stegger, Marc; Hasman, Henrik

2017-08-01

To describe the changing epidemiology of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis in clinical samples in Denmark 2005-15 according to species and van type, and, furthermore, to investigate the genetic relatedness of the clinical E. faecium isolates from 2015. During 2005-14, all clinical VRE isolates were tested for the presence of vanA/B/C genes by PCR. In 2015, all clinical VRE isolates were whole-genome sequenced. From the WGS data, the presence of van genes and MLST STs were extracted in silico . Core-genome MLST (cgMLST) analysis was performed for the vancomycin-resistant E. faecium isolates. During 2005-15, 1043 vanA E. faecium , 25 vanB E. faecium , 4 vanA E. faecalis and 28 vanB E. faecalis were detected. The number of VRE was <50 isolates/year until 2012 to > 200 isolates/year in 2013-15. In 2015, 368 vanA E. faecium and 1 vanB E. faecium were detected along with 1 vanA E. faecalis and 1 vanB E. faecalis . cgMLST subdivided the 368 vanA E. faecium isolates into 33 cluster types (CTs), whereas the vanB E. faecium isolate belonged to a different CT. ST203-CT859 was most prevalent (51%), followed by ST80-CT14 (22%), ST117-CT24 (6%), ST80-CT866 (4%) and ST80-CT860 (2%). Comparison with the cgMLST.org database, previous studies and personal communications with neighbouring countries revealed that the novel cluster ST203-CT859 emerged in December 2014 and spread to the south of Sweden and the Faroe Islands during 2015. VRE increased in Denmark during 2005-15 due to the emergence of several vanA E. faecium clones. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Simultaneous occurrence of MRSA and ESBL-producing Enterobacteriaceae on pig farms and in nasal and stool samples from farmers.

PubMed

Fischer, Julia; Hille, Katja; Ruddat, Inga; Mellmann, Alexander; Köck, Robin; Kreienbrock, Lothar

2017-02-01

Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase (ESBL) producing enterobacteria (ESBL-E) have emerged in livestock. This study prospectively investigates the prevalence of MRSA and ESBL-E on pig farms and in nasal and stool samples from farmers and compares molecular characteristics of these ESBL-E isolates. In 2014, samples were derived at 51 pig farms in Germany. Per farm, five dust and five fecal samples were collected; one nasal and one stool sample were retrieved from farmers. ESBL-E isolates from humans and environmental isolates from the respective farms were characterized using whole genome sequencing for classical multilocus sequence typing (MLST), determination of ESBL-encoding genes and an ad hoc core genome MLST (cgMLST) analysis. MRSA and ESBL-E were detected on 49 (96%) and 31 (61%) of the farms, respectively; in most cases (59%) simultaneously. Nasal MRSA carriage was detected in 72 of 85 (84.7%) farmers and five of 84 (6.0%) farmers carried ESBL-E. ESBL-Escherichia coli isolates from farmers belonged to MLST STs/ESBL-genes ST10/CTX-M-1, ST196/TEM-52, ST278/TEM-52, ST410/CTX-M-15 and ST453/CTX-M-1. In one case, the human ESBL-E isolate was clonally identical to isolates from the farm environment; in the other four cases typing results indicated potential exchange of resistance determinants between human and environmental isolates, but, comparing the isolates within a minimum spanning tree indicated differences in cgMLST-patterns between the farms (p=0.076). This study demonstrated rectal ESBL-E carriage rates among farmers, which were similar to those in the general population. Molecular typing suggested that cross-transmission between the farmers and the farm environment is possible. Copyright © 2016 Elsevier B.V. All rights reserved.

How Often Do They Have Sex? A Comparative Analysis of the Population Structure of Seven Eukaryotic Microbial Pathogens

PubMed Central

Tomasini, Nicolás; Lauthier, Juan José; Ayala, Francisco José; Tibayrenc, Michel; Diosque, Patricio

2014-01-01

The model of predominant clonal evolution (PCE) proposed for micropathogens does not state that genetic exchange is totally absent, but rather, that it is too rare to break the prevalent PCE pattern. However, the actual impact of this “residual” genetic exchange should be evaluated. Multilocus Sequence Typing (MLST) is an excellent tool to explore the problem. Here, we compared online available MLST datasets for seven eukaryotic microbial pathogens: Trypanosoma cruzi, the Fusarium solani complex, Aspergillus fumigatus, Blastocystis subtype 3, the Leishmania donovani complex, Candida albicans and Candida glabrata. We first analyzed phylogenetic relationships among genotypes within each dataset. Then, we examined different measures of branch support and incongruence among loci as signs of genetic structure and levels of past recombination. The analyses allow us to identify three types of genetic structure. The first was characterized by trees with well-supported branches and low levels of incongruence suggesting well-structured populations and PCE. This was the case for the T. cruzi and F. solani datasets. The second genetic structure, represented by Blastocystis spp., A. fumigatus and the L. donovani complex datasets, showed trees with weakly-supported branches but low levels of incongruence among loci, whereby genetic structuration was not clearly defined by MLST. Finally, trees showing weakly-supported branches and high levels of incongruence among loci were observed for Candida species, suggesting that genetic exchange has a higher evolutionary impact in these mainly clonal yeast species. Furthermore, simulations showed that MLST may fail to show right clustering in population datasets even in the absence of genetic exchange. In conclusion, these results make it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens. Moreover, our results reveal different problems of MLST to determine the genetic structure in these organisms that should be considered. PMID:25054834

A rural worker infected with a bovine-prevalent genotype of Campylobacter fetus subsp. fetus supports zoonotic transmission and inconsistency of MLST and whole-genome typing.

PubMed

Iraola, G; Betancor, L; Calleros, L; Gadea, P; Algorta, G; Galeano, S; Muxi, P; Greif, G; Pérez, R

2015-08-01

Whole-genome characterisation in clinical microbiology enables to detect trends in infection dynamics and disease transmission. Here, we report a case of bacteraemia due to Campylobacter fetus subsp. fetus in a rural worker under cancer treatment that was diagnosed with cellulitis; the patient was treated with antibiotics and recovered. The routine typing methods were not able to identify the microorganism causing the infection, so it was further analysed by molecular methods and whole-genome sequencing. The multi-locus sequence typing (MLST) revealed the presence of the bovine-associated ST-4 genotype. Whole-genome comparisons with other C. fetus strains revealed an inconsistent phylogenetic position based on the core genome, discordant with previous ST-4 strains. To the best of our knowledge, this is the first C. fetus subsp. fetus carrying the ST-4 isolated from humans and represents a probable case of zoonotic transmission from cattle.

Prevalence of GBS serotype III and identification of a ST 17-like genotype from neonates with invasive diseases in Guangzhou, China.

PubMed

Liu, Junyan; Xu, Ruiru; Zhong, Huamin; Zhong, Yukui; Xie, Yongqiang; Li, Lin; Li, Bing; Chen, Dingqiang; Xu, Zhenbo

2018-05-03

The aim of this study is to understand the surveillance of Streptococcus agalactiae (GBS) serotype and genotype which help developing specific vaccine for GBS infection. The GBS serotype was determined by strep-B-Latex rapid agglutination method and multiplex PCR assay based on the differences among serotypes. Alleles and multilocus sequence types (MLST) were determined using the GBS MLST Web site. Four GBS serotypes (Ia, Ib, III, and V) were identified, with serotype III which was intimately associated with purulent meningitis and sepsis, as the dominance. In EOD cases, sepsis and pneumonia showed dominance, but purulent meningitis was dominant in LOD cases. Also, a new ST-17 like type which might be a clone derive from ST-17 and emerge among neonatal disease cases was identified. The prevalence of GBS serotype and genotype and their relation with GBS diseases guide the development of capsular polysaccharide vaccine. Copyright © 2018. Published by Elsevier Ltd.

Nonclonal emergence of colistin-resistant Klebsiella pneumoniae isolates from blood samples in South Korea.

PubMed

Suh, Ji-Yoeun; Son, Jun Seong; Chung, Doo Ryeon; Peck, Kyong Ran; Ko, Kwan Soo; Song, Jae-Hoon

2010-01-01

In vitro activities of colistin and other drugs were tested against 221 Klebsiella pneumoniae isolates that were collected between 2006 and 2007 in nine tertiary care South Korean hospitals from patients with bacteremia. The clonality of colistin-resistant K. pneumoniae (CRKP) isolates was assessed by multilocus sequence typing (MLST). We found that 15 isolates (6.8%) were resistant to colistin. MLST showed that CRKP isolates were nonclonal, with colistin resistance in K. pneumoniae occurring independently and not by clonal spreading.

Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem.

PubMed

Singh, Pallavi; Sha, Qiong; Lacher, David W; Del Valle, Jacquelyn; Mosci, Rebekah E; Moore, Jennifer A; Scribner, Kim T; Manning, Shannon D

2015-01-01

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST) was performed on a subset. STEC and EHEC were cultured from 12 to 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March vs. June where the frequency of STEC, EHEC, and EPEC was 1, 6, and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds.

Assignment of Streptococcus agalactiae isolates to clonal complexes using a small set of single nucleotide polymorphisms.

PubMed

Honsa, Erin; Fricke, Thomas; Stephens, Alex J; Ko, Danny; Kong, Fanrong; Gilbert, Gwendolyn L; Huygens, Flavia; Giffard, Philip M

2008-08-19

Streptococcus agalactiae (Group B Streptococcus (GBS)) is an important human pathogen, particularly of newborns. Emerging evidence for a relationship between genotype and virulence has accentuated the need for efficient and well-defined typing methods. The objective of this study was to develop a single nucleotide polymorphism (SNP) based method for assigning GBS isolates to multilocus sequence typing (MLST)-defined clonal complexes. It was found that a SNP set derived from the MLST database on the basis of maximization of Simpsons Index of Diversity provided poor resolution and did not define groups concordant with the population structure as defined by eBURST analysis of the MLST database. This was interpreted as being a consequence of low diversity and high frequency horizontal gene transfer. Accordingly, a different approach to SNP identification was developed. This entailed use of the "Not-N" bioinformatic algorithm that identifies SNPs diagnostic for groups of known sequence variants, together with an empirical process of SNP testing. This yielded a four member SNP set that divides GBS into 10 groups that are concordant with the population structure. A fifth SNP was identified that increased the sensitivity for the clinically significant clonal complex 17 to 100%. Kinetic PCR methods for the interrogation of these SNPs were developed, and used to genotype 116 well characterized isolates. A five SNP method for dividing GBS into biologically valid groups has been developed. These SNPs are ideal for high throughput surveillance activities, and combining with more rapidly evolving loci when additional resolution is required.

Assignment of Streptococcus agalactiae isolates to clonal complexes using a small set of single nucleotide polymorphisms

PubMed Central

Honsa, Erin; Fricke, Thomas; Stephens, Alex J; Ko, Danny; Kong, Fanrong; Gilbert, Gwendolyn L; Huygens, Flavia; Giffard, Philip M

2008-01-01

Background Streptococcus agalactiae (Group B Streptococcus (GBS)) is an important human pathogen, particularly of newborns. Emerging evidence for a relationship between genotype and virulence has accentuated the need for efficient and well-defined typing methods. The objective of this study was to develop a single nucleotide polymorphism (SNP) based method for assigning GBS isolates to multilocus sequence typing (MLST)-defined clonal complexes. Results It was found that a SNP set derived from the MLST database on the basis of maximisation of Simpsons Index of Diversity provided poor resolution and did not define groups concordant with the population structure as defined by eBURST analysis of the MLST database. This was interpreted as being a consequence of low diversity and high frequency horizontal gene transfer. Accordingly, a different approach to SNP identification was developed. This entailed use of the "Not-N" bioinformatic algorithm that identifies SNPs diagnostic for groups of known sequence variants, together with an empirical process of SNP testing. This yielded a four member SNP set that divides GBS into 10 groups that are concordant with the population structure. A fifth SNP was identified that increased the sensitivity for the clinically significant clonal complex 17 to 100%. Kinetic PCR methods for the interrogation of these SNPs were developed, and used to genotype 116 well characterized isolates. Conclusion A five SNP method for dividing GBS into biologically valid groups has been developed. These SNPs are ideal for high throughput surveillance activities, and combining with more rapidly evolving loci when additional resolution is required. PMID:18710585

Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem

PubMed Central

Singh, Pallavi; Sha, Qiong; Lacher, David W.; Del Valle, Jacquelyn; Mosci, Rebekah E.; Moore, Jennifer A.; Scribner, Kim T.; Manning, Shannon D.

2015-01-01

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST) was performed on a subset. STEC and EHEC were cultured from 12 to 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March vs. June where the frequency of STEC, EHEC, and EPEC was 1, 6, and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds. PMID:25883908

Genetic Analysis of Vibrio parahaemolyticus O3:K6 Strains That Have Been Isolated in Mexico Since 1998

PubMed Central

Licea-Navarro, Alexei Fedorovish; Revilla-Castellanos, Valeria Jeanette; Wong-Chang, Irma; González-Sánchez, Ricardo

2017-01-01

Vibrio parahaemolyticus is an important human pathogen that has been isolated worldwide from clinical cases, most of which have been associated with seafood consumption. Environmental and clinical toxigenic strains of V. parahaemolyticus that were isolated in Mexico from 1998 to 2012, including those from the only outbreak that has been reported in this country, were characterized genetically to assess the presence of the O3:K6 pandemic clone, and their genetic relationship to strains that are related to the pandemic clonal complex (CC3). Pathogenic tdh+ and tdh+/trh+ strains were analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Also, the entire genome of a Mexican O3:K6 strain was sequenced. Most of the strains were tdh/ORF8-positive and corresponded to the O3:K6 serotype. By PFGE and MLST, there was very close genetic relationship between ORF8/O3:K6 strains, and very high genetic diversities from non-pandemic strains. The genetic relationship is very close among O3:K6 strains that were isolated in Mexico and sequences that were available for strains in the CC3, based on the PubMLST database. The whole-genome sequence of CICESE-170 strain had high similarity with that of the reference RIMD 2210633 strain, and harbored 7 pathogenicity islands, including the 4 that denote O3:K6 pandemic strains. These results indicate that pandemic strains that have been isolated in Mexico show very close genetic relationship among them and with those isolated worldwide. PMID:28099500

Multi-locus sequence typing provides epidemiological insights for diseased sharks infected with fungi belonging to the Fusarium solani species complex.

PubMed

Desoubeaux, Guillaume; Debourgogne, Anne; Wiederhold, Nathan P; Zaffino, Marie; Sutton, Deanna; Burns, Rachel E; Frasca, Salvatore; Hyatt, Michael W; Cray, Carolyn

2018-07-01

Fusarium spp. are saprobic moulds that are responsible for severe opportunistic infections in humans and animals. However, we need epidemiological tools to reliably trace the circulation of such fungal strains within medical or veterinary facilities, to recognize environmental contaminations that might lead to infection and to improve our understanding of factors responsible for the onset of outbreaks. In this study, we used molecular genotyping to investigate clustered cases of Fusarium solani species complex (FSSC) infection that occurred in eight Sphyrnidae sharks under managed care at a public aquarium. Genetic relationships between fungal strains were determined by multi-locus sequence typing (MLST) analysis based on DNA sequencing at five loci, followed by comparison with sequences of 50 epidemiologically unrelated FSSC strains. Our genotyping approach revealed that F. keratoplasticum and F. solani haplotype 9x were most commonly isolated. In one case, the infection proved to be with another Hypocrealian rare opportunistic pathogen Metarhizium robertsii. Twice, sharks proved to be infected with FSSC strains with the same MLST sequence type, supporting the hypothesis the hypothesis that common environmental populations of fungi existed for these sharks and would suggest the longtime persistence of the two clonal strains within the environment, perhaps in holding pools and life support systems of the aquarium. This study highlights how molecular tools like MLST can be used to investigate outbreaks of microbiological disease. This work reinforces the need for regular controls of water quality to reduce microbiological contamination due to waterborne microorganisms.

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 ...

Emerging Tick-borne Rickettsia and Ehrlichia at Joint Base Langley-Eustis, Fort Eustis, Virginia.

PubMed

Miller, Melissa K; Jiang, Ju; Truong, Melissa; Yarina, Tamasin; Evans, Holly; Christensen, Timothy P; Richards, Allen L

2016-01-01

Four species of ticks known to parasitize humans (Amblyomma americanum (lone star tick), Dermacentor variabilis (American dog tick), Amblyomma maculatum (Gulf Coast tick), and Ixodes scapularis (black-legged tick)) were collected at Joint Base Langley-Eustis, Fort Eustis, Virginia during 2009. These ticks were tested individually (adults and nymphs) and in pools of 15 (larvae) for pathogens of public health importance within the genera: Rickettsia, Borrelia, and Ehrlichia, by quantitative real-time polymerase chain reaction (qPCR) assays and, where appropriate, multilocus sequence typing (MLST). Of the 340 A americanum ticks tested, a minimum of 65 (19%), 4 (1%), 4 (1%), and one (<1%) were positive for Rickettsia amblyommii, B lonestari, E ewingii and E chaffeensis, respectively. One of 2 (50%) A maculatum ticks collected was found to be positive for R parkeri by MLST and qPCR analyses. All 33 D variabilis ticks were negative for evidence of rickettsial infections. Likewise, no pathogenic organisms were detected from the single Ixodes scapularis tick collected. Pathogenic rickettsiae and ehrlichiae are likely emerging and cause under-recognized diseases, which threaten people who live, work, train, or otherwise engage in outdoor activities at, or in the vicinity of, Fort Eustis, Virginia.

Friction and wear on laser textured Ti6Al4V surface subjected to laser shock peening with contacting foil

NASA Astrophysics Data System (ADS)

Dai, F. Z.; Geng, J.; Tan, W. S.; Ren, X. D.; Lu, J. Z.; Huang, Shu

2018-07-01

The Ti6Al4V micro-dimple surfaces fabricated by a masked laser surface texturing (MLST) technique within water were subjected to soft contact laser shock peening (SCLSP) and hard contact laser shock peening (HCLSP). The effects of these two LSP methods on topography, micro-hardness and residual stress distribution were studied. The friction and wear performance under dry friction and oil lubrication were also studied. The enclosure of micro cracks in the micro-dimple bottom was observed when treated by SCLSP and HCLSP. The dry friction and wear test showed that the MLST+HCLSP surfaces had the best wear resistance performance. In the oil lubricated friction test, the occurrence of the hydrodynamic lubrication effect occurred on the micro-dimple surfaces. The MLST+HCLSP exhibited the best friction and wear resistance performance. These were due to the micro-hardness increase, the producing of compressive residual stress and the surface roughness reduction of as treated surfaces.

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.

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

Isolation and identification of Salmonella spp. in drinking water, streams, and swine wastewater by molecular techniques in Taiwan

NASA Astrophysics Data System (ADS)

Kuo, C.; Hsu, B.; Shen, T.; Tseng, S.; Tsai, J.; Huang, K.; Kao, P.; Chen, J.

2013-12-01

Salmonella spp. is a common water-borne pathogens and its genus comprises more than 2,500 serotypes. Major pathogenic genotypes which cause typhoid fever, enteritis and other intestinal-type diseases are S. Typhimurium, S. Enteritidis, S. Stanley, S. Agona, S.Albany, S. Schwarzengrund, S. Newport, S. Choleraesuis, and S. Derby. Hence, the identification of the serotypes of Salmonella spp. is important. In the present study, the analytical procedures include direct concentration method, non-selective pre-enrichment method and selective enrichment method of Salmonella spp.. Both selective enrichment method and cultured bacteria were detected with specific primers of Salmonella spp. by polymerase chain reaction (PCR). At last, the serotypes of Salmonella were confirmed by using MLST (multilocus sequence typing) with aroC, dnaN, hemD, hisD, purE, sucA, thrA housekeeping genes to identify the strains of positive samples. This study contains 121 samples from three different types of water sources including the drinking water (51), streams (45), and swine wastewater (25). Thirteen samples with positive invA gene are separated from culture method. The strains of these positive samples which identified from MLST method are S. Albany, S. Typhimurium, S. Newport, S. Bareilly, and S. Derby. Some of the serotypes, S. Albany, S. Typhimurium and S. Newport, are highly pathogenic which correlated to human diarrhea. In our results, MLST is a useful method to identify the strains of Salmonella spp.. Keywords: Salmonella, PCR, MLST.

Inferring a Population Structure for Staphylococcus epidermidis from Multilocus Sequence Typing Data▿

PubMed Central

Miragaia, M.; Thomas, J. C.; Couto, I.; Enright, M. C.; de Lencastre, H.

2007-01-01

Despite its importance as a human pathogen, information on population structure and global epidemiology of Staphylococcus epidermidis is scarce and the relative importance of the mechanisms contributing to clonal diversification is unknown. In this study, we addressed these issues by analyzing a representative collection of S. epidermidis isolates from diverse geographic and clinical origins using multilocus sequence typing (MLST). Additionally, we characterized the mobile element (SCCmec) carrying the genetic determinant of methicillin resistance. The 217 S. epidermidis isolates from our collection were split by MLST into 74 types, suggesting a high level of genetic diversity. Analysis of MLST data using the eBURST algorithm revealed the existence of nine epidemic clonal lineages that were disseminated worldwide. One single clonal lineage (clonal complex 2) comprised 74% of the isolates, whereas the remaining isolates were clustered into 8 minor clonal lineages and 13 singletons. According to our evolutionary model, SCCmec was acquired at least 56 times by S. epidermidis. Although geographic dissemination of S. epidermidis strains and the value of the index of association between the alleles, 0.2898 (P < 0.05), support the clonality of S. epidermidis species, examination of the sequence changes at MLST loci during clonal diversification showed that recombination gives rise to new alleles approximately twice as frequently as point mutations. We suggest that S. epidermidis has a population with an epidemic structure, in which nine clones have emerged upon a recombining background and evolved quickly through frequent transfer of genetic mobile elements, including SCCmec. PMID:17220222

High-throughput informative single nucleotide polymorphism-based typing of Neisseria gonorrhoeae using the Sequenom MassARRAY iPLEX platform.

PubMed

Trembizki, Ella; Smith, Helen; Lahra, Monica M; Chen, Marcus; Donovan, Basil; Fairley, Christopher K; Guy, Rebecca; Kaldor, John; Regan, David; Ward, James; Nissen, Michael D; Sloots, Theo P; Whiley, David M

2014-06-01

Neisseria gonorrhoeae antimicrobial resistance (AMR) is a global problem heightened by emerging resistance to ceftriaxone. Appropriate molecular typing methods are important for understanding the emergence and spread of N. gonorrhoeae AMR. We report on the development, validation and testing of a Sequenom MassARRAY iPLEX method for multilocus sequence typing (MLST)-style genotyping of N. gonorrhoeae isolates. An iPLEX MassARRAY method (iPLEX14SNP) was developed targeting 14 informative gonococcal single nucleotide polymorphisms (SNPs) previously shown to predict MLST types. The method was initially validated using 24 N. gonorrhoeae control isolates and was then applied to 397 test isolates collected throughout Queensland, Australia in the first half of 2012. The iPLEX14SNP method provided 100% accuracy for the control isolates, correctly identifying all 14 SNPs for all 24 isolates (336/336). For the 397 test isolates, the iPLEX14SNP assigned results for 5461 of the possible 5558 SNPs (SNP call rate 98.25%), with complete 14 SNP profiles obtained for 364 isolates. Based on the complete SNP profile data, there were 49 different sequence types identified in Queensland, with 11 of the 49 SNP profiles accounting for the majority (n = 280; 77%) of isolates. AMR was dominated by several geographically clustered sequence types. Using the iPLEX14SNP method, up to 384 isolates could be tested within 1 working day for less than Aus$10 per isolate. The iPLEX14SNP offers an accurate and high-throughput method for the MLST-style genotyping of N. gonorrhoeae and may prove particularly useful for large-scale studies investigating the emergence and spread of gonococcal AMR. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

[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.

Nonclonal Emergence of Colistin-Resistant Klebsiella pneumoniae Isolates from Blood Samples in South Korea ▿

PubMed Central

Suh, Ji-Yoeun; Son, Jun Seong; Chung, Doo Ryeon; Peck, Kyong Ran; Ko, Kwan Soo; Song, Jae-Hoon

2010-01-01

In vitro activities of colistin and other drugs were tested against 221 Klebsiella pneumoniae isolates that were collected between 2006 and 2007 in nine tertiary care South Korean hospitals from patients with bacteremia. The clonality of colistin-resistant K. pneumoniae (CRKP) isolates was assessed by multilocus sequence typing (MLST). We found that 15 isolates (6.8%) were resistant to colistin. MLST showed that CRKP isolates were nonclonal, with colistin resistance in K. pneumoniae occurring independently and not by clonal spreading. PMID:19752282

Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from “Pseudo-Cronobacter” to New Enterobacteriaceae Genera

PubMed Central

Vlach, Jiří; Junková, Petra; Karamonová, Ludmila; Blažková, Martina; Fukal, Ladislav

2017-01-01

ABSTRACT In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter. Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method. IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter. While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease. PMID:28455327

Helicobacter pylori from gastric cancer and duodenal ulcer show same phylogeographic origin in the Andean region in Colombia.

PubMed

Shiota, Seiji; Suzuki, Rumiko; Matsuo, Yuichi; Miftahussurur, Muhammad; Tran, Trang Thu Huyen; Binh, Tran Thanh; Yamaoka, Yoshio

2014-01-01

A recent report has shown that the phylogenetic origin of Helicobacter pylori based on multi-locus sequence typing (MLST) was significantly associated with the severity of gastritis in Colombia. However, the potential relationship between phylogenetic origin and clinical outcomes was not examined in that study. If the phylogenetic origin rather than virulence factors were truly associated with clinical outcomes, identifying a population at high risk for gastric cancer in Colombia would be relatively straightforward. In this study, we examined the phylogenetic origins of strains from gastric cancer and duodenal ulcer patients living in Bogota, Colombia. We included 35 gastric cancer patients and 31 duodenal ulcer patients, which are considered the variant outcomes. The genotypes of cagA and vacA were determined by polymerase chain reaction. The genealogy of these Colombian strains was analyzed by MLST. Bacterial population structure was analyzed using STRUCTURE software. H. pylori strains from gastric cancer and duodenal ulcer patients were scattered in the phylogenetic tree; thus, we did not detect any difference in phylogenetic distribution between gastric cancer and duodenal ulcer strains in the hpEurope group in Colombia. Sixty-six strains, with one exception, were classified as hpEurope irrespective of the cagA and vacA genotypes, and type of disease. STRUCTURE analysis revealed that Colombian hpEurope strains have a phylogenetic connection to Spanish strains. Our study showed that a phylogeographic origin determined by MLST was insufficient for distinguishing between gastric cancer and duodenal ulcer risk among hpEurope strains in the Andean region in Colombia. Our analysis also suggests that hpEurope strains in Colombia were primarily introduced by Spanish immigrants.

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

Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales, Australia

PubMed Central

Gray, Timothy J.; Wang, Qinning; Ng, Jimmy; Hicks, Leanne; Nguyen, Trang; Yuen, Marion; Hill-Cawthorne, Grant A.; Sintchenko, Vitali

2015-01-01

Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont/B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST; however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by >3,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST. PMID:26109442

Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales, Australia.

PubMed

McCallum, Nadine; Gray, Timothy J; Wang, Qinning; Ng, Jimmy; Hicks, Leanne; Nguyen, Trang; Yuen, Marion; Hill-Cawthorne, Grant A; Sintchenko, Vitali

2015-09-01

Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont/B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST; however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by >3,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant.

PubMed

Palma, Federica; Pasquali, Frédérique; Lucchi, Alex; Cesare, Alessandra De; Manfreda, Gerardo

2017-08-16

Listeria monocytogenes is a food-borne pathogen able to survive and grow in different environments including food processing plants where it can persist for month or years. In the present study the discriminatory power of Whole Genome Sequencing (WGS)-based analysis (cgMLST) was compared to that of molecular typing methods on 34 L. monocytogenes isolates collected over one year in the same rabbit meat processing plant and belonging to three genotypes (ST14, ST121, ST224). Each genotype included isolates indistinguishable by standard molecular typing methods. The virulence potential of all isolates was assessed by Multi Virulence-Locus Sequence Typing (MVLST) and the investigation of a representative database of virulence determinant genes. The whole genome of each isolate was sequenced on a MiSeq platform. The cgMLST, MVLST, and in silico identification of virulence genes were performed using publicly available tools. Draft genomes included a number of contigs ranging from 13 to 28 and N50 ranging from 456298 to 580604. The coverage ranged from 41 to 187X. The cgMLST showed a significantly superior discriminatory power only in comparison to ribotyping, nevertheless it allows the detection of two singletons belonging to ST14 that were not observed by other molecular methods. All ST14 isolates belonged to VT107, which 7-loci concatenated sequence differs for only 4 nucleotides to VT1 (Epidemic clone III). Analysis of virulence genes showed the presence of a fulllength inlA version in all ST14 isolates and of a mutated version including a premature stop codon (PMSC) associated to attenuated virulence in all ST121 isolates.

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.

Comparative genomics of Enterococcus faecalis from healthy Norwegian infants

PubMed Central

Solheim, Margrete; Aakra, Ågot; Snipen, Lars G; Brede, Dag A; Nes, Ingolf F

2009-01-01

Background Enterococcus faecalis, traditionally considered a harmless commensal of the intestinal tract, is now ranked among the leading causes of nosocomial infections. In an attempt to gain insight into the genetic make-up of commensal E. faecalis, we have studied genomic variation in a collection of community-derived E. faecalis isolated from the feces of Norwegian infants. Results The E. faecalis isolates were first sequence typed by multilocus sequence typing (MLST) and characterized with respect to antibiotic resistance and properties associated with virulence. A subset of the isolates was compared to the vancomycin resistant strain E. faecalis V583 (V583) by whole genome microarray comparison (comparative genomic hybridization (CGH)). Several of the putative enterococcal virulence factors were found to be highly prevalent among the commensal baby isolates. The genomic variation as observed by CGH was less between isolates displaying the same MLST sequence type than between isolates belonging to different evolutionary lineages. Conclusion The variations in gene content observed among the investigated commensal E. faecalis is comparable to the genetic variation previously reported among strains of various origins thought to be representative of the major E. faecalis lineages. Previous MLST analysis of E. faecalis have identified so-called high-risk enterococcal clonal complexes (HiRECC), defined as genetically distinct subpopulations, epidemiologically associated with enterococcal infections. The observed correlation between CGH and MLST presented here, may offer a method for the identification of lineage-specific genes, and may therefore add clues on how to distinguish pathogenic from commensal E. faecalis. In this work, information on the core genome of E. faecalis is also substantially extended. PMID:19393078

Clonal structure in Ichthyobacterium seriolicida, the causative agent of bacterial haemolytic jaundice in yellowtail, Seriola quinqueradiata, inferred from molecular epidemiological analysis.

PubMed

Matsuyama, T; Fukuda, Y; Sakai, T; Tanimoto, N; Nakanishi, M; Nakamura, Y; Takano, T; Nakayasu, C

2017-08-01

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I. seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Twenty-eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I. seriolicida populations share a common ancestor, which may be a recently introduced strain. © 2016 John Wiley & Sons Ltd.

Genotyping of clinical and environmental multidrug resistant Enterococcus faecium strains.

PubMed

Shokoohizadeh, Leili; Mobarez, Ashraf Mohabati; Alebouyeh, Masoud; Zali, Mohammad Reza; Ranjbar, Reza

2017-01-01

Multidrug resistant (MDR) Enterococcus faecium is a nosocomial pathogen and clonal complex 17 (CC17) is the main genetic subpopulation of E. faecium in hospitals worldwide. There has thus far been no report of major E. faecium clones in Iranian hospitals. The present study analyzed strains of MDR E. faecium obtained from patients and the Intensive Care Unit environments using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to determine the antibiotic resistance patterns and genetic features of the dominant. clones of E. faecium. PFGE and MLST analysis revealed the presence of 17and 15 different subtypes, respectively. Of these, 18 (86%) isolates belonged toCC17. Most strains in this clonal complex harbored the esp gene and exhibited resistance to vancomycin, teicoplanin, ampicillin, ciprofloxacin, gentamicin, and erythromycin. The MLST results revealed 12 new sequence types (ST) for the first time. Approximately 50% of the STs were associated with ST203. Detection of E. faecium strains belonging to CC17 on medical equipment and in clinical specimens verified the circulation of high-risk MDR clones among the patients and in hospital environments in Iran.

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry to identify MLST clade 4 Clostridium difficile isolates.

PubMed

Cheng, Jing-Wei; Liu, Chang; Kudinha, Timothy; Xiao, Meng; Yu, Shu-Ying; Yang, Chun-Xia; Wei, Ming; Liang, Guo-Wei; Shao, Dong-Hua; Kong, Fanrong; Tong, Zhao-Hui; Xu, Ying-Chun

2018-04-26

Clostridium difficile is the leading cause of health care-associated infections. Previous studies suggest that C. difficile MLST clade 4 strains with higher drug resistance rates constitute the major clone spreading in China. Thus development of a rapid and accurate typing method for these strains is needed to monitor the epidemiology of this clone and to guide clinical treatment. A total of 160 non-duplicate C. difficile isolates recovered from three large teaching hospitals in Beijing were studied. All the 41 clade 4 C. difficile isolates clustered together on the PCA dendrogram. Spectra peak statistics revealed that five markers (2691.43Da, 2704.91Da, 2711.93Da, 3247.27Da and 3290.76Da) can easily and reliably distinguish between clade 4 and non-clade 4 isolates, with area under the curve (AUC) values of 0.991, 0.997, 0.973, 1 and 1, respectively. In conclusion, MALDI-TOF MS is a very simple and accurate method for identifying C. difficile MLST clade 4 strains. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular Epidemiology Reveals Genetic Diversity amongst Isolates of the Cryptococcus neoformans/C. gattii Species Complex in Thailand

PubMed Central

Kaocharoen, Sirada; Ngamskulrungroj, Popchai; Firacative, Carolina; Trilles, Luciana; Piyabongkarn, Dumrongdej; Banlunara, Wijit; Poonwan, Natteewan; Chaiprasert, Angkana; Meyer, Wieland; Chindamporn, Ariya

2013-01-01

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported. PMID:23861989

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.

Common genomic features of Campylobacter jejuni subsp. doylei strains distinguish them from C. jejuni subsp. jejuni

PubMed Central

Parker, Craig T; Miller, William G; Horn, Sharon T; Lastovica, Albert J

2007-01-01

Background Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains. Results A geographically diverse collection of eight Cjd strains was examined by MLST and determined to be phylogenetically distinct from Cjj strains. Microarray-based CGI approach also supported this. We were able to demonstrate that Cjd strains exhibited divergence from Cjj strains NCTC 11168 and RM1221 in many of the intraspecies hypervariable regions. Moreover, multiple metabolic, transport and virulence functions (e.g. cytolethal distending toxin) were shown to be absent in the Cjd strains examined. Conclusion Our data demonstrate that Cjd are phylogenetically distinct from Cjj strains. Using the CGI approach, we identified subsets of absent genes from amongst the C. jejuni genes that provide clues as to the potential evolutionary origin and unusual pathogenicity of Cjd. PMID:17535437

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

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.

Population analysis of clinical and environmental Vibrio parahaemolyticus isolated from eastern provinces in China by removing the recombinant SNPs in the MLST loci.

PubMed

Lu, Xin; Zhou, Haijian; Du, Xiaoli; Liu, Sha; Xu, Jialiang; Cui, Zhigang; Pang, Bo; Kan, Biao

2016-11-01

Vibrio parahaemolyticus is a common seafood-borne pathogenic bacterium which causes gastroenteritis in humans. Continuous surveillance on the molecular characters of the clinical and environmental V. parahaemolyticus strains needs to be conducted for the epidemiological and genetic purposes. To generate a picture of the population distribution of V. parahaemolyticus in eastern China isolated from clinical cases of gastroenteritis and environmental samples, we investigated the genetic and evolutionary relationships of the strains using the commonly used multi-locus sequence typing (MLST, in which seven house-keeping genes are used in the protocol). A highly genetic diversity within the V. parahaemolyticus population was observed but ST3 was still dominant in the clinical strains, and 103 new sequence types (ST) were found in the clinical strains by searching in the global V. parahaemolyticus MLST database. With these genetically diverse strains, we estimated the recombination rates of the loci in MLST analysis. The locus recA was found to be subject to exceptionally high rate of recombination, and the recombinant single nucleotide polymorphisms (SNPs) were also identified within the seven loci. The phylogenetic tree of the strains was re-constructed using the maximum likelihood method by removing the recombination SNPs of the seven loci, and the minimum spanning tree was re-constructed with the six loci without recA. Some changes were observed in comparison with the previously used methods, suggesting that the homologous recombination has roles in shaping the clonal structure of V. parahaemolyticus. We propose the recombination-free SNPs strategy in the clonality analysis of V. parahaemolyticus, especially when using the maximum likelihood method. Copyright © 2016. Published by Elsevier B.V.

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.

Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei.

PubMed

Hall, Carina M; Busch, Joseph D; Shippy, Kenzie; Allender, Christopher J; Kaestli, Mirjam; Mayo, Mark; Sahl, Jason W; Schupp, James M; Colman, Rebecca E; Keim, Paul; Currie, Bart J; Wagner, David M

2015-01-01

The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States.

Variable Number of Tandem Repeats (VNTR) analysis of Flavobacterium psychrophilum from salmonids in Chile and Norway.

PubMed

Apablaza, Patricia; Brevik, Øyvind J; Mjøs, Svein; Valdebenito, Samuel; Ilardi, Pedro; Battaglia, Juan; Dalsgaard, Inger; Nylund, Are

2015-07-14

Flavobacterium psychrophilum causes serious fish diseases such RTFS and BCWD, affecting the aquaculture industry worldwide. Commercial vaccines are not available and control of the disease depends on the use of antibiotics. Reliable methods for detection and identification of different isolates of this bacterium could play an important role in the development of good management strategies. The aim of this study was to identify genetic markers for discrimination between isolates. A selection of eight VNTRs from 53 F. psychrophilum isolates from Norway, Chile, Denmark and Scotland were analyzed. The results were compared with previous work on the same pathogen using MLST for genetic differentiation. The VNTR analysis gave a separation between the F. psychrophilum isolates supporting the results of previous MLST work. A higher diversity was found among the Chilean isolates compared to those from Norway, which suggests a more homogenous reservoir in Norway. Transgenerational transmission of F. psychrophilum from other countries, exporting salmon embryos to Chile, may explain the differences in diversity. The same transmission mechanisms could also explain the wide geographical distribution of identical isolates in Norway. But, this could also be a result of movement of smolts and embryos. The selected VNTRs are stable genetic markers and no variation was observed after several passages on agar plates at different temperatures. These VNTRs are important additions for genotyping of F. psychrophilum isolates. Future studies on VNTRs of F. psychrophilum should include isolates from more host species from a wider geographical area. To get a more robust genotyping the VNTRs should be used in concert with MLST. Future studies of isolates with high and low virulence should focus on identifying virulence markers using VTNRs and MLST.

MLST typing of antimicrobial-resistant Propionibacterium acnes isolates from patients with moderate to severe acne vulgaris.

PubMed

Giannopoulos, Lambros; Papaparaskevas, Joseph; Refene, Eirini; Daikos, Georgios; Stavrianeas, Nikolaos; Tsakris, Athanassios

2015-02-01

Molecular typing data on antimicrobial-resistant Propionibacterium strains are limited in the literature. We examined antimicrobial resistance profiles and the underlying resistance mechanisms in Propionibacterium spp. isolates recovered from patients with moderate to severe acne vulgaris in Greece. The clonallity of the resistant Propionibacterium acnes isolates was also investigated. Propionibacterium spp. isolates were detected using Tryptone-Yeast Extract-Glucose (TYG) agar plates supplemented with 4% furazolidone. Erythromycin, clindamycin, vancomycin, penicillin, co-trimoxazole, doxycycline, minocycline and ciprofloxacin MICs were determined using the gradient strip method. Erythromycin, clindamycin and tetracycline mechanisms of resistance were determined using PCR and sequencing of the domain V of 23S rRNA and 16S rRNA, as well as the presence of the ermX gene. Typing was performed using the multi locus sequence typing (MLST) methodology. Seventy nine isolates from 76 patients were collected. Twenty-three isolates (29.1%) exhibited resistance to erythromycin and clindamycin, while two additional isolates (2.5%) were resistant only to erythromycin. Resistance to tetracycline was not detected. The underlying molecular mechanisms were point mutations A2059G and A2058G. MLST typing of the P. acnes resistant isolates revealed that lineage type IA1 (ST-1, 3 and 52) prevailed (12/18; 66.7%), whilst lineage type IA2 (ST-2 and 22) accounted for five more isolates (27.8%). Susceptible isolates were more evenly distributed between ST types. Propionibacterium spp. from moderate to severe acne vulgaris in Greece are frequently resistant to erythromycin/clindamycin but not to tetracyclines, mainly due to the point mutations A2059G and A2058G. P. acnes resistant isolates were more clonally related than susceptible ones and belonged to a limited number of MLST types. Copyright © 2014 Elsevier Ltd. All rights reserved.

Infection Density Dynamics and Phylogeny of Wolbachia Associated with Coconut Hispine Beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), by Multilocus Sequence Type (MLST) Genotyping.

PubMed

Ali, Habib; Muhammad, Abrar; Hou, Youming

2018-05-28

The intracellular bacterium Wolbachia pipientis is widespread in arthropods. Recently, possibilities of novel Wolbachia -mediated hosts, their distribution, and natural rate have been anticipated, and the coconut leaf beetle Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), which has garnered attention as a serious pest of palms, was subjected to this interrogation. By adopting Wolbachia surface protein ( wsp ) and multilocus sequence type (MLST) genotypic systems, we determined the Wolbachia infection density within host developmental stages, body parts, and tissues, and the results revealed that all the tested samples of B. longissima were infected with the same Wolbachia strain (wLog), suggesting complete vertical transmission. The MLST profile elucidated two new alleles ( ftsZ -234 and coxA-266) that define a new sequence type (ST-483), which indicates the particular genotypic association of B. longissima and Wolbachia . The quantitative real-time PCR analysis revealed a higher infection density in the eggs and adult stage, followed by the abdomen and reproductive tissues, respectively. However, no significant differences were observed in the infection density between sexes. Moreover, the wsp and concatenated MLST alignment analysis of this study with other known Wolbachia-mediated arthropods revealed similar clustering with distinct monophyletic supergroup B. This is the first comprehensive report on the prevalence, infection dynamics, and phylogeny of the Wolbachia endosymbiont in B. longissima , which demonstrated that Wolbachia is ubiquitous across all developmental stages and distributed in the entire body of B. longissima . Understanding the Wolbachia infection dynamics would provide useful insight to build a framework for future investigations, understand its impacts on host physiology, and exploit it as a potential biocontrol agent.

Comparison of Uniform Flux and Uniform Head Wellbore Boundary for the Multilevel Slug Test

NASA Astrophysics Data System (ADS)

Chen, C.

2012-12-01

The multilevel slug test (MLST) is useful in characterizing the vertical distribution of hydraulic conductivity K(z) around a well. Most MLST models assume a uniform flux (UF) distribution along the screen length ls during the test. This assumption leads to a nonuniform head distribution along ls, which is of question under the field conditions. To this end, the head distribution along ls is assumed to be uniform (UH). The MLST model associated with the UH assumption is mathematically more complicated and thus is less used. The difference of using UF and UH in modeling the MLST is investigated here for confined aquifers. For the low-K conditions of monotonic recovery of well water level, it is found that the well water level recovery predicted by the UH model is faster than that predicted by the UF model, and the discrepancy is more pronounced for a larger aspect ratio of rw/ls with rw being the well radius, a smaller partial penetration ratio of ls/b with b being aquifer thickness, and/or a smaller anisotropy ratio of Kz/Kr. For the high-K condition where oscillatory well water level recovery is oscillatory about its initial position, it is found that amplitude of the oscillatory recovery predicted by the UH model is larger than that by the UF model, and the discrepancy gets larger for a larger aspect ratio, a smaller partial penetration ratio, or a smaller anisotropy ratio. For the fully penetrating condition, both the UH and UF models give the same results, regardless of low- or high-K conditions. For the same set of data, the K value estimated by the UH model will be greater than that by the UF model.

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans, Chickens, and Pigs in Malaysia

PubMed Central

Getachew, Yitbarek; Zakaria, Zunita; Abdul Aziz, Saleha

2013-01-01

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals. PMID:23666337

Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei

PubMed Central

Hall, Carina M.; Busch, Joseph D.; Shippy, Kenzie; Allender, Christopher J.; Kaestli, Mirjam; Mayo, Mark; Sahl, Jason W.; Schupp, James M.; Colman, Rebecca E.; Keim, Paul; Currie, Bart J.; Wagner, David M.

2015-01-01

The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States. PMID:26600238

Novel sequence types (STs) of Staphylococcus aureus isolates causing clinical and subclinical mastitis in flocks of sheep in the northeast of Brazil.

PubMed

de Almeida, Lara M; de Almeida, Mayra Zilta P R B; de Mendonça, Carla L; Mamizuka, Elsa M

2011-08-01

Staphylococcus aureus is one of the most important infectious mastitis causative agents in small ruminants. In order to know the distribution of Staph. aureus strains associated with infectious mastitis in flocks of sheep in the northeast of Brazil and establish whether these clones are related to the strains distributed internationally, this study analysed the genetic diversity of Staph. aureus isolates from cases of clinical and subclinical mastitis in ewes by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In this research, 135 ewes with mastitis from 31 sheep flocks distributed in 15 districts were examined. Staph. aureus was isolated from sheep milk in 9 (29%) out of 31 herds located in 47% of the districts surveyed. MLST analysis allowed the identification of four STs (ST750, ST1728, ST1729 and ST1730). The last three with their respective novel alleles (glp-220; pta-182 and yqil-180) were recently reported in the Staph. aureus MLST database (http://www.mlst.net). Each novel allele showed only a nucleotide different from those already described. The occurrence of CC133 (ST750 and ST1729) in this study is in agreement with other reports that only a few clones of Staph. aureus seem to be responsible for most cases of mastitis in dairy farms and that some of these clones may have broad geographic distribution. However, the prevalence of CC5 (ST1728 and ST1730)--an important group related to cases of colonization or infection in humans--differs from previous studies by its widespread occurrence and may suggest human contamination followed by selective pressures of the allelic diversifications presented for these STs.

High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature.

PubMed

Tamburro, M; Ripabelli, G

2017-01-01

Rapid, reliable and accurate molecular typing methods are essential for outbreaks detection and infectious diseases control, for monitoring the evolution and dynamics of microbial populations, and for effective epidemiological surveillance. The introduction of a novel method based on the analysis of melting temperature of amplified products, known as High Resolution Melting (HRM) since 2002, has found applications in epidemiological studies, either for identification of bacterial species or molecular typing, as well as an extensive and increasing use in many research fields. HRM method is based on the use of saturating third generation dyes, advanced real-time PCR platforms, and bioinformatics tools. To describe, by a comphrehensive review of the literature, the use, application and usefulness of HRM for the genotyping of bacterial pathogens in the context of epidemiological surveillance and public health. A literature search was carried out during July-August 2016, by consulting the biomedical databases PubMed/Medline, Scopus, EMBASE, and ISI Web of Science without limits. The search strategy was performed according to the following keywords: high resolution melting analysis and bacteria and genotyping or molecular typing. All the articles evaluating the application of HRM for bacterial pathogen genotyping were selected and reviewed, taking into account the objective of each study, the rationale explaining the use of this technology, and the main results obtained in comparison with gold standards and/or alternative methods, when available. HRM method was extensively used for molecular typing of both Gram-positive and Gram-negative bacterial pathogens, representing a versatile genetic tool: a) to evaluate genetic diversity and subtype at species/subspecies level, based also on allele discrimination/identification and mutation screening; b) to recognize phylogenetic groupings (lineage, sublineage, subgroups); c) to identify antimicrobial resistance; d) to detect and screen for mutations related to drug-resistance; e) to discriminate gene isoforms. HRM method showed, in almost all instances, excellent typeability and discriminatory power, with high concordance of typing results obtained with gold standards or comparable methods. Conversely, for the evaluation of genetic determinants associated to antibiotic-resistance or for screening of associated mutations in key gene fragments, the sensitivity and specificity was not optimal, because the targeted amplicons did not encompass all the crucial mutations. Despite the recent introduction of sequencing-based methods, the HRM method deserves consideration in research fields of infectious diseases, being characterized by low cost, rapidity, flexibility and versatility. However, there are some limitations to HRM assays development, which should be carefully considered. The most common application of HRM for bacterial typing is related to Single Nucleotide Polymorphism (SNP)-based genotyping with the analysis of gene fragments within the multilocus sequence typing (MLST) loci, following an approach termed mini-MLST or Minim typing. Although the resolving power is not totally correspondent to MLST, the Simpson's Index of Diversity provided by HRM method typically >0.95. Furthermore, the cost of this approach is less than MLST, enabling low cost surveillance and rapid response for outbreak control. Hence, the potential of HRM technology can strongly facilitate routine research and diagnostics in the epidemiological studies, as well as advance and streamline the genetic characterization of bacterial pathogens.

Molecular Characterization and Panton-Valentine Leucocidin Typing of Community-Acquired Methicillin-Sensitive Staphylococcus aureus Clinical Isolates

PubMed Central

Sloan, Tim; Kearns, Angela M.; James, Richard

2012-01-01

Limited comprehensive molecular typing data exist currently for Panton-Valentine leucocidin (PVL)-positive, methicillin-sensitive Staphylococcus aureus (PVL-MSSA) clinical isolates. Characterization of PVL-MSSA isolates by multilocus sequence typing (MLST) and spa typing in this study showed a genetic similarity to PVL-positive, methicillin-resistant S. aureus (PVL-MRSA) strains, although three novel spa types and a novel MLST (ST1518) were detected. Furthermore, the detection of PVL phages and haplotypes in PVL-MSSA identical to those previously found in PVL-MRSA isolates highlights the role these strains may play as precursors of emerging lineages of clinical significance. PMID:22718937

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.

Genetic variants near MLST8 and DHX57 affect the epigenetic age of the cerebellum

NASA Astrophysics Data System (ADS)

Lu, Ake T.; Hannon, Eilis; Levine, Morgan E.; Hao, Ke; Crimmins, Eileen M.; Lunnon, Katie; Kozlenkov, Alexey; Mill, Jonathan; Dracheva, Stella; Horvath, Steve

2016-02-01

DNA methylation (DNAm) levels lend themselves for defining an epigenetic biomarker of aging known as the `epigenetic clock'. Our genome-wide association study (GWAS) of cerebellar epigenetic age acceleration identifies five significant (P<5.0 × 10-8) SNPs in two loci: 2p22.1 (inside gene DHX57) and 16p13.3 near gene MLST8 (a subunit of mTOR complex 1 and 2). We find that the SNP in 16p13.3 has a cis-acting effect on the expression levels of MLST8 (P=6.9 × 10-18) in most brain regions. In cerebellar samples, the SNP in 2p22.1 has a cis-effect on DHX57 (P=4.4 × 10-5). Gene sets found by our GWAS analysis of cerebellar age acceleration exhibit significant overlap with those of Alzheimer's disease (P=4.4 × 10-15), age-related macular degeneration (P=6.4 × 10-6), and Parkinson's disease (P=2.6 × 10-4). Overall, our results demonstrate the utility of a new paradigm for understanding aging and age-related diseases: it will be fruitful to use epigenetic tissue age as endophenotype in GWAS.

Population Genetic Structure of Listeria monocytogenes Strains as Determined by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

PubMed Central

Henri, Clémentine; Félix, Benjamin; Guillier, Laurent; Leekitcharoenphon, Pimlapas; Michelon, Damien; Mariet, Jean-François; Aarestrup, Frank M.; Mistou, Michel-Yves; Hendriksen, René S.

2016-01-01

ABSTRACT Listeria monocytogenes is a ubiquitous bacterium that may cause the foodborne illness listeriosis. Only a small amount of data about the population genetic structure of strains isolated from food is available. This study aimed to provide an accurate view of the L. monocytogenes food strain population in France. From 1999 to 2014, 1,894 L. monocytogenes strains were isolated from food at the French National Reference Laboratory for L. monocytogenes and classified according to the five risk food matrices defined by the European Food Safety Authority (EFSA). A total of 396 strains were selected on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France. The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i) there was not a clear association between ST9 and ST121 and the food matrices, (ii) serotype IIc, ST8, and ST4 were associated with meat products, and (iii) ST13 was associated with dairy products. Of the two major STs, ST121 was the ST that included the fewest clinical strains, which might indicate lower virulence. This observation may be directly relevant for refining risk analysis models for the better management of food safety. IMPORTANCE This study showed a very useful backward compatibility between PFGE and MLST for surveillance. The results enabled better understanding of the population structure of L. monocytogenes strains isolated from food and management of the health risks associated with L. monocytogenes food strains. Moreover, this work provided an accurate view of L. monocytogenes strain populations associated with specific food matrices. We clearly showed that some STs were associated with food matrices, such as meat, meat products, and dairy products. We opened the way to source attribution modeling in order to quantify the relative importance of the main food matrices. PMID:27235443

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.

Comparison of advanced whole genome sequence-based methods to distinguish strains of Salmonella enterica serovar Heidelberg involved in foodborne outbreaks in Québec.

PubMed

Vincent, Caroline; Usongo, Valentine; Berry, Chrystal; Tremblay, Denise M; Moineau, Sylvain; Yousfi, Khadidja; Doualla-Bell, Florence; Fournier, Eric; Nadon, Céline; Goodridge, Lawrence; Bekal, Sadjia

2018-08-01

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145 S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

Based Upon Repeat Pattern (BURP): an algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms.

PubMed

Mellmann, Alexander; Weniger, Thomas; Berssenbrügge, Christoph; Rothgänger, Jörg; Sammeth, Michael; Stoye, Jens; Harmsen, Dag

2007-10-29

For typing of Staphylococcus aureus, DNA sequencing of the repeat region of the protein A (spa) gene is a well established discriminatory method for outbreak investigations. Recently, it was hypothesized that this region also reflects long-term epidemiology. However, no automated and objective algorithm existed to cluster different repeat regions. In this study, the Based Upon Repeat Pattern (BURP) implementation that is a heuristic variant of the newly described EDSI algorithm was investigated to infer the clonal relatedness of different spa types. For calibration of BURP parameters, 400 representative S. aureus strains with different spa types were characterized by MLST and clustered using eBURST as "gold standard" for their phylogeny. Typing concordance analysis between eBURST and BURP clustering (spa-CC) were performed using all possible BURP parameters to determine their optimal combination. BURP was subsequently evaluated with a strain collection reflecting the breadth of diversity of S. aureus (JCM 2002; 40:4544). In total, the 400 strains exhibited 122 different MLST types. eBURST grouped them into 23 clonal complexes (CC; 354 isolates) and 33 singletons (46 isolates). BURP clustering of spa types using all possible parameter combinations and subsequent comparison with eBURST CCs resulted in concordances ranging from 8.2 to 96.2%. However, 96.2% concordance was reached only if spa types shorter than 8 repeats were excluded, which resulted in 37% excluded spa types. Therefore, the optimal combination of the BURP parameters was "exclude spa types shorter than 5 repeats" and "cluster spa types into spa-CC if cost distances are less than 4" exhibiting 95.3% concordance to eBURST. This algorithm identified 24 spa-CCs, 40 singletons, and excluded only 7.8% spa types. Analyzing the natural population with these parameters, the comparison of whole-genome micro-array groupings (at the level of 0.31 Pearson correlation index) and spa-CCs gave a concordance of 87.1%; BURP spa-CCs vs. manually grouped spa types resulted in 95.7% concordance. BURP is the first automated and objective tool to infer clonal relatedness from spa repeat regions. It is able to extract an evolutionary signal rather congruent to MLST and micro-array data.

Discrimination of multilocus sequence typing-based Campylobacter jejuni subgroups by MALDI-TOF mass spectrometry.

PubMed

Zautner, Andreas Erich; Masanta, Wycliffe Omurwa; Tareen, Abdul Malik; Weig, Michael; Lugert, Raimond; Groß, Uwe; Bader, Oliver

2013-11-07

Campylobacter jejuni, the most common bacterial pathogen causing gastroenteritis, shows a wide genetic diversity. Previously, we demonstrated by the combination of multi locus sequence typing (MLST)-based UPGMA-clustering and analysis of 16 genetic markers that twelve different C. jejuni subgroups can be distinguished. Among these are two prominent subgroups. The first subgroup contains the majority of hyperinvasive strains and is characterized by a dimeric form of the chemotaxis-receptor Tlp7(m+c). The second has an extended amino acid metabolism and is characterized by the presence of a periplasmic asparaginase (ansB) and gamma-glutamyl-transpeptidase (ggt). Phyloproteomic principal component analysis (PCA) hierarchical clustering of MALDI-TOF based intact cell mass spectrometry (ICMS) spectra was able to group particular C. jejuni subgroups of phylogenetic related isolates in distinct clusters. Especially the aforementioned Tlp7(m+c)(+) and ansB+/ ggt+ subgroups could be discriminated by PCA. Overlay of ICMS spectra of all isolates led to the identification of characteristic biomarker ions for these specific C. jejuni subgroups. Thus, mass peak shifts can be used to identify the C. jejuni subgroup with an extended amino acid metabolism. Although the PCA hierarchical clustering of ICMS-spectra groups the tested isolates into a different order as compared to MLST-based UPGMA-clustering, the isolates of the indicator-groups form predominantly coherent clusters. These clusters reflect phenotypic aspects better than phylogenetic clustering, indicating that the genes corresponding to the biomarker ions are phylogenetically coupled to the tested marker genes. Thus, PCA clustering could be an additional tool for analyzing the relatedness of bacterial isolates.

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States

PubMed Central

O'Hara, F. Patrick; Suaya, Jose A.; Ray, G. Thomas; Baxter, Roger; Brown, Megan L.; Mera, Robertino M.; Close, Nicole M.; Thomas, Elizabeth

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants. PMID:26669861

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States.

PubMed

O'Hara, F Patrick; Suaya, Jose A; Ray, G Thomas; Baxter, Roger; Brown, Megan L; Mera, Robertino M; Close, Nicole M; Thomas, Elizabeth; Amrine-Madsen, Heather

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants.

Occurrence and molecular characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses in an equine clinic.

PubMed

Apostolakos, Ilias; Franz, Eelco; van Hoek, Angela H A M; Florijn, Alice; Veenman, Christiaan; Sloet-van Oldruitenborgh-Oosterbaan, Marianne M; Dierikx, Cindy; van Duijkeren, Engeline

2017-07-01

To investigate the occurrence and characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses at one equine clinic in the Netherlands. A total of 91 horses, including residents and patients, were sampled. ESBL/AmpC-producing E. coli were identified by a combination disc diffusion test. Phylogenetic groups and MLST were determined. ESBL/AmpC genes were analysed using PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid MLST. At least one E. coli isolate with a confirmed ESBL/AmpC gene was found in samples from 76 horses (84%). Although phylogenetic group B1 E. coli bla CTX-M-1 predominated, a diverse E. coli population was found, indicating that clonal nosocomial spread was not the only reason for the high occurrence found. MLST analysis revealed the presence of 47 E. coli STs, organized in four clusters of genetically related strains. ST10, ST641, ST1079 and ST1250 were most commonly found. With regard to the genes, bla CTX-M-1 was most prevalent ( n  =   91), followed by bla CTX-M-2 ( n  =   26). The most frequently found plasmid type was IncHI1, but plasmids belonging to the IncF, IncI1 and IncN groups were also identified. A high occurrence of ESBL-producing E. coli in faecal samples was found among horses in an equine clinic and the variety of STs, ESBL genes and plasmid types suggests nosocomial transmission. ESBL E. coli can cause difficult-to-treat infections in horses and prudent use of antimicrobials is warranted. A further assessment of the risks of transmission to persons in close contact with horses, such as caretakers or veterinarians, is crucial. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Population structure of clinical Pseudomonas aeruginosa from West and Central African countries.

PubMed

Cholley, Pascal; Ka, Roughyatou; Guyeux, Christophe; Thouverez, Michelle; Guessennd, Nathalie; Ghebremedhin, Beniam; Frank, Thierry; Bertrand, Xavier; Hocquet, Didier

2014-01-01

Pseudomonas aeruginosa (PA) has a non-clonal, epidemic population with a few widely distributed and frequently encountered sequence types (STs) called 'high-risk clusters'. Clinical P. aeruginosa (clinPA) has been studied in all inhabited continents excepted in Africa, where a very few isolates have been analyzed. Here, we characterized a collection of clinPA isolates from four countries of West and Central Africa. 184 non-redundant isolates of clinPA from hospitals of Senegal, Ivory Coast, Nigeria, and Central African Republic were genotyped by MLST. We assessed their resistance level to antibiotics by agar diffusion and identified the extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) by sequencing. The population structure of the species was determined by a nucleotide-based analysis of the entire PA MLST database and further localized on the phylogenetic tree (i) the sequence types (STs) of the present collection, (ii) the STs by continents, (iii) ESBL- and MBL-producing STs from the MLST database. We found 80 distinct STs, of which 24 had no relationship with any known STs. 'High-risk' international clonal complexes (CC155, CC244, CC235) were frequently found in West and Central Africa. The five VIM-2-producing isolates belonged to CC233 and CC244. GES-1 and GES-9 enzymes were produced by one CC235 and one ST1469 isolate, respectively. We showed the spread of 'high-risk' international clonal complexes, often described as multidrug-resistant on other continents, with a fully susceptible phenotype. The MBL- and ESBL-producing STs were scattered throughout the phylogenetic tree and our data suggest a poor association between a continent and a specific phylogroup. ESBL- and MBL-encoding genes are borne by both successful international clonal complexes and distinct local STs in clinPA of West and Central Africa. Furthermore, our data suggest that the spread of a ST could be either due to its antibiotic resistance or to features independent from the resistance to antibiotics.

MLST and Whole-Genome-Based Population Analysis of Cryptococcus gattii VGIII Links Clinical, Veterinary and Environmental Strains, and Reveals Divergent Serotype Specific Sub-populations and Distant Ancestors

PubMed Central

Firacative, Carolina; Roe, Chandler C.; Malik, Richard; Ferreira-Paim, Kennio; Escandón, Patricia; Sykes, Jane E.; Castañón-Olivares, Laura Rocío; Contreras-Peres, Cudberto; Samayoa, Blanca; Sorrell, Tania C.; Castañeda, Elizabeth; Lockhart, Shawn R.; Engelthaler, David M.; Meyer, Wieland

2016-01-01

The emerging pathogen Cryptococcus gattii causes life-threatening disease in immunocompetent and immunocompromised hosts. Of the four major molecular types (VGI-VGIV), the molecular type VGIII has recently emerged as cause of disease in otherwise healthy individuals, prompting a need to investigate its population genetic structure to understand if there are potential genotype-dependent characteristics in its epidemiology, environmental niche(s), host range and clinical features of disease. Multilocus sequence typing (MLST) of 122 clinical, environmental and veterinary C. gattii VGIII isolates from Australia, Colombia, Guatemala, Mexico, New Zealand, Paraguay, USA and Venezuela, and whole genome sequencing (WGS) of 60 isolates representing all established MLST types identified four divergent sub-populations. The majority of the isolates belong to two main clades, corresponding either to serotype B or C, indicating an ongoing species evolution. Both major clades included clinical, environmental and veterinary isolates. The C. gattii VGIII population was genetically highly diverse, with minor differences between countries, isolation source, serotype and mating type. Little to no recombination was found between the two major groups, serotype B and C, at the whole and mitochondrial genome level. C. gattii VGIII is widespread in the Americas, with sporadic cases occurring elsewhere, WGS revealed Mexico and USA as a likely origin of the serotype B VGIII population and Colombia as a possible origin of the serotype C VGIII population. Serotype B isolates are more virulent than serotype C isolates in a murine model of infection, causing predominantly pulmonary cryptococcosis. No specific link between genotype and virulence was observed. Antifungal susceptibility testing against six antifungal drugs revealed that serotype B isolates are more susceptible to azoles than serotype C isolates, highlighting the importance of strain typing to guide effective treatment to improve the disease outcome. PMID:27494185

A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination.

PubMed

Ankarklev, Johan; Lebbad, Marianne; Einarsson, Elin; Franzén, Oscar; Ahola, Harri; Troell, Karin; Svärd, Staffan G

2018-06-01

Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia. Copyright © 2018. Published by Elsevier B.V.

Analysis of Typing Methods for Epidemiological Surveillance of both Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains▿ †

PubMed Central

Faria, Nuno A.; Carrico, João A.; Oliveira, Duarte C.; Ramirez, Mário; de Lencastre, Hermínia

2008-01-01

Sequence-based methods for typing Staphylococcus aureus, such as multilocus sequence typing (MLST) and spa typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for S. aureus, using a diverse collection of 198 S. aureus strains previously characterized by PFGE, spa typing, MLST, and, in the case of methicillin-resistant S. aureus (MRSA), SCCmec typing in order to establish the quantitative congruence between the typing methods. The results of most typing methods agree in that MRSA and methicillin-susceptible S. aureus (MSSA) differ in terms of diversity of genetic backgrounds, with MSSA being more diverse. Our results show that spa typing has a very good predictive power over the clonal relationships defined by eBURST, while PFGE is less accurate for that purpose but nevertheless provides better typeability and discriminatory power. The combination of PFGE and spa typing provided even better results. Based on these observations, we suggest the use of the conjugation of spa typing and PFGE typing for epidemiological surveillance studies, since this combination provides the ability to infer long-term relationships while maintaining the discriminatory power and typeability needed in short-term studies. PMID:17989188

A Novel IncA/C1 Group Conjugative Plasmid, Encoding VIM-1 Metallo-Beta-Lactamase, Mediates the Acquisition of Carbapenem Resistance in ST104 Klebsiella pneumoniae Isolates from Neonates in the Intensive Care Unit of V. Monaldi Hospital in Naples

PubMed Central

Esposito, Eliana P.; Gaiarsa, Stefano; Del Franco, Mariateresa; Crivaro, Valeria; Bernardo, Mariano; Cuccurullo, Susanna; Pennino, Francesca; Triassi, Maria; Marone, Piero; Sassera, Davide; Zarrilli, Raffaele

2017-01-01

The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of β-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim–sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum β-lactamase blaSHV -12 and carbapenem-hydrolyzing metallo-β-lactamase blaV IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with blaV IM-1 into a class I integron, blaSHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2′-phosphotransferase clusters, ant(3″), aph(3″), aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaV IM-1 and blaSHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples. PMID:29163422

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.

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.

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.

Natural coinfection by Streptococcus agalactiae and Francisella noatunensis subsp. orientalis in farmed Nile tilapia (Oreochromis niloticus L.).

PubMed

Assis, G B N; Tavares, G C; Pereira, F L; Figueiredo, H C P; Leal, C A G

2017-01-01

Streptococcus agalactiae and Francisella noatunensis subsp. orientalis (Fno) are important pathogens for farm-raised tilapia worldwide. There are no reports of coinfection caused by S. agalactiae and Fno in fish. This study aimed to determine the aetiology of atypical mortalities in a cage farm of Nile tilapia and to characterize the genetic diversity of the isolates. Fifty-two fish were sampled and subjected to parasitological and bacteriological examination. The S. agalactiae and Fno isolates were genotyped using MLST and REP-PCR, respectively. Whole-genome sequencing was performed to confirm the MLST results. Seven fish were shown coinfected by S. agalactiae and Fno. Chronic hypoxia and a reduction in the water temperature were determined as risk factors for coinfection. Fno isolates were shown clonally related in REP-PCR. The MLST analysis revealed that the S. agalactiae isolates from seven coinfected fish were negative for the glcK gene; however, these were determined to be members of clonal complex CC-552. This is the first description of coinfection by S. agalactiae and Fno in farm-raised Nile tilapia. The coinfection was predisposed by chronic hypoxia and was caused by the main genotypes of S. agalactiae and Fno reported in Brazil. Finally, a new S. agalactiae genotype with glcK gene partially deleted was described. © 2016 John Wiley & Sons Ltd.

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 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.

MLVA and MLST typing of Brucella from Qinghai, China.

PubMed

Ma, Jun-Ying; Wang, Hu; Zhang, Xue-Fei; Xu, Li-Qing; Hu, Gui-Ying; Jiang, Hai; Zhao, Fang; Zhao, Hong-Yan; Piao, Dong-Ri; Qin, Yu-Min; Cui, Bu-Yun; Lin, Gong-Hua

2016-04-13

The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection. A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates. Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species. There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans.

Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex.

PubMed

Matray, Olivier; Mouhajir, Abdelmounaim; Giraud, Sandrine; Godon, Charlotte; Gargala, Gilles; Labbé, Franck; Rougeron, Amandine; Ballet, Jean-Jacques; Zouhair, Rachid; Bouchara, Jean-Philippe; Favennec, Loïc

2016-05-01

The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization. Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST). Within the panel of isolates studied,S. apiospermum sensu stricto and Scedosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium aurantiacum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type. These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this complex.Abstract presentation: Part of this work was presented during the 18th Congress of the International Society for Human and Animal Mycology, Berlin (Germany), June 2012.S. Giraud, C. Godon, A. Rougeron, J.P. Bouchara and L. Favennec are members of the ECMM/ISHAM working group on Fungal respiratory infections in Cystic Fibrosis(Fri-CF). © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Population Structure of Clinical Pseudomonas aeruginosa from West and Central African Countries

PubMed Central

Cholley, Pascal; Ka, Roughyatou; Guyeux, Christophe; Thouverez, Michelle; Guessennd, Nathalie; Ghebremedhin, Beniam; Frank, Thierry; Bertrand, Xavier; Hocquet, Didier

2014-01-01

Background Pseudomonas aeruginosa (PA) has a non-clonal, epidemic population with a few widely distributed and frequently encountered sequence types (STs) called ‘high-risk clusters’. Clinical P. aeruginosa (clinPA) has been studied in all inhabited continents excepted in Africa, where a very few isolates have been analyzed. Here, we characterized a collection of clinPA isolates from four countries of West and Central Africa. Methodology 184 non-redundant isolates of clinPA from hospitals of Senegal, Ivory Coast, Nigeria, and Central African Republic were genotyped by MLST. We assessed their resistance level to antibiotics by agar diffusion and identified the extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) by sequencing. The population structure of the species was determined by a nucleotide-based analysis of the entire PA MLST database and further localized on the phylogenetic tree (i) the sequence types (STs) of the present collection, (ii) the STs by continents, (iii) ESBL- and MBL-producing STs from the MLST database. Principal Findings We found 80 distinct STs, of which 24 had no relationship with any known STs. ‘High-risk’ international clonal complexes (CC155, CC244, CC235) were frequently found in West and Central Africa. The five VIM-2-producing isolates belonged to CC233 and CC244. GES-1 and GES-9 enzymes were produced by one CC235 and one ST1469 isolate, respectively. We showed the spread of ‘high-risk’ international clonal complexes, often described as multidrug-resistant on other continents, with a fully susceptible phenotype. The MBL- and ESBL-producing STs were scattered throughout the phylogenetic tree and our data suggest a poor association between a continent and a specific phylogroup. Conclusions ESBL- and MBL-encoding genes are borne by both successful international clonal complexes and distinct local STs in clinPA of West and Central Africa. Furthermore, our data suggest that the spread of a ST could be either due to its antibiotic resistance or to features independent from the resistance to antibiotics. PMID:25187957

Extensive Genetic Diversity Identified among Sporadic Methicillin-Resistant Staphylococcus aureus Isolates Recovered in Irish Hospitals between 2000 and 2012

PubMed Central

Kinnevey, Peter M.; Shore, Anna C.; Brennan, Grainne I.; Sullivan, Derek J.; Ehricht, Ralf; Monecke, Stefan

2014-01-01

Clonal replacement of predominant nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains has occurred several times in Ireland during the last 4 decades. However, little is known about sporadically occurring MRSA in Irish hospitals or in other countries. Eighty-eight representative pvl-negative sporadic MRSA isolates recovered in Irish hospitals between 2000 and 2012 were investigated. These yielded unusual pulsed-field gel electrophoresis and antibiogram-resistogram typing patterns distinct from those of the predominant nosocomial MRSA clone, ST22-MRSA-IV, during the study period. Isolates were characterized by spa typing and DNA microarray profiling for multilocus sequence type (MLST) clonal complex (CC) and/or sequence type (ST) and SCCmec type assignment, as well as for detection of virulence and antimicrobial resistance genes. Conventional PCR-based SCCmec subtyping was undertaken when necessary. Extensive diversity was detected, including 38 spa types, 13 MLST-CCs (including 18 STs among 62 isolates assigned to STs), and 25 SCCmec types (including 2 possible novel SCCmec elements and 7 possible novel SCCmec subtypes). Fifty-four MLST-spa-SCCmec type combinations were identified. Overall, 68.5% of isolates were assigned to nosocomial lineages, with ST8-t190-MRSA-IID/IIE ± SCCM1 predominating (17.4%), followed by CC779/ST779-t878-MRSA-ψSCCmec-SCC-SCCCRISPR (7.6%) and CC22/ST22-t032-MRSA-IVh (5.4%). Community-associated clones, including CC1-t127/t386/t2279-MRSA-IV, CC59-t216-MRSA-V, CC8-t008-MRSA-IVa, and CC5-t002/t242-MRSA-IV/V, and putative animal-associated clones, including CC130-t12399-MRSA-XI, ST8-t064-MRSA-IVa, ST398-t011-MRSA-IVa, and CC6-t701-MRSA-V, were also identified. In total, 53.3% and 47.8% of isolates harbored genes for resistance to two or more classes of antimicrobial agents and two or more mobile genetic element-encoded virulence-associated factors, respectively. Effective ongoing surveillance of sporadic nosocomial MRSA is warranted for early detection of emerging clones and reservoirs of virulence, resistance, and SCCmec genes. PMID:24395241

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination

PubMed Central

Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

2015-01-01

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. PMID:26712553

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).

A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis

PubMed Central

De Los Santos, Maxy; Soberón, Valeria; Lucas, Carmen M.; Matlashewski, Greg; Llanos-Cuentas, Alejandro; Ore, Marianela; Baldeviano, G. Christian; Edgel, Kimberly A.; Lescano, Andres G.; Graf, Paul C. F.; Bacon, David J.

2013-01-01

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America. PMID:23301111

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.

Clonal dissemination of multilocus sequence type ST15 KPC-2-producing Klebsiella pneumoniae in Bulgaria.

PubMed

Markovska, Rumyana; Stoeva, Temenuga; Schneider, Ines; Boyanova, Lyudmila; Popova, Valentina; Dacheva, Daniela; Kaneva, Radka; Bauernfeind, Adolf; Mitev, Vanyo; Mitov, Ivan

2015-10-01

A total of 36 consecutive clinical and two fecal-screening carbapenem-resistant Klebsiella pneumoniae isolates from two Bulgarian university hospitals (Varna and Pleven) were investigated. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and multilocus sequence typing (MLST). Most of the isolates demonstrated multidrug resistance profile. Amikacin and tigecycline retained good activity with susceptibility rates of 95 and 87%, respectively. The resistance rate to colistin was 63%. Six RAPD- and MLST-types were identified: the dominating MLST-type was ST15 (27 isolates), followed by ST76 (six isolates), and ST1350 (two isolates). ST101, ST258, and ST151 were detected once. All except one of the K. pneumoniae produced KPC-2, mostly in combination with CTX-M-15, while for one isolate (ST101) the enzymes OXA-48 and CTX-M-14 were found. All KPC-2-producing transconjugants revealed the presence of IncFII plasmid. The OXA-48- and CTX-M-14-producing isolate showed the presence of L/M replicon type. The dissemination of KPC-2-producing K.pneumoniae in Bulgaria is mainly due to the sustained spread of successful ST15 clone and to a lesser extent of ST76 clone. This is the first report of OXA-48 producing ST101 K. pneumoniae in Bulgaria. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Prevalence and genetic diversity of Trichomonas vaginalis in the general population of Granada and co-infections with Gardnerella vaginalis and Candida species.

PubMed

Carrillo-Ávila, José Antonio; Serrano-García, María Luisa; Fernández-Parra, Jorge; Sorlózano-Puerto, Antonio; Navarro-Marí, José María; Stensvold, C Rune; Gutiérrez-Fernández, Jose

2017-10-01

Purulent or exudative genitourinary infections are a frequent cause of consultation in primary and specialized healthcare. The objectives of this study were: to determine the prevalence of Trichomonas vaginalis and co-infections with Candida spp. and Gardnerella vaginalis in vaginal secretion; and to use multilocus sequence typing (MLST) to analyse the genetic diversity of T. vaginalis strains. The samples were submitted for analysis (n=5230) to a third-level hospital in Granada (Southern Spain) between 2011 and 2014; eight T. vaginalis strains isolated during 2015 were randomly selected for MLST analysis. Culture and nucleic acid hybridization techniques were used to detect microorganisms in the samples. The prevalence of T. vaginalis was 2.4 % between 2011 and 2014, being higher during the first few months of both 2011 and 2012. Among samples positive for T. vaginalis, co-infection with G. vaginalis was detected in 29 samples and co-infection with Candida spp. in 6, while co-infection with all three pathogens was observed in 3 samples. The only statistically significant between-year difference in co-infection rates was observed for T. vaginalis with G. vaginalis due to an elevated rate in 2011. MLST analysis results demonstrated a high genetic variability among strains circulating in our setting. These findings emphasize the need for the routine application of diagnostic procedures to avoid the spread of this sexually transmitted infection.

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 and strain-specific characteristics of Streptococcus agalactiae at the herd and cow level.

PubMed

Mahmmod, Y S; Klaas, I C; Katholm, J; Lutton, M; Zadoks, R N

2015-10-01

Host-adaptation of Streptococcus agalactiae subpopulations has been described whereby strains that are commonly associated with asymptomatic carriage or disease in people differ phenotypically and genotypically from those causing mastitis in dairy cattle. Based on multilocus sequence typing (MLST), the most common strains in dairy herds in Denmark belong to sequence types (ST) that are also frequently found in people. The aim of this study was to describe epidemiological and diagnostic characteristics of such strains in relation to bovine mastitis. Among 1,199 cattle from 6 herds, cow-level prevalence of S. agalactiae was estimated to be 27.4% based on PCR and 7.8% based on bacteriological culture. Quarter-level prevalence was estimated at 2.8% based on bacteriological culture. Per herd, between 2 and 26 isolates were characterized by pulsed-field gel electrophoresis (PFGE) and MLST. Within each herd, a single PFGE type and ST predominated, consistent with a contagious mode of transmission or point source infection within herds. Evidence of within-herd evolution of S. agalactiae was detected with both typing methods, although ST belonged to a single clonal complex (CC) per herd. Detection of CC23 (3 herds) was associated with significantly lower approximate count (colony-forming units) at the quarter level and significantly lower cycle threshold value at the cow level than detection of CC1 (2 herds) or CC19 (1 herd), indicating a lower bacterial load in CC23 infections. Median values for the number of infected quarters and somatic cell count (SCC) were numerically but not significantly lower for cows infected with CC23 than for cows with CC1 or CC19. For all CC, an SCC threshold of 200,000 cells/mL was an unreliable indicator of infection status, and prescreening of animals based on SCC as part of S. agalactiae detection and eradication campaigns should be discouraged. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Identifying the seasonal origins of human campylobacteriosis

PubMed Central

STRACHAN, N. J. C.; ROTARIU, O.; SMITH-PALMER, A.; COWDEN, J.; SHEPPARD, S. K.; O’BRIEN, S. J.; MAIDEN, M. C. J.; MACRAE, M.; BESSELL, P. R.; MATTHEWS, L.; REID, S. W. J.; INNOCENT, G. T.; OGDEN, I. D.; FORBES, K. J.

2014-01-01

SUMMARY Human campylobacteriosis exhibits a distinctive seasonality in temperate regions. This paper aims to identify the origins of this seasonality. Clinical isolates [typed by multi-locus sequence typing (MLST)] and epidemiological data were collected from Scotland. Young rural children were found to have an increased burden of disease in the late spring due to strains of non-chicken origin (e.g. ruminant and wild bird strains from environmental sources). In contrast the adult population had an extended summer peak associated with chicken strains. Travel abroad and UK mainland travel were associated with up to 17% and 18% of cases, respectively. International strains were associated with chicken, had a higher diversity than indigenous strains and a different spectrum of MLST types representative of these countries. Integrating empirical epidemiology and molecular subtyping can successfully elucidate the seasonal components of human campylobacteriosis. The findings will enable public health officials to focus strategies to reduce the disease burden. PMID:22989449

Comparison of four molecular methods to type Salmonella Enteritidis strains.

PubMed

Campioni, Fábio; Pitondo-Silva, André; Bergamini, Alzira M M; Falcão, Juliana P

2015-05-01

This study compared the pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), multilocus variable-number of tanden-repeat analysis (MLVA), and multilocus sequence typing (MLST) methods for typing 188 Salmonella Enteritidis strains from different sources isolated over a 24-year period in Brazil. PFGE and ERIC-PCR were more efficient than MLVA for subtyping the strains. However, MLVA provided additional epidemiological information for those strains. In addition, MLST showed the Brazilian strains as belonging to the main clonal complex of S. Enteritidis, CC11, and provided the first report of two new STs in the S. enterica database but could not properly subtype the strains. Our results showed that the use of PFGE or ERIC-PCR together with MLVA is suitable to efficiently subtype S. Enteritidis strains and provide important epidemiological information. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Genotypes and virulence in serotype K2 Klebsiella pneumoniae from liver abscess and non-infectious carriers in Hong Kong, Singapore and Taiwan.

PubMed

Lin, Jung-Chung; Koh, Tse Hsien; Lee, Nelson; Fung, Chang-Phone; Chang, Feng-Yee; Tsai, Yu-Kuo; Ip, Margaret; Siu, L Kristopher

2014-01-01

In Klebsiella pneumoniae liver abscess (KP-LA), K. pneumoniae K2 is the most frequently isolated serotype after K1, but this serotype has been much less studied. In the present study, the molecular types sequences type (MLST) of serotype K2 isolates from three different regions in Asia were identified and the virulence of these isolates was investigated. Eight different MLSTs were found among 26 isolates (ST 65, 66, 86, 373, 374, 375, 380, and 434). There were two major MLST groups, ST-65-like (42%) and ST86-like (46%). No isolates contained allS while all isolates contained rmpA. The prevalence of aerobactin gene and kfu were 25/26 (96%) and 3/26 (11.5%) respectively. Although liver abscess isolates were generally more resistant (11/15 isolates) to serum killing, there was no specific distribution of serum killing resistant or susceptible ST types between stool carriage and liver abscess isolates. Neutrophil phagocytosis showed that the liver abscess and carriage isolates varied in their susceptibility to phagocytosis. Strains with resistance to both neutrophil phagocytosis and serum killing were generally hypervirulent with lethality at LD50 < 10(3) colony forming units by intraperitoneal injection. In conclusion, Anti-phagocytosis and resistance to serum killing are two parameters that most predict hyperviurlence in serotype K2 isolates. Unlike serotype K1 KP-LA that mainly belong to ST-23, ST-65-like and -86-like are the two major MLST types among serotype K2 isolates from Singapore, Hong Kong and Taiwan.

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

Multilocus sequence typing (MLST) of leptospiral strains isolated from two geographic locations of Tamil Nadu, India.

PubMed

Kanagavel, Murugesan; Princy Margreat, Alphonse Asirvatham; Arunkumar, Manivel; Prabhakaran, Shanmugarajan Gnanasekaran; Shanmughapriya, Santhanam; Natarajaseenivasan, Kalimuthusamy

2016-01-01

Here the rodent carrier status for the transmission of human leptospirosis in Tiruchirappalli, district, Tamil Nadu, India was assessed. The predominantly circulating leptospiral STs were recognized by multilocus sequence typing (MLST). A total of 113 rodents were trapped from different provinces of the Tiruchirappalli district. The most prevalent rodent was Bandicota bengalensis (37.2%), and of the total, 52.2% (n=59) rodents were found to be positive for leptospiral 16S rRNA. These results were validated with a leptospiral culture positivity of 45.8% (n=27). Three isolates from Chennai (2 rodents and 1 human) and 1 human isolate from Tiruchirappalli were included to understand the spatial variations and to track the source of human leptospirosis. The serogroup, serovar, and species level identification of all 31 isolates identified 28 to be Leptospira borgpetersenii serovar Javanica and three as Leptospira interrogans serovar Autumnalis. MLST analysis defined all isolates to the existing ST profiles (ST145 and ST27) with the exception of 6 L. borgpetersenii (ST DR) isolates that showed variations in the sucA and pfkB loci. The DR ST was locally confined to Chatram province of Tiruchirappalli suggesting an epidemiological link. The predominant STs, ST145 and ST-DR form a group, indicating the presence of original strain that subsequently diverged evolutionarily into two STs. The variations between L. borgpetersenii in sucA and pfkB loci may be an indication that evolutionary changes transpired in Tiruchirappalli. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular characterization of endocarditis-associated Staphylococcus aureus.

PubMed

Nethercott, Cara; Mabbett, Amanda N; Totsika, Makrina; Peters, Paul; Ortiz, Juan C; Nimmo, Graeme R; Coombs, Geoffrey W; Walker, Mark J; Schembri, Mark A

2013-07-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.

Molecular Characterization of Endocarditis-Associated Staphylococcus aureus

PubMed Central

Nethercott, Cara; Mabbett, Amanda N.; Totsika, Makrina; Peters, Paul; Ortiz, Juan C.; Nimmo, Graeme R.; Coombs, Geoffrey W.

2013-01-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted. PMID:23616460

Genotyping of Indian antigenic, vaccine, and field Brucella spp. using multilocus sequence typing.

PubMed

Shome, Rajeswari; Krithiga, Natesan; Shankaranarayana, Padmashree B; Jegadesan, Sankarasubramanian; Udayakumar S, Vishnu; Shome, Bibek Ranjan; Saikia, Girin Kumar; Sharma, Narendra Kumar; Chauhan, Harshad; Chandel, Bharat Singh; Jeyaprakash, Rajendhran; Rahman, Habibur

2016-03-31

Brucellosis is one of the most important zoonotic diseases that affects multiple livestock species and causes great economic losses. The highly conserved genomes of Brucella, with > 90% homology among species, makes it important to study the genetic diversity circulating in the country. A total of 26 Brucella spp. (4 reference strains and 22 field isolates) and 1 B. melitensis draft genome sequence from India (B. melitensis Bm IND1) were included for sequence typing. The field isolates were identified by biochemical tests and confirmed by both conventional and quantitative polymerase chain reaction (qPCR) targeting bcsp 31Brucella genus-specific marker. Brucella speciation and biotyping was done by Bruce ladder, probe qPCR, and AMOS PCRs, respectively, and genotyping was done by multilocus sequence typing (MLST). The MLST typing of 27 Brucella spp. revealed five distinct sequence types (STs); the B. abortus S99 reference strain and 21 B. abortus field isolates belonged to ST1. On the other hand, the vaccine strain B. abortus S19 was genotyped as ST5. Similarly, B. melitensis 16M reference strain and one B. melitensis field isolate were grouped into ST7. Another B. melitensis field isolate belonged to ST8 (draft genome sequence from India), and only B. suis 1330 reference strain was found to be ST14. The sequences revealed genetic similarity of the Indian strains to the global reference and field strains. The study highlights the usefulness of MLST for typing of field isolates and validation of reference strains used for diagnosis and vaccination against brucellosis.

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.

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

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolates in the Gulf Cooperation Council States: Dominance of OXA-23-Type Producers

PubMed Central

Sartor, Anna L.; Sidjabat, Hanna E.; Balkhy, Hanan H.; Walsh, Timothy R.; Al Johani, Sameera M.; AlJindan, Reem Y.; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al Salman, Jameela; Dashti, Ali A.; Johani, Khalid; Paterson, David L.

2015-01-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Acinetobacter baumannii (CRAB) were determined in hospitals in the states of the Cooperation Council for the Arab States of the Gulf (Gulf Cooperation Council [GCC]), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic resistance genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Selected isolates were subjected to multilocus sequence typing (MLST). We investigated 117 isolates resistant to carbapenem antibiotics (either imipenem or meropenem). All isolates were positive for OXA-51. The most common carbapenemases were the OXA-23-type, found in 107 isolates, followed by OXA-40-type (OXA-24-type), found in 5 isolates; 3 isolates carried the ISAba1 element upstream of blaOXA-51-type. No OXA-58-type, NDM-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with 16 clusters of clonally related CRAB. Some clusters involved hospitals in different states. MLST analysis of 15 representative isolates from different clusters identified seven different sequence types (ST195, ST208, ST229, ST436, ST450, ST452, and ST499), as well as three novel STs. The vast majority (84%) of the isolates in this study were associated with health care exposure. Awareness of multidrug-resistant organisms in GCC states has important implications for optimizing infection control practices; establishing antimicrobial stewardship programs within hospital, community, and agricultural settings; and emphasizing the need for establishing regional active surveillance systems. This will help to control the spread of CRAB in the Middle East and in hospitals accommodating transferred patients from this region. PMID:25568439

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates in the Gulf Cooperation Council States: dominance of OXA-23-type producers.

PubMed

Zowawi, Hosam M; Sartor, Anna L; Sidjabat, Hanna E; Balkhy, Hanan H; Walsh, Timothy R; Al Johani, Sameera M; AlJindan, Reem Y; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al Salman, Jameela; Dashti, Ali A; Johani, Khalid; Paterson, David L

2015-03-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Acinetobacter baumannii (CRAB) were determined in hospitals in the states of the Cooperation Council for the Arab States of the Gulf (Gulf Cooperation Council [GCC]), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic resistance genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Selected isolates were subjected to multilocus sequence typing (MLST). We investigated 117 isolates resistant to carbapenem antibiotics (either imipenem or meropenem). All isolates were positive for OXA-51. The most common carbapenemases were the OXA-23-type, found in 107 isolates, followed by OXA-40-type (OXA-24-type), found in 5 isolates; 3 isolates carried the ISAba1 element upstream of blaOXA-51-type. No OXA-58-type, NDM-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with 16 clusters of clonally related CRAB. Some clusters involved hospitals in different states. MLST analysis of 15 representative isolates from different clusters identified seven different sequence types (ST195, ST208, ST229, ST436, ST450, ST452, and ST499), as well as three novel STs. The vast majority (84%) of the isolates in this study were associated with health care exposure. Awareness of multidrug-resistant organisms in GCC states has important implications for optimizing infection control practices; establishing antimicrobial stewardship programs within hospital, community, and agricultural settings; and emphasizing the need for establishing regional active surveillance systems. This will help to control the spread of CRAB in the Middle East and in hospitals accommodating transferred patients from this region. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination.

PubMed

Jacques, Marie-Agnès; Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

2015-12-28

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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.

Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes

PubMed Central

Barnard, Emma; Nagy, István; Hunyadkürti, Judit; Patrick, Sheila

2015-01-01

The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterized by MLST and representing types IA1 (n = 145), IA2 (n = 20), IB (n = 65), IC (n = 7), II (n = 45), and III (n = 30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses. PMID:25631794

Epidemiologic and Genotypic Review of Carbapenemase-Producing Organisms in British Columbia, Canada, between 2008 and 2014

PubMed Central

Sekirov, Inna; Croxen, Matthew A.; Ng, Corrinne; Azana, Robert; Chang, Yin; Mataseje, Laura; Boyd, David; Mangat, Chand; Mack, Benjamin; Tadros, Manal; Brodkin, Elizabeth; Kibsey, Pamela; Stefanovic, Aleksandra; Champagne, Sylvie; Mulvey, Michael R.

2015-01-01

Carbapenemase-producing organisms (CPOs) are a serious emerging problem for health care facilities worldwide. Owing to their resistance to most antimicrobial therapies, CPOs are difficult to treat and pose a challenge for infection prevention and control. Since 2010, lab-based surveillance for CPOs and PCR-based testing were implemented in British Columbia (BC), Canada. A review of CPOs in BC from 2008 to March 2014 was done to characterize the resistance mechanisms and possible clonal strain transmission and to compare pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid restriction fragment length polymorphism (RFLP) as molecular typing tools. During this study period, a total of 177 CPO cases were identified. Patient demographics and travel history were reviewed, and a descriptive analysis was carried out. PFGE profiles, MLST, and plasmid RFLP analysis for a subset of Escherichia coli, Klebsiella pneumoniae, and Enterobacter species isolates were obtained and analyzed. Our findings demonstrate that CPOs have been increasing in number in BC over time, from 1 isolate/year retrospectively identified in 2008 and 2009 to 82 isolates in 2013 and 30 isolates in the first quarter of 2014. Overall, K. pneumoniae isolates lack clonality, although some seemingly related clusters have been found. Plasmid analysis showed evidence of the spread of plasmids carrying carbapenemase-encoding genes between the examined isolates. Analysis of Enterobacter cloacae isolates revealed a more clonal nature of these CPOs in BC. The presence of related clusters provides evidence of interpatient organism transmission both within and between institutions. Although in our study, NDM-harboring E. cloacae isolates appeared to spread clonally, the spread of carbapenem resistance in K. pneumoniae seems to be plasmid mediated. PMID:26607987

Methicillin-Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types.

PubMed

Oliveira, C J B; Tiao, N; de Sousa, F G C; de Moura, J F P; Santos Filho, L; Gebreyes, W A

2016-03-01

The aim of this study was to investigate the phenotypic and genotypic diversity and anti-microbial resistance among staphylococci of dairy herds that originated from Paraiba State, north-eastern Brazil, a region where such studies are rare. Milk samples (n = 552) were collected from 15 dairy farms. Isolates were evaluated for anti-microbial susceptibility by Kirby-Bauer disc diffusion method. Confirmation of methicillin-resistant Staphylococcus aureus (MRSA) was performed using multiplex PCR targeting mecA and nuc genes in addition to phenotypic assay based on PBP-2a latex agglutination. Clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) genotyping. Staphylococci were detected in 269 (49%) of the samples. Among these, 65 (24%) were S. aureus. The remaining 204 isolates were either coagulase-negative staphylococci (n = 188; 70%) or coagulase positive other than S. aureus (n = 16; 6%). Staphylococci were cultured in seven (35%) of the 20 hand swab samples, from which five isolates were S. aureus. The isolates were most commonly resistant against penicillin (43%), ampicillin (38%) and oxacillin (27%). The gene mecA was detected in 21 S. aureus from milk and in one isolate from a milker's hand. None of the isolates were resistant to vancomycin. PFGE findings showed high clonal diversity among the isolates. Based on MLST, we identified a total of 11 different sequence types (STs 1, 5, 6, 83, 97, 126, 1583, 1622, 1623, 1624 and 1625) with four novel STs (ST1622-ST1625). The findings show that MRSA is prevalent in milk from semi-extensive dairy cows in north-eastern Brazil, and further investigation on its extent in various types of milk production systems and the farm-to-table continuum is warranted. © 2015 Blackwell Verlag GmbH.

The Evolution of Campylobacter jejuni and Campylobacter coli

PubMed Central

Sheppard, Samuel K.; Maiden, Martin C.J.

2015-01-01

The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure. PMID:26101080

Clones of Streptococcus zooepidemicus from Outbreaks of Hemorrhagic Canine Pneumonia and Associated Immune Responses

PubMed Central

Velineni, Sridhar; Russell, Kim; Hamlen, Heidi J.; Pesavento, Patricia; Fortney, William D.; Crawford, P. Cynda

2014-01-01

Acute hemorrhagic pneumonia caused by Streptococcus zooepidemicus has emerged as a major disease of shelter dogs and greyhounds. S. zooepidemicus strains differing in multilocus sequence typing (MLST), protective protein (SzP), and M-like protein (SzM) sequences were identified from 9 outbreaks in Texas, Kansas, Florida, Nevada, New Mexico, and Pennsylvania. Clonality based on 2 or more isolates was evident for 7 of these outbreaks. The Pennsylvania and Nevada outbreaks also involved cats. Goat antisera against acutely infected lung tissue as well as convalescent-phase sera reacted with a mucinase (Sz115), hyaluronidase (HylC), InlA domain-containing cell surface-anchored protein (INLA), membrane-anchored protein (MAP), SzP, SzM, and extracellular oligopeptide-binding protein (OppA). The amino acid sequences of SzP and SzM of the isolates varied greatly. The szp and szm alleles of the closely related Kansas clone (sequence type 129 [ST-129]) and United Kingdom isolate BHS5 (ST-123) were different, indicating that MLST was unreliable as a predictor of virulence phenotype. Combinations of conserved HylC and serine protease (ScpC) and variable SzM and SzP proteins of S. zooepidemicus strain NC78 were protectively immunogenic for mice challenged with a virulent canine strain. Thus, although canine pneumonia outbreaks are caused by different strains of S. zooepidemicus, protective immune responses were elicited in mice by combinations of conserved or variable S. zooepidemicus proteins from a single strain. PMID:24990905

Dissemination of the ST-103 clonal complex serogroup C meningococci in Salvador, Brazil.

PubMed

Cordeiro, Soraia Machado; Cardoso, Cristiane Wanderley; de Araújo, Lorena Galvão; Ribeiro, Luis Eduardo; Azevedo, Jailton; Silva, Rita de Cassia Vilasboas; Dos Reis, Mitermayer Galvão; Ko, Albert Icksang; Reis, Joice Neves

2018-01-01

Invasive meningococcal disease (IMD) is a major public health problem worldwide. An epidemic of serogroup C (NmC) IMD occurred in 2010 in the city of Salvador. In this study, we describe the antigenic and genetic characterization of meningococcal isolates collected from meningitis cases in Salvador from 2001 to 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed for the analysis of IMD isolates. A total of 733 cases were identified, and the serogroup was determined for 391 (53.0%) of these. Most cases were caused by NmC (53%) or B (47%). The most prevalent strains were B:4,7:P1.19,15 (32.9%; 129/391) and C:23:P1.14-6 (28.6%; 112/391). Based on PFGE/MLST analysis, 71.3% (77/108 PFGE-tested isolates) clustered as two clones of sequence type ST-3779 and ST-3780, both belonging to the ST-103 clonal complex. ST-3779 has been detected in Salvador since 1996 and together with ST-3780 became predominant after 2005. There was a predominance of C:23:P1.14-6, ST-3779/3780 in Salvador during the period of 2007-2012, establishing a major clonal lineage, which remained in the community for a long time; this has serious implications for public health, particularly in terms of prevention and control strategies of IMD. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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.

Molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients with bacteremia based on MLST, SCCmec, spa, and agr locus types analysis.

PubMed

Goudarzi, Mehdi; Seyedjavadi, Sima Sadat; Nasiri, Mohammad Javad; Goudarzi, Hossein; Sajadi Nia, Raheleh; Dabiri, Hossein

2017-03-01

The widespread emergence of methicillin resistant Staphylococcus aureus, as a common cause of nosocomial infections, is becoming a serious concern in global public health. The objective of the present study was to investigate antimicrobial susceptibility pattern, frequency of virulence genes and molecular characteristics of methicillin-resistant Staphylococcus aureus strains isolated from patients with bacteremia. A total of 128 methicillin-resistant Staphylococcus aureus isolates were collected during February 2015 to January 2016. In vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion method. Conventional PCR was performed for the detection of adhesion (can, bbp, ebp, fnbB, fnbA, clfB, clfA) and toxin (etb, eta, pvl, tst) encoding genes, determining the agr type, SCCmec, MLST and spa typing of the isolates. All the methicillin-resistant Staphylococcus aureus isolates were found to be sensitive to linezolid, teicoplanin, and vancomycin. Resistance to the tested antibiotics varied from 97.7% for penicillin to 24.2% for mupirocin. The rate of multi drug resistance (MDR) in the present study was 97.7%. The most commonly detected toxin and adhesion genes were tst (58.6%), and clfB (100%), respectively. The majority of SCCmec III isolates were found in agr group I while SCCmec IV and II isolates were distributed among agr group III. Multilocus Sequence Typing (MLST) of the MRSA isolates showed five different sequence types: ST239 (43%), ST22 (39.8%), ST585 (10.9%), ST45 (3.9%) and ST240 (2.3%). All of the pvl positive strains belonged to ST22-SCCmec IV/t790 clone and were MDR. Among different 7 spa types, the most common were t790 (27.3%), t037 (21.9%), and t030 (14.1%). spa types t016, t924 and spa type t383 were reported for the first time from Asia and Iran, respectively. It was shown that spa types circulating in the studied hospitals varied which support the need to perform future surveillance studies in order to understand methicillin-resistant Staphylococcus aureus distribution and thus more effective infection control. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic Diversity, Antimicrobial Susceptibility, and Biofilm Formation of Cronobacter spp. Recovered from Spices and Cereals

PubMed Central

Li, Yuanhong; Yu, Huan; Jiang, Hua; Jiao, Yang; Zhang, Yaodong; Shao, Jihong

2017-01-01

Cronobacter species are important food-borne opportunistic pathogens which have been implicated in the cause of necrotizing enterocolitis, sepsis, and meningitis in neonates and infants. However, these bacteria are routinely found in foodstuffs, clinical specimens, and environmental samples. This study investigated the genetic diversity, antimicrobial susceptibility, and biofilm formation of Cronobacter isolates (n = 40) recovered from spices and cereals in China during 2014–2015. Based on the fusA sequencing analysis, we found that the majority (23/40, 57.5%) of Cronobacter isolates in spices and cereals were C. sakazakii, while the remaining strains were C. dublinensis (6/40, 15.0%), C. malonaticus (5/40, 12.5%), C. turicensis (4/40, 10.0%), and C. universalis (2/40, 5.0%). Multilocus sequence typing (MLST) analysis produced 30 sequence types (STs) among the 40 Cronobacter isolates, with 5 STs (ST4, ST13, ST50, ST129, and ST158) related to neonatal meningitis. The pattern of the overall ST distribution was diverse; in particular, it was revealed that ST148 was the predominant ST, presenting 12.5% within the whole population. MLST assigned 12 isolates to 7 different clonal complexes (CCs), 4, 13, 16, 17, 72, 129, and 143, respectively. The results of O-antigen serotyping indicated that C. sakazakii serotype O1 and O2 were the most two prevalent serotypes. The antimicrobial susceptibility testing showed that the 40 Cronobacter isolates were susceptible to most of the antibiotics tested except for ceftriaxone, meropenem, and aztreona. Of the 40 Cronobacter strains tested, 13 (32.5%) were assessed as weak bioflim producers, one (2.5%) was a moderate biofilm producer, one (2.5%) was strong biofilm producer, and the others (62.5%) were non-biofilm producers. MLST and O-antigen serotyping have indicated that Cronobacter strains recovered from spices and cereals were genetically diverse. Isolates of clinical origin, particularly the C. sakazakii ST4 neonatal meningitic pathovar, have been identified from spices and cereals. Moreover, antimicrobial resistance of Cronobacter strains was observed, which may imply a potential public health risk. Therefore, the surveillance of Cronobacter spp. in spices and cereals should be strengthened to improve epidemiological understandings of Cronobacter infections. PMID:29312246

Optimal rotated staggered-grid finite-difference schemes for elastic wave modeling in TTI media

NASA Astrophysics Data System (ADS)

Yang, Lei; Yan, Hongyong; Liu, Hong

2015-11-01

The rotated staggered-grid finite-difference (RSFD) is an effective approach for numerical modeling to study the wavefield characteristics in tilted transversely isotropic (TTI) media. But it surfaces from serious numerical dispersion, which directly affects the modeling accuracy. In this paper, we propose two different optimal RSFD schemes based on the sampling approximation (SA) method and the least-squares (LS) method respectively to overcome this problem. We first briefly introduce the RSFD theory, based on which we respectively derive the SA-based RSFD scheme and the LS-based RSFD scheme. Then different forms of analysis are used to compare the SA-based RSFD scheme and the LS-based RSFD scheme with the conventional RSFD scheme, which is based on the Taylor-series expansion (TE) method. The contrast in numerical accuracy analysis verifies the greater accuracy of the two proposed optimal schemes, and indicates that these schemes can effectively widen the wavenumber range with great accuracy compared with the TE-based RSFD scheme. Further comparisons between these two optimal schemes show that at small wavenumbers, the SA-based RSFD scheme performs better, while at large wavenumbers, the LS-based RSFD scheme leads to a smaller error. Finally, the modeling results demonstrate that for the same operator length, the SA-based RSFD scheme and the LS-based RSFD scheme can achieve greater accuracy than the TE-based RSFD scheme, while for the same accuracy, the optimal schemes can adopt shorter difference operators to save computing time.

Aspergillus and Penicillium identification using DNA sequences: Barcode or MLST?

USDA-ARS?s Scientific Manuscript database

Current methods in DNA technology can detect single nucleotide polymorphisms with measurable accuracy using several different approaches appropriate for different uses. If there are even single nucleotide differences that are invariant markers of the species, we can accomplish identification through...

Changes in the population structure of canine methicillin-resistant Staphylococcus pseudintermedius in Poland.

PubMed

Kizerwetter-Świda, Magdalena; Chrobak-Chmiel, Dorota; Rzewuska, Magdalena; Binek, Marian

2017-09-01

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is being reported with an increasing frequency in small animal veterinary practice. The molecular typing of MRSP isolates revealed that the dominating European multidrug-resistant lineage is the sequence type 71 (ST71), associated with staphylococcal chromosomal cassette SCCmec type II-III. However, the recent reports indicated the emergence of other clones. The study aimed to determine the genetic properties of MRSP isolates obtained from dogs in Poland over a ten-year period. A total of 42 clinical MRSP isolates were subjected to multilocus-sequence typing (MLST) and SCCmec typing. MLST typing of 42 MRSP isolates yielded six STs belonging to two major clonal complexes (CCs): CC71 and CC551, associated with SCCmec element II-III and V, respectively. CC71 comprising ST71 and its newly described single locus variant (SLV) ST680. The second dominating CC551was represented by ST551 and newly described SLV ST771. The other, ST258 and ST85 were detected in single MRSP isolates. This is the first report concerning MLST typing of MRSP isolates in Poland. The results confirmed the domination of ST71 among MRSP until 2015, and the emergence of ST551 in 2015. Furthermore, in 2016 ST551 was identified in the majority of the strains, indicating the changes in the population structure of MRSP in Poland. Polish clinical MRSP isolates showed a shift in the population structure during the period of 2007 and 2016. The dominating MRSP lineage until 2015 was multidrug-resistant ST71-SCCmecII-III. The other lineage ST551-SCCmecV emerged in Poland since 2015, and in 2016 was found in the majority of MRSP isolates. Copyright © 2017 Elsevier B.V. All rights reserved.

Multi-locus variable-number tandem repeat analysis of Bordetella pertussis isolates circulating in Poland in the period 1959-2013.

PubMed

Mosiej, Ewa; Krysztopa-Grzybowska, Katarzyna; Polak, Maciej; Prygiel, Marta; Lutyńska, Anna

2017-06-01

Despite the long history of pertussis vaccination and high vaccination coverage in Poland and many other developed countries, pertussis incidence rates have increased substantially, making whooping cough one of the most prevalent vaccine-preventable diseases. Among the factors potentially involved in pertussis resurgence, the adaptation of the Bordetella pertussis population to country-specific vaccine-induced immunity through selection of non-vaccine-type strains still needs detailed studies. Multi-locus variable-number tandem repeat analysis (MLVA), also linked to MLST and PFGE profiling, was applied to trace the genetic changes in the B. pertussis population circulating in Poland in the period 1959-2013 versus country-specific vaccine strains. Generally, among 174 B. pertussis isolates, 31 MLVA types were detected, of which 11 were not described previously. The predominant MLVA types of recent isolates in Poland were different from those of the typical isolates circulating in other European countries. The MT27 type, currently predominant in Europe, was rarely seen and detected in only five isolates among all studied. The features of the vaccine strains used for production of the pertussis component of a national whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, as studied by MLVA and MLST tools, were found to not match those observed in the currently circulating B. pertussis isolates in Poland. Differences traced by MLVA in relation to the MLST and PFGE profiling confirmed that the B. pertussis strain types currently observed elsewhere in Europe, even if appearing in Poland, were not able to successfully disseminate within a human population in Poland that has been vaccinated with a whole-cell pertussis vaccine not used in other countries.

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.

Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus from Ready-to-Eat Foods in China

PubMed Central

Xie, Tengfei; Xu, Xiaoke; Wu, Qingping; Zhang, Jumei; Cheng, Jianheng

2016-01-01

Vibrio parahaemolyticus is the leading cause of foodborne outbreaks, particularly outbreaks associated with consumption of fish and shellfish, and represents a major threat to human health worldwide. This bacterium harbors two main virulence factors: the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Additionally, various serotypes have been identified. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant V. parahaemolyticus. In the current study, we aimed to determine the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods in China. We found 39 V. parahaemolyticus strains on Chinese RTE foods through investigation of 511 RTE foods samples from 24 cities in China. All isolates were analyzed for the presence of tdh and trh gene by PCR, serotyping was performed using multiplex PCR, antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing and multilocus sequence typing (MLST). The results showed that none of the isolates were positive for tdh and trh. Most of the isolates (33.3%) were serotype O2. Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (89.7%), cefazolin (51.3%), and ampicillin (51.3%). The isolates were grouped into five clusters by ERIC-PCR and four clusters by MLST. We updated 10 novel loci and 33 sequence types (STs) in the MLST database. Thus, our findings demonstrated the presence of V. parahaemolyticus in Chinese RTE foods, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of methods of microbiological risk assessment in RTE foods. PMID:27148231

Resolution of a Protracted Serogroup B Meningococcal Outbreak with Whole-Genome Sequencing Shows Interspecies Genetic Transfer

PubMed Central

Brehony, Carina; O'Connor, Lois; Meyler, Kenneth; Jolley, Keith A.; Bray, James; Bennett, Desiree; Maiden, Martin C. J.; Cunney, Robert

2016-01-01

A carriage study was undertaken (n = 112) to ascertain the prevalence of Neisseria spp. following the eighth case of invasive meningococcal disease in young children (5 to 46 months) and members of a large extended indigenous ethnic minority Traveller family (n = 123), typically associated with high-occupancy living conditions. Nested multilocus sequence typing (MLST) was employed for case specimen extracts. Isolates were genome sequenced and then were assembled de novo and deposited into the Bacterial Isolate Genome Sequencing Database (BIGSdb). This facilitated an expanded MLST approach utilizing large numbers of loci for isolate characterization and discrimination. A rare sequence type, ST-6697, predominated in disease specimens and isolates that were carried (n = 8/14), persisting for at least 44 months, likely driven by the high population density of houses (n = 67/112) and trailers (n = 45/112). Carriage for Neisseria meningitidis (P < 0.05) and Neisseria lactamica (P < 0.002) (2-sided Fisher's exact test) was more likely in the smaller, more densely populated trailers. Meningococcal carriage was highest in 24- to 39-year-olds (45%, n = 9/20). Evidence of horizontal gene transfer (HGT) was observed in four individuals cocolonized by Neisseria lactamica and Neisseria meningitidis. One HGT event resulted in the acquisition of 26 consecutive N. lactamica alleles. This study demonstrates how housing density can drive meningococcal transmission and carriage, which likely facilitated the persistence of ST-6697 and prolonged the outbreak. Whole-genome MLST effectively distinguished between highly similar outbreak strain isolates, including those isolated from person-to-person transmission, and also highlighted how a few HGT events can distort the true phylogenetic relationship between highly similar clonal isolates. PMID:27629899

Enrichment of Multilocus Sequence Typing Clade 1 with Oral Candida albicans Isolates in Patients with Untreated Periodontitis

PubMed Central

McManus, Brenda A.; Maguire, Rory; Cashin, Phillipa J.; Claffey, Noel; Flint, Stephen; Abdulrahim, Mohammed H.

2012-01-01

This study investigated the prevalence and cell density of Candida species in periodontal pockets, healthy subgingival sites, and oral rinse samples of patients with untreated periodontitis. Twenty-one periodontitis patients underwent sampling at two periodontitis sites, and 19/21 of these patients underwent sampling at one periodontally healthy site. Both paper point and curette sampling techniques were employed. The periodontitis patients and 50 healthy subjects were also sampled by oral rinse. Candida isolates were recovered on CHROMagar Candida medium, and representative isolates were identified. Candida spp. were recovered from 10/21 (46.7%) periodontitis patients and from 16/50 (32%) healthy subjects. C. albicans predominated in both groups and was recovered from all Candida-positive subjects. Candida-positive periodontitis patients yielded Candida from periodontal pockets with average densities of 3,528 and 3,910 CFU/sample from curette and paper point samples, respectively, and 1,536 CFU/ml from oral rinse samples. The majority (18/19) of the healthy sites sampled from periodontitis patients were Candida negative. The 16 Candida-positive healthy subjects yielded an average of 279 CFU/ml from oral rinse samples. C. albicans isolates were investigated by multilocus sequence typing (MLST) to determine if specific clonal groups were associated with periodontitis. MLST analysis of 31 C. albicans isolates from periodontitis patients yielded 19 sequence types (STs), 13 of which were novel. Eleven STs belonged to MLST clade 1. In contrast, 16 C. albicans isolates from separate healthy subjects belonged to 16 STs, with 4 isolates belonging to clade 1. The distributions of STs between both groups were significantly different (P = 0.04) and indicated an enrichment of C. albicans isolates in periodontal pockets, which warrants a larger study. PMID:22875886

Transmission and genetic diversity of Enterococcus faecalis among layer chickens during hatch

PubMed Central

2011-01-01

Background Studies on transmission of Enterococcus faecalis among chickens during hatch have not been carried out so far. Information about vertical transmission and subsequent spreading and colonization of the cloacal mucosa through cloacal 'drinking' during hatch are important to understand the epidemiology of E. faecalis infections. In the present investigation vertical transmission and subsequent spreading and colonization of the cloacal mucosa of chickens by E. faecalis through cloacal 'drinking' were examined. Methods Two different batches of layer chickens originating from 45 weeks old Brown and White Lohmann parents, respectively from the same farm were sampled in the hatcher. Isolates were confirmed to be E. faecalis by polymerase chain reaction (PCR) and further by multilocus sequence typing (MLST) to state their population structure and comparison made to sequence types previously obtained from chicken. Results A total of 480 chickens were swabbed from the cloacae just after hatch and after 24 hours. A total of 101 isolates were confirmed as E. faecalis by a species specific PCR. The prevalence of E. faecalis increased from 14% at 0 h to 97% after 24 h for the Brown Lohmann chickens and from 0.5% to 23% for the White Lohmann flock. The 84 isolates analysed by MLST were distributed on 14 sequence types (ST). Three ST (401, 82 and 249) accounted for 64% of all isolates analysed by MLST after 24 h. ST 82 has previously been reported from amyloid arthropathy and other lesions in poultry. Conclusions The present findings demonstrated a high potential of a few contaminated eggs or embryos to rapidly facilitate the spread of E. faecalis to almost all chickens during hatch. PMID:22017822

Evidence of genotypic diversity among Candida auris isolates by multilocus sequence typing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and amplified fragment length polymorphism.

PubMed

Prakash, A; Sharma, C; Singh, A; Kumar Singh, P; Kumar, A; Hagen, F; Govender, N P; Colombo, A L; Meis, J F; Chowdhary, A

2016-03-01

Candida auris is a multidrug-resistant nosocomial bloodstream pathogen that has been reported from Asian countries and South Africa. Herein, we studied the population structure and genetic relatedness among 104 global C. auris isolates from India, South Africa and Brazil using multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RPB1, RPB2 and internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal DNA were sequenced for MLST. Further, genetic variation and proteomic assessment was carried out using AFLP and MALDI-TOF MS, respectively. Both MLST and AFLP typing clearly demarcated two major clusters comprising Indian and Brazilian isolates. However, the South African isolates were randomly distributed, suggesting different genotypes. MALDI-TOF MS spectral profiling also revealed evidence of geographical clustering but did not correlate fully with the genotyping methods. Notably, overall the population structure of C. auris showed evidence of geographical clustering by all the three techniques analysed. Antifungal susceptibility testing by the CLSI microbroth dilution method revealed that fluconazole had limited activity against 87% of isolates (MIC90, 64 mg/L). Also, MIC90 of AMB was 4 mg/L. Candida auris is emerging as an important yeast pathogen globally and requires reproducible laboratory methods for identification and typing. Evaluation of MALDI-TOF MS as a typing method for this yeast is warranted. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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.

Fluorescent Amplified-Fragment Length Polymorphism Genotyping of Neisseria meningitidis Identifies Clones Associated with Invasive Disease

PubMed Central

Goulding, Jonathan N.; Hookey, John V.; Stanley, John; Olver, Will; Neal, Keith R.; Ala'Aldeen, Dlawer A. A.; Arnold, Catherine

2000-01-01

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures. PMID:11101599

Molecular Typing and Epidemiology of Human Listeriosis Cases, Denmark, 2002-2012.

PubMed

Jensen, Anne Kvistholm; Björkman, Jonas T; Ethelberg, Steen; Kiil, Kristoffer; Kemp, Michael; Nielsen, Eva Møller

2016-04-01

Denmark has a high incidence of invasive listeriosis (0.9 cases/100,000 population in 2012). We analyzed patient data, clinical outcome, and trends in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of Listeria monocytogenes strains isolated in Denmark during 2002-2012. We performed 2-enzyme PFGE and serotyping on 559 isolates and MLST on 92 isolates and identified some correlation between molecular type and clinical outcome and patient characteristics. We found 178 different PFGE types, but isolates from 122 cases belonged to just 2 closely related PFGE types, clonal complex 8 and sequence type 8. These 2 types were the main cause of a peak in incidence of invasive listeriosis during 2005-2009, possibly representing an outbreak or the presence of a highly prevalent clone. However, current typing methods could not fully confirm these possibilities, highlighting the need for more refined discriminatory typing methods to identify outbreaks within frequently occurring L. monocytogenes PFGE types.

Molecular Typing and Epidemiology of Human Listeriosis Cases, Denmark, 2002–20121

PubMed Central

Björkman, Jonas T.; Ethelberg, Steen; Kiil, Kristoffer; Kemp, Michael; Nielsen, Eva Møller

2016-01-01

Denmark has a high incidence of invasive listeriosis (0.9 cases/100,000 population in 2012). We analyzed patient data, clinical outcome, and trends in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of Listeria monocytogenes strains isolated in Denmark during 2002–2012. We performed 2-enzyme PFGE and serotyping on 559 isolates and MLST on 92 isolates and identified some correlation between molecular type and clinical outcome and patient characteristics. We found 178 different PFGE types, but isolates from 122 cases belonged to just 2 closely related PFGE types, clonal complex 8 and sequence type 8. These 2 types were the main cause of a peak in incidence of invasive listeriosis during 2005–2009, possibly representing an outbreak or the presence of a highly prevalent clone. However, current typing methods could not fully confirm these possibilities, highlighting the need for more refined discriminatory typing methods to identify outbreaks within frequently occurring L. monocytogenes PFGE types. PMID:26982714

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.

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.

Resolving the Mortierellaceae phylogeny through Multi-Locus Sequence Typing (MLST) and phylogenomics

USDA-ARS?s Scientific Manuscript database

The Mortierellaceae (Mortierellomycotina) are a diverse family of fungi that are of evolutionary and ecological relevance. They are the closest lineage to the arbuscular mycorrhizae (Glomeromycotina) and include some of the first species to evolve fruiting body production. The Mortierellaceae are es...

Prevalence and molecular typing of Vibrio parahaemolyticus isolated from seafood in Shanghai using multilocus sequence typing (MLST)

USDA-ARS?s Scientific Manuscript database

Vibrio parahaemolyticus is a gram-negative bacterium that inhabits coastal and marine environments. Thermostable direct hemolysin (tdh), tdh-related hemolysin (trh) and the type III secretion system are considered the potential virulent factors of pathogenic V. parahaemolyticus. The frequency of str...

Novel, host-restricted genotypes of Bordetella bronchiseptica associated with phocine respiratory tract isolates.

PubMed

Register, Karen B; Ivanov, Yury V; Harvill, Eric T; Davison, Nick; Foster, Geoffrey

2015-03-01

During a succession of phocine morbillivirus outbreaks spanning the past 25 years, Bordetella bronchiseptica was identified as a frequent secondary invader and cause of death. The goal of this study was to evaluate genetic diversity and the molecular basis for host specificity among seal isolates from these outbreaks. MLST and PvuII ribotyping of 54 isolates from Scottish, English or Danish coasts of the Atlantic or North Sea revealed a single, host-restricted genotype. A single, novel genotype, unique from that of the Atlantic and North Sea isolates, was found in isolates from an outbreak in the Caspian Sea. Phylogenetic analysis based either on MLST sequence, ribotype patterns or genome-wide SNPs consistently placed both seal-specific genotypes within the same major clade but indicates a distinct evolutionary history for each. An additional isolate from the intestinal tract of a seal on the south-west coast of England has a genotype otherwise found in rabbit, guinea pig and pig isolates. To investigate the molecular basis for host specificity, DNA and predicted protein sequences of virulence genes that mediate host interactions were used in comparisons between a North Sea isolate, a Caspian Sea isolate and each of their closest relatives as inferred from genome-wide SNP analysis. Despite their phylogenetic divergence, fewer nucleotide and amino acid substitutions were found in comparisons of the two seal isolates than in comparisons with closely related strains. These data indicate isolates of B. bronchiseptica associated with respiratory disease in seals comprise unique, host-adapted and highly clonal populations. © 2015 The Authors.

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.

Identification of accessory genome regions in poultry Clostridium perfringens isolates carrying the netB plasmid.

PubMed

Lepp, D; Gong, J; Songer, J G; Boerlin, P; Parreira, V R; Prescott, J F

2013-03-01

Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance.

Prevalence and Characterization of Oxacillin Susceptible mecA-Positive Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis in India.

PubMed

Mistry, Hiral; Sharma, Paresh; Mahato, Sudipta; Saravanan, R; Kumar, P Anand; Bhandari, Vasundhra

2016-01-01

Bovine mastitis caused by multidrug resistant Staphylococcus aureus is a huge problem reported worldwide, resulting in prolonged antibiotic treatment and death of livestock. The current study is focused on surveillance of antibiotic susceptibility along with genotypic and phenotypic characterization of the pathogenic S. aureus strains causing mastitis in India. One hundred and sixty seven milk samples were collected from mastitis-affected cows from different farms in India resulting in thirty nine isolated S. aureus strains. Antibiotic sensitivity profiling revealed the majority of the strains (n = 24) to be multidrug resistant and eleven strains showed reduced susceptibility to vancomycin (MICs = 2μg/ml). All strains were oxacillin sensitive, but 19 strains were positive for the mecA gene, which revealed the occurrence of oxacillin susceptible mecA positive strains (OS-MRSA) for the first time from India. Additionally, 32 strains were positive for the pvl gene, a virulence determinant; of these 17 were also OS-MRSA strains. Molecular characterization based on multilocus sequence typing (MLST), spa typing, agr typing and SCCmec classification revealed strains belonging to different groups. Moreover, strains showed spa types (t2526, t9602) and MLST sequence types, ST-72, ST-88 and ST-239 which have been earlier reported in human infections. The prevalence of OS-MRSA strains indicates the importance of including both the genetic and phenotypic tests in characterizing S. aureus strains. Increased genotypic variability with strain related to human infections and pvl positive isolates indicates a worrisome situation with the possibility of bilateral transfer.

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order

PubMed Central

Javůrková, Barbora; Vlach, Jiří; Göselová, Sandra; Karamonová, Ludmila; Ogrodzki, Pauline; Forsythe, Stephen; Fukal, Ladislav

2015-01-01

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases. PMID:26070668

Human, food and animal Campylobacter spp. isolated in Portugal: high genetic diversity and antibiotic resistance rates.

PubMed

Duarte, Andreia; Santos, Andrea; Manageiro, Vera; Martins, Ana; Fraqueza, Maria J; Caniça, Manuela; Domingues, Fernanda C; Oleastro, Mónica

2014-10-01

Infections by Campylobacter jejuni and Campylobacter coli are considered the major cause of bacterial gastroenteritis in humans, with food being the main source of infection. In this study, a total of 196 Campylobacter strains (125 isolates from humans, 39 from retail food and 32 from food animal sources) isolated in Portugal between 2009 and 2012 were characterised by multilocus sequence typing (MLST) and flaA short variable region (SVR) typing. Susceptibility to six antibiotics as well as the mechanisms underlying antibiotic resistance phenotypes was also studied. Based on MLST typing, C. coli strains were genetically more conserved, with a predominant clonal complex (CC828), than C. jejuni strains. In contrast, C. coli isolates were genetically more variable than C. jejuni with regard to flaA-SVR typing. A high rate of resistance was observed for quinolones (100% to nalidixic acid, >90% to ciprofloxacin) and, in general, resistance was more common among C. coli, especially for erythromycin (40.2% vs. 6.7%). In addition, most isolates (86%) were resistant to multiple antimicrobial families. Besides the expected point mutations associated with antibiotic resistance, detected polymorphisms in the cmeABC locus likely play a role in the multiresistant phenotype. This study provides for the first time an overview of the genetic diversity of Campylobacter strains from Portugal. It also shows a worrying antibiotic multiresistance rate and the emergence of Campylobacter strains resistant to antibiotics of human use. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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

Burkholderia cepacia complex in cystic fibrosis in the post-epidemic period: multilocus sequence typing-based approach.

PubMed

Fila, Libor; Dřevínek, Pavel

2017-11-01

Cystic fibrosis (CF) patients in the Czech Republic suffered in the late 1990s from an epidemic with ST32 strain of Burkholderia cepacia complex (Bcc). Cohort segregation of Bcc and of ST32 positive patients was introduced in 1999 and 2002, respectively. We performed a study to evaluate the molecular epidemiology of Bcc infection after implementation of these infection control measures. Patients attending the Prague CF adult Centre from 2000 to 2015 were included in the present study. Demographic data and microbial statuses were collected from patient records. All Bcc isolates were analyzed using multilocus sequence typing (MLST). The prevalences of epidemic strain ST32 and of other Bcc strains were calculated. Ninety out of 227 CF patients were infected with Bcc during the study period. The prevalence of ST32 cases significantly decreased from 46.5% in 2000-2001 to 10.4% in 2014-2015 (P < 0.001) due to occurrence of only one new case in 2003, as well as to the death of 72% of ST32-infected patients. Conversely, there was a significant increase in prevalence of other Bcc strains, which rose from 0 to 14.9% (P = 0.015) and of transient infections. A micro-epidemic of infection with ST630 strain was observed in 2014 in lung transplant patients hospitalized in intensive care unit. The prevalence of epidemic strain ST32 decreased, whereas that of non-clonal strains of Bcc increased. Routine use of MLST allowed early detection of new and potentially epidemic strains.

Molecular typing of environmental and clinical strains of Vibrio vulnificus isolated in the northeastern USA.

PubMed

Reynaud, Yann; Pitchford, Steven; De Decker, Sophie; Wikfors, Gary H; Brown, Christopher L

2013-01-01

Vibrio vulnificus is a ubiquitous marine bacterium that is responsible for infections and some seafood-related illnesses and deaths in the United States, mainly in individuals with compromised health status in the Gulf of Mexico region. Most phylogenetic studies focus on V. vulnificus strains isolated in the southern United States, but almost no genetic data are available on northeastern bacterial isolates of clinical or environmental origin. Our goal in this study was to examine the genetic diversity of environmental strains isolated from commercially-produced oysters and in clinical strains of known pathogenicity in northeastern United States. We conducted analyses of a total of eighty-three strains of V. vulnificus, including 18 clinical strains known to be pathogenic. A polyphasic, molecular-typing approach was carried out, based upon established biotypes, vcg, CPS, 16S rRNA types and three other genes possibly associated with virulence (arylsulfatase A, mtlABC, and nanA). An established Multi Locus Sequence Typing (MLST) method was also performed. Phylogenetic analyses of these markers and MLST results produced similar patterns of clustering of strains into two main lineages (we categorized as 'LI' and 'LII'), with clinical and environmental strains clustering together in both lineages. Lineage LII was comprised primarily but not entirely of clinical bacterial isolates. Putative virulence markers were present in both clinical and environmental strains. These results suggest that some northeastern environmental strains of V. vulnificus are phylogenetically close to clinical strains and probably are capable of virulence. Further studies are necessary to assess the risk of human illness from consuming raw oysters harvested in the northeastern US.

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

Adaptive Packet Combining Scheme in Three State Channel Model

NASA Astrophysics Data System (ADS)

Saring, Yang; Bulo, Yaka; Bhunia, Chandan Tilak

2018-01-01

The two popular techniques of packet combining based error correction schemes are: Packet Combining (PC) scheme and Aggressive Packet Combining (APC) scheme. PC scheme and APC scheme have their own merits and demerits; PC scheme has better throughput than APC scheme, but suffers from higher packet error rate than APC scheme. The wireless channel state changes all the time. Because of this random and time varying nature of wireless channel, individual application of SR ARQ scheme, PC scheme and APC scheme can't give desired levels of throughput. Better throughput can be achieved if appropriate transmission scheme is used based on the condition of channel. Based on this approach, adaptive packet combining scheme has been proposed to achieve better throughput. The proposed scheme adapts to the channel condition to carry out transmission using PC scheme, APC scheme and SR ARQ scheme to achieve better throughput. Experimentally, it was observed that the error correction capability and throughput of the proposed scheme was significantly better than that of SR ARQ scheme, PC scheme and APC scheme.

Control of parallel manipulators using force feedback

NASA Technical Reports Server (NTRS)

Nanua, Prabjot

1994-01-01

Two control schemes are compared for parallel robotic mechanisms actuated by hydraulic cylinders. One scheme, the 'rate based scheme', uses the position and rate information only for feedback. The second scheme, the 'force based scheme' feeds back the force information also. The force control scheme is shown to improve the response over the rate control one. It is a simple constant gain control scheme better suited to parallel mechanisms. The force control scheme can be easily modified for the dynamic forces on the end effector. This paper presents the results of a computer simulation of both the rate and force control schemes. The gains in the force based scheme can be individually adjusted in all three directions, whereas the adjustment in just one direction of the rate based scheme directly affects the other two directions.

Cluster analysis of Scedosporium boydii infections in a single hospital.

PubMed

Bernhardt, Anne; Seibold, Michael; Rickerts, Volker; Tintelnot, Kathrin

2015-10-01

Scedosporiosis is a rare, but often fatal mycotic infection occurring in immunosuppressed as well as in immunocompetent patients. Over a period of 14 months, Scedosporium boydii isolates were sent to our reference laboratory from six immunocompetent patients treated at a single hospital in Germany. In analogy to the EORTC/MSG criteria, four patients were classified as proven invasive scedosporiosis cases, and two patients as probable or possible cases. Of note, in five patients scedosporiosis was diagnosed between 1 and 14 months (median 5.0 months) after cardiac surgery. Despite antimycotic treatment two patients died, and three were lost for long-term follow-up. All clinical S. boydii isolates were characterized by molecular analysis using multilocus sequence typing (MLST). An identical MLST type was found in five patients who had been treated in the surgery unit, suggesting a link between these infections. The source of S. boydii has not been identified. Within an observation period of 2 years before and after this cluster of infections no further cases of scedosporiosis were reported from this hospital. Copyright © 2015 Elsevier GmbH. All rights reserved.

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

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

An outbreak of Burkholderia stabilis colonization in a nasal ward.

PubMed

Wang, Lijun; Wang, Mei; Zhang, Junyi; Wu, Wei; Lu, Yuan; Fan, Yanyan

2015-04-01

The aim of this study was to describe an outbreak of Burkholderia stabilis colonization among patients in a nasal ward. Multilocus sequence typing (MLST) was used for the molecular typing of B. stabilis isolates. Microbiological records were reviewed to delineate the colonization outbreak period. One hundred seventy-one cultures of environment and equipment samples from the nasal ward were performed to trace the source of contamination. Infection control measures were taken in order to end the outbreak. All B. stabilis isolates were identified as a new MLST type, ST821. A total of 53 patients carried this B. stabilis in the nasal ward between March and September 2013, which was defined as the outbreak period. The source of the colonization was not determined because all environment cultures were negative for Burkholderia cepacia complex. No further B. stabilis carriers have been found in the ward since the implementation of interventions. Attention must be paid to asymptomatic colonization in order to identify outbreaks early. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Risk Factors for Campylobacteriosis of Chicken, Ruminant, and Environmental Origin: A Combined Case-Control and Source Attribution Analysis

PubMed Central

Wagenaar, Jaap A.; de Boer, Albert G.; Havelaar, Arie H.; Friesema, Ingrid H. M.; French, Nigel P.; Busani, Luca; van Pelt, Wilfrid

2012-01-01

Background Campylobacteriosis contributes strongly to the disease burden of food-borne pathogens. Case-control studies are limited in attributing human infections to the different reservoirs because they can only trace back to the points of exposure, which may not point to the original reservoirs because of cross-contamination. Human Campylobacter infections can be attributed to specific reservoirs by estimating the extent of subtype sharing between strains from humans and reservoirs using multilocus sequence typing (MLST). Methodology/Principal Findings We investigated risk factors for human campylobacteriosis caused by Campylobacter strains attributed to different reservoirs. Sequence types (STs) were determined for 696 C. jejuni and 41 C. coli strains from endemic human cases included in a case-control study. The asymmetric island model, a population genetics approach for modeling Campylobacter evolution and transmission, attributed these cases to four putative animal reservoirs (chicken, cattle, sheep, pig) and to the environment (water, sand, wild birds) considered as a proxy for other unidentified reservoirs. Most cases were attributed to chicken (66%) and cattle (21%), identified as the main reservoirs in The Netherlands. Consuming chicken was a risk factor for campylobacteriosis caused by chicken-associated STs, whereas consuming beef and pork were protective. Risk factors for campylobacteriosis caused by ruminant-associated STs were contact with animals, barbecuing in non-urban areas, consumption of tripe, and never/seldom chicken consumption. Consuming game and swimming in a domestic swimming pool during springtime were risk factors for campylobacteriosis caused by environment-associated STs. Infections with chicken- and ruminant-associated STs were only partially explained by food-borne transmission; direct contact and environmental pathways were also important. Conclusion/Significance This is the first case-control study in which risk factors for campylobacteriosis are investigated in relation to the attributed reservoirs based on MLST profiles. Combining epidemiological and source attribution data improved campylobacteriosis risk factor identification and characterization, generated hypotheses, and showed that genotype-based source attribution is epidemiologically sensible. PMID:22880049

Fine typing of methicillin-resistant Staphylococcus aureus isolates using direct repeat unit and staphylococcal interspersed repeat unit typing methods.

PubMed

Ho, Cheng-Mao; Ho, Mao-Wang; Li, Chi-Yuan; Lu, Jang-Jih

2015-08-01

Methicillin-resistant Staphylococcus aureus (MRSA) typing is an important epidemiologic tool for monitoring trends and preventing outbreaks. However, the efficiency of various MRSA typing methods for each SCCmec MRSA isolate is rarely evaluated. A total of 157 MRSA isolates from four different regions in Taiwan were typed with five different molecular methods, including SCCmec typing, multilocus sequence typing (MLST), spa typing, mec-associated direct repeat unit (dru) copy number determination, and staphylococcal interspersed repeat unit (SIRU) profiling. There were four SCCmec types, eight MLST types, 15 spa types, 11 dru types, and 31 SIRU profiles. The most common type determined by each molecular typing method was SCCmec III (115 isolates, 73.2%), ST239 (99 isolates, 63.1%), t037 (107 isolates, 68.2%), 14 dru copies (76 isolates, 48.4%), and SIRU profile 3013722 (102 isolates, 65%), respectively. When using the combination of MLST, spa typing, and dru copy number, ST5-t002-4 (n = 8), ST239-t037-14 (n = 68), ST59-t437-9 (n = 9), and ST59-t437-11 (n = 6) were found to be the most common types of SCCmec types II (n = 9), III (n = 115), IV (n = 21), and VT (n = 11) isolates, respectively. SCCmec type III isolates were further classified into 11 dru types. Of the 21 SCCmec type IV isolates, 14 SIRU profiles were found. Seven SIRU patterns were observed in the 11 SCCmec type VT isolates. Different typing methods showed a similar Hunter-Gaston discrimination index among the 157 MRSA isolates. However, dru and SIRU typing methods had a better discriminatory power for SCCmec type III and SCCmec types IV and VT isolates, respectively, suggesting that dru and SIRU can be used to further type these isolates. Copyright © 2013. Published by Elsevier B.V.

Environmental Isolation of Cryptococcus gattii VGII from Indoor Dust from Typical Wooden Houses in the Deep Amazonas of the Rio Negro Basin

PubMed Central

Brito-Santos, Fábio; Barbosa, Gláucia Gonçalves; Trilles, Luciana; Nishikawa, Marília Martins; Wanke, Bodo; Meyer, Wieland; Carvalho-Costa, Filipe Anibal; Lazéra, Márcia dos Santos

2015-01-01

Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and its agents, C. neoformans and C. gattii, present eight major molecular types—VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern to this molecular type. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Canada, and is expanding to the Northwest of the United States, two temperate regions. Exposure to propagules dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study investigated the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas state. Indoor dust was collected from 51 houses, diluted and plated on bird seed agar. Dark brown colonies were identified phenotypically, and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and MATa, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and MATa were isolated from the environment and correlates with endemic cryptococcosis in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and MATa. PMID:25688971

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.

Prevalence, antibiotic resistance, and MLST typing of Helicobacter pylori in Algiers, Algeria.

PubMed

Raaf, Naïma; Amhis, Wahiba; Saoula, Houria; Abid, Ahmed; Nakmouche, Mhamed; Balamane, Abdelmalek; Ali Arous, Nassima; Ouar-Korichi, Mounira; Vale, Filipa F; Bénéjat, Lucie; Mégraud, Francis

2017-12-01

Helicobacter pylori infection is common in Algeria, but there are few data on the characterization of isolated strains. The aim of this study was to update data on the prevalence of H. pylori in patients submitted to endoscopy, antibiotic resistance, and phylogeography of H. pylori strains isolated in Algiers. This is a prospective study carried out between November 2015 and August 2016. The culture of H. pylori was performed on antral and fundic gastric biopsies of adult patients from 3 hospitals. A real-time PCR using the fluorescence resonance energy transfer (FRET) principle for the detection of H. pylori followed by a melting curve analysis for the detection of mutations associated with resistance to clarithromycin was applied. Differentiation between antral and fundic isolates of the same patient was also determined by RAPD, and an MLST typing was performed for characterization of the phylogeographic group of H. pylori. By real-time PCR, the prevalence of H. pylori infection among the 147 patients included was 57%. Culture was positive in only 29% of the cases. Twenty-seven percent of patients had received H. pylori eradication treatment. The primary and secondary resistance rates to clarithromycin were 23% and 36%, respectively, and to metronidazole, 45% and 71%, respectively. Only one isolate was resistant to levofloxacin, and no resistance to amoxicillin, tetracycline, and rifampicin was detected. A double population was present in 14 patients. The MLST analysis classified the 42 H. pylori strains from 38 patients in 2 haplotypes: hpEurope (33) and hpNEAfrica (9). The prevalence of H. pylori remains high in Algeria but appears to be decreasing in recent years. High resistance to clarithromycin requires increased monitoring of the evolution of antibiotic resistance and adaptation of eradication therapy. © 2017 John Wiley & Sons Ltd.

Multilocus sequence typing of Trichomonas vaginalis clinical samples from Amsterdam, the Netherlands

PubMed Central

van der Veer, C; Himschoot, M; Bruisten, S M

2016-01-01

Objectives In this cross-sectional epidemiological study we aimed to identify molecular profiles for Trichomonas vaginalis and to determine how these molecular profiles were related to patient demographic and clinical characteristics. Setting Molecular typing methods previously identified two genetically distinct subpopulations for T. vaginalis; however, few molecular epidemiological studies have been performed. We now increased the sensitivity of a previously described multilocus sequence typing (MLST) tool for T. vaginalis by using nested PCR. This enabled the typing of direct patient samples. Participants From January to December 2014, we collected all T. vaginalis positive samples as detected by routine laboratory testing. Samples from patients either came from general practitioners offices or from the sexually transmitted infections (STI) clinic in Amsterdam. Epidemiological data for the STI clinic patients were retrieved from electronic patient files. Primary and secondary outcome measures The primary outcome was the success rate of genotyping direct T. vaginalis positive samples. The secondary outcome was the relation between T. vaginalis genotypes and risk factors for STI. Results All 7 MLST loci were successfully typed for 71/87 clinical samples. The 71 typed samples came from 69 patients, the majority of whom were women (n=62; 90%) and half (n=34; 49%) were STI clinic patients. Samples segregated into a two population structure for T. vaginalis representing genotypes I and II. Genotype I was most common (n=40; 59.7%). STI clinic patients infected with genotype II reported more sexual partners in the preceding 6 months than patients infected with genotype I (p=0.028). No other associations for gender, age, ethnicity, urogenital discharge or co-occurring STIs with T. vaginalis genotype were found. Conclusions MLST with nested PCR is a sensitive typing method that allows typing of direct (uncultured) patient material. Genotype II is possibly more prevalent in high-risk sexual networks. PMID:27737887

Multilocus sequence typing of Trichomonas vaginalis clinical samples from Amsterdam, the Netherlands.

PubMed

van der Veer, C; Himschoot, M; Bruisten, S M

2016-10-13

In this cross-sectional epidemiological study we aimed to identify molecular profiles for Trichomonas vaginalis and to determine how these molecular profiles were related to patient demographic and clinical characteristics. Molecular typing methods previously identified two genetically distinct subpopulations for T. vaginalis; however, few molecular epidemiological studies have been performed. We now increased the sensitivity of a previously described multilocus sequence typing (MLST) tool for T. vaginalis by using nested PCR. This enabled the typing of direct patient samples. From January to December 2014, we collected all T. vaginalis positive samples as detected by routine laboratory testing. Samples from patients either came from general practitioners offices or from the sexually transmitted infections (STI) clinic in Amsterdam. Epidemiological data for the STI clinic patients were retrieved from electronic patient files. The primary outcome was the success rate of genotyping direct T. vaginalis positive samples. The secondary outcome was the relation between T. vaginalis genotypes and risk factors for STI. All 7 MLST loci were successfully typed for 71/87 clinical samples. The 71 typed samples came from 69 patients, the majority of whom were women (n=62; 90%) and half (n=34; 49%) were STI clinic patients. Samples segregated into a two population structure for T. vaginalis representing genotypes I and II. Genotype I was most common (n=40; 59.7%). STI clinic patients infected with genotype II reported more sexual partners in the preceding 6 months than patients infected with genotype I (p=0.028). No other associations for gender, age, ethnicity, urogenital discharge or co-occurring STIs with T. vaginalis genotype were found. MLST with nested PCR is a sensitive typing method that allows typing of direct (uncultured) patient material. Genotype II is possibly more prevalent in high-risk sexual networks. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Population Genetics and Antimicrobial Susceptibility of Canine Campylobacter Isolates Collected before and after a Raw Feeding Experiment

PubMed Central

Roine, Johanna; Hänninen, Marja-Liisa; Hielm-Björkman, Anna; Kivistö, Rauni

2015-01-01

In recent years, increasing numbers of consumers have become interested in feeding raw food for their pet dogs as opposed to commercial dry food, in the belief of health advantages. However, raw meat and internal organs, possibly contaminated by pathogens such as Campylobacter spp., may pose a risk of transmission of zoonoses to the pet owners. Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans but C. upsaliensis has also been associated with human disease. In this study we investigated the effect of different feeding strategies on the prevalence of Campylobacter spp. in Finnish dogs. We further characterized the isolates using multilocus sequence typing (MLST), whole-genome (wg) MLST and antimicrobial susceptibility testing. Dogs were sampled before and after a feeding period consisting of commercial raw feed or dry pellet feed. Altogether 56% (20/36) of the dogs yielded at least one Campylobacter-positive fecal sample. C. upsaliensis was the major species detected from 39% of the dogs before and 30% after the feeding period. Two C. jejuni isolates were recovered, both from raw-fed dogs after the dietary regimen. The isolates represented the same genotype (ST-1326), suggesting a common infection source. However, no statistically significant correlation was found between the feeding strategies and Campylobacter spp. carriage. The global genealogy of MLST types of dog and human C. upsaliensis isolates revealed weakly clonal population structure as most STs were widely dispersed. Major antimicrobial resistance among C. upsaliensis isolates was against streptomycin (STR MIC > 4mg/l). Apart from that, all isolates were highly susceptible against the antimicrobials tested. Mutations were found in the genes rpsL or rpsL and rsmG in streptomycin resistant isolates. In conclusion, increasing trend to feed dogs with raw meat warrants more studies to evaluate the risk associated with raw feeding of pets in transmission of zoonoses to humans. PMID:26172151

Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing.

PubMed

Killgore, George; Thompson, Angela; Johnson, Stuart; Brazier, Jon; Kuijper, Ed; Pepin, Jacques; Frost, Eric H; Savelkoul, Paul; Nicholson, Brad; van den Berg, Renate J; Kato, Haru; Sambol, Susan P; Zukowski, Walter; Woods, Christopher; Limbago, Brandi; Gerding, Dale N; McDonald, L Clifford

2008-02-01

Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

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

Diversity and distribution of Listeria monocytogenes in meat processing plants.

PubMed

Martín, Belén; Perich, Adriana; Gómez, Diego; Yangüela, Javier; Rodríguez, Alicia; Garriga, Margarita; Aymerich, Teresa

2014-12-01

Listeria monocytogenes is a major concern for the meat processing industry because many listeriosis outbreaks have been linked to meat product consumption. The aim of this study was to elucidate L. monocytogenes diversity and distribution across different Spanish meat processing plants. L. monocytogenes isolates (N = 106) collected from food contact surfaces of meat processing plants and meat products were serotyped and then characterised by multilocus sequence typing (MLST). The isolates were serotyped as 1/2a (36.8%), 1/2c (34%), 1/2b (17.9%) and 4b (11.3%). MLST identified ST9 as the most predominant allelic profile (33% of isolates) followed by ST121 (16%), both of which were detected from several processing plants and meat products sampled in different years, suggesting that those STs are highly adapted to the meat processing environment. Food contact surfaces during processing were established as an important source of L. monocytogenes in meat products because the same STs were obtained in isolates recovered from surfaces and products. L. monocytogenes was recovered after cleaning and disinfection procedures in two processing plants, highlighting the importance of thorough cleaning and disinfection procedures. Epidemic clone (EC) marker ECI was identified in 8.5%, ECIII was identified in 2.8%, and ECV was identified in 7.5% of the 106 isolates. Furthermore, a selection of presumably unrelated ST9 isolates was analysed by multi-virulence-locus sequence typing (MVLST). Most ST9 isolates had the same virulence type (VT11), confirming the clonal origin of ST9 isolates; however, one ST9 isolate was assigned to a new VT (VT95). Consequently, MLST is a reliable tool for identification of contamination routes and niches in processing plants, and MVLST clearly differentiates EC strains, which both contribute to the improvement of L. monocytogenes control programs in the meat industry. Copyright © 2014 Elsevier Ltd. All rights reserved.

Predominance of Cryptococcus neoformans var. grubii multilocus sequence type 5 and emergence of isolates with non-wild-type minimum inhibitory concentrations to fluconazole: a multi-centre study in China.

PubMed

Fan, X; Xiao, M; Chen, S; Kong, F; Dou, H-T; Wang, H; Xiao, Y-L; Kang, M; Sun, Z-Y; Hu, Z-D; Wan, Z; Chen, S-L; Liao, K; Chu, Y-Z; Hu, T-S; Zou, G-L; Hou, X; Zhang, L; Zhao, Y-P; Xu, Y-C; Liu, Z-Y

2016-10-01

There are few data on the molecular epidemiology of cryptococcosis in China. Here we investigated the species distribution, molecular types and antifungal susceptibilities of 312 Cryptococcus neoformans species complex isolates from ten hospitals over 5 years. Isolates were identified by internal transcribed spacer (ITS) sequencing and by two matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems. Multilocus sequence typing (MLST) was used to verify species/variety and to designate molecular types. Susceptibility to six antifungal drugs was determined by the Sensititre YeastOne™ method. Cryptococcus neoformans was the predominant species (305/312 isolates (97.8%), all were ITS type 1, serotype A), of which 89.2% (272/305) were C. neoformans var. grubii MLST sequence type (ST) 5 and 6.2% (19/305) were ST31. Other C. neoformans var. grubii STs were rare but included six novel STs. Only two strains were C. neoformans var. neoformans (both serotype AD). Cryptococcus gattii was uncommon (n = 7, four ITS types) and comprised five MLST STs including one novel ST. For C. neoformans var. grubii, the proportion of isolates with non-wild-type MICs to fluconazole significantly rose in the fourth study year (from 0% (0/56 isolates) in the first year to 23.9% (17/71) in the fourth year), including five isolates with fluconazole MICs of ≥32 mg/L. The study has provided useful data on the species epidemiology and their genetic diversity and antifungal susceptibility. The proportional increase in isolates with non-wild-type MICs to fluconazole is noted. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Major globally disseminated clonal complexes of antimicrobial resistant enterococci associated with infections in cancer patients in Brazil.

PubMed

Santos, Barbara A; Oliveira, Jéssica S; Cardoso, Nayara T; Barbosa, André V; Superti, Silvana V; Teixeira, Lúcia M; Neves, Felipe P G

2017-11-01

Cancer and hematological malignancies constitute major comorbidities in enterococcal infections, but little is known about the characteristics of enterococci affecting cancer patients. The aim of this study was to characterize 132 enterococcal clinical isolates obtained from cancer patients attending a Cancer Reference Center in Brazil between April 2013 and March 2014. Susceptibility to 17 antimicrobial agents was assessed by disk diffusion method. Resistance and virulence genes were investigated by PCR. Multilocus sequence typing (MLST) was performed for selected Enterococcus faecalis and Enterococcus faecium isolates. The predominant species was E. faecalis (108 isolates), followed by E. faecium (18), Enterococcus gallinarum (3), Enterococcus avium (2) and Enterococcus durans (1). Multidrug-resistant (MDR) isolates made up 44.7%, but all isolates were susceptible to fosfomycin, linezolid and glycopeptides. The most prevalent genes associated with erythromycin- and tetracycline-non susceptible isolates were erm(B) (47/71; 66.2%) and tet(M) (24/68; 35.3%), respectively. High-level resistance (HLR) to gentamicin was found in 22 (16.7%) isolates and 13 (59.1%) of them carried the aac(6')-Ie-aph(2″)-Ia gene. HLR to streptomycin was detected in 34 (25.8%) isolates, of which 15 (44.1%) isolates had the ant(6')-Ia gene. The most common virulence genes were gelE (48.9%), esp (30.5%) and asa1 (29.8%). MLST performed for 26 E. faecalis isolates revealed 18 different sequence-types (STs), with seven corresponding to novel STs (625, 626, 627, 628, 629, 630, and 635). On the other hand, nine of 10 E. faecium isolates analyzed by MLST belonged to a single clonal complex, comprised of mostly ST412, which emerged worldwide after mid-2000s, but also two novel STs (963 and 964). We detected major globally disseminated E. faecalis and E. faecium clonal complexes along with novel closely related STs, indicating the fitness and continuous evolution of these hospital-adapted lineages. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic Relatedness and Novel Sequence Types of Non-O157 Shiga Toxin-Producing Escherichia coli Strains Isolated in Argentina.

PubMed

Cadona, Jimena S; Bustamante, Ana V; González, Juliana; Sanso, A Mariel

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen responsible for severe disease in humans such as hemolytic uremic syndrome (HUS) and cattle, the principal reservoir. Identification of the clones/lineages is important as several characteristics, among them propensity to cause disease varies with STEC phylogenetic origin. At present, we do not know what STEC clones, especially of non-O157:H7, are circulating in Argentina. To fill this knowledge gap we assessed the genetic diversity of STEC strains isolated in Argentina from various sources, mostly cattle and food, using multilocus sequence typing (MLST). Our objectives were to determine the phylogenetic relationships among strains and to compare them with strains from different geographic origins, especially with those from clinical human cases, in order to evaluate their potential health risk. A total of 59 STEC isolates from 41 serotypes were characterized by MLST. Analysis using EcMLST database identified 38 sequence types (ST), 17 (45%) of which were new STs detected in 18 serotypes. Fifteen out of 38 STs identified were grouped into 11 clonal groups (CGs) and, 23 not grouped in any of the defined CGs. Different STs were found in the same serotype. Results highlighted a high degree of phylogenetic heterogeneity among Argentinean strains and they showed that several cattle and food isolates belonged to the same STs that are commonly associated with clinical human cases in several geographical areas. STEC is a significant public health concern. Argentina has the highest incidence of HUS in the world and this study provides the first data about which STEC clones are circulating. Data showed that most of them might pose a serious zoonotic risk and this information is important for developing public health initiatives. However, the actual potential risk will be defined by the virulence profiles, which may differ among isolates belonging to the same ST.

Global distribution and diversity of ovine-associated Staphylococcus aureus.

PubMed

Smith, Edward M; Needs, Polly F; Manley, Grace; Green, Laura E

2014-03-01

Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n=153), Africa (n=28), South America (n=14) and Australia (n=1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Genetic algorithms with memory- and elitism-based immigrants in dynamic environments.

PubMed

Yang, Shengxiang

2008-01-01

In recent years the genetic algorithm community has shown a growing interest in studying dynamic optimization problems. Several approaches have been devised. The random immigrants and memory schemes are two major ones. The random immigrants scheme addresses dynamic environments by maintaining the population diversity while the memory scheme aims to adapt genetic algorithms quickly to new environments by reusing historical information. This paper investigates a hybrid memory and random immigrants scheme, called memory-based immigrants, and a hybrid elitism and random immigrants scheme, called elitism-based immigrants, for genetic algorithms in dynamic environments. In these schemes, the best individual from memory or the elite from the previous generation is retrieved as the base to create immigrants into the population by mutation. This way, not only can diversity be maintained but it is done more efficiently to adapt genetic algorithms to the current environment. Based on a series of systematically constructed dynamic problems, experiments are carried out to compare genetic algorithms with the memory-based and elitism-based immigrants schemes against genetic algorithms with traditional memory and random immigrants schemes and a hybrid memory and multi-population scheme. The sensitivity analysis regarding some key parameters is also carried out. Experimental results show that the memory-based and elitism-based immigrants schemes efficiently improve the performance of genetic algorithms in dynamic environments.

A Decade of Invasive Meningococcal Disease Surveillance in Poland

PubMed Central

Skoczyńska, Anna; Waśko, Izabela; Kuch, Alicja; Kadłubowski, Marcin; Gołębiewska, Agnieszka; Foryś, Małgorzata; Markowska, Marlena; Ronkiewicz, Patrycja; Wasiak, Katarzyna; Kozińska, Aleksandra; Matynia, Bożena; Hryniewicz, Waleria

2013-01-01

Background Neisseria meningitidis is a leading etiologic agent of severe invasive disease. The objective of the study was to characterise invasive meningococcal disease (IMD) epidemiology in Poland during the last decade, based on laboratory confirmed cases. Methods The study encompassed all invasive meningococci collected between 2002 and 2011 in the National Reference Centre for Bacterial Meningitis. The isolates were re-identified and characterised by susceptibility testing, MLST analysis, porA and fetA sequencing. A PCR technique was used for meningococcal identification directly from clinical materials. Results In the period studied, 1936 cases of IMD were confirmed, including 75.6% identified by culture. Seven IMD outbreaks, affecting mostly adolescents, were reported; all were caused by serogroup C meningococci of ST-11. The highest incidence was observed among children under one year of age (15.71/100,000 in 2011). The general case fatality rate in the years 2010–2011 was 10.0%. Meningococci of serogroup B, C, Y and W-135 were responsible for 48.8%, 36.6%, 1.2% and 1.2% of cases, respectively. All isolates were susceptible to third generation cephalosporins, chloramphenicol, ciprofloxacin, and 84.2% were susceptible to penicillin. MLST analysis (2009–2011) revealed that among serogroup B isolates the most represented were clonal complexes (CC) ST-32CC, ST-18CC, ST-41/44CC, ST-213CC and ST-269CC, and among serogroup C: ST-103CC, ST-41/44CC and ST-11CC. Conclusions The detection of IMD in Poland has changed over time, but observed increase in the incidence of the disease was mostly attributed to changes in the surveillance system including an expanded case definition and inclusion of data from non-culture diagnostics. PMID:23977184

Direct Repeat Unit (dru) Typing of Methicillin-Resistant Staphylococcus pseudintermedius from Dogs and Cats.

PubMed

Kadlec, Kristina; Schwarz, Stefan; Goering, Richard V; Weese, J Scott

2015-12-01

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged in a remarkable manner as an important problem in dogs and cats. However, limited molecular epidemiological information is available. The aims of this study were to apply direct repeat unit (dru) typing in a large collection of well-characterized MRSP isolates and to use dru typing to analyze a collection of previously uncharacterized MRSP isolates. Two collections of MRSP isolates from dogs and cats were included in this study. The first collection comprised 115 well-characterized MRSP isolates from North America and Europe. The data for these isolates included multilocus sequence typing (MLST) and staphylococcal protein A gene (spa) typing results as well as SmaI macrorestriction patterns after pulsed-field gel electrophoresis (PFGE). The second collection was a convenience sample of 360 isolates from North America. The dru region was amplified by PCR, sequenced, and analyzed. For the first collection, the discriminatory indices of the typing methods were calculated. All isolates were successfully dru typed. The discriminatory power for dru typing (D = 0.423) was comparable to that of spa typing (D = 0.445) and of MLST (D = 0.417) in the first collection. Occasionally, dru typing was able to further discriminate between isolates that shared the same spa type. Among all 475 isolates, 26 different dru types were identified, with 2 predominant types (dt9a and dt11a) among 349 (73.4%) isolates. The results of this study underline that dru typing is a useful tool for MRSP typing, being an objective, standardized, sequence-based method that is relatively cost-efficient and easy to perform. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A reliable combination method to identification and typing of epidemic and endemic clones among clinical isolates of Acinetobacter baumannii.

PubMed

Piran, Arezoo; Shahcheraghi, Fereshteh; Solgi, Hamid; Rohani, Mahdi; Badmasti, Farzad

2017-10-01

The multi-drug resistant (MDR) Acinetobacter baumannii as an important nosocomial pathogen has emerged a global health concern in recent years. In this study, we applied three easier, faster, and cost-effective methods including PCR-based open reading frames (ORFs) typing, sequence typing of bla OXA-51-like and RAPD-PCR method to rapid typing of A. baumannii strains. Taken together in the present study the results of ORFs typing, PCR-sequencing of bla OXA-51-like genes and MLST sequence typing revealed there was a high prevalence (62%, 35/57) of ST2 as international and successful clone which detected among clinical isolates of multi-drug resistant A. baumannii with ORF pattern B and bla OXA-66 gene. Only 7% (4/57) of MDR isolates belonged to ST1 with ORF pattern A and bla OXA-69 gene. Interestingly, we detected singleton ST513 (32%, 18/57) that encoded bla OXA-90 and showed the ORF pattern H as previously isolated in Middle East. Moreover, our data showed RAPD-PCR method can detect divergent strains of the STs. The Cl-1, Cl-2, Cl-3, Cl-4, Cl-10, Cl-11, Cl-12, Cl-13 and Cl-14 belonged to ST2. While the Cl-6, Cl-7, Cl-8 and Cl-9 belonged to ST513. Only Cl-5 belonged to ST1. It seems that the combination of these methods have more discriminatory than any method separately and could be effectively applied to rapid detection of the clonal complex (CC) of A. baumannii strains without performing of MLST or PFGE. Copyright © 2017 Elsevier B.V. All rights reserved.

[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.

Clonal spread of carbapenem-resistant Acinetobacter baumannii across a community hospital and its affiliated long-term care facilities: A cross sectional study.

PubMed

Chen, Chang-Hua; Kuo, Han-Yueh; Hsu, Po-Jui; Chang, Chien-Min; Chen, Jiann-Yuan; Lu, Henry Horng-Shing; Chen, Hsin-Yao; Liou, Ming-Li

2018-06-01

The global spread of carbapenem-resistant Acinetobacter baumannii (CRAB) is now a public health problem. In Taiwan, the relationship of the CRAB circulation between long-term care facilities (LTCFs) and acute care hospitals remains unclear. Here, we use molecular epidemiologic methods to describe the transmission of CRAB isolates between a community hospital and its affiliated LTCFs. Subjects localized in eight LTCFs who were not admitted acute care hospitals in recent a year were enrolled in this study. CRAB isolates were collected during June 1, 2015 and December 31, 2015. DNA fingerprinting was performed by repetitive extragenic palindromic sequence-based polymerase chain reaction (Rep-PCR) and multilocus sequence typing (MLST). Multiplex-PCR amplification for the detection of bla OXA genes and beta-lactamase genes was performed. Twenty one subjects were enrolled. The major hospital admission diagnoses among the 21 subjects were pneumonia (71.4%). Genotyping of CRAB isolates by Rep-PCR revealed that a major clone, designated as type III, comprised fifteen of 21 (71.4%) isolates taken from 5 LTCFs and one study hospital. The isolates with type III were subtyped by PubMLST into 4 ST types. The most prevalent bla OXA genes in these isolates were bla OXA-23 -like (85.70%, 18/21). Twenty isolates carried bla SHV. CONCLUSION: Clonal spread of bla OxA-23 -carrying CRABs was found around LTCFs and the affiliated hospital. In Taiwan, it is important for the government to focus attention on the importance of identifying and tracing CRAB infections in LTCFs. Copyright © 2017. Published by Elsevier B.V.

Clostridium difficile colonization and disease in patients undergoing hematopoietic stem cell transplantation.

PubMed

Bruminhent, Jackrapong; Wang, Zi-Xuan; Hu, Carol; Wagner, John; Sunday, Richard; Bobik, Brent; Hegarty, Sarah; Keith, Scott; Alpdogan, Seyfettin; Carabasi, Matthew; Filicko-O'Hara, Joanne; Flomenberg, Neal; Kasner, Margaret; Outschoorn, Ubaldo Martinez; Weiss, Mark; Flomenberg, Phyllis

2014-09-01

There was an increase in the Clostridium difficile infection (CDI) rate in our bone marrow transplantation unit. To evaluate the role of unit-based transmission, C. difficile screening was performed on adult patients admitted for hematopoietic stem cell transplantation (HSCT) over a 2-year period, and C. difficile isolates were typed. C. difficile testing was performed using a 2-step C. difficile glutamate dehydrogenase antigen plus toxin A/B enzyme immunoassay (EIA) and cytotoxin assay (or molecular toxin assay). Multilocus sequence typing (MLST) was performed on toxin-positive whole stool samples. A retrospective chart review was performed on all patients with a positive toxin assay. Sixteen of 150 patients (10.7%) had toxigenic C. difficile colonization (CDC) on admission. The overall incidence of CDI within 100 days after HSCT was 24.7% (37 of 150). The median time to diagnosis of CDI was 3.5 days after HSCT. In an adjusted logistic regression model, CDC on admission was a significant risk factor for CDI (odds ratio, 68.5; 95% confidence interval, 11.4 to 416.2). MLST on 22 unit patient toxin-positive stool specimens revealed 15 distinct strain types. Further analysis identified at least 1 potential cross-transmission event; some events may have been missed because of incomplete typing from other specimens. Despite aggressive infection control interventions, there was no decline in the number of CDI cases during the study period. These data suggest that prior CDC plays a major role in CDI rates in this high-risk patient population. It remains unclear if CDI was cross-transmitted in the unit. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

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.

Anthropogenic antibiotic resistance genes mobilization to the polar regions.

PubMed

Hernández, Jorge; González-Acuña, Daniel

2016-01-01

Anthropogenic influences in the southern polar region have been rare, but lately microorganisms associated with humans have reached Antarctica, possibly from military bases, fishing boats, scientific expeditions, and/or ship-borne tourism. Studies of seawater in areas of human intervention and proximal to fresh penguin feces revealed the presence of Escherichia coli strains least resistant to antibiotics in penguins, whereas E. coli from seawater elsewhere showed resistance to one or more of the following antibiotics: ampicillin, tetracycline, streptomycin, and trim-sulfa. In seawater samples, bacteria were found carrying extended-spectrum β-lactamase (ESBL)-type CTX-M genes in which multilocus sequencing typing (MLST) showed different sequence types (STs), previously reported in humans. In the Arctic, on the contrary, people have been present for a long time, and the presence of antibiotic resistance genes (ARGs) appears to be much more wide-spread than was previously reported. Studies of E coli from Arctic birds (Bering Strait) revealed reduced susceptibility to antibiotics, but one globally spreading clone of E. coli genotype O25b-ST131, carrying genes of ESBL-type CTX-M, was identified. In the few years between sample collections in the same area, differences in resistance pattern were observed, with E. coli from birds showing resistance to a maximum of five different antibiotics. Presence of resistance-type ESBLs (TEM, SHV, and CTX-M) in E. coli and Klebsiella pneumoniae was also confirmed by specified PCR methods. MLST revealed that those bacteria carried STs that connect them to previously described strains in humans. In conclusion, bacteria previously related to humans could be found in relatively pristine environments, and presently human-associated, antibiotic-resistant bacteria have reached a high global level of distribution that they are now found even in the polar regions.

Clinical, Bacteriologic, and Geographic Stratification of Melioidosis Emerges from the Sri Lankan National Surveillance Program.

PubMed

Sathkumara, Harindra D; Merritt, Adam J; Corea, Enoka M; Krishnananthasivam, Shivankari; Natesan, Mohan; Inglis, Timothy J J; De Silva, Aruna Dharshan

2018-02-01

Melioidosis, a potentially fatal tropical infection, is said to be underdiagnosed in low-income countries. An increase in melioidosis cases in Sri Lanka allowed us to analyze the relationship among clinical outcome, bacteriology, epidemiology, and geography in the first 108 laboratory-confirmed cases of melioidosis from a nationwide surveillance program. The additional 76 cases of laboratory-confirmed melioidosis confirmed further associations between Burkholderia pseudomallei multilocus sequence typing (MLST) and infection phenotype; ST1137/unifocal bacteremic infection (χ 2 = 3.86, P < 0.05), ST1136/multifocal infection without bacteremia (χ 2 = 15.8, P < 0.001), and ST1132/unifocal nonbacteremic infection (χ 2 = 6.34, P = 0.02). ST1137 infections were predominantly seen in the Western Province, whereas ST1132, 1135, and 1136 infections predominated in the Northwestern Province. Early participating centers in the surveillance program had a lower melioidosis-associated mortality than later participants (χ 2 = 3.99, P < 0.05). The based upon related sequence types (eBURST) algorithm, a MLST clustering method that infers founding genotypes and patterns of descent for related isolates and clonal complexes in an unrooted tree, showed uneven distribution of sequence types (STs). There was spatial clustering of the commonest STs (ST1132, 1136, and 1137) in the Western, Northwestern, and Central provinces. The recent increase in melioidosis in Sri Lanka uncovered by laboratory-enhanced surveillance is likely to be the result of a combination of improved laboratory detection, increased clinician awareness, recruitment of clinical centers, and small outbreaks. Further development of the surveillance program into a national genotyping-supported melioidosis registry will improve melioidosis diagnosis, treatment, and prevention where underdiagnosis and mortality rates remain high.

Performance of a PCR assay for the rapid identification of the Klebsiella pneumoniae ST258 epidemic clone in Latin American clinical isolates.

PubMed

Gomez, S A; Rapoport, M; Piergrossi, N; Faccone, D; Pasteran, F; De Belder, D; ReLAVRA-Group; Petroni, A; Corso, A

2016-10-01

The worldwide dissemination of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae ST258 demands a rapid PCR-based typing method to detect unique genes of the ST258 clone. This study evaluates a PCR developed by Adler et al. (2014) for the detection of ST258 in K. pneumoniae clinical isolates centered on the identification of the pilv-I and prp genes. We tested 143 clinical isolates from Argentina (n=109), Chile (n=1), Colombia (n=1), Costa Rica (n=2), Ecuador (n=5), El Salvador (n=2), Nicaragua (n=5), Panamá (n=2), Paraguay (n=2), Perú (n=3) and Trinidad and Tobago (n=11) recovered from 2006 to 2015. blaKPC, pilv-l and prp genes were detected by PCR and sequenced by standard procedures. ST258 and non-ST258 were defined by PFGE and/or MLST. Isolates were grouped according to PFGE patterns: 58 were compatible with ST258 (group 1) and 85 with non-ST258 (group 2). MLST study was done on an arbitrary selection of isolates. The pilv-l gene was present only in ST258 isolates, regardless of the presence of the blaKPC gene. Results for the prp gene were variable. Its presence did not define ST258. The pilv-I PCR had a sensitivity and specificity of 100%, respectively, for the detection of ST258 in the isolates under investigation. Given our findings, the pilv-I PCR could replace more time and resource consuming methods, allowing for more rapid detection of the circulating high risk K. pneumoniae clone ST258 in Latin American (LA) countries. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular and comparative analysis of Salmonella enterica Senftenberg from humans and animals using PFGE, MLST and NARMS.

PubMed

Stepan, Ryan M; Sherwood, Julie S; Petermann, Shana R; Logue, Catherine M

2011-06-27

Salmonella species are recognized worldwide as a significant cause of human and animal disease. In this study the molecular profiles and characteristics of Salmonella enterica Senftenberg isolated from human cases of illness and those recovered from healthy or diagnostic cases in animals were assessed. Included in the study was a comparison with our own sequenced strain of S. Senfteberg recovered from production turkeys in North Dakota. Isolates examined in this study were subjected to antimicrobial susceptibility profiling using the National Antimicrobial Resistance Monitoring System (NARMS) panel which tested susceptibility to 15 different antimicrobial agents. The molecular profiles of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) and the sequence types of the strains were obtained using Multi-Locus Sequence Type (MLST) analysis based on amplification and sequence interrogation of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA). PFGE data was input into BioNumerics analysis software to generate a dendrogram of relatedness among the strains. The study found 93 profiles among 98 S. Senftenberg isolates tested and there were primarily two sequence types associated with humans and animals (ST185 and ST14) with overlap observed in all host types suggesting that the distribution of S. Senftenberg sequence types is not host dependent. Antimicrobial resistance was observed among the animal strains, however no resistance was detected in human isolates suggesting that animal husbandry has a significant influence on the selection and promotion of antimicrobial resistance. The data demonstrates the circulation of at least two strain types in both animal and human health suggesting that S. Senftenberg is relatively homogeneous in its distribution. The data generated in this study could be used towards defining a pathotype for this serovar.

Genetic Structure, Linkage Disequilibrium and Signature of Selection in Sorghum: Lessons from Physically Anchored DArT Markers

PubMed Central

Bouchet, Sophie; Pot, David; Deu, Monique; Rami, Jean-François; Billot, Claire; Perrier, Xavier; Rivallan, Ronan; Gardes, Laëtitia; Xia, Ling; Wenzl, Peter; Kilian, Andrzej; Glaszmann, Jean-Christophe

2012-01-01

Population structure, extent of linkage disequilibrium (LD) as well as signatures of selection were investigated in sorghum using a core sample representative of worldwide diversity. A total of 177 accessions were genotyped with 1122 informative physically anchored DArT markers. The properties of DArTs to describe sorghum genetic structure were compared to those of SSRs and of previously published RFLP markers. Model-based (STRUCTURE software) and Neighbor-Joining diversity analyses led to the identification of 6 groups and confirmed previous evolutionary hypotheses. Results were globally consistent between the different marker systems. However, DArTs appeared more robust in terms of data resolution and bayesian group assignment. Whole genome linkage disequilibrium as measured by mean r2 decreased from 0.18 (between 0 to 10 kb) to 0.03 (between 100 kb to 1 Mb), stabilizing at 0.03 after 1 Mb. Effects on LD estimations of sample size and genetic structure were tested using i. random sampling, ii. the Maximum Length SubTree algorithm (MLST), and iii. structure groups. Optimizing population composition by the MLST reduced the biases in small samples and seemed to be an efficient way of selecting samples to make the best use of LD as a genome mapping approach in structured populations. These results also suggested that more than 100,000 markers may be required to perform genome-wide association studies in collections covering worldwide sorghum diversity. Analysis of DArT markers differentiation between the identified genetic groups pointed out outlier loci potentially linked to genes controlling traits of interest, including disease resistance genes for which evidence of selection had already been reported. In addition, evidence of selection near a homologous locus of FAR1 concurred with sorghum phenotypic diversity for sensitivity to photoperiod. PMID:22428056

Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Cronobacter sakazakii Isolates from Powdered Infant Formula Collected from Chinese Retail Markets

PubMed Central

Fei, Peng; Jiang, Yichao; Jiang, Yan; Yuan, Xiujuan; Yang, Tongxiang; Chen, Junliang; Wang, Ziyuan; Kang, Huaibin; Forsythe, Stephen J.

2017-01-01

Cronobacter sakazakii is an opportunistic pathogen that causes severe infections in neonates and infants through contaminated powdered infant formula (PIF). Therefore, the aim of this study was a large-scale study on determine the prevalence, molecular characterization and antibiotic susceptibility of C. sakazakii isolates from PIF purchased from Chinese retail markets. Two thousand and twenty PIF samples were collected from different institutions. Fifty-six C. sakazakii strains were isolated, and identified using fusA sequencing analysis, giving a contamination rate of 2.8%. Multilocus sequence typing (MLST) was more discriminatory than other genotyping methods. The C. sakazakii isolates were divided into 14 sequence types (STs) by MLST, compared with only seven clusters by ompA and rpoB sequence analysis, and four C. sakazakii serotypes by PCR-based O-antigen serotyping. C. sakazakii ST4 (19/56, 33.9%), ST1 (12/56, 21.4%), and ST64 (11/56, 16.1%) were the dominant sequence types isolated. C. sakazakii serotype O2 (34/56, 60.7%) was the primary serotype, along with ompA6 and rpoB1 as the main allele profiles, respectively. Antibiotic susceptibility testing indicated that all C. sakazakii isolates were susceptible to ampicillin-sulbactam, cefotaxime, ciprofloxacin, meropenem, tetracycline, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole. The majority of C. sakazakii strains were susceptible to chloramphenicol and gentamicin (87.5 and 92.9%, respectively). In contrast, 55.4% C. sakazakii strains were resistant to cephalothin. In conclusion, this large-scale study revealed the prevalence and characteristics of C. sakazakii from PIF in Chinese retail markets, demonstrating a potential risk for neonates and infants, and provide a guided to effective control the contamination of C. sakazakii in production process. PMID:29089940

Molecular characterisation of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from hospital and ambulatory patients in Germany.

PubMed

Pietsch, Michael; Eller, Christoph; Wendt, Constanze; Holfelder, Martin; Falgenhauer, Linda; Fruth, Angelika; Grössl, Tobias; Leistner, Rasmus; Valenza, Giuseppe; Werner, Guido; Pfeifer, Yvonne

2017-02-01

The increase of Escherichia coli producing extended-spectrum β-lactamases (ESBL) in hospitals and their emergence as intestinal colonisers of healthy humans is of concern. Transmission ways and the extent of spread of distinct E. coli clones or ESBL genes among humans and animals via the food chain or the environment is a matter of debate. In this study we determined ESBL genotypes in E. coli isolates (n=233) resistant to 3rd generation cephalosporins from hospitals and medical practices using PCR and sequencing. Bacterial strain typing was performed by PCR-based phylogrouping, multilocus sequence typing (MLST) and a ST131-specific PCR. Results showed that CTX-M-15 (50.4%), CTX-M-1 (28.4%) and CTX-M-14 (5.6%) were the most common ESBL types. Especially, CTX-M-15 was associated with E. coli ST131 of phylogenetic group B2, which was the dominant sequence type among our isolates (35.8%). MLST typing revealed 40 different sequence types (STs), with ST131, ST410, ST10 and ST38 as the most prevalent ones. Our findings give an overview of the current distribution of ESBL-producing E. coli isolates from humans in Germany. E. coli O25b:H4-ST131 was confirmed to be the most common clone, which is known for its successful dissemination worldwide. Although heterogeneity among the isolates was found, several successful clones previously described in animals (ST410, ST10) also occurred in our isolate collection. Further detailed investigations of ESBL-producing isolates from different habitats are needed to evaluate possible transfer ways. Copyright © 2015 Elsevier B.V. All rights reserved.

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China

PubMed Central

Fei, Peng; Man, Chaoxin; Lou, Binbin; Forsythe, Stephen J.; Chai, Yunlei; Li, Ran; Niu, Jieting

2015-01-01

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product. PMID:26048942

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China.

PubMed

Fei, Peng; Man, Chaoxin; Lou, Binbin; Forsythe, Stephen J; Chai, Yunlei; Li, Ran; Niu, Jieting; Jiang, Yujun

2015-08-15

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Homologous Recombination and Xylella fastidiosa Host-Pathogen Associations in South America.

PubMed

Coletta-Filho, Helvécio D; Francisco, Carolina S; Lopes, João R S; Muller, Christiane; Almeida, Rodrigo P P

2017-03-01

Homologous recombination affects the evolution of bacteria such as Xylella fastidiosa, a naturally competent plant pathogen that requires insect vectors for dispersal. This bacterial species is taxonomically divided into subspecies, with phylogenetic clusters within subspecies that are host specific. One subspecies, pauca, is primarily limited to South America, with the exception of recently reported strains in Europe and Costa Rica. Despite the economic importance of X. fastidiosa subsp. pauca in South America, little is known about its genetic diversity. Multilocus sequence typing (MLST) has previously identified six sequence types (ST) among plant samples collected in Brazil (both subsp. pauca and multiplex). Here, we report on a survey of X. fastidiosa genetic diversity (MLST based) performed in six regions in Brazil and two in Argentina, by sampling five different plant species. In addition to the six previously reported ST, seven new subsp. pauca and two new subsp. multiplex ST were identified. The presence of subsp. multiplex in South America is considered to be the consequence of a single introduction from its native range in North America more than 80 years ago. Different phylogenetic approaches clustered the South American ST into four groups, with strains infecting citrus (subsp. pauca); coffee and olive (subsp. pauca); coffee, hibiscus, and plum (subsp. pauca); and plum (subsp. multiplex). In areas where these different genetic clusters occurred sympatrically, we found evidence of homologous recombination in the form of bidirectional allelic exchange between subspp. pauca and multiplex. In fact, the only strain of subsp. pauca isolated from a plum host had an allele that originated from subsp. multiplex. These signatures of bidirectional homologous recombination between endemic and introduced ST indicate that gene flow occurs in short evolutionary time frames in X. fastidiosa, despite the ecological isolation (i.e., host plant species) of genotypes.

"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.

Genetic structure, linkage disequilibrium and signature of selection in Sorghum: lessons from physically anchored DArT markers.

PubMed

Bouchet, Sophie; Pot, David; Deu, Monique; Rami, Jean-François; Billot, Claire; Perrier, Xavier; Rivallan, Ronan; Gardes, Laëtitia; Xia, Ling; Wenzl, Peter; Kilian, Andrzej; Glaszmann, Jean-Christophe

2012-01-01

Population structure, extent of linkage disequilibrium (LD) as well as signatures of selection were investigated in sorghum using a core sample representative of worldwide diversity. A total of 177 accessions were genotyped with 1122 informative physically anchored DArT markers. The properties of DArTs to describe sorghum genetic structure were compared to those of SSRs and of previously published RFLP markers. Model-based (STRUCTURE software) and Neighbor-Joining diversity analyses led to the identification of 6 groups and confirmed previous evolutionary hypotheses. Results were globally consistent between the different marker systems. However, DArTs appeared more robust in terms of data resolution and bayesian group assignment. Whole genome linkage disequilibrium as measured by mean r(2) decreased from 0.18 (between 0 to 10 kb) to 0.03 (between 100 kb to 1 Mb), stabilizing at 0.03 after 1 Mb. Effects on LD estimations of sample size and genetic structure were tested using i. random sampling, ii. the Maximum Length SubTree algorithm (MLST), and iii. structure groups. Optimizing population composition by the MLST reduced the biases in small samples and seemed to be an efficient way of selecting samples to make the best use of LD as a genome mapping approach in structured populations. These results also suggested that more than 100,000 markers may be required to perform genome-wide association studies in collections covering worldwide sorghum diversity. Analysis of DArT markers differentiation between the identified genetic groups pointed out outlier loci potentially linked to genes controlling traits of interest, including disease resistance genes for which evidence of selection had already been reported. In addition, evidence of selection near a homologous locus of FAR1 concurred with sorghum phenotypic diversity for sensitivity to photoperiod.

Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age

PubMed Central

Vinueza-Burgos, Christian; Wautier, Magali; Martiny, Delphine; Cisneros, Marco; Van Damme, Inge; De Zutter, Lieven

2017-01-01

Abstract Thermotolerant Campylobacter spp. are a major cause of foodborne gastrointestinal infections worldwide. The linkage of human campylobacteriosis and poultry has been widely described. In this study we aimed to investigate the prevalence, antimicrobial resistance and genetic diversity of C. coli and C. jejuni in broilers from Ecuador. Caecal content from 379 randomly selected broiler batches originating from 115 farms were collected from 6 slaughterhouses located in the province of Pichincha during 1 year. Microbiological isolation was performed by direct plating on mCCDA agar. Identification of Campylobacter species was done by PCR. Minimum inhibitory concentration (MIC) values for gentamicin, ciprofloxacin, nalidixic acid, tetracycline, streptomycin, and erythromycin were obtained. Genetic variation was assessed by RFLP-flaA typing and Multilocus Sequence Typing (MLST) of selected isolates. Prevalence at batch level was 64.1%. Of the positive batches 68.7% were positive for C. coli, 18.9% for C. jejuni, and 12.4% for C. coli and C. jejuni. Resistance rates above 67% were shown for tetracycline, ciprofloxacin, and nalidixic acid. The resistance pattern tetracycline, ciprofloxin, and nalidixic acid was the dominant one in both Campylobacter species. RFLP-flaA typing analysis showed that C. coli and C. jejuni strains belonged to 38 and 26 profiles respectively. On the other hand MLST typing revealed that C. coli except one strain belonged to CC-828, while C. jejuni except 2 strains belonged to 12 assigned clonal complexes (CCs). Furthermore 4 new sequence types (STs) for both species were described, whereby 2 new STs for C. coli were based on new allele sequences. Further research is necessary to estimate the impact of the slaughter of Campylobacter positive broiler batches on the contamination level of carcasses in slaughterhouses and at retail in Ecuador. PMID:28339716

Comparison of the co-gasification of sewage sludge and food wastes and cost-benefit analysis of gasification- and incineration-based waste treatment schemes.

PubMed

You, Siming; Wang, Wei; Dai, Yanjun; Tong, Yen Wah; Wang, Chi-Hwa

2016-10-01

The compositions of food wastes and their co-gasification producer gas were compared with the existing data of sewage sludge. Results showed that food wastes are more favorable than sewage sludge for co-gasification based on residue generation and energy output. Two decentralized gasification-based schemes were proposed to dispose of the sewage sludge and food wastes in Singapore. Monte Carlo simulation-based cost-benefit analysis was conducted to compare the proposed schemes with the existing incineration-based scheme. It was found that the gasification-based schemes are financially superior to the incineration-based scheme based on the data of net present value (NPV), benefit-cost ratio (BCR), and internal rate of return (IRR). Sensitivity analysis was conducted to suggest effective measures to improve the economics of the schemes. Copyright © 2016 Elsevier Ltd. All rights reserved.

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...

PMD compensation in multilevel coded-modulation schemes with coherent detection using BLAST algorithm and iterative polarization cancellation.

PubMed

Djordjevic, Ivan B; Xu, Lei; Wang, Ting

2008-09-15

We present two PMD compensation schemes suitable for use in multilevel (M>or=2) block-coded modulation schemes with coherent detection. The first scheme is based on a BLAST-type polarization-interference cancellation scheme, and the second scheme is based on iterative polarization cancellation. Both schemes use the LDPC codes as channel codes. The proposed PMD compensations schemes are evaluated by employing coded-OFDM and coherent detection. When used in combination with girth-10 LDPC codes those schemes outperform polarization-time coding based OFDM by 1 dB at BER of 10(-9), and provide two times higher spectral efficiency. The proposed schemes perform comparable and are able to compensate even 1200 ps of differential group delay with negligible penalty.

Report on Pairing-based Cryptography.

PubMed

Moody, Dustin; Peralta, Rene; Perlner, Ray; Regenscheid, Andrew; Roginsky, Allen; Chen, Lily

2015-01-01

This report summarizes study results on pairing-based cryptography. The main purpose of the study is to form NIST's position on standardizing and recommending pairing-based cryptography schemes currently published in research literature and standardized in other standard bodies. The report reviews the mathematical background of pairings. This includes topics such as pairing-friendly elliptic curves and how to compute various pairings. It includes a brief introduction to existing identity-based encryption (IBE) schemes and other cryptographic schemes using pairing technology. The report provides a complete study of the current status of standard activities on pairing-based cryptographic schemes. It explores different application scenarios for pairing-based cryptography schemes. As an important aspect of adopting pairing-based schemes, the report also considers the challenges inherent in validation testing of cryptographic algorithms and modules. Based on the study, the report suggests an approach for including pairing-based cryptography schemes in the NIST cryptographic toolkit. The report also outlines several questions that will require further study if this approach is followed.

Report on Pairing-based Cryptography

PubMed Central

Moody, Dustin; Peralta, Rene; Perlner, Ray; Regenscheid, Andrew; Roginsky, Allen; Chen, Lily

2015-01-01

This report summarizes study results on pairing-based cryptography. The main purpose of the study is to form NIST’s position on standardizing and recommending pairing-based cryptography schemes currently published in research literature and standardized in other standard bodies. The report reviews the mathematical background of pairings. This includes topics such as pairing-friendly elliptic curves and how to compute various pairings. It includes a brief introduction to existing identity-based encryption (IBE) schemes and other cryptographic schemes using pairing technology. The report provides a complete study of the current status of standard activities on pairing-based cryptographic schemes. It explores different application scenarios for pairing-based cryptography schemes. As an important aspect of adopting pairing-based schemes, the report also considers the challenges inherent in validation testing of cryptographic algorithms and modules. Based on the study, the report suggests an approach for including pairing-based cryptography schemes in the NIST cryptographic toolkit. The report also outlines several questions that will require further study if this approach is followed. PMID:26958435

A secure smart-card based authentication and key agreement scheme for telecare medicine information systems.

PubMed

Lee, Tian-Fu; Liu, Chuan-Ming

2013-06-01

A smart-card based authentication scheme for telecare medicine information systems enables patients, doctors, nurses, health visitors and the medicine information systems to establish a secure communication platform through public networks. Zhu recently presented an improved authentication scheme in order to solve the weakness of the authentication scheme of Wei et al., where the off-line password guessing attacks cannot be resisted. This investigation indicates that the improved scheme of Zhu has some faults such that the authentication scheme cannot execute correctly and is vulnerable to the attack of parallel sessions. Additionally, an enhanced authentication scheme based on the scheme of Zhu is proposed. The enhanced scheme not only avoids the weakness in the original scheme, but also provides users' anonymity and authenticated key agreements for secure data communications.

Cryptanalysis and Improvement of a Biometric-Based Multi-Server Authentication and Key Agreement Scheme.

PubMed

Wang, Chengqi; Zhang, Xiao; Zheng, Zhiming

2016-01-01

With the security requirements of networks, biometrics authenticated schemes which are applied in the multi-server environment come to be more crucial and widely deployed. In this paper, we propose a novel biometric-based multi-server authentication and key agreement scheme which is based on the cryptanalysis of Mishra et al.'s scheme. The informal and formal security analysis of our scheme are given, which demonstrate that our scheme satisfies the desirable security requirements. The presented scheme provides a variety of significant functionalities, in which some features are not considered in the most of existing authentication schemes, such as, user revocation or re-registration and biometric information protection. Compared with several related schemes, our scheme has more secure properties and lower computation cost. It is obviously more appropriate for practical applications in the remote distributed networks.

ID-based encryption scheme with revocation

NASA Astrophysics Data System (ADS)

Othman, Hafizul Azrie; Ismail, Eddie Shahril

2017-04-01

In 2015, Meshram proposed an efficient ID-based cryptographic encryption based on the difficulty of solving discrete logarithm and integer-factoring problems. The scheme was pairing free and claimed to be secure against adaptive chosen plaintext attacks (CPA). Later, Tan et al. proved that the scheme was insecure by presenting a method to recover the secret master key and to obtain prime factorization of modulo n. In this paper, we propose a new pairing-free ID-based encryption scheme with revocation based on Meshram's ID-based encryption scheme, which is also secure against Tan et al.'s attacks.

A secure biometrics-based authentication scheme for telecare medicine information systems.

PubMed

Yan, Xiaopeng; Li, Weiheng; Li, Ping; Wang, Jiantao; Hao, Xinhong; Gong, Peng

2013-10-01

The telecare medicine information system (TMIS) allows patients and doctors to access medical services or medical information at remote sites. Therefore, it could bring us very big convenient. To safeguard patients' privacy, authentication schemes for the TMIS attracted wide attention. Recently, Tan proposed an efficient biometrics-based authentication scheme for the TMIS and claimed their scheme could withstand various attacks. However, in this paper, we point out that Tan's scheme is vulnerable to the Denial-of-Service attack. To enhance security, we also propose an improved scheme based on Tan's work. Security and performance analysis shows our scheme not only could overcome weakness in Tan's scheme but also has better performance.

Linking payment to health outcomes: a taxonomy and examination of performance-based reimbursement schemes between healthcare payers and manufacturers.

PubMed

Carlson, Josh J; Sullivan, Sean D; Garrison, Louis P; Neumann, Peter J; Veenstra, David L

2010-08-01

To identify, categorize and examine performance-based health outcomes reimbursement schemes for medical technology. We performed a review of performance-based health outcomes reimbursement schemes over the past 10 years (7/98-010/09) using publicly available databases, web and grey literature searches, and input from healthcare reimbursement experts. We developed a taxonomy of scheme types by inductively organizing the schemes identified according to the timing, execution, and health outcomes measured in the schemes. Our search yielded 34 coverage with evidence development schemes, 10 conditional treatment continuation schemes, and 14 performance-linked reimbursement schemes. The majority of schemes are in Europe and Australia, with an increasing number in Canada and the U.S. These schemes have the potential to alter the reimbursement and pricing landscape for medical technology, but significant challenges, including high transaction costs and insufficient information systems, may limit their long-term impact. Future studies regarding experiences and outcomes of implemented schemes are necessary. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Understanding security failures of two authentication and key agreement schemes for telecare medicine information systems.

PubMed

Mishra, Dheerendra

2015-03-01

Smart card based authentication and key agreement schemes for telecare medicine information systems (TMIS) enable doctors, nurses, patients and health visitors to use smart cards for secure login to medical information systems. In recent years, several authentication and key agreement schemes have been proposed to present secure and efficient solution for TMIS. Most of the existing authentication schemes for TMIS have either higher computation overhead or are vulnerable to attacks. To reduce the computational overhead and enhance the security, Lee recently proposed an authentication and key agreement scheme using chaotic maps for TMIS. Xu et al. also proposed a password based authentication and key agreement scheme for TMIS using elliptic curve cryptography. Both the schemes provide better efficiency from the conventional public key cryptography based schemes. These schemes are important as they present an efficient solution for TMIS. We analyze the security of both Lee's scheme and Xu et al.'s schemes. Unfortunately, we identify that both the schemes are vulnerable to denial of service attack. To understand the security failures of these cryptographic schemes which are the key of patching existing schemes and designing future schemes, we demonstrate the security loopholes of Lee's scheme and Xu et al.'s scheme in this paper.

Robust and efficient biometrics based password authentication scheme for telecare medicine information systems using extended chaotic maps.

PubMed

Lu, Yanrong; Li, Lixiang; Peng, Haipeng; Xie, Dong; Yang, Yixian

2015-06-01

The Telecare Medicine Information Systems (TMISs) provide an efficient communicating platform supporting the patients access health-care delivery services via internet or mobile networks. Authentication becomes an essential need when a remote patient logins into the telecare server. Recently, many extended chaotic maps based authentication schemes using smart cards for TMISs have been proposed. Li et al. proposed a secure smart cards based authentication scheme for TMISs using extended chaotic maps based on Lee's and Jiang et al.'s scheme. In this study, we show that Li et al.'s scheme has still some weaknesses such as violation the session key security, vulnerability to user impersonation attack and lack of local verification. To conquer these flaws, we propose a chaotic maps and smart cards based password authentication scheme by applying biometrics technique and hash function operations. Through the informal and formal security analyses, we demonstrate that our scheme is resilient possible known attacks including the attacks found in Li et al.'s scheme. As compared with the previous authentication schemes, the proposed scheme is more secure and efficient and hence more practical for telemedical environments.

Rapid pulsed-field gel electrophoresis protocol for subtyping of Streptococcus suis serotype 2.

PubMed

Luey, Cindy K Y; Chu, Yiu Wai; Cheung, Terence K M; Law, Catherine C P; Chu, Man Yu; Cheung, Danny T L; Kam, Kai Man

2007-03-01

A rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping of Streptococcus suis serotype 2 was developed and evaluated using 27 clinical isolates from 22 epidemiologically unrelated patients. Results were matched against antibiogram, virulence genotyping and multi locus sequence typing (MLST). PFGE appeared to be the most discriminatory with numerical index of discrimination (D) equal to 0.87.

Dissemination of successful international clone ST15 and clonal complex 17 among Bulgarian CTX-M-15 producing K. pneumoniae isolates.

PubMed

Markovska, Rumyana; Stoeva, Temenuga; Boyanova, Lyudmila; Stankova, Petya; Pencheva, Daniela; Keuleyan, Emma; Murjeva, Marianna; Sredkova, Marya; Ivanova, Dobrinka; Lazarova, Grozdanka; Nedelcheva, Gergana; Kaneva, Radka; Mitov, Ivan

2017-12-01

A total of 82 extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae and 4 Klebsiella oxytoca isolates were collected in 2014 from four geographical areas in Bulgaria and their multilocus sequence type (MLST) and transferability of the ESBL encoding genes were investigated. The predominant type was CTX-M-15 (87%), followed by CTX-M-3 (9%), SHV-12 or SHV-2 (2%) and CTX-M-14 (1%). The CTX-M-15 producers belonged to ST15 (34.1%) and to a lesser extent to CC17 (ST16, ST17, ST336). The CTX-M-15 transconjugants showed a presence of R, A/C 2 and F replicons. The CTX-M-3 producers were assigned to ST29, ST70, ST432, ST542 and ST15 types and the transconjugants carried M 2 replicons. To the best of our knowledge, this is the first report that fully describes the MLST types among Bulgarian ESBL producing K. pneumoniae and the first report of the detection of IncR plasmid replicon type in our country. Copyright © 2017 Elsevier Inc. All rights reserved.

Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates.

PubMed

Bier, Nadja; Jäckel, Claudia; Dieckmann, Ralf; Brennholt, Nicole; Böer, Simone I; Strauch, Eckhard

2015-12-15

Vibrio vulnificus is a halophilic bacterium of coastal environments known for sporadically causing severe foodborne or wound infections. Global warming is expected to lead to a rising occurrence of V. vulnificus and an increasing incidence of human infections in Northern Europe. So far, infections in Germany were exclusively documented for the Baltic Sea coast, while no cases from the North Sea region have been reported. Regional variations in the prevalence of infections may be influenced by differences in the pathogenicity of V. vulnificus populations in both areas. This study aimed to compare the distribution of virulence-associated traits and genotypes among 101 V. vulnificus isolates from the Baltic Sea and North Sea in order to assess their pathogenicity potential. Furthermore, genetic relationships were examined by multilocus sequence typing (MLST). A high diversity of MLST sequences (74 sequence types) and differences regarding the presence of six potential pathogenicity markers were observed in the V. vulnificus populations of both areas. Strains with genotypes and markers associated with pathogenicity are not restricted to a particular geographic region. This indicates that lack of reported cases in the North Sea region is not caused by the absence of potentially pathogenic strains.

Biodiversity of dairy Propionibacterium isolated from dairy farms in Minas Gerais, Brazil.

PubMed

de Freitas, Rosangela; Chuat, Victoria; Madec, Marie-Noelle; Nero, Luis Augusto; Thierry, Anne; Valence, Florence; de Carvalho, Antonio Fernandes

2015-06-16

Dairy propionibacteria are used as ripening cultures for the production of Swiss-type cheeses, and some strains have potential for use as probiotics. This study investigated the biodiversity of wild dairy Propionibacteria isolates in dairy farms that produce Swiss-type cheeses in Minas Gerais State, Brazil. RAPD and PFGE were used for molecular typing of strains and MLST was applied for phylogenetic analysis of strains of Propionibacterium freudenreichii. The results showed considerable genetic diversity of the wild dairy propionibacteria, since three of the main species were observed to be randomly distributed among the samples collected from different farms in different biotopes (raw milk, sillage, soil and pasture). Isolates from different farms showed distinct genetic profiles, suggesting that each location represented a specific niche. Furthermore, the STs identified for the strains of P. freudenreichii by MLST were not related to any specific origin. The environment of dairy farms and milk production proved to be a reservoir for Propionibacterium strains, which are important for future use as possible starter cultures or probiotics, as well as in the study of prevention of cheese defects. Copyright © 2015 Elsevier B.V. All rights reserved.

Campylobacter jejuni survival in a poultry processing plant environment.

PubMed

García-Sánchez, Lourdes; Melero, Beatriz; Jaime, Isabel; Hänninen, Marja-Liisa; Rossi, Mirko; Rovira, Jordi

2017-08-01

Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Consumption of poultry, especially chicken's meat is considered the most common route for human infection. The aim of this study was to determine if Campylobacter spp. might persist in the poultry plant environment before and after cleaning and disinfection procedures and the distribution and their genetic relatedness. During one month from a poultry plant were analyzed a total of 494 samples -defeathering machine, evisceration machine, floor, sink, conveyor belt, shackles and broiler meat- in order to isolate C. jejuni and C. coli. Results showed that C. jejuni and C. coli prevalence was 94.5% and 5.5% respectively. Different typing techniques as PFGE, MLST established seven C. jejuni genotypes. Whole genome MLST strongly suggest that highly clonal populations of C. jejuni can survive in adverse environmental conditions, even after cleaning and disinfection, and persist for longer periods than previous thought (at least 21 days) in the poultry plant environment. Even so, it might act as a source of contamination independently of the contamination level of the flock entering the slaughter line. Copyright © 2017 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.

Multistate outbreak of Listeria monocytogenes infections linked to whole apples used in commercially produced, prepackaged caramel apples: United States, 2014-2015.

PubMed

Angelo, K M; Conrad, A R; Saupe, A; Dragoo, H; West, N; Sorenson, A; Barnes, A; Doyle, M; Beal, J; Jackson, K A; Stroika, S; Tarr, C; Kucerova, Z; Lance, S; Gould, L H; Wise, M; Jackson, B R

2017-04-01

Whole apples have not been previously implicated in outbreaks of foodborne bacterial illness. We investigated a nationwide listeriosis outbreak associated with caramel apples. We defined an outbreak-associated case as an infection with one or both of two outbreak strains of Listeria monocytogenes highly related by whole-genome multilocus sequence typing (wgMLST) from 1 October 2014 to 1 February 2015. Single-interviewer open-ended interviews identified the source. Outbreak-associated cases were compared with non-outbreak-associated cases and traceback and environmental investigations were performed. We identified 35 outbreak-associated cases in 12 states; 34 (97%) were hospitalized and seven (20%) died. Outbreak-associated ill persons were more likely to have eaten commercially produced, prepackaged caramel apples (odds ratio 326·7, 95% confidence interval 32·2-3314). Environmental samples from the grower's packing facility and distribution-chain whole apples yielded isolates highly related to outbreak isolates by wgMLST. This outbreak highlights the importance of minimizing produce contamination with L. monocytogenes. Investigators should perform single-interviewer open-ended interviews when a food is not readily identified.

An overview of Mycoplasma bovis mastitis in Israel (2004-2014).

PubMed

Lysnyansky, Inna; Freed, Mor; Rosales, Ruben S; Mikula, Inna; Khateb, Nihaya; Gerchman, Irena; van Straten, Michael; Levisohn, Sharon

2016-01-01

The prevalence of Mycoplasma bovis in milk samples submitted to the Israeli National Service for Udder Health and Milk Quality was determined during the period 2004-2014 and the genetic pattern of the obtained isolates was assessed by multilocus sequence typing (MLST). Mycoplasma spp. were identified in 66 herds including M. bovis (n = 60), M. cottewii (n = 3), M. bovigenitalium (n = 2), M. alkalescens (n = 2) and M. yeatsii (n = 1). The proportion of M. bovis infected herds was relatively low (0-0.68%) in 2004-2007, increased to 3.77% during the 2008 outbreak, and ranged from 0.77 to 2.77% during the 2009-2014 period. Since 2008, about eight M. bovis positive dairy herds have been identified in Israel annually, with six of which on average being newly infected. MLST of 57 M. bovis isolates revealed that sequence type 10 was the dominant genotype identified in 60% of the herds. In conclusion, these data show that M. bovis is the main mycoplasmal mastitic pathogen in Israel. Copyright © 2015 Elsevier Ltd. 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.

Enhanced smartcard-based password-authenticated key agreement using extended chaotic maps.

PubMed

Lee, Tian-Fu; Hsiao, Chia-Hung; Hwang, Shi-Han; Lin, Tsung-Hung

2017-01-01

A smartcard based password-authenticated key agreement scheme enables a legal user to log in to a remote authentication server and access remote services through public networks using a weak password and a smart card. Lin recently presented an improved chaotic maps-based password-authenticated key agreement scheme that used smartcards to eliminate the weaknesses of the scheme of Guo and Chang, which does not provide strong user anonymity and violates session key security. However, the improved scheme of Lin does not exhibit the freshness property and the validity of messages so it still fails to withstand denial-of-service and privileged-insider attacks. Additionally, a single malicious participant can predetermine the session key such that the improved scheme does not exhibit the contributory property of key agreements. This investigation discusses these weaknesses and proposes an enhanced smartcard-based password-authenticated key agreement scheme that utilizes extended chaotic maps. The session security of this enhanced scheme is based on the extended chaotic map-based Diffie-Hellman problem, and is proven in the real-or-random and the sequence of games models. Moreover, the enhanced scheme ensures the freshness of communicating messages by appending timestamps, and thereby avoids the weaknesses in previous schemes.

Enhanced smartcard-based password-authenticated key agreement using extended chaotic maps

PubMed Central

Lee, Tian-Fu; Hsiao, Chia-Hung; Hwang, Shi-Han

2017-01-01

A smartcard based password-authenticated key agreement scheme enables a legal user to log in to a remote authentication server and access remote services through public networks using a weak password and a smart card. Lin recently presented an improved chaotic maps-based password-authenticated key agreement scheme that used smartcards to eliminate the weaknesses of the scheme of Guo and Chang, which does not provide strong user anonymity and violates session key security. However, the improved scheme of Lin does not exhibit the freshness property and the validity of messages so it still fails to withstand denial-of-service and privileged-insider attacks. Additionally, a single malicious participant can predetermine the session key such that the improved scheme does not exhibit the contributory property of key agreements. This investigation discusses these weaknesses and proposes an enhanced smartcard-based password-authenticated key agreement scheme that utilizes extended chaotic maps. The session security of this enhanced scheme is based on the extended chaotic map-based Diffie-Hellman problem, and is proven in the real-or-random and the sequence of games models. Moreover, the enhanced scheme ensures the freshness of communicating messages by appending timestamps, and thereby avoids the weaknesses in previous schemes. PMID:28759615

Cryptanalysis and Improvement of a Biometric-Based Multi-Server Authentication and Key Agreement Scheme

PubMed Central

Wang, Chengqi; Zhang, Xiao; Zheng, Zhiming

2016-01-01

With the security requirements of networks, biometrics authenticated schemes which are applied in the multi-server environment come to be more crucial and widely deployed. In this paper, we propose a novel biometric-based multi-server authentication and key agreement scheme which is based on the cryptanalysis of Mishra et al.’s scheme. The informal and formal security analysis of our scheme are given, which demonstrate that our scheme satisfies the desirable security requirements. The presented scheme provides a variety of significant functionalities, in which some features are not considered in the most of existing authentication schemes, such as, user revocation or re-registration and biometric information protection. Compared with several related schemes, our scheme has more secure properties and lower computation cost. It is obviously more appropriate for practical applications in the remote distributed networks. PMID:26866606

An efficient and provable secure revocable identity-based encryption scheme.

PubMed

Wang, Changji; Li, Yuan; Xia, Xiaonan; Zheng, Kangjia

2014-01-01

Revocation functionality is necessary and crucial to identity-based cryptosystems. Revocable identity-based encryption (RIBE) has attracted a lot of attention in recent years, many RIBE schemes have been proposed in the literature but shown to be either insecure or inefficient. In this paper, we propose a new scalable RIBE scheme with decryption key exposure resilience by combining Lewko and Waters' identity-based encryption scheme and complete subtree method, and prove our RIBE scheme to be semantically secure using dual system encryption methodology. Compared to existing scalable and semantically secure RIBE schemes, our proposed RIBE scheme is more efficient in term of ciphertext size, public parameters size and decryption cost at price of a little looser security reduction. To the best of our knowledge, this is the first construction of scalable and semantically secure RIBE scheme with constant size public system parameters.

Efficiently Multi-User Searchable Encryption Scheme with Attribute Revocation and Grant for Cloud Storage

PubMed Central

Wang, Shangping; Zhang, Xiaoxue; Zhang, Yaling

2016-01-01

Cipher-policy attribute-based encryption (CP-ABE) focus on the problem of access control, and keyword-based searchable encryption scheme focus on the problem of finding the files that the user interested in the cloud storage quickly. To design a searchable and attribute-based encryption scheme is a new challenge. In this paper, we propose an efficiently multi-user searchable attribute-based encryption scheme with attribute revocation and grant for cloud storage. In the new scheme the attribute revocation and grant processes of users are delegated to proxy server. Our scheme supports multi attribute are revoked and granted simultaneously. Moreover, the keyword searchable function is achieved in our proposed scheme. The security of our proposed scheme is reduced to the bilinear Diffie-Hellman (BDH) assumption. Furthermore, the scheme is proven to be secure under the security model of indistinguishability against selective ciphertext-policy and chosen plaintext attack (IND-sCP-CPA). And our scheme is also of semantic security under indistinguishability against chosen keyword attack (IND-CKA) in the random oracle model. PMID:27898703

Efficiently Multi-User Searchable Encryption Scheme with Attribute Revocation and Grant for Cloud Storage.

PubMed

Wang, Shangping; Zhang, Xiaoxue; Zhang, Yaling

2016-01-01

Cipher-policy attribute-based encryption (CP-ABE) focus on the problem of access control, and keyword-based searchable encryption scheme focus on the problem of finding the files that the user interested in the cloud storage quickly. To design a searchable and attribute-based encryption scheme is a new challenge. In this paper, we propose an efficiently multi-user searchable attribute-based encryption scheme with attribute revocation and grant for cloud storage. In the new scheme the attribute revocation and grant processes of users are delegated to proxy server. Our scheme supports multi attribute are revoked and granted simultaneously. Moreover, the keyword searchable function is achieved in our proposed scheme. The security of our proposed scheme is reduced to the bilinear Diffie-Hellman (BDH) assumption. Furthermore, the scheme is proven to be secure under the security model of indistinguishability against selective ciphertext-policy and chosen plaintext attack (IND-sCP-CPA). And our scheme is also of semantic security under indistinguishability against chosen keyword attack (IND-CKA) in the random oracle model.

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk.

PubMed

Shome, Bibek Ranjan; Bhuvana, Mani; Mitra, Susweta Das; Krithiga, Natesan; Shome, Rajeswari; Velu, Dhanikachalam; Banerjee, Apala; Barbuddhe, Sukhadeo B; Prabhudas, Krishnamshetty; Rahman, Habibar

2012-12-01

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A-D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.

A provably-secure ECC-based authentication scheme for wireless sensor networks.

PubMed

Nam, Junghyun; Kim, Moonseong; Paik, Juryon; Lee, Youngsook; Won, Dongho

2014-11-06

A smart-card-based user authentication scheme for wireless sensor networks (in short, a SUA-WSN scheme) is designed to restrict access to the sensor data only to users who are in possession of both a smart card and the corresponding password. While a significant number of SUA-WSN schemes have been suggested in recent years, their intended security properties lack formal definitions and proofs in a widely-accepted model. One consequence is that SUA-WSN schemes insecure against various attacks have proliferated. In this paper, we devise a security model for the analysis of SUA-WSN schemes by extending the widely-accepted model of Bellare, Pointcheval and Rogaway (2000). Our model provides formal definitions of authenticated key exchange and user anonymity while capturing side-channel attacks, as well as other common attacks. We also propose a new SUA-WSN scheme based on elliptic curve cryptography (ECC), and prove its security properties in our extended model. To the best of our knowledge, our proposed scheme is the first SUA-WSN scheme that provably achieves both authenticated key exchange and user anonymity. Our scheme is also computationally competitive with other ECC-based (non-provably secure) schemes.

A Provably-Secure ECC-Based Authentication Scheme for Wireless Sensor Networks

PubMed Central

Nam, Junghyun; Kim, Moonseong; Paik, Juryon; Lee, Youngsook; Won, Dongho

2014-01-01

A smart-card-based user authentication scheme for wireless sensor networks (in short, a SUA-WSN scheme) is designed to restrict access to the sensor data only to users who are in possession of both a smart card and the corresponding password. While a significant number of SUA-WSN schemes have been suggested in recent years, their intended security properties lack formal definitions and proofs in a widely-accepted model. One consequence is that SUA-WSN schemes insecure against various attacks have proliferated. In this paper, we devise a security model for the analysis of SUA-WSN schemes by extending the widely-accepted model of Bellare, Pointcheval and Rogaway (2000). Our model provides formal definitions of authenticated key exchange and user anonymity while capturing side-channel attacks, as well as other common attacks. We also propose a new SUA-WSN scheme based on elliptic curve cryptography (ECC), and prove its security properties in our extended model. To the best of our knowledge, our proposed scheme is the first SUA-WSN scheme that provably achieves both authenticated key exchange and user anonymity. Our scheme is also computationally competitive with other ECC-based (non-provably secure) schemes. PMID:25384009

A soft-hard combination-based cooperative spectrum sensing scheme for cognitive radio networks.

PubMed

Do, Nhu Tri; An, Beongku

2015-02-13

In this paper we propose a soft-hard combination scheme, called SHC scheme, for cooperative spectrum sensing in cognitive radio networks. The SHC scheme deploys a cluster based network in which Likelihood Ratio Test (LRT)-based soft combination is applied at each cluster, and weighted decision fusion rule-based hard combination is utilized at the fusion center. The novelties of the SHC scheme are as follows: the structure of the SHC scheme reduces the complexity of cooperative detection which is an inherent limitation of soft combination schemes. By using the LRT, we can detect primary signals in a low signal-to-noise ratio regime (around an average of -15 dB). In addition, the computational complexity of the LRT is reduced since we derive the closed-form expression of the probability density function of LRT value. The SHC scheme also takes into account the different effects of large scale fading on different users in the wide area network. The simulation results show that the SHC scheme not only provides the better sensing performance compared to the conventional hard combination schemes, but also reduces sensing overhead in terms of reporting time compared to the conventional soft combination scheme using the LRT.

A Novel Passive Tracking Scheme Exploiting Geometric and Intercept Theorems

PubMed Central

Zhou, Biao; Sun, Chao; Ahn, Deockhyeon; Kim, Youngok

2018-01-01

Passive tracking aims to track targets without assistant devices, that is, device-free targets. Passive tracking based on Radio Frequency (RF) Tomography in wireless sensor networks has recently been addressed as an emerging field. The passive tracking scheme using geometric theorems (GTs) is one of the most popular RF Tomography schemes, because the GT-based method can effectively mitigate the demand for a high density of wireless nodes. In the GT-based tracking scheme, the tracking scenario is considered as a two-dimensional geometric topology and then geometric theorems are applied to estimate crossing points (CPs) of the device-free target on line-of-sight links (LOSLs), which reveal the target’s trajectory information in a discrete form. In this paper, we review existing GT-based tracking schemes, and then propose a novel passive tracking scheme by exploiting the Intercept Theorem (IT). To create an IT-based CP estimation scheme available in the noisy non-parallel LOSL situation, we develop the equal-ratio traverse (ERT) method. Finally, we analyze properties of three GT-based tracking algorithms and the performance of these schemes is evaluated experimentally under various trajectories, node densities, and noisy topologies. Analysis of experimental results shows that tracking schemes exploiting geometric theorems can achieve remarkable positioning accuracy even under rather a low density of wireless nodes. Moreover, the proposed IT scheme can provide generally finer tracking accuracy under even lower node density and noisier topologies, in comparison to other schemes. PMID:29562621

An Improved Biometrics-Based Remote User Authentication Scheme with User Anonymity

PubMed Central

Kumari, Saru

2013-01-01

The authors review the biometrics-based user authentication scheme proposed by An in 2012. The authors show that there exist loopholes in the scheme which are detrimental for its security. Therefore the authors propose an improved scheme eradicating the flaws of An's scheme. Then a detailed security analysis of the proposed scheme is presented followed by its efficiency comparison. The proposed scheme not only withstands security problems found in An's scheme but also provides some extra features with mere addition of only two hash operations. The proposed scheme allows user to freely change his password and also provides user anonymity with untraceability. PMID:24350272

An improved biometrics-based remote user authentication scheme with user anonymity.

PubMed

Khan, Muhammad Khurram; Kumari, Saru

2013-01-01

The authors review the biometrics-based user authentication scheme proposed by An in 2012. The authors show that there exist loopholes in the scheme which are detrimental for its security. Therefore the authors propose an improved scheme eradicating the flaws of An's scheme. Then a detailed security analysis of the proposed scheme is presented followed by its efficiency comparison. The proposed scheme not only withstands security problems found in An's scheme but also provides some extra features with mere addition of only two hash operations. The proposed scheme allows user to freely change his password and also provides user anonymity with untraceability.

Provably secure identity-based identification and signature schemes from code assumptions

PubMed Central

Zhao, Yiming

2017-01-01

Code-based cryptography is one of few alternatives supposed to be secure in a post-quantum world. Meanwhile, identity-based identification and signature (IBI/IBS) schemes are two of the most fundamental cryptographic primitives, so several code-based IBI/IBS schemes have been proposed. However, with increasingly profound researches on coding theory, the security reduction and efficiency of such schemes have been invalidated and challenged. In this paper, we construct provably secure IBI/IBS schemes from code assumptions against impersonation under active and concurrent attacks through a provably secure code-based signature technique proposed by Preetha, Vasant and Rangan (PVR signature), and a security enhancement Or-proof technique. We also present the parallel-PVR technique to decrease parameter values while maintaining the standard security level. Compared to other code-based IBI/IBS schemes, our schemes achieve not only preferable public parameter size, private key size, communication cost and signature length due to better parameter choices, but also provably secure. PMID:28809940

Provably secure identity-based identification and signature schemes from code assumptions.

PubMed

Song, Bo; Zhao, Yiming

2017-01-01

Code-based cryptography is one of few alternatives supposed to be secure in a post-quantum world. Meanwhile, identity-based identification and signature (IBI/IBS) schemes are two of the most fundamental cryptographic primitives, so several code-based IBI/IBS schemes have been proposed. However, with increasingly profound researches on coding theory, the security reduction and efficiency of such schemes have been invalidated and challenged. In this paper, we construct provably secure IBI/IBS schemes from code assumptions against impersonation under active and concurrent attacks through a provably secure code-based signature technique proposed by Preetha, Vasant and Rangan (PVR signature), and a security enhancement Or-proof technique. We also present the parallel-PVR technique to decrease parameter values while maintaining the standard security level. Compared to other code-based IBI/IBS schemes, our schemes achieve not only preferable public parameter size, private key size, communication cost and signature length due to better parameter choices, but also provably secure.

A secure and efficient chaotic map-based authenticated key agreement scheme for telecare medicine information systems.

PubMed

Mishra, Dheerendra; Srinivas, Jangirala; Mukhopadhyay, Sourav

2014-10-01

Advancement in network technology provides new ways to utilize telecare medicine information systems (TMIS) for patient care. Although TMIS usually faces various attacks as the services are provided over the public network. Recently, Jiang et al. proposed a chaotic map-based remote user authentication scheme for TMIS. Their scheme has the merits of low cost and session key agreement using Chaos theory. It enhances the security of the system by resisting various attacks. In this paper, we analyze the security of Jiang et al.'s scheme and demonstrate that their scheme is vulnerable to denial of service attack. Moreover, we demonstrate flaws in password change phase of their scheme. Further, our aim is to propose a new chaos map-based anonymous user authentication scheme for TMIS to overcome the weaknesses of Jiang et al.'s scheme, while also retaining the original merits of their scheme. We also show that our scheme is secure against various known attacks including the attacks found in Jiang et al.'s scheme. The proposed scheme is comparable in terms of the communication and computational overheads with Jiang et al.'s scheme and other related existing schemes. Moreover, we demonstrate the validity of the proposed scheme through the BAN (Burrows, Abadi, and Needham) logic.

Research to Assembly Scheme for Satellite Deck Based on Robot Flexibility Control Principle

NASA Astrophysics Data System (ADS)

Guo, Tao; Hu, Ruiqin; Xiao, Zhengyi; Zhao, Jingjing; Fang, Zhikai

2018-03-01

Deck assembly is critical quality control point in final satellite assembly process, and cable extrusion and structure collision problems in assembly process will affect development quality and progress of satellite directly. Aimed at problems existing in deck assembly process, assembly project scheme for satellite deck based on robot flexibility control principle is proposed in this paper. Scheme is introduced firstly; secondly, key technologies on end force perception and flexible docking control in the scheme are studied; then, implementation process of assembly scheme for satellite deck is described in detail; finally, actual application case of assembly scheme is given. Result shows that compared with traditional assembly scheme, assembly scheme for satellite deck based on robot flexibility control principle has obvious advantages in work efficiency, reliability and universality aspects etc.

A keyword searchable attribute-based encryption scheme with attribute update for cloud storage.

PubMed

Wang, Shangping; Ye, Jian; Zhang, Yaling

2018-01-01

Ciphertext-policy attribute-based encryption (CP-ABE) scheme is a new type of data encryption primitive, which is very suitable for data cloud storage for its fine-grained access control. Keyword-based searchable encryption scheme enables users to quickly find interesting data stored in the cloud server without revealing any information of the searched keywords. In this work, we provide a keyword searchable attribute-based encryption scheme with attribute update for cloud storage, which is a combination of attribute-based encryption scheme and keyword searchable encryption scheme. The new scheme supports the user's attribute update, especially in our new scheme when a user's attribute need to be updated, only the user's secret key related with the attribute need to be updated, while other user's secret key and the ciphertexts related with this attribute need not to be updated with the help of the cloud server. In addition, we outsource the operation with high computation cost to cloud server to reduce the user's computational burden. Moreover, our scheme is proven to be semantic security against chosen ciphertext-policy and chosen plaintext attack in the general bilinear group model. And our scheme is also proven to be semantic security against chosen keyword attack under bilinear Diffie-Hellman (BDH) assumption.

A keyword searchable attribute-based encryption scheme with attribute update for cloud storage

PubMed Central

Wang, Shangping; Zhang, Yaling

2018-01-01

Ciphertext-policy attribute-based encryption (CP-ABE) scheme is a new type of data encryption primitive, which is very suitable for data cloud storage for its fine-grained access control. Keyword-based searchable encryption scheme enables users to quickly find interesting data stored in the cloud server without revealing any information of the searched keywords. In this work, we provide a keyword searchable attribute-based encryption scheme with attribute update for cloud storage, which is a combination of attribute-based encryption scheme and keyword searchable encryption scheme. The new scheme supports the user's attribute update, especially in our new scheme when a user's attribute need to be updated, only the user's secret key related with the attribute need to be updated, while other user's secret key and the ciphertexts related with this attribute need not to be updated with the help of the cloud server. In addition, we outsource the operation with high computation cost to cloud server to reduce the user's computational burden. Moreover, our scheme is proven to be semantic security against chosen ciphertext-policy and chosen plaintext attack in the general bilinear group model. And our scheme is also proven to be semantic security against chosen keyword attack under bilinear Diffie-Hellman (BDH) assumption. PMID:29795577

Exponential Arithmetic Based Self-Healing Group Key Distribution Scheme with Backward Secrecy under the Resource-Constrained Wireless Networks

PubMed Central

Guo, Hua; Zheng, Yandong; Zhang, Xiyong; Li, Zhoujun

2016-01-01

In resource-constrained wireless networks, resources such as storage space and communication bandwidth are limited. To guarantee secure communication in resource-constrained wireless networks, group keys should be distributed to users. The self-healing group key distribution (SGKD) scheme is a promising cryptographic tool, which can be used to distribute and update the group key for the secure group communication over unreliable wireless networks. Among all known SGKD schemes, exponential arithmetic based SGKD (E-SGKD) schemes reduce the storage overhead to constant, thus is suitable for the the resource-constrained wireless networks. In this paper, we provide a new mechanism to achieve E-SGKD schemes with backward secrecy. We first propose a basic E-SGKD scheme based on a known polynomial-based SGKD, where it has optimal storage overhead while having no backward secrecy. To obtain the backward secrecy and reduce the communication overhead, we introduce a novel approach for message broadcasting and self-healing. Compared with other E-SGKD schemes, our new E-SGKD scheme has the optimal storage overhead, high communication efficiency and satisfactory security. The simulation results in Zigbee-based networks show that the proposed scheme is suitable for the resource-restrained wireless networks. Finally, we show the application of our proposed scheme. PMID:27136550

Zinc resistance within swine associated methicillin resistant Staphylococcus aureus (MRSA) isolates in the USA is associated with MLST lineage

USDA-ARS?s Scientific Manuscript database

Zinc resistance in livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) is mediated by the czrC gene co-located with the mecA gene, encoding methicillin resistance, on the type V SCCmec element. Since the czrC gene and the mecA gene are co-located on the SCCmec element, it has ...

Characterisation by multilocus sequence and porA and flaA typing of Campylobacter jejuni isolated from samples of dog faeces collected in one city in New Zealand.

PubMed

Mohan, V; Stevenson, M A; Marshall, J C; French, N P

2017-07-01

To investigate the prevalence of Campylobacter spp. and C. jejuni in dog faecal material collected from dog walkways in the city of Palmerston North, New Zealand, and to characterise the C. jejuni isolates by multilocus sequence typing (MLST) and porA and flaA antigen gene typing. A total of 355 fresh samples of dogs faeces were collected from bins provided for the disposal of dog faeces in 10 walkways in Palmerston North, New Zealand, between August 2008-July 2009. Presumptive Campylobacter colonies, cultured on modified charcoal cefoperazone deoxycholate plates, were screened for genus Campylobacter and C. jejuni by PCR. The C. jejuni isolates were subsequently characterised by MLST and porA and flaA typing, and C. jejuni sequence types (ST) were assigned. Of the 355 samples collected, 72 (20 (95% CI=16-25)%) were positive for Campylobacter spp. and 22 (6 (95% CI=4-9)%) were positive for C. jejuni. Of the 22 C. jejuni isolates, 19 were fully typed by MLST. Ten isolates were assigned to the clonal complex ST-45 and three to ST-52. The allelic combinations of ST-45/flaA 21/porA 44 (n=3), ST-45/flaA 22/porA 53 (n=3) and ST-52/ flaA 57/porA 905 (n=3) were most frequent. The successful isolation of C. jejuni from canine faecal samples collected from faecal bins provides evidence that Campylobacter spp. may survive outside the host for at least several hours despite requiring fastidious growth conditions in culture. The results show that dogs carry C. jejuni genotypes (ST-45, ST-50, ST-52 and ST-696) that have been reported in human clinical cases. Although these results do not provide any evidence either for the direction of infection or for dogs being a potential risk factor for human campylobacteriosis, dog owners are advised to practice good hygiene with respect to their pets to reduce potential exposure to infection.

Orthogonal typing methods identify genetic diversity among Belgian Campylobacter jejuni strains isolated over a decade from poultry and cases of sporadic human illness.

PubMed

Elhadidy, Mohamed; Arguello, Hector; Álvarez-Ordóñez, Avelino; Miller, William G; Duarte, Alexandra; Martiny, Delphine; Hallin, Marie; Vandenberg, Olivier; Dierick, Katelijne; Botteldoorn, Nadine

2018-06-20

Campylobacter jejuni is a zoonotic pathogen commonly associated with human gastroenteritis. Retail poultry meat is a major food-related transmission source of C. jejuni to humans. The present study investigated the genetic diversity, clonal relationship, and strain risk-analysis of 403 representative C. jejuni isolates from chicken broilers (n = 204) and sporadic cases of human diarrhea (n = 199) over a decade (2006-2015) in Belgium, using multilocus sequence typing (MLST), PCR binary typing (P-BIT), and identification of lipooligosaccharide (LOS) biosynthesis locus classes. A total of 123 distinct sequence types (STs), clustered in 28 clonal complexes (CCs) were assigned, including ten novel sequence types that were not previously documented in the international database. Sequence types ST-48, ST-21, ST-50, ST-45, ST-464, ST-2274, ST-572, ST-19, ST-257 and ST-42 were the most prevalent. Clonal complex 21 was the main clonal complex in isolates from humans and chickens. Among observed STs, a total of 35 STs that represent 72.2% (291/403) of the isolates were identified in both chicken and human isolates confirming considerable epidemiological relatedness; these 35 STs also clustered together in the most prevalent CCs. A majority of the isolates harbored sialylated LOS loci associated with potential neuropathic outcomes in humans. Although the concordance between MLST and P-BIT, determined by the adjusted Rand and Wallace coefficients, showed low congruence between both typing methods. The discriminatory power of P-BIT and MLST was similar, with Simpson's diversity indexes of 0.978 and 0.975, respectively. Furthermore, P-BIT could provide additional epidemiological information that would provide further insights regarding the potential association to human health from each strain. In addition, certain clones could be linked to specific clinical symptoms. Indeed, LOS class E was associated with less severe infections. Moreover, ST-572 was significantly associated with clinical infections occurring after travelling abroad. Ultimately, the data generated from this study will help to better understand the molecular epidemiology of C. jejuni infection. Copyright © 2018. Published by Elsevier B.V.

Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates from retail meat and humans in Georgia.

PubMed

Jackson, Charlene R; Davis, Johnnie A; Barrett, John B

2013-04-01

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl(+). Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat.

Prevalence and Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Retail Meat and Humans in Georgia

PubMed Central

Davis, Johnnie A.; Barrett, John B.

2013-01-01

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl+. Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat. PMID:23363837

An improved biometrics-based authentication scheme for telecare medical information systems.

PubMed

Guo, Dianli; Wen, Qiaoyan; Li, Wenmin; Zhang, Hua; Jin, Zhengping

2015-03-01

Telecare medical information system (TMIS) offers healthcare delivery services and patients can acquire their desired medical services conveniently through public networks. The protection of patients' privacy and data confidentiality are significant. Very recently, Mishra et al. proposed a biometrics-based authentication scheme for telecare medical information system. Their scheme can protect user privacy and is believed to resist a range of network attacks. In this paper, we analyze Mishra et al.'s scheme and identify that their scheme is insecure to against known session key attack and impersonation attack. Thereby, we present a modified biometrics-based authentication scheme for TMIS to eliminate the aforementioned faults. Besides, we demonstrate the completeness of the proposed scheme through BAN-logic. Compared to the related schemes, our protocol can provide stronger security and it is more practical.

Parallelised photoacoustic signal acquisition using a Fabry-Perot sensor and a camera-based interrogation scheme

NASA Astrophysics Data System (ADS)

Saeb Gilani, T.; Villringer, C.; Zhang, E.; Gundlach, H.; Buchmann, J.; Schrader, S.; Laufer, J.

2018-02-01

Tomographic photoacoustic (PA) images acquired using a Fabry-Perot (FP) based scanner offer high resolution and image fidelity but can result in long acquisition times due to the need for raster scanning. To reduce the acquisition times, a parallelised camera-based PA signal detection scheme is developed. The scheme is based on using a sCMOScamera and FPI sensors with high homogeneity of optical thickness. PA signals were acquired using the camera-based setup and the signal to noise ratio (SNR) was measured. A comparison of the SNR of PA signal detected using 1) a photodiode in a conventional raster scanning detection scheme and 2) a sCMOS camera in parallelised detection scheme is made. The results show that the parallelised interrogation scheme has the potential to provide high speed PA imaging.

Surface reconstruction and deformation monitoring of stratospheric airship based on laser scanning technology

NASA Astrophysics Data System (ADS)

Guo, Kai; Xie, Yongjie; Ye, Hu; Zhang, Song; Li, Yunfei

2018-04-01

Due to the uncertainty of stratospheric airship's shape and the security problem caused by the uncertainty, surface reconstruction and surface deformation monitoring of airship was conducted based on laser scanning technology and a √3-subdivision scheme based on Shepard interpolation was developed. Then, comparison was conducted between our subdivision scheme and the original √3-subdivision scheme. The result shows our subdivision scheme could reduce the shrinkage of surface and the number of narrow triangles. In addition, our subdivision scheme could keep the sharp features. So, surface reconstruction and surface deformation monitoring of airship could be conducted precisely by our subdivision scheme.

MRSA clonal complex 22 strains harboring toxic shock syndrome toxin (TSST-1) are endemic in the primary hospital in Gaza, Palestine.

PubMed

Al Laham, Nahed; Mediavilla, José R; Chen, Liang; Abdelateef, Nahed; Elamreen, Farid Abu; Ginocchio, Christine C; Pierard, Denis; Becker, Karsten; Kreiswirth, Barry N

2015-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in both community and healthcare-related settings worldwide. Current knowledge regarding the epidemiology of S. aureus and MRSA in Gaza is based on a single community-based carriage study. Here we describe a cross-sectional analysis of 215 clinical isolates collected from Al-Shifa Hospital in Gaza during 2008 and 2012. All isolates were characterized by spa typing, SCCmec typing, and detection of genes encoding Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST-1). Representative genotypes were also subjected to multilocus sequence typing (MLST). Antibiotic susceptibility testing was performed using VITEK2 and MicroScan. MRSA represented 56.3% of all S. aureus strains, and increased in frequency from 2008 (54.8%) to 2012 (58.4%). Aside from beta-lactams, resistance was observed to tetracycline, erythromycin, clindamycin, gentamicin, and fluoroquinolones. Molecular typing identified 35 spa types representing 17 MLST clonal complexes (CC), with spa 998 (Ridom t223, CC22) and spa 70 (Ridom t044, CC80) being the most prevalent. SCCmec types I, III, IV, V and VI were identified among MRSA isolates, while type II was not detected. PVL genes (lukF/S-PV) were detected in 40.0% of all isolates, while the TSST-1 gene (tst) was detected in 27.4% of all isolates, with surprisingly high frequency within CC22 (70.4%). Both PVL and TSST-1 genes were found in several isolates from 2012. Molecular typing of clinical isolates from Gaza hospitals revealed unusually high prevalence of TSST-1 genes among CC22 MRSA, which is noteworthy given a recent community study describing widespread carriage of a CC22 MRSA clone known as the 'Gaza strain'. While the latter did not address TSST-1, tst-positive spa 998 (Ridom t223) has been detected in several neighboring countries, and described as endemic in an Italian NICU, suggesting international spread of a 'Middle Eastern variant' of pandemic CC22 strain EMRSA-15.

New Insights into the Diversity of the Genus Faecalibacterium.

PubMed

Benevides, Leandro; Burman, Sriti; Martin, Rebeca; Robert, Véronique; Thomas, Muriel; Miquel, Sylvie; Chain, Florian; Sokol, Harry; Bermudez-Humaran, Luis G; Morrison, Mark; Langella, Philippe; Azevedo, Vasco A; Chatel, Jean-Marc; Soares, Siomar

2017-01-01

Faecalibacterium prausnitzii is a commensal bacterium, ubiquitous in the gastrointestinal tracts of animals and humans. This species is a functionally important member of the microbiota and studies suggest it has an impact on the physiology and health of the host. F. prausnitzii is the only identified species in the genus Faecalibacterium , but a recent study clustered strains of this species in two different phylogroups. Here, we propose the existence of distinct species in this genus through the use of comparative genomics. Briefly, we performed analyses of 16S rRNA gene phylogeny, phylogenomics, whole genome Multi-Locus Sequence Typing (wgMLST), Average Nucleotide Identity (ANI), gene synteny, and pangenome to better elucidate the phylogenetic relationships among strains of Faecalibacterium . For this, we used 12 newly sequenced, assembled, and curated genomes of F. prausnitzii , which were isolated from feces of healthy volunteers from France and Australia, and combined these with published data from 5 strains downloaded from public databases. The phylogenetic analysis of the 16S rRNA sequences, together with the wgMLST profiles and a phylogenomic tree based on comparisons of genome similarity, all supported the clustering of Faecalibacterium strains in different genospecies. Additionally, the global analysis of gene synteny among all strains showed a highly fragmented profile, whereas the intra-cluster analyses revealed larger and more conserved collinear blocks. Finally, ANI analysis substantiated the presence of three distinct clusters-A, B, and C-composed of five, four, and four strains, respectively. The pangenome analysis of each cluster corroborated the classification of these clusters into three distinct species, each containing less variability than that found within the global pangenome of all strains. Here, we propose that comparison of pangenome subsets and their associated α values may be used as an alternative approach, together with ANI, in the in silico classification of new species. Altogether, our results provide evidence not only for the reconsideration of the phylogenetic and genomic relatedness among strains currently assigned to F. prausnitzii , but also the need for lineage (strain-based) differentiation of this taxon to better define how specific members might be associated with positive or negative host interactions.

An Identity-Based Anti-Quantum Privacy-Preserving Blind Authentication in Wireless Sensor Networks.

PubMed

Zhu, Hongfei; Tan, Yu-An; Zhu, Liehuang; Wang, Xianmin; Zhang, Quanxin; Li, Yuanzhang

2018-05-22

With the development of wireless sensor networks, IoT devices are crucial for the Smart City; these devices change people's lives such as e-payment and e-voting systems. However, in these two systems, the state-of-art authentication protocols based on traditional number theory cannot defeat a quantum computer attack. In order to protect user privacy and guarantee trustworthy of big data, we propose a new identity-based blind signature scheme based on number theorem research unit lattice, this scheme mainly uses a rejection sampling theorem instead of constructing a trapdoor. Meanwhile, this scheme does not depend on complex public key infrastructure and can resist quantum computer attack. Then we design an e-payment protocol using the proposed scheme. Furthermore, we prove our scheme is secure in the random oracle, and satisfies confidentiality, integrity, and non-repudiation. Finally, we demonstrate that the proposed scheme outperforms the other traditional existing identity-based blind signature schemes in signing speed and verification speed, outperforms the other lattice-based blind signature in signing speed, verification speed, and signing secret key size.

An Identity-Based Anti-Quantum Privacy-Preserving Blind Authentication in Wireless Sensor Networks

PubMed Central

Zhu, Hongfei; Tan, Yu-an; Zhu, Liehuang; Wang, Xianmin; Zhang, Quanxin; Li, Yuanzhang

2018-01-01

With the development of wireless sensor networks, IoT devices are crucial for the Smart City; these devices change people’s lives such as e-payment and e-voting systems. However, in these two systems, the state-of-art authentication protocols based on traditional number theory cannot defeat a quantum computer attack. In order to protect user privacy and guarantee trustworthy of big data, we propose a new identity-based blind signature scheme based on number theorem research unit lattice, this scheme mainly uses a rejection sampling theorem instead of constructing a trapdoor. Meanwhile, this scheme does not depend on complex public key infrastructure and can resist quantum computer attack. Then we design an e-payment protocol using the proposed scheme. Furthermore, we prove our scheme is secure in the random oracle, and satisfies confidentiality, integrity, and non-repudiation. Finally, we demonstrate that the proposed scheme outperforms the other traditional existing identity-based blind signature schemes in signing speed and verification speed, outperforms the other lattice-based blind signature in signing speed, verification speed, and signing secret key size. PMID:29789475

Adaptive Numerical Dissipative Control in High Order Schemes for Multi-D Non-Ideal MHD

NASA Technical Reports Server (NTRS)

Yee, H. C.; Sjoegreen, B.

2004-01-01

The goal is to extend our adaptive numerical dissipation control in high order filter schemes and our new divergence-free methods for ideal MHD to non-ideal MHD that include viscosity and resistivity. The key idea consists of automatic detection of different flow features as distinct sensors to signal the appropriate type and amount of numerical dissipation/filter where needed and leave the rest of the region free of numerical dissipation contamination. These scheme-independent detectors are capable of distinguishing shocks/shears, flame sheets, turbulent fluctuations and spurious high-frequency oscillations. The detection algorithm is based on an artificial compression method (ACM) (for shocks/shears), and redundant multi-resolution wavelets (WAV) (for the above types of flow feature). These filter approaches also provide a natural and efficient way for the minimization of Div(B) numerical error. The filter scheme consists of spatially sixth order or higher non-dissipative spatial difference operators as the base scheme for the inviscid flux derivatives. If necessary, a small amount of high order linear dissipation is used to remove spurious high frequency oscillations. For example, an eighth-order centered linear dissipation (AD8) might be included in conjunction with a spatially sixth-order base scheme. The inviscid difference operator is applied twice for the viscous flux derivatives. After the completion of a full time step of the base scheme step, the solution is adaptively filtered by the product of a 'flow detector' and the 'nonlinear dissipative portion' of a high-resolution shock-capturing scheme. In addition, the scheme independent wavelet flow detector can be used in conjunction with spatially compact, spectral or spectral element type of base schemes. The ACM and wavelet filter schemes using the dissipative portion of a second-order shock-capturing scheme with sixth-order spatial central base scheme for both the inviscid and viscous MHD flux derivatives and a fourth-order Runge-Kutta method are denoted.

Error function attack of chaos synchronization based encryption schemes.

PubMed

Wang, Xingang; Zhan, Meng; Lai, C-H; Gang, Hu

2004-03-01

Different chaos synchronization based encryption schemes are reviewed and compared from the practical point of view. As an efficient cryptanalysis tool for chaos encryption, a proposal based on the error function attack is presented systematically and used to evaluate system security. We define a quantitative measure (quality factor) of the effective applicability of a chaos encryption scheme, which takes into account the security, the encryption speed, and the robustness against channel noise. A comparison is made of several encryption schemes and it is found that a scheme based on one-way coupled chaotic map lattices performs outstandingly well, as judged from quality factor. Copyright 2004 American Institute of Physics.

Security analysis and enhancements of an effective biometric-based remote user authentication scheme using smart cards.

PubMed

An, Younghwa

2012-01-01

Recently, many biometrics-based user authentication schemes using smart cards have been proposed to improve the security weaknesses in user authentication system. In 2011, Das proposed an efficient biometric-based remote user authentication scheme using smart cards that can provide strong authentication and mutual authentication. In this paper, we analyze the security of Das's authentication scheme, and we have shown that Das's authentication scheme is still insecure against the various attacks. Also, we proposed the enhanced scheme to remove these security problems of Das's authentication scheme, even if the secret information stored in the smart card is revealed to an attacker. As a result of security analysis, we can see that the enhanced scheme is secure against the user impersonation attack, the server masquerading attack, the password guessing attack, and the insider attack and provides mutual authentication between the user and the server.

Security Analysis and Enhancements of an Effective Biometric-Based Remote User Authentication Scheme Using Smart Cards

PubMed Central

An, Younghwa

2012-01-01

Recently, many biometrics-based user authentication schemes using smart cards have been proposed to improve the security weaknesses in user authentication system. In 2011, Das proposed an efficient biometric-based remote user authentication scheme using smart cards that can provide strong authentication and mutual authentication. In this paper, we analyze the security of Das's authentication scheme, and we have shown that Das's authentication scheme is still insecure against the various attacks. Also, we proposed the enhanced scheme to remove these security problems of Das's authentication scheme, even if the secret information stored in the smart card is revealed to an attacker. As a result of security analysis, we can see that the enhanced scheme is secure against the user impersonation attack, the server masquerading attack, the password guessing attack, and the insider attack and provides mutual authentication between the user and the server. PMID:22899887

An efficient chaotic maps-based authentication and key agreement scheme using smartcards for telecare medicine information systems.

PubMed

Lee, Tian-Fu

2013-12-01

A smartcard-based authentication and key agreement scheme for telecare medicine information systems enables patients, doctors, nurses and health visitors to use smartcards for secure login to medical information systems. Authorized users can then efficiently access remote services provided by the medicine information systems through public networks. Guo and Chang recently improved the efficiency of a smartcard authentication and key agreement scheme by using chaotic maps. Later, Hao et al. reported that the scheme developed by Guo and Chang had two weaknesses: inability to provide anonymity and inefficient double secrets. Therefore, Hao et al. proposed an authentication scheme for telecare medicine information systems that solved these weaknesses and improved performance. However, a limitation in both schemes is their violation of the contributory property of key agreements. This investigation discusses these weaknesses and proposes a new smartcard-based authentication and key agreement scheme that uses chaotic maps for telecare medicine information systems. Compared to conventional schemes, the proposed scheme provides fewer weaknesses, better security, and more efficiency.

Lightweight ECC based RFID authentication integrated with an ID verifier transfer protocol.

PubMed

He, Debiao; Kumar, Neeraj; Chilamkurti, Naveen; Lee, Jong-Hyouk

2014-10-01

The radio frequency identification (RFID) technology has been widely adopted and being deployed as a dominant identification technology in a health care domain such as medical information authentication, patient tracking, blood transfusion medicine, etc. With more and more stringent security and privacy requirements to RFID based authentication schemes, elliptic curve cryptography (ECC) based RFID authentication schemes have been proposed to meet the requirements. However, many recently published ECC based RFID authentication schemes have serious security weaknesses. In this paper, we propose a new ECC based RFID authentication integrated with an ID verifier transfer protocol that overcomes the weaknesses of the existing schemes. A comprehensive security analysis has been conducted to show strong security properties that are provided from the proposed authentication scheme. Moreover, the performance of the proposed authentication scheme is analyzed in terms of computational cost, communicational cost, and storage requirement.

Searchable attribute-based encryption scheme with attribute revocation in cloud storage.

PubMed

Wang, Shangping; Zhao, Duqiao; Zhang, Yaling

2017-01-01

Attribute based encryption (ABE) is a good way to achieve flexible and secure access control to data, and attribute revocation is the extension of the attribute-based encryption, and the keyword search is an indispensable part for cloud storage. The combination of both has an important application in the cloud storage. In this paper, we construct a searchable attribute-based encryption scheme with attribute revocation in cloud storage, the keyword search in our scheme is attribute based with access control, when the search succeeds, the cloud server returns the corresponding cipher text to user and the user can decrypt the cipher text definitely. Besides, our scheme supports multiple keywords search, which makes the scheme more practical. Under the assumption of decisional bilinear Diffie-Hellman exponent (q-BDHE) and decisional Diffie-Hellman (DDH) in the selective security model, we prove that our scheme is secure.

Simple scheme to implement decoy-state reference-frame-independent quantum key distribution

NASA Astrophysics Data System (ADS)

Zhang, Chunmei; Zhu, Jianrong; Wang, Qin

2018-06-01

We propose a simple scheme to implement decoy-state reference-frame-independent quantum key distribution (RFI-QKD), where signal states are prepared in Z, X, and Y bases, decoy states are prepared in X and Y bases, and vacuum states are set to no bases. Different from the original decoy-state RFI-QKD scheme whose decoy states are prepared in Z, X and Y bases, in our scheme decoy states are only prepared in X and Y bases, which avoids the redundancy of decoy states in Z basis, saves the random number consumption, simplifies the encoding device of practical RFI-QKD systems, and makes the most of the finite pulses in a short time. Numerical simulations show that, considering the finite size effect with reasonable number of pulses in practical scenarios, our simple decoy-state RFI-QKD scheme exhibits at least comparable or even better performance than that of the original decoy-state RFI-QKD scheme. Especially, in terms of the resistance to the relative rotation of reference frames, our proposed scheme behaves much better than the original scheme, which has great potential to be adopted in current QKD systems.

Work, Train, Win: Work-Based Learning Design and Management for Productivity Gains. OECD Education Working Papers, No. 135

ERIC Educational Resources Information Center

Kis, Viktoria

2016-01-01

Realising the potential of work-based learning schemes as a driver of productivity requires careful design and support. The length of work-based learning schemes should be adapted to the profile of productivity gains. A scheme that is too long for a given skill set might be unattractive for learners and waste public resources, but a scheme that is…

Cryptanalysis and improvement of Yan et al.'s biometric-based authentication scheme for telecare medicine information systems.

PubMed

Mishra, Dheerendra; Mukhopadhyay, Sourav; Chaturvedi, Ankita; Kumari, Saru; Khan, Muhammad Khurram

2014-06-01

Remote user authentication is desirable for a Telecare Medicine Information System (TMIS) for the safety, security and integrity of transmitted data over the public channel. In 2013, Tan presented a biometric based remote user authentication scheme and claimed that his scheme is secure. Recently, Yan et al. demonstrated some drawbacks in Tan's scheme and proposed an improved scheme to erase the drawbacks of Tan's scheme. We analyze Yan et al.'s scheme and identify that their scheme is vulnerable to off-line password guessing attack, and does not protect anonymity. Moreover, in their scheme, login and password change phases are inefficient to identify the correctness of input where inefficiency in password change phase can cause denial of service attack. Further, we design an improved scheme for TMIS with the aim to eliminate the drawbacks of Yan et al.'s scheme.

An Improvement of Robust and Efficient Biometrics Based Password Authentication Scheme for Telecare Medicine Information Systems Using Extended Chaotic Maps.

PubMed

Moon, Jongho; Choi, Younsung; Kim, Jiye; Won, Dongho

2016-03-01

Recently, numerous extended chaotic map-based password authentication schemes that employ smart card technology were proposed for Telecare Medical Information Systems (TMISs). In 2015, Lu et al. used Li et al.'s scheme as a basis to propose a password authentication scheme for TMISs that is based on biometrics and smart card technology and employs extended chaotic maps. Lu et al. demonstrated that Li et al.'s scheme comprises some weaknesses such as those regarding a violation of the session-key security, a vulnerability to the user impersonation attack, and a lack of local verification. In this paper, however, we show that Lu et al.'s scheme is still insecure with respect to issues such as a violation of the session-key security, and that it is vulnerable to both the outsider attack and the impersonation attack. To overcome these drawbacks, we retain the useful properties of Lu et al.'s scheme to propose a new password authentication scheme that is based on smart card technology and requires the use of chaotic maps. Then, we show that our proposed scheme is more secure and efficient and supports security properties.

A privacy preserving secure and efficient authentication scheme for telecare medical information systems.

PubMed

Mishra, Raghavendra; Barnwal, Amit Kumar

2015-05-01

The Telecare medical information system (TMIS) presents effective healthcare delivery services by employing information and communication technologies. The emerging privacy and security are always a matter of great concern in TMIS. Recently, Chen at al. presented a password based authentication schemes to address the privacy and security. Later on, it is proved insecure against various active and passive attacks. To erase the drawbacks of Chen et al.'s anonymous authentication scheme, several password based authentication schemes have been proposed using public key cryptosystem. However, most of them do not present pre-smart card authentication which leads to inefficient login and password change phases. To present an authentication scheme with pre-smart card authentication, we present an improved anonymous smart card based authentication scheme for TMIS. The proposed scheme protects user anonymity and satisfies all the desirable security attributes. Moreover, the proposed scheme presents efficient login and password change phases where incorrect input can be quickly detected and a user can freely change his password without server assistance. Moreover, we demonstrate the validity of the proposed scheme by utilizing the widely-accepted BAN (Burrows, Abadi, and Needham) logic. The proposed scheme is also comparable in terms of computational overheads with relevant schemes.

U.S. EPA, Pesticide Product Label, NALCO VISCO 3990, 05/24/1989

EPA Pesticide Factsheets

2011-04-19

... MaJI., , •• hllc. I .. ",, s~.nl If ""IS ,utlcUI. I"., .lxI"I, ., '11I.t. is , .il,ti ••• f f ••• ". L,.. If ti.st Mlst.s c .... t ••• Is ,.S ••• , " c.,.i., t. 1 ••• 1 ilSt"ct ... 'IS,'" ., il •••• i tart ... ...

Suppurative peritonitis by Klebsiella pneumoniae in captive gold-handed tamarin (Saguinus midas midas).

PubMed

Guerra, Maria F L; Teixeira, Rodrigo H F; Ribeiro, Vanessa L; Cunha, Marcos P V; Oliveira, Maria G X; Davies, Yamê M; Silva, Ketrin C; Silva, Ana P S; Lincopan, Nilton; Moreno, Andrea M; Knöbl, Terezinha

2016-02-01

This report describes an outbreak of suppurative peritonitis caused by Klebsiella pneumoniae in an adult female of captive golden-handed tamarin (Saguinus midas midas). Two virulent and multidrug-resistant strains were isolated and classified through MLST as ST60 and ST1263. The microbiological diagnosis works as a support tool for preventive measures. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

Wolbachia endosymbionts in haplodiploid and diploid scolytine beetles (Coleoptera: Curculionidae: Scolytinae).

PubMed

Kawasaki, Yuuki; Schuler, Hannes; Stauffer, Christian; Lakatos, Ferenc; Kajimura, Hisashi

2016-05-19

Haplodiploidy is a sex determination system in which fertilized diploid eggs develop into females and unfertilized haploid eggs develop into males. The evolutionary explanations for this phenomenon include the possibility that haplodiploidy can be reinforced by infection with endosymbiotic bacteria, such as Wolbachia. The subfamily Scolytinae contains species with haplodiploid and diploid sex determination systems. Thus, we studied the association with Wolbachia in 12 diploid and 11 haplodiploid scolytine beetles by analyzing wsp and multilocus sequence typing (MLST) of five loci in this endosymbiont. Wolbachia genotypes were compared with mitochondrial (COI) and nuclear (EF) genotypes in the scolytines. Eight of the 23 scolytine species were infected with Wolbachia, with haplodiploids at significantly higher rates than diploid species. Cloning and sequencing detected multiple infections with up to six Wolbachia strains in individual species. Phylogenetic analyses of wsp and five MLST genes revealed different Wolbachia strains in scolytines. Comparisons between the beetle and Wolbachia phylogenies revealed that closely related beetles were infected with genetically different Wolbachia strains. These results suggest the horizontal transmission of multiple Wolbachia strains between scolytines. We discuss these results in terms of the evolution of different sex determination systems in scolytine beetles. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Strains of Pseudomonas syringae pv. syringae from pea are phylogenetically and pathogenically diverse.

PubMed

Martín-Sanz, Alberto; de la Vega, Marcelino Pérez; Murillo, Jesús; Caminero, Constantino

2013-07-01

Pseudomonas syringae pv. syringae causes extensive yield losses in the pea crop worldwide, although there is little information on its host specialization and its interactions with pea. A collection of 88 putative P. syringae pv. syringae strains (including 39 strains isolated from pea) was characterized by repetitive polymerase chain reaction (rep-PCR), multilocus sequence typing (MLST), and syrB amplification and evaluated for pathogenicity and virulence. rep-PCR data grouped the strains from pea into two groups (1B and 1C) together with strains from other hosts; a third group (1A) was formed exclusively with strains isolated from non-legume species. MLST data included all strains from pea in the genomospecies 1 of P. syringae pathovars defined in previous studies; they were distributed in the same three groups defined by rep-PCR. The inoculations performed in two pea cultivars showed that P. syringae pv. syringae strains from groups 1A and 1C were less virulent than strains from group 1B, suggesting a possible pathogenic specialization in this group. This study shows the existence of genetically and pathogenically distinct P. syringae pv. syringae strain groups from pea, which will be useful for the diagnostic and epidemiology of this pathogen and for disease resistance breeding.

Molecular Epidemiology of Invasive Listeriosis due to Listeria monocytogenes in a Spanish Hospital over a Nine-Year Study Period, 2006-2014.

PubMed

Ariza-Miguel, Jaime; Fernández-Natal, María Isabel; Soriano, Francisco; Hernández, Marta; Stessl, Beatrix; Rodríguez-Lázaro, David

2015-01-01

We investigated the pathogenicity, invasiveness, and genetic relatedness of 17 clinical Listeria monocytogenes stains isolated over a period of nine years (2006-2014). All isolates were phenotypically characterised and growth patterns were determined. The antimicrobial susceptibility of L. monocytogenes isolates was determined in E-tests. Invasion assays were performed with epithelial HeLa cells. Finally, L. monocytogenes isolates were subtyped by PFGE and MLST. All isolates had similar phenotypic characteristics (β-haemolysis and lecithinase activity), and three types of growth curve were observed. Bacterial recovery rates after invasion assays ranged from 0.09% to 7.26% (1.62 ± 0.46). MLST identified 11 sequence types (STs), and 14 PFGE profiles were obtained, indicating a high degree of genetic diversity. Genetic studies unequivocally revealed the occurrence of one outbreak of listeriosis in humans that had not previously been reported. This outbreak occurred in October 2009 and affected three patients from neighbouring towns. In conclusion, the molecular epidemiological analysis clearly revealed a cluster (three human cases, all ST1) of not previously reported listeriosis cases in northwestern Spain. Our findings indicate that molecular subtyping, in combination with epidemiological case analysis, is essential and should be implemented in routine diagnosis, to improve the tracing of the sources of outbreaks.

Increase of genetic diversity and clonal replacement of epidemic methicillin-resistant Staphylococcus aureus strains in South-East Austria.

PubMed

Zarfel, Gernot; Luxner, Josefa; Folli, Bettina; Leitner, Eva; Feierl, Gebhard; Kittinger, Clemens; Grisold, Andrea

2016-07-01

Spa-typing and microarray techniques were used to study epidemiological changes in methicillin-resistant Staphylococcus aureus (MRSA) in South-East Austria. The population structure of 327 MRSA isolated between 2002 and 2012 was investigated. MRSA was assigned to 58 different spa types and 14 different MLST CC (multilocus sequence type clonal complexes); in particular, between 2007 and 2012, an increasing diversity in MRSA clones could be observed. The most abundant clonal complex was CC5. On the respective SCCmec cassettes, the CC5 isolates differed clearly within this decade and CC5/SCCmecI, the South German MRSA, predominant in 2002, was replaced by CC5/SCCmecII, the Rhine-Hesse MRSA in 2012. Whereas in many European countries MLST CC22-MRSA (EMRSA 15, the Barnim epidemic MRSA) is predominant, this clone occurred in Austria nearly 10 years later than in neighbouring countries. CC45, the Berlin EMRSA, epidemic in Germany, was only sporadically found in South-East Austria. The Irish ST8-MRSA-II represented by spa-type t190 was frequently found in 2002 and 2007, but disappeared in 2012. Our results demonstrate clonal replacement of MRSA clones within the last years in Austria. Ongoing surveillance is warranted for detection of changes within the MRSA population. © FEMS 2016.

Occurrence of Carbapenemase-Producing Enterobacteriaceae Isolates in the Wildlife: First Report of OXA-48 in Wild Boars in Algeria.

PubMed

Bachiri, Taous; Bakour, Sofiane; Lalaoui, Rym; Belkebla, Nadia; Allouache, Meriem; Rolain, Jean Marc; Touati, Abdelaziz

2018-04-01

The aim of the present study was to screen for the presence of carbapenemase-producing Enterobacteriaceae (CPE) isolates from wild boars and Barbary macaques in Algeria. Fecal samples were collected from wild boars (n = 168) and Barbary macaques (n = 212), in Bejaia, Algeria, between September 2014 and April 2016. The isolates were identified and antimicrobial susceptibility was determined. Carbapenem resistance determinants were studied using PCR and sequencing, while clonal relatedness was performed using multilocus sequence typing (MLST). PCR was used to investigate certain virulence genes. Three CPE isolates from three different samples (1.8%) recovered from wild boars were identified as Escherichia coli (two isolates) and Klebsiella pneumoniae (one isolate). These isolates were resistant to amoxicillin, amoxicillin-clavulanate, tobramycin, ertapenem, and meropenem. The results of PCR and sequencing analysis showed that all three isolates produced the OXA-48 enzyme. The MLST showed that the two E. coli isolates were assigned to the same sequence type, ST635, and belonged to phylogroup A, whereas K. pneumoniae strain belonged to ST13. The K. pneumoniae strain was positive for multiple virulence factors, whereas no virulence determinants were found in E. coli isolates. This is the first report of OXA-48-producing Enterobacteriaceae in wild animals from Algeria and Africa.

[Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b].

PubMed

Platonov, A E; Mironov, K O; Iatsyshina, S B; Koroleva, I S; Platonova, O V; Gushchin, A E; Shipulin, G A

2003-01-01

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.

Analysis of group B streptococcal isolates from infants and pregnant women in Portugal revealing two lineages with enhanced invasiveness.

PubMed

Martins, E R; Pessanha, M A; Ramirez, M; Melo-Cristino, J

2007-10-01

The populations of group B streptococcus (GBS) associated with vaginal carriage in pregnant women and invasive neonatal infections in Portugal were compared. GBS isolates were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) profiling, and multilocus sequence typing (MLST). Serotypes III and V accounted for 44% of all colonization isolates (n = 269), whereas serotypes III and Ia amounted to 69% of all invasive isolates (n = 64). Whereas serotype Ia was associated with early-onset disease (EOD), serotype III was associated with late-onset disease (LOD). Characterization by PFGE and MLST identified very diverse populations in carriage and invasive disease. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and the infrequent ST24. In contrast, serotype III was found in a large number of PFGE clusters and STs, but a single PFGE cluster defined by ST17 was found to be associated with invasive disease. Although serotype III was associated only with LOD, ST17 showed an enhanced capacity to cause both EOD and LOD. Our data reinforce the evidence for enhanced invasiveness of ST17 and identify a lineage expressing serotype Ia capsule and represented by ST23 and ST24 as having enhanced potential to cause EOD.

An outbreak of pneumonia associated with S. pneumoniae at a military training facility in Finland in 2006.

PubMed

Vainio, Anni; Lyytikäinen, Outi; Sihvonen, Reetta; Kaijalainen, Tarja; Teirilä, Laura; Rantala, Merja; Lehtinen, Pirkko; Ruuska, Pekka; Virolainen, Anni

2009-07-01

Streptococcus pneumoniae is a well-known cause of community-acquired bacterial pneumonia. The purpose of this study was to assess the cause and extent of the outbreak of pneumonia which occurred among military recruits following a 1-week hard encampment in Finland. We also assessed the carriage rate and molecular characteristics of the S. pneumoniae isolates. All pneumococcal isolates were studied for antibiotic susceptibility, serotyped, genotyped by multilocus sequence typing (MLST), and the presence of pneumococcal rlrA pilus islet was detected. The genotype results defined by MLST corresponded with the serotype results. S. pneumoniae serotype 7F, ST2331, seemed to be associated with an outbreak of pneumonia and nasopharyngeal carriage among 43 military recruits. Of the 43 military recruits, five (12%) were hospitalized with pneumonia and two (40%) of them were positive for S. pneumoniae serotype 7F, ST2331 by blood culture. Eighteen (42%) of the 43 men were found to be positive for S. pneumoniae by nasopharyngeal culture, and nine (50%) of them carried pneumococcal serotype 7F, ST2331. The outbreak strain covered 55% of all the pneumococcal findings. Outbreaks of invasive pneumococcal disease seem to occur in a crowded environment such as a military training facility even among previously healthy young men.

Molecular epidemiology, antimicrobial susceptibility, and characterization of macrolide-resistant Streptococcus pyogenes in Japan.

PubMed

Tanaka, Yuhei; Gotoh, Kenji; Teramachi, Mariko; Ishimoto, Kazuhisa; Tsumura, Naoki; Shindou, Shizuo; Yamashita, Yushiro

2016-11-01

Here we report the molecular epidemiology of macrolide-resistant Streptococcus pyogenes (group A streptococci, GAS) isolated from children with pharyngotonsillitis between 2011 and 2013 in Japan. In 299 isolates, 124 (41.5%) isolates were macrolide-resistant. We characterized the isolates by emm typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Of 299 isolates, 124 (41.5%) were macrolide-resistant isolates, 76 (61.3%) possessed mefA and 46 (37.1%) possessed ermB. All 76 isolates with mefA possessed msrD. There were no isolates possessed ermTR in this study. Eight emm/MLST types were observed. The predominant type was emm1/ST28 (57 isolates, 46.0%), which possessed the mefA/msrD complex, presenting as the M phenotype. The second most predominant type was emm12/ST467, which possessed ermB, presenting as the cMLS B phenotype. Of the cMLS B phenotype isolates, types emm28/ST52 and emm12/ST36 had multiple genetic backgrounds. We found high proportions of macrolide-resistant GAS in the southwestern areas of Japan. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Antimicrobial Resistance and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus Isolates from Child Patients of High-Risk Wards in Shenzhen, China.

PubMed

Qin, Yang; Wen, Feiqiu; Zheng, Yuejie; Zhao, Ruizhen; Hu, Qinghua; Zhang, Renli

2017-09-25

Methicillin-resistant Staphylococcus aureus (MRSA) strains are responsible for high rates of mortality and thus pose a substantial burden to public health worldwide. Here, we investigated the antimicrobial susceptibility and molecular characteristics of MRSA isolated from child patients at Shenzhen Children's Hospital. We characterized 140 MRSA strains through antimicrobial susceptibility testing. We further performed spa typing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) analysis, pvl gene analysis, and pulsed-field gel electrophoresis (PFGE). The analyzed MRSA strains were found to be sensitive to most non-β-lactam antimicrobial agents. Sequence type (ST) 59 was found to be the most common MLST lineage (54.3%). Most MRSA isolates belonged to the SCCmec IV (64.3%) and V (22.8%) types. The MRSA-ST59-SCCmec IV-t437 clone was the most predominant strain that infected 28.6% of all patients studied. Moreover, 50.7% of MRSA isolates were found to be pvl-positive. We report preliminary data on the prevalence and distribution of MRSA genotypes in Shenzhen Children's Hospital. We characterized MRSA colonization dynamics in child patients in China, and our findings can serve as the basis for the development of strategies to prevent MRSA infection and transmission.

Tropism and virulence of Cutibacterium (formerly Propionibacterium) acnes involved in implant-associated infection.

PubMed

Aubin, Guillaume Ghislain; Lavigne, Jean-Philippe; Foucher, Yohan; Dellière, Sarah; Lepelletier, Didier; Gouin, François; Corvec, Stéphane

2017-10-01

The recognition of the pathogenicity of Cutibacterium acnes in implant-associated infection is not always obvious. In this paper, we aimed to distinguish pathogenic and non-pathogenic C. acnes isolates. To reach this goal, we investigated the clonal complex (CC) of a large collection of C. acnes clinical isolates through Multi-Locus Sequence Typing (MLST), we established a Caenorhabditis elegans model to assess C. acnes virulence and we investigated the presence of virulence factors in our collection. Ours results showed that CC36 and CC53 C. acnes isolates were more frequently observed in prosthetic joint infections (PJI) than CC18 and CC28 C. acnes isolates (p = 0.021). The C. elegans model developed here showed two distinct virulence groups of C. acnes (p < 0.05). These groups were not correlated to CC or clinical origin. Whole genome sequencing allowed us to identify a putative gene linked to low virulent strains. In conclusion, MLST remains a good method to screen pathogenic C. acnes isolates according to their clinical context but mechanisms of C. acnes virulence need to be assess thought transcriptomic analysis to investigate regulatory process. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular epidemiological characteristics of Salmonella enterica serovars Enteritidis, Typhimurium and Livingstone strains isolated in a Tunisian university hospital.

PubMed

Ktari, Sonia; Ksibi, Boutheina; Gharsallah, Houda; Mnif, Basma; Maalej, Sonda; Rhimi, Fouzia; Hammami, Adnene

2016-03-01

Enteritidis, Typhimurium and Livingstone are the main Salmonella enterica serovars recovered in Tunisia. Here, we aimed to assess the genetic diversity of fifty-seven Salmonella enterica strains from different sampling periods, origins and settings using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA). Salmonella Enteritidis, isolated from human and food sources from two regions in Sfax in 2007, were grouped into one cluster using PFGE. However, using MLVA these strains were divided into two clusters. Salmonella Typhimurium strains, recovered in 2012 and represent sporadic cases of human clinical isolates, were included in one PFGE cluster. Nevertheless, the MLVA technique, divided Salmonella Typhimurium isolates into six clusters with diversity index reaching (DI = 0.757). For Salmonella Livingstone which was responsible of two nosocomial outbreaks during 2000-2003, the PFGE and MLVA methods showed that these strains were genetically closely related. Salmonella Enteritidis and Salmonella Livingstone populations showed a single ST lineage ST11 and ST543 respectively. For Salmonella Typhimurium, two MLST sequence types ST19 and ST328 were defined. Salmonella Enteritidis and Salmonella Typhimurium strains were clearly differentiated by MLVA which was not the case using PFGE. © 2015 APMIS. Published by John Wiley & Sons Ltd.

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.

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.

Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates

PubMed Central

Bier, Nadja; Jäckel, Claudia; Dieckmann, Ralf; Brennholt, Nicole; Böer, Simone I.; Strauch, Eckhard

2015-01-01

Vibrio vulnificus is a halophilic bacterium of coastal environments known for sporadically causing severe foodborne or wound infections. Global warming is expected to lead to a rising occurrence of V. vulnificus and an increasing incidence of human infections in Northern Europe. So far, infections in Germany were exclusively documented for the Baltic Sea coast, while no cases from the North Sea region have been reported. Regional variations in the prevalence of infections may be influenced by differences in the pathogenicity of V. vulnificus populations in both areas. This study aimed to compare the distribution of virulence-associated traits and genotypes among 101 V. vulnificus isolates from the Baltic Sea and North Sea in order to assess their pathogenicity potential. Furthermore, genetic relationships were examined by multilocus sequence typing (MLST). A high diversity of MLST sequences (74 sequence types) and differences regarding the presence of six potential pathogenicity markers were observed in the V. vulnificus populations of both areas. Strains with genotypes and markers associated with pathogenicity are not restricted to a particular geographic region. This indicates that lack of reported cases in the North Sea region is not caused by the absence of potentially pathogenic strains. PMID:26694432

Use of multilocus sequence typing for the investigation of colonisation by Candida albicans in intensive care unit patients.

PubMed

Cliff, P R; Sandoe, J A T; Heritage, J; Barton, R C

2008-05-01

A prospective study was performed to determine the prevalence of candidal colonisation on the general intensive care unit at a large teaching hospital. Colonisation with Candida spp. was found to be common, occurring in 79% of patients on the unit. C. albicans was the commonest species, colonising 64% of patients, followed by C. glabrata (18%) and C. parapsilosis (14%). Most of the members of staff tested carried Candida spp. at some point, although carriage appeared to be transient. C. parapsilosis was the most commonly isolated species from staff hands, whereas C. albicans was the most commonly isolated species from the mouth. The molecular epidemiology of C. albicans was investigated using Ca3 typing and multilocus sequence typing (MLST). MLST proved to be a reproducible typing method and a useful tool for the investigation of the molecular epidemiology of C. albicans. The results of the molecular typing provided evidence for the presence of an endemic strain on the unit, which was isolated repeatedly from patients and staff. This finding suggests horizontal transmission of C. albicans on the unit though it may also reflect the relative frequency of C. albicans strain types colonising patients on admission. This study has important implications for the epidemiology of systemic candidal infections.

Microbial Genomics of a Host-Associated Commensal Bacterium in Fragmented Populations of Endangered Takahe.

PubMed

Grange, Zoë L; Gartrell, Brett D; Biggs, Patrick J; Nelson, Nicola J; Anderson, Marti; French, Nigel P

2016-05-01

Isolation of wildlife into fragmented populations as a consequence of anthropogenic-mediated environmental change may alter host-pathogen relationships. Our understanding of some of the epidemiological features of infectious disease in vulnerable populations can be enhanced by the use of commensal bacteria as a proxy for invasive pathogens in natural ecosystems. The distinctive population structure of a well-described meta-population of a New Zealand endangered flightless bird, the takahe (Porphyrio hochstetteri), provided a unique opportunity to investigate the influence of host isolation on enteric microbial diversity. The genomic epidemiology of a prevalent rail-associated endemic commensal bacterium was explored using core genome and ribosomal multilocus sequence typing (rMLST) of 70 Campylobacter sp. nova 1 isolated from one third of the takahe population resident in multiple locations. While there was evidence of recombination between lineages, bacterial divergence appears to have occurred and multivariate analysis of 52 rMLST genes revealed location-associated differentiation of C. sp. nova 1 sequence types. Our results indicate that fragmentation and anthropogenic manipulation of populations can influence host-microbial relationships, with potential implications for niche adaptation and the evolution of micro-organisms in remote environments. This study provides a novel framework in which to explore the complex genomic epidemiology of micro-organisms in wildlife populations.

Color encryption scheme based on adapted quantum logistic map

NASA Astrophysics Data System (ADS)

Zaghloul, Alaa; Zhang, Tiejun; Amin, Mohamed; Abd El-Latif, Ahmed A.

2014-04-01

This paper presents a new color image encryption scheme based on quantum chaotic system. In this scheme, a new encryption scheme is accomplished by generating an intermediate chaotic key stream with the help of quantum chaotic logistic map. Then, each pixel is encrypted by the cipher value of the previous pixel and the adapted quantum logistic map. The results show that the proposed scheme has adequate security for the confidentiality of color images.

A Hash Based Remote User Authentication and Authenticated Key Agreement Scheme for the Integrated EPR Information System.

PubMed

Li, Chun-Ta; Weng, Chi-Yao; Lee, Cheng-Chi; Wang, Chun-Cheng

2015-11-01

To protect patient privacy and ensure authorized access to remote medical services, many remote user authentication schemes for the integrated electronic patient record (EPR) information system have been proposed in the literature. In a recent paper, Das proposed a hash based remote user authentication scheme using passwords and smart cards for the integrated EPR information system, and claimed that the proposed scheme could resist various passive and active attacks. However, in this paper, we found that Das's authentication scheme is still vulnerable to modification and user duplication attacks. Thereafter we propose a secure and efficient authentication scheme for the integrated EPR information system based on lightweight hash function and bitwise exclusive-or (XOR) operations. The security proof and performance analysis show our new scheme is well-suited to adoption in remote medical healthcare services.

A Practical and Secure Coercion-Resistant Scheme for Internet Voting

NASA Astrophysics Data System (ADS)

Araújo, Roberto; Foulle, Sébastien; Traoré, Jacques

Juels, Catalano, and Jakobsson (JCJ) proposed at WPES 2005 the first voting scheme that considers real-world threats and that is more realistic for Internet elections. Their scheme, though, has a quadratic work factor and thereby is not efficient for large scale elections. Based on the work of JCJ, Smith proposed an efficient scheme that has a linear work factor. In this paper we first show that Smith's scheme is insecure. Then we present a new coercion-resistant election scheme with a linear work factor that overcomes the flaw of Smith's proposal. Our solution is based on the group signature scheme of Camenisch and Lysyanskaya (Crypto 2004).

[Molecular typing methods for Pasteurella multocida-A review].

PubMed

Peng, Zhong; Liang, Wan; Wu, Bin

2016-10-04

Pasteurella multocida is an important gram-negative pathogenic bacterium that could infect wide ranges of animals. Humans could also be infected by P. multocida via animal bite or scratching. Current typing methods for P. multocida include serological typing methods and molecular typing methods. Of them, serological typing methods are based on immunological assays, which are too complicated for clinical bacteriological studies. However, the molecular methods including multiple PCRs and multilocus sequence typing (MLST) methods are more suitable for bacteriological studies of P. multocida in clinic, with their simple operation, high efficiency and accurate detection compared to the traditional serological typing methods, they are therefore widely used. In the current review, we briefly describe the molecular typing methods for P. multocida. Our aim is to provide a knowledge-foundation for clinical bacteriological investigation especially the molecular investigation for P. multocida.

FIELD TESTS OF GEOGRAPHICALLY-DEPENDENT VS. THRESHOLD-BASED WATERSHED CLASSIFICATION SCHEMES IN THE GREAT LAKES BASIN

EPA Science Inventory

We compared classification schemes based on watershed storage (wetland + lake area/watershed area) and forest fragmentation with a geographically-based classification scheme for two case studies involving 1) Lake Superior tributaries and 2) watersheds of riverine coastal wetlands...

FIELD TESTS OF GEOGRAPHICALLY-DEPENDENT VS. THRESHOLD-BASED WATERSHED CLASSIFICATION SCHEMED IN THE GREAT LAKES BASIN

EPA Science Inventory

We compared classification schemes based on watershed storage (wetland + lake area/watershed area) and forest fragmentation with a geographically-based classification scheme for two case studies involving 1)Lake Superior tributaries and 2) watersheds of riverine coastal wetlands ...

An optimal implicit staggered-grid finite-difference scheme based on the modified Taylor-series expansion with minimax approximation method for elastic modeling

NASA Astrophysics Data System (ADS)

Yang, Lei; Yan, Hongyong; Liu, Hong

2017-03-01

Implicit staggered-grid finite-difference (ISFD) scheme is competitive for its great accuracy and stability, whereas its coefficients are conventionally determined by the Taylor-series expansion (TE) method, leading to a loss in numerical precision. In this paper, we modify the TE method using the minimax approximation (MA), and propose a new optimal ISFD scheme based on the modified TE (MTE) with MA method. The new ISFD scheme takes the advantage of the TE method that guarantees great accuracy at small wavenumbers, and keeps the property of the MA method that keeps the numerical errors within a limited bound at the same time. Thus, it leads to great accuracy for numerical solution of the wave equations. We derive the optimal ISFD coefficients by applying the new method to the construction of the objective function, and using a Remez algorithm to minimize its maximum. Numerical analysis is made in comparison with the conventional TE-based ISFD scheme, indicating that the MTE-based ISFD scheme with appropriate parameters can widen the wavenumber range with high accuracy, and achieve greater precision than the conventional ISFD scheme. The numerical modeling results also demonstrate that the MTE-based ISFD scheme performs well in elastic wave simulation, and is more efficient than the conventional ISFD scheme for elastic modeling.

An effective and secure key-management scheme for hierarchical access control in E-medicine system.

PubMed

Odelu, Vanga; Das, Ashok Kumar; Goswami, Adrijit

2013-04-01

Recently several hierarchical access control schemes are proposed in the literature to provide security of e-medicine systems. However, most of them are either insecure against 'man-in-the-middle attack' or they require high storage and computational overheads. Wu and Chen proposed a key management method to solve dynamic access control problems in a user hierarchy based on hybrid cryptosystem. Though their scheme improves computational efficiency over Nikooghadam et al.'s approach, it suffers from large storage space for public parameters in public domain and computational inefficiency due to costly elliptic curve point multiplication. Recently, Nikooghadam and Zakerolhosseini showed that Wu-Chen's scheme is vulnerable to man-in-the-middle attack. In order to remedy this security weakness in Wu-Chen's scheme, they proposed a secure scheme which is again based on ECC (elliptic curve cryptography) and efficient one-way hash function. However, their scheme incurs huge computational cost for providing verification of public information in the public domain as their scheme uses ECC digital signature which is costly when compared to symmetric-key cryptosystem. In this paper, we propose an effective access control scheme in user hierarchy which is only based on symmetric-key cryptosystem and efficient one-way hash function. We show that our scheme reduces significantly the storage space for both public and private domains, and computational complexity when compared to Wu-Chen's scheme, Nikooghadam-Zakerolhosseini's scheme, and other related schemes. Through the informal and formal security analysis, we further show that our scheme is secure against different attacks and also man-in-the-middle attack. Moreover, dynamic access control problems in our scheme are also solved efficiently compared to other related schemes, making our scheme is much suitable for practical applications of e-medicine systems.

High-Order Central WENO Schemes for Multi-Dimensional Hamilton-Jacobi Equations

NASA Technical Reports Server (NTRS)

Bryson, Steve; Levy, Doron; Biegel, Bryan (Technical Monitor)

2002-01-01

We present new third- and fifth-order Godunov-type central schemes for approximating solutions of the Hamilton-Jacobi (HJ) equation in an arbitrary number of space dimensions. These are the first central schemes for approximating solutions of the HJ equations with an order of accuracy that is greater than two. In two space dimensions we present two versions for the third-order scheme: one scheme that is based on a genuinely two-dimensional Central WENO reconstruction, and another scheme that is based on a simpler dimension-by-dimension reconstruction. The simpler dimension-by-dimension variant is then extended to a multi-dimensional fifth-order scheme. Our numerical examples in one, two and three space dimensions verify the expected order of accuracy of the schemes.

A Quantum Proxy Signature Scheme Based on Genuine Five-qubit Entangled State

NASA Astrophysics Data System (ADS)

Cao, Hai-Jing; Huang, Jun; Yu, Yao-Feng; Jiang, Xiu-Li

2014-09-01

In this paper a very efficient and secure proxy signature scheme is proposed. It is based on controlled quantum teleportation. Genuine five-qubit entangled state functions as quantum channel. The scheme uses the physical characteristics of quantum mechanics to implement delegation, signature and verification. Quantum key distribution and one-time pad are adopted in our scheme, which could guarantee not only the unconditional security of the scheme but also the anonymity of the messages owner.

An improved and effective secure password-based authentication and key agreement scheme using smart cards for the telecare medicine information system.

PubMed

Das, Ashok Kumar; Bruhadeshwar, Bezawada

2013-10-01

Recently Lee and Liu proposed an efficient password based authentication and key agreement scheme using smart card for the telecare medicine information system [J. Med. Syst. (2013) 37:9933]. In this paper, we show that though their scheme is efficient, their scheme still has two security weaknesses such as (1) it has design flaws in authentication phase and (2) it has design flaws in password change phase. In order to withstand these flaws found in Lee-Liu's scheme, we propose an improvement of their scheme. Our improved scheme keeps also the original merits of Lee-Liu's scheme. We show that our scheme is efficient as compared to Lee-Liu's scheme. Further, through the security analysis, we show that our scheme is secure against possible known attacks. In addition, we simulate our scheme for the formal security verification using the widely-accepted AVISPA (Automated Validation of Internet Security Protocols and Applications) tool to show that our scheme is secure against passive and active attacks.

Improving Biometric-Based Authentication Schemes with Smart Card Revocation/Reissue for Wireless Sensor Networks.

PubMed

Moon, Jongho; Lee, Donghoon; Lee, Youngsook; Won, Dongho

2017-04-25

User authentication in wireless sensor networks is more difficult than in traditional networks owing to sensor network characteristics such as unreliable communication, limited resources, and unattended operation. For these reasons, various authentication schemes have been proposed to provide secure and efficient communication. In 2016, Park et al. proposed a secure biometric-based authentication scheme with smart card revocation/reissue for wireless sensor networks. However, we found that their scheme was still insecure against impersonation attack, and had a problem in the smart card revocation/reissue phase. In this paper, we show how an adversary can impersonate a legitimate user or sensor node, illegal smart card revocation/reissue and prove that Park et al.'s scheme fails to provide revocation/reissue. In addition, we propose an enhanced scheme that provides efficiency, as well as anonymity and security. Finally, we provide security and performance analysis between previous schemes and the proposed scheme, and provide formal analysis based on the random oracle model. The results prove that the proposed scheme can solve the weaknesses of impersonation attack and other security flaws in the security analysis section. Furthermore, performance analysis shows that the computational cost is lower than the previous scheme.

Improving Biometric-Based Authentication Schemes with Smart Card Revocation/Reissue for Wireless Sensor Networks

PubMed Central

Moon, Jongho; Lee, Donghoon; Lee, Youngsook; Won, Dongho

2017-01-01

User authentication in wireless sensor networks is more difficult than in traditional networks owing to sensor network characteristics such as unreliable communication, limited resources, and unattended operation. For these reasons, various authentication schemes have been proposed to provide secure and efficient communication. In 2016, Park et al. proposed a secure biometric-based authentication scheme with smart card revocation/reissue for wireless sensor networks. However, we found that their scheme was still insecure against impersonation attack, and had a problem in the smart card revocation/reissue phase. In this paper, we show how an adversary can impersonate a legitimate user or sensor node, illegal smart card revocation/reissue and prove that Park et al.’s scheme fails to provide revocation/reissue. In addition, we propose an enhanced scheme that provides efficiency, as well as anonymity and security. Finally, we provide security and performance analysis between previous schemes and the proposed scheme, and provide formal analysis based on the random oracle model. The results prove that the proposed scheme can solve the weaknesses of impersonation attack and other security flaws in the security analysis section. Furthermore, performance analysis shows that the computational cost is lower than the previous scheme. PMID:28441331

Phenotypic and molecular characterization of antimicrobial resistance in Enterobacter spp. isolates from companion animals in Japan.

PubMed

Harada, Kazuki; Shimizu, Takae; Mukai, Yujiro; Kuwajima, Ken; Sato, Tomomi; Kajino, Akari; Usui, Masaru; Tamura, Yutaka; Kimura, Yui; Miyamoto, Tadashi; Tsuyuki, Yuzo; Ohki, Asami; Kataoka, Yasushi

2017-01-01

The emergence of antimicrobial resistance among Enterobacter spp., including resistance to extended-spectrum cephalosporins (ESC), is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance among 60 isolates of Enterobacter spp., including E. cloacae (n = 44), E. aerogenes (n = 10), and E. asburiae (n = 6), from clinical specimens of dogs and cats from 15 prefectures in Japan. Furthermore, we characterized the resistance mechanisms harbored by these isolates, including extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR); and assessed the genetic relatedness of ESC-resistant Enterobacter spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated the resistance rates to ampicillin (93.3%), amoxicillin-clavulanic acid (93.3%), cefmetazole (93.3%), chloramphenicol (46.7%), ciprofloxacin (43.3%), tetracycline (40.0%), ceftazidime (33.3%), cefotaxime (33.3%), trimethoprim/sulfamethoxazole (28.3%), gentamicin (23.3%), and meropenem (0%). Phenotypic testing detected ESBLs in 16 of 18 ESC-resistant E. cloacae isolates but not in the other species. The most frequent ESBL was CTX-M-15 (n = 8), followed by SHV-12 (n = 7), and CTX-M-3 (n = 1). As for AmpC β-lactamases, CMY-2 (n = 2) and DHA-1 (n = 2) were identified in ESC-resistant E. cloacae strains with or without ESBLs. All of the ESC-resistant E. cloacae strains also harbored one or two PMQRs, including qnrB (n = 15), aac(6')-Ib-cr (n = 8), and qnrS (n = 2). Based on MLST and PFGE analysis, E. cloacae clones of ST591-SHV-12, ST171-CTX-M-15, and ST121-CTX-M-15 were detected in one or several hospitals. These results suggested intra- and inter-hospital dissemination of E. cloacae clones co-harboring ESBLs and PMQRs among companion animals. This is the first report on the large-scale monitoring of antimicrobial-resistant isolates of Enterobacter spp. from companion animals in Japan.

MALDI-TOF MS as a Tool To Detect a Nosocomial Outbreak of Extended-Spectrum-β-Lactamase- and ArmA Methyltransferase-Producing Enterobacter cloacae Clinical Isolates in Algeria

PubMed Central

Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi

2015-01-01

Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6′)-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France. PMID:26239991

Molecular and comparative analysis of Salmonella enterica Senftenberg from humans and animals using PFGE, MLST and NARMS

PubMed Central

2011-01-01

Background Salmonella species are recognized worldwide as a significant cause of human and animal disease. In this study the molecular profiles and characteristics of Salmonella enterica Senftenberg isolated from human cases of illness and those recovered from healthy or diagnostic cases in animals were assessed. Included in the study was a comparison with our own sequenced strain of S. Senfteberg recovered from production turkeys in North Dakota. Isolates examined in this study were subjected to antimicrobial susceptibility profiling using the National Antimicrobial Resistance Monitoring System (NARMS) panel which tested susceptibility to 15 different antimicrobial agents. The molecular profiles of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) and the sequence types of the strains were obtained using Multi-Locus Sequence Type (MLST) analysis based on amplification and sequence interrogation of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA). PFGE data was input into BioNumerics analysis software to generate a dendrogram of relatedness among the strains. Results The study found 93 profiles among 98 S. Senftenberg isolates tested and there were primarily two sequence types associated with humans and animals (ST185 and ST14) with overlap observed in all host types suggesting that the distribution of S. Senftenberg sequence types is not host dependent. Antimicrobial resistance was observed among the animal strains, however no resistance was detected in human isolates suggesting that animal husbandry has a significant influence on the selection and promotion of antimicrobial resistance. Conclusion The data demonstrates the circulation of at least two strain types in both animal and human health suggesting that S. Senftenberg is relatively homogeneous in its distribution. The data generated in this study could be used towards defining a pathotype for this serovar. PMID:21708021

Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands.

PubMed

Voets, Guido M; Fluit, Ad C; Scharringa, Jelle; Schapendonk, Claudia; van den Munckhof, Thijs; Leverstein-van Hall, Maurine A; Stuart, James Cohen

2013-11-01

The increasing prevalence of third-generation cephalosporin-resistant Enterobacteriaceae is a worldwide problem. Recent studies showed that poultry meat and humans share identical Extended-Spectrum Beta-Lactamase genes, plasmid types, and Escherichia coli strain types, suggesting that transmission from poultry meat to humans may occur. The aim of this study was to compare plasmid-encoded Ambler class C beta-lactamase (pAmpC) genes, their plasmids, and bacterial strain types between E. coli isolates from retail chicken meat and clinical isolates in the Netherlands. In total, 98 Dutch retail chicken meat samples and 479 third-generation cephalosporin non-susceptible human clinical E. coli isolates from the same period were screened for pAmpC production. Plasmid typing was performed using PCR-based replicon typing (PBRT). E coli strains were compared using Multi-Locus-Sequence-Typing (MLST). In 12 of 98 chicken meat samples (12%), pAmpC producing E. coli were detected (all blaCMY-2). Of the 479 human E. coli, 25 (5.2%) harboured pAmpC genes (blaCMY-2 n = 22, blaACT n = 2, blaMIR n = 1). PBRT showed that 91% of poultry meat isolates harboured blaCMY-2 on an IncK plasmid, and 9% on an IncI1 plasmid. Of the human blaCMY-2 producing isolates, 42% also harboured blaCMY-2 on an IncK plasmid, and 47% on an IncI1 plasmid. Thus, 68% of human pAmpC producing E. coli have the same AmpC gene (blaCMY-2) and plasmid type (IncI1 or IncK) as found in poultry meat. MLST showed one cluster containing one human isolate and three meat isolates, with an IncK plasmid. These findings imply that a foodborne transmission route of blaCMY-2 harbouring plasmids cannot be excluded and that further evaluation is required. © 2013.

Multilocus Sequence Typing of an Emerging Cryptosporidium hominis Subtype in the United States

PubMed Central

Tiao, Narry; Li, Na; Hlavsa, Michele

2014-01-01

The United States has experienced a substantial increase in the reported incidence of cryptosporidiosis since 2005. Accompanying this is the emergence of a new subtype of Cryptosporidium hominis based on variation at the 60-kDa glycoprotein (gp60) locus, IaA28R4, which has become a frequently identified subtype in both sporadic and outbreak-related cases. In this study, using multilocus sequence typing (MLST) at eight genetic loci, we characterized 62 specimens of IaA28R4 and 33 specimens of three other gp60 subtypes of C. hominis from four U.S. states with increased cryptosporidiosis incidences during the summer of 2008. Extensive genetic heterogeneity was seen within the gp60 subtype IaA28R4, but specimens from Ohio and southwestern states formed two distinct subpopulations, suggesting that there were at least two origins of IaA28R4 within the United States. Discordance in typing results was observed between gp60 and other genetic markers, especially DZ-HRGP, and this discordance was largely the result of genetic recombination within the gp60 subtype IaA28R4. The results of population genetic analyses supported the presence of two subpopulations of IaA28R4 and the occurrence of genetic recombination within this gp60 subtype. Thus, the IaA28R4 subtype at gp60 is likely a fitness marker for C. hominis, and genetic recombination is potentially a driving force in the emergence of the virulent IaA28R4 subtype in the United States. A rapid evolution of IaA28R4 was indicated by the observation of multiple MLST subtypes of IaA28R4 within two large outbreaks that lasted for extended periods and involved multiple swimming pools. PMID:24478483

“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

Draft Genomes, Phylogenetic Reconstruction, and Comparative Genomics of Two Novel Cohabiting Bacterial Symbionts Isolated from Frankliniella occidentalis

PubMed Central

Facey, Paul D.; Méric, Guillaume; Hitchings, Matthew D.; Pachebat, Justin A.; Hegarty, Matt J.; Chen, Xiaorui; Morgan, Laura V.A.; Hoeppner, James E.; Whitten, Miranda M.A.; Kirk, William D.J.; Dyson, Paul J.; Sheppard, Sam K.; Sol, Ricardo Del

2015-01-01

Obligate bacterial symbionts are widespread in many invertebrates, where they are often confined to specialized host cells and are transmitted directly from mother to progeny. Increasing numbers of these bacteria are being characterized but questions remain about their population structure and evolution. Here we take a comparative genomics approach to investigate two prominent bacterial symbionts (BFo1 and BFo2) isolated from geographically separated populations of western flower thrips, Frankliniella occidentalis. Our multifaceted approach to classifying these symbionts includes concatenated multilocus sequence analysis (MLSA) phylogenies, ribosomal multilocus sequence typing (rMLST), construction of whole-genome phylogenies, and in-depth genomic comparisons. We showed that the BFo1 genome clusters more closely to species in the genus Erwinia, and is a putative close relative to Erwinia aphidicola. BFo1 is also likely to have shared a common ancestor with Erwinia pyrifoliae/Erwinia amylovora and the nonpathogenic Erwinia tasmaniensis and genetic traits similar to Erwinia billingiae. The BFo1 genome contained virulence factors found in the genus Erwinia but represented a divergent lineage. In contrast, we showed that BFo2 belongs within the Enterobacteriales but does not group closely with any currently known bacterial species. Concatenated MLSA phylogenies indicate that it may have shared a common ancestor to the Erwinia and Pantoea genera, and based on the clustering of rMLST genes, it was most closely related to Pantoea ananatis but represented a divergent lineage. We reconstructed a core genome of a putative common ancestor of Erwinia and Pantoea and compared this with the genomes of BFo bacteria. BFo2 possessed none of the virulence determinants that were omnipresent in the Erwinia and Pantoea genera. Taken together, these data are consistent with BFo2 representing a highly novel species that maybe related to known Pantoea. PMID:26185096

Draft Genomes, Phylogenetic Reconstruction, and Comparative Genomics of Two Novel Cohabiting Bacterial Symbionts Isolated from Frankliniella occidentalis.

PubMed

Facey, Paul D; Méric, Guillaume; Hitchings, Matthew D; Pachebat, Justin A; Hegarty, Matt J; Chen, Xiaorui; Morgan, Laura V A; Hoeppner, James E; Whitten, Miranda M A; Kirk, William D J; Dyson, Paul J; Sheppard, Sam K; Del Sol, Ricardo

2015-07-15

Obligate bacterial symbionts are widespread in many invertebrates, where they are often confined to specialized host cells and are transmitted directly from mother to progeny. Increasing numbers of these bacteria are being characterized but questions remain about their population structure and evolution. Here we take a comparative genomics approach to investigate two prominent bacterial symbionts (BFo1 and BFo2) isolated from geographically separated populations of western flower thrips, Frankliniella occidentalis. Our multifaceted approach to classifying these symbionts includes concatenated multilocus sequence analysis (MLSA) phylogenies, ribosomal multilocus sequence typing (rMLST), construction of whole-genome phylogenies, and in-depth genomic comparisons. We showed that the BFo1 genome clusters more closely to species in the genus Erwinia, and is a putative close relative to Erwinia aphidicola. BFo1 is also likely to have shared a common ancestor with Erwinia pyrifoliae/Erwinia amylovora and the nonpathogenic Erwinia tasmaniensis and genetic traits similar to Erwinia billingiae. The BFo1 genome contained virulence factors found in the genus Erwinia but represented a divergent lineage. In contrast, we showed that BFo2 belongs within the Enterobacteriales but does not group closely with any currently known bacterial species. Concatenated MLSA phylogenies indicate that it may have shared a common ancestor to the Erwinia and Pantoea genera, and based on the clustering of rMLST genes, it was most closely related to Pantoea ananatis but represented a divergent lineage. We reconstructed a core genome of a putative common ancestor of Erwinia and Pantoea and compared this with the genomes of BFo bacteria. BFo2 possessed none of the virulence determinants that were omnipresent in the Erwinia and Pantoea genera. Taken together, these data are consistent with BFo2 representing a highly novel species that maybe related to known Pantoea. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

An improved scheme for Flip-OFDM based on Hartley transform in short-range IM/DD systems.

PubMed

Zhou, Ji; Qiao, Yaojun; Cai, Zhuo; Ji, Yuefeng

2014-08-25

In this paper, an improved Flip-OFDM scheme is proposed for IM/DD optical systems, where the modulation/demodulation processing takes advantage of the fast Hartley transform (FHT) algorithm. We realize the improved scheme in one symbol period while conventional Flip-OFDM scheme based on fast Fourier transform (FFT) in two consecutive symbol periods. So the complexity of many operations in improved scheme is half of that in conventional scheme, such as CP operation, polarity inversion and symbol delay. Compared to FFT with complex input constellation, the complexity of FHT with real input constellation is halved. The transmission experiment over 50-km SSMF has been realized to verify the feasibility of improved scheme. In conclusion, the improved scheme has the same BER performance with conventional scheme, but great superiority on complexity.

Chain-Based Communication in Cylindrical Underwater Wireless Sensor Networks

PubMed Central

Javaid, Nadeem; Jafri, Mohsin Raza; Khan, Zahoor Ali; Alrajeh, Nabil; Imran, Muhammad; Vasilakos, Athanasios

2015-01-01

Appropriate network design is very significant for Underwater Wireless Sensor Networks (UWSNs). Application-oriented UWSNs are planned to achieve certain objectives. Therefore, there is always a demand for efficient data routing schemes, which can fulfill certain requirements of application-oriented UWSNs. These networks can be of any shape, i.e., rectangular, cylindrical or square. In this paper, we propose chain-based routing schemes for application-oriented cylindrical networks and also formulate mathematical models to find a global optimum path for data transmission. In the first scheme, we devise four interconnected chains of sensor nodes to perform data communication. In the second scheme, we propose routing scheme in which two chains of sensor nodes are interconnected, whereas in third scheme single-chain based routing is done in cylindrical networks. After finding local optimum paths in separate chains, we find global optimum paths through their interconnection. Moreover, we develop a computational model for the analysis of end-to-end delay. We compare the performance of the above three proposed schemes with that of Power Efficient Gathering System in Sensor Information Systems (PEGASIS) and Congestion adjusted PEGASIS (C-PEGASIS). Simulation results show that our proposed 4-chain based scheme performs better than the other selected schemes in terms of network lifetime, end-to-end delay, path loss, transmission loss, and packet sending rate. PMID:25658394

Public-key quantum digital signature scheme with one-time pad private-key

NASA Astrophysics Data System (ADS)

Chen, Feng-Lin; Liu, Wan-Fang; Chen, Su-Gen; Wang, Zhi-Hua

2018-01-01

A quantum digital signature scheme is firstly proposed based on public-key quantum cryptosystem. In the scheme, the verification public-key is derived from the signer's identity information (such as e-mail) on the foundation of identity-based encryption, and the signature private-key is generated by one-time pad (OTP) protocol. The public-key and private-key pair belongs to classical bits, but the signature cipher belongs to quantum qubits. After the signer announces the public-key and generates the final quantum signature, each verifier can verify publicly whether the signature is valid or not with the public-key and quantum digital digest. Analysis results show that the proposed scheme satisfies non-repudiation and unforgeability. Information-theoretic security of the scheme is ensured by quantum indistinguishability mechanics and OTP protocol. Based on the public-key cryptosystem, the proposed scheme is easier to be realized compared with other quantum signature schemes under current technical conditions.

Gas-Kinetic Theory Based Flux Splitting Method for Ideal Magnetohydrodynamics

NASA Technical Reports Server (NTRS)

Xu, Kun

1998-01-01

A gas-kinetic solver is developed for the ideal magnetohydrodynamics (MHD) equations. The new scheme is based on the direct splitting of the flux function of the MHD equations with the inclusion of "particle" collisions in the transport process. Consequently, the artificial dissipation in the new scheme is much reduced in comparison with the MHD Flux Vector Splitting Scheme. At the same time, the new scheme is compared with the well-developed Roe-type MHD solver. It is concluded that the kinetic MHD scheme is more robust and efficient than the Roe- type method, and the accuracy is competitive. In this paper the general principle of splitting the macroscopic flux function based on the gas-kinetic theory is presented. The flux construction strategy may shed some light on the possible modification of AUSM- and CUSP-type schemes for the compressible Euler equations, as well as to the development of new schemes for a non-strictly hyperbolic system.

Universal block diagram based modeling and simulation schemes for fractional-order control systems.

PubMed

Bai, Lu; Xue, Dingyü

2017-05-08

Universal block diagram based schemes are proposed for modeling and simulating the fractional-order control systems in this paper. A fractional operator block in Simulink is designed to evaluate the fractional-order derivative and integral. Based on the block, the fractional-order control systems with zero initial conditions can be modeled conveniently. For modeling the system with nonzero initial conditions, the auxiliary signal is constructed in the compensation scheme. Since the compensation scheme is very complicated, therefore the integrator chain scheme is further proposed to simplify the modeling procedures. The accuracy and effectiveness of the schemes are assessed in the examples, the computation results testify the block diagram scheme is efficient for all Caputo fractional-order ordinary differential equations (FODEs) of any complexity, including the implicit Caputo FODEs. Copyright © 2017 ISA. Published by Elsevier Ltd. All rights reserved.

Secure Communications in CIoT Networks with a Wireless Energy Harvesting Untrusted Relay

PubMed Central

Hu, Hequn; Liao, Xuewen

2017-01-01

The Internet of Things (IoT) represents a bright prospect that a variety of common appliances can connect to one another, as well as with the rest of the Internet, to vastly improve our lives. Unique communication and security challenges have been brought out by the limited hardware, low-complexity, and severe energy constraints of IoT devices. In addition, a severe spectrum scarcity problem has also been stimulated by the use of a large number of IoT devices. In this paper, cognitive IoT (CIoT) is considered where an IoT network works as the secondary system using underlay spectrum sharing. A wireless energy harvesting (EH) node is used as a relay to improve the coverage of an IoT device. However, the relay could be a potential eavesdropper to intercept the IoT device’s messages. This paper considers the problem of secure communication between the IoT device (e.g., sensor) and a destination (e.g., controller) via the wireless EH untrusted relay. Since the destination can be equipped with adequate energy supply, secure schemes based on destination-aided jamming are proposed based on power splitting (PS) and time splitting (TS) policies, called intuitive secure schemes based on PS (Int-PS), precoded secure scheme based on PS (Pre-PS), intuitive secure scheme based on TS (Int-TS) and precoded secure scheme based on TS (Pre-TS), respectively. The secure performances of the proposed schemes are evaluated through the metric of probability of successfully secure transmission (PSST), which represents the probability that the interference constraint of the primary user is satisfied and the secrecy rate is positive. PSST is analyzed for the proposed secure schemes, and the closed form expressions of PSST for Pre-PS and Pre-TS are derived and validated through simulation results. Numerical results show that the precoded secure schemes have better PSST than the intuitive secure schemes under similar power consumption. When the secure schemes based on PS and TS polices have similar PSST, the average transmit power consumption of the secure scheme based on TS is lower. The influences of power splitting and time slitting ratios are also discussed through simulations. PMID:28869540

Efficient and Provable Secure Pairing-Free Security-Mediated Identity-Based Identification Schemes

PubMed Central

Chin, Ji-Jian; Tan, Syh-Yuan; Heng, Swee-Huay; Phan, Raphael C.-W.

2014-01-01

Security-mediated cryptography was first introduced by Boneh et al. in 2001. The main motivation behind security-mediated cryptography was the capability to allow instant revocation of a user's secret key by necessitating the cooperation of a security mediator in any given transaction. Subsequently in 2003, Boneh et al. showed how to convert a RSA-based security-mediated encryption scheme from a traditional public key setting to an identity-based one, where certificates would no longer be required. Following these two pioneering papers, other cryptographic primitives that utilize a security-mediated approach began to surface. However, the security-mediated identity-based identification scheme (SM-IBI) was not introduced until Chin et al. in 2013 with a scheme built on bilinear pairings. In this paper, we improve on the efficiency results for SM-IBI schemes by proposing two schemes that are pairing-free and are based on well-studied complexity assumptions: the RSA and discrete logarithm assumptions. PMID:25207333

Efficient and provable secure pairing-free security-mediated identity-based identification schemes.

PubMed

Chin, Ji-Jian; Tan, Syh-Yuan; Heng, Swee-Huay; Phan, Raphael C-W

2014-01-01

Security-mediated cryptography was first introduced by Boneh et al. in 2001. The main motivation behind security-mediated cryptography was the capability to allow instant revocation of a user's secret key by necessitating the cooperation of a security mediator in any given transaction. Subsequently in 2003, Boneh et al. showed how to convert a RSA-based security-mediated encryption scheme from a traditional public key setting to an identity-based one, where certificates would no longer be required. Following these two pioneering papers, other cryptographic primitives that utilize a security-mediated approach began to surface. However, the security-mediated identity-based identification scheme (SM-IBI) was not introduced until Chin et al. in 2013 with a scheme built on bilinear pairings. In this paper, we improve on the efficiency results for SM-IBI schemes by proposing two schemes that are pairing-free and are based on well-studied complexity assumptions: the RSA and discrete logarithm assumptions.

Privacy Protection for Telecare Medicine Information Systems Using a Chaotic Map-Based Three-Factor Authenticated Key Agreement Scheme.

PubMed

Zhang, Liping; Zhu, Shaohui; Tang, Shanyu

2017-03-01

Telecare medicine information systems (TMIS) provide flexible and convenient e-health care. However, the medical records transmitted in TMIS are exposed to unsecured public networks, so TMIS are more vulnerable to various types of security threats and attacks. To provide privacy protection for TMIS, a secure and efficient authenticated key agreement scheme is urgently needed to protect the sensitive medical data. Recently, Mishra et al. proposed a biometrics-based authenticated key agreement scheme for TMIS by using hash function and nonce, they claimed that their scheme could eliminate the security weaknesses of Yan et al.'s scheme and provide dynamic identity protection and user anonymity. In this paper, however, we demonstrate that Mishra et al.'s scheme suffers from replay attacks, man-in-the-middle attacks and fails to provide perfect forward secrecy. To overcome the weaknesses of Mishra et al.'s scheme, we then propose a three-factor authenticated key agreement scheme to enable the patient to enjoy the remote healthcare services via TMIS with privacy protection. The chaotic map-based cryptography is employed in the proposed scheme to achieve a delicate balance of security and performance. Security analysis demonstrates that the proposed scheme resists various attacks and provides several attractive security properties. Performance evaluation shows that the proposed scheme increases efficiency in comparison with other related schemes.

PHACK: An Efficient Scheme for Selective Forwarding Attack Detection in WSNs.

PubMed

Liu, Anfeng; Dong, Mianxiong; Ota, Kaoru; Long, Jun

2015-12-09

In this paper, a Per-Hop Acknowledgement (PHACK)-based scheme is proposed for each packet transmission to detect selective forwarding attacks. In our scheme, the sink and each node along the forwarding path generate an acknowledgement (ACK) message for each received packet to confirm the normal packet transmission. The scheme, in which each ACK is returned to the source node along a different routing path, can significantly increase the resilience against attacks because it prevents an attacker from compromising nodes in the return routing path, which can otherwise interrupt the return of nodes' ACK packets. For this case, the PHACK scheme also has better potential to detect abnormal packet loss and identify suspect nodes as well as better resilience against attacks. Another pivotal issue is the network lifetime of the PHACK scheme, as it generates more acknowledgements than previous ACK-based schemes. We demonstrate that the network lifetime of the PHACK scheme is not lower than that of other ACK-based schemes because the scheme just increases the energy consumption in non-hotspot areas and does not increase the energy consumption in hotspot areas. Moreover, the PHACK scheme greatly simplifies the protocol and is easy to implement. Both theoretical and simulation results are given to demonstrate the effectiveness of the proposed scheme in terms of high detection probability and the ability to identify suspect nodes.

PHACK: An Efficient Scheme for Selective Forwarding Attack Detection in WSNs

PubMed Central

Liu, Anfeng; Dong, Mianxiong; Ota, Kaoru; Long, Jun

2015-01-01

In this paper, a Per-Hop Acknowledgement (PHACK)-based scheme is proposed for each packet transmission to detect selective forwarding attacks. In our scheme, the sink and each node along the forwarding path generate an acknowledgement (ACK) message for each received packet to confirm the normal packet transmission. The scheme, in which each ACK is returned to the source node along a different routing path, can significantly increase the resilience against attacks because it prevents an attacker from compromising nodes in the return routing path, which can otherwise interrupt the return of nodes’ ACK packets. For this case, the PHACK scheme also has better potential to detect abnormal packet loss and identify suspect nodes as well as better resilience against attacks. Another pivotal issue is the network lifetime of the PHACK scheme, as it generates more acknowledgements than previous ACK-based schemes. We demonstrate that the network lifetime of the PHACK scheme is not lower than that of other ACK-based schemes because the scheme just increases the energy consumption in non-hotspot areas and does not increase the energy consumption in hotspot areas. Moreover, the PHACK scheme greatly simplifies the protocol and is easy to implement. Both theoretical and simulation results are given to demonstrate the effectiveness of the proposed scheme in terms of high detection probability and the ability to identify suspect nodes. PMID:26690178

Secure anonymity-preserving password-based user authentication and session key agreement scheme for telecare medicine information systems.

PubMed

Sutrala, Anil Kumar; Das, Ashok Kumar; Odelu, Vanga; Wazid, Mohammad; Kumari, Saru

2016-10-01

Information and communication and technology (ICT) has changed the entire paradigm of society. ICT facilitates people to use medical services over the Internet, thereby reducing the travel cost, hospitalization cost and time to a greater extent. Recent advancements in Telecare Medicine Information System (TMIS) facilitate users/patients to access medical services over the Internet by gaining health monitoring facilities at home. Amin and Biswas recently proposed a RSA-based user authentication and session key agreement protocol usable for TMIS, which is an improvement over Giri et al.'s RSA-based user authentication scheme for TMIS. In this paper, we show that though Amin-Biswas's scheme considerably improves the security drawbacks of Giri et al.'s scheme, their scheme has security weaknesses as it suffers from attacks such as privileged insider attack, user impersonation attack, replay attack and also offline password guessing attack. A new RSA-based user authentication scheme for TMIS is proposed, which overcomes the security pitfalls of Amin-Biswas's scheme and also preserves user anonymity property. The careful formal security analysis using the two widely accepted Burrows-Abadi-Needham (BAN) logic and the random oracle models is done. Moreover, the informal security analysis of the scheme is also done. These security analyses show the robustness of our new scheme against the various known attacks as well as attacks found in Amin-Biswas's scheme. The simulation of the proposed scheme using the widely accepted Automated Validation of Internet Security Protocols and Applications (AVISPA) tool is also done. We present a new user authentication and session key agreement scheme for TMIS, which fixes the mentioned security pitfalls found in Amin-Biswas's scheme, and we also show that the proposed scheme provides better security than other existing schemes through the rigorous security analysis and verification tool. Furthermore, we present the formal security verification of our scheme using the widely accepted AVISPA tool. High security and extra functionality features allow our proposed scheme to be applicable for telecare medicine information systems which is used for e-health care medical applications. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Regolith thermal energy storage for lunar nighttime power

NASA Technical Reports Server (NTRS)

Tillotson, Brian

1992-01-01

A scheme for providing nighttime electric power to a lunar base is described. This scheme stores thermal energy in a pile of regolith. Any such scheme must somehow improve on the poor thermal conductivity of lunar regolith in vacuum. Two previous schemes accomplish this by casting or melting the regolith. The scheme described here wraps the regolith in a gas-tight bag and introduces a light gas to enhance thermal conductivity. This allows the system to be assembled with less energy and equipment than schemes which require melting of regolith. A point design based on the new scheme is presented. Its mass from Earth compares favorably with the mass of a regenerative fuel cell of equal capacity.

A new semiclassical decoupling scheme for electronic transitions in molecular collisions - Application to vibrational-to-electronic energy transfer

NASA Technical Reports Server (NTRS)

Lee, H.-W.; Lam, K. S.; Devries, P. L.; George, T. F.

1980-01-01

A new semiclassical decoupling scheme (the trajectory-based decoupling scheme) is introduced in a computational study of vibrational-to-electronic energy transfer for a simple model system that simulates collinear atom-diatom collisions. The probability of energy transfer (P) is calculated quasiclassically using the new scheme as well as quantum mechanically as a function of the atomic electronic-energy separation (lambda), with overall good agreement between the two sets of results. Classical mechanics with the new decoupling scheme is found to be capable of predicting resonance behavior whereas an earlier decoupling scheme (the coordinate-based decoupling scheme) failed. Interference effects are not exhibited in P vs lambda results.

Modeling and performance analysis of an improved movement-based location management scheme for packet-switched mobile communication systems.

PubMed

Chung, Yun Won; Kwon, Jae Kyun; Park, Suwon

2014-01-01

One of the key technologies to support mobility of mobile station (MS) in mobile communication systems is location management which consists of location update and paging. In this paper, an improved movement-based location management scheme with two movement thresholds is proposed, considering bursty data traffic characteristics of packet-switched (PS) services. The analytical modeling for location update and paging signaling loads of the proposed scheme is developed thoroughly and the performance of the proposed scheme is compared with that of the conventional scheme. We show that the proposed scheme outperforms the conventional scheme in terms of total signaling load with an appropriate selection of movement thresholds.

Comparison of wavelet based denoising schemes for gear condition monitoring: An Artificial Neural Network based Approach

NASA Astrophysics Data System (ADS)

Ahmed, Rounaq; Srinivasa Pai, P.; Sriram, N. S.; Bhat, Vasudeva

2018-02-01

Vibration Analysis has been extensively used in recent past for gear fault diagnosis. The vibration signals extracted is usually contaminated with noise and may lead to wrong interpretation of results. The denoising of extracted vibration signals helps the fault diagnosis by giving meaningful results. Wavelet Transform (WT) increases signal to noise ratio (SNR), reduces root mean square error (RMSE) and is effective to denoise the gear vibration signals. The extracted signals have to be denoised by selecting a proper denoising scheme in order to prevent the loss of signal information along with noise. An approach has been made in this work to show the effectiveness of Principal Component Analysis (PCA) to denoise gear vibration signal. In this regard three selected wavelet based denoising schemes namely PCA, Empirical Mode Decomposition (EMD), Neighcoeff Coefficient (NC), has been compared with Adaptive Threshold (AT) an extensively used wavelet based denoising scheme for gear vibration signal. The vibration signals acquired from a customized gear test rig were denoised by above mentioned four denoising schemes. The fault identification capability as well as SNR, Kurtosis and RMSE for the four denoising schemes have been compared. Features extracted from the denoised signals have been used to train and test artificial neural network (ANN) models. The performances of the four denoising schemes have been evaluated based on the performance of the ANN models. The best denoising scheme has been identified, based on the classification accuracy results. PCA is effective in all the regards as a best denoising scheme.

Implementation analysis of RC5 algorithm on Preneel-Govaerts-Vandewalle (PGV) hashing schemes using length extension attack

NASA Astrophysics Data System (ADS)

Siswantyo, Sepha; Susanti, Bety Hayat

2016-02-01

Preneel-Govaerts-Vandewalle (PGV) schemes consist of 64 possible single-block-length schemes that can be used to build a hash function based on block ciphers. For those 64 schemes, Preneel claimed that 4 schemes are secure. In this paper, we apply length extension attack on those 4 secure PGV schemes which use RC5 algorithm in its basic construction to test their collision resistance property. The attack result shows that the collision occurred on those 4 secure PGV schemes. Based on the analysis, we indicate that Feistel structure and data dependent rotation operation in RC5 algorithm, XOR operations on the scheme, along with selection of additional message block value also give impact on the collision to occur.

Enhancing the LVRT Capability of PMSG-Based Wind Turbines Based on R-SFCL

NASA Astrophysics Data System (ADS)

Xu, Lin; Lin, Ruixing; Ding, Lijie; Huang, Chunjun

2018-03-01

A novel low voltage ride-through (LVRT) scheme for PMSG-based wind turbines based on the Resistor Superconducting Fault Current Limiter (R-SFCL) is proposed in this paper. The LVRT scheme is mainly formed by R-SFCL in series between the transformer and the Grid Side Converter (GSC), and basic modelling has been discussed in detail. The proposed LVRT scheme is implemented to interact with PMSG model in PSCAD/EMTDC under three phase short circuit fault condition, which proves that the proposed scheme based on R-SFCL can improve the transient performance and LVRT capability to consolidate grid connection with wind turbines.

A robust anonymous biometric-based authenticated key agreement scheme for multi-server environments

PubMed Central

Huang, Yuanfei; Ma, Fangchao

2017-01-01

In order to improve the security in remote authentication systems, numerous biometric-based authentication schemes using smart cards have been proposed. Recently, Moon et al. presented an authentication scheme to remedy the flaws of Lu et al.’s scheme, and claimed that their improved protocol supports the required security properties. Unfortunately, we found that Moon et al.’s scheme still has weaknesses. In this paper, we show that Moon et al.’s scheme is vulnerable to insider attack, server spoofing attack, user impersonation attack and guessing attack. Furthermore, we propose a robust anonymous multi-server authentication scheme using public key encryption to remove the aforementioned problems. From the subsequent formal and informal security analysis, we demonstrate that our proposed scheme provides strong mutual authentication and satisfies the desirable security requirements. The functional and performance analysis shows that the improved scheme has the best secure functionality and is computational efficient. PMID:29121050

A robust anonymous biometric-based authenticated key agreement scheme for multi-server environments.

PubMed

Guo, Hua; Wang, Pei; Zhang, Xiyong; Huang, Yuanfei; Ma, Fangchao

2017-01-01

In order to improve the security in remote authentication systems, numerous biometric-based authentication schemes using smart cards have been proposed. Recently, Moon et al. presented an authentication scheme to remedy the flaws of Lu et al.'s scheme, and claimed that their improved protocol supports the required security properties. Unfortunately, we found that Moon et al.'s scheme still has weaknesses. In this paper, we show that Moon et al.'s scheme is vulnerable to insider attack, server spoofing attack, user impersonation attack and guessing attack. Furthermore, we propose a robust anonymous multi-server authentication scheme using public key encryption to remove the aforementioned problems. From the subsequent formal and informal security analysis, we demonstrate that our proposed scheme provides strong mutual authentication and satisfies the desirable security requirements. The functional and performance analysis shows that the improved scheme has the best secure functionality and is computational efficient.

An Efficient Quantum Somewhat Homomorphic Symmetric Searchable Encryption

NASA Astrophysics Data System (ADS)

Sun, Xiaoqiang; Wang, Ting; Sun, Zhiwei; Wang, Ping; Yu, Jianping; Xie, Weixin

2017-04-01

In 2009, Gentry first introduced an ideal lattices fully homomorphic encryption (FHE) scheme. Later, based on the approximate greatest common divisor problem, learning with errors problem or learning with errors over rings problem, FHE has developed rapidly, along with the low efficiency and computational security. Combined with quantum mechanics, Liang proposed a symmetric quantum somewhat homomorphic encryption (QSHE) scheme based on quantum one-time pad, which is unconditional security. And it was converted to a quantum fully homomorphic encryption scheme, whose evaluation algorithm is based on the secret key. Compared with Liang's QSHE scheme, we propose a more efficient QSHE scheme for classical input states with perfect security, which is used to encrypt the classical message, and the secret key is not required in the evaluation algorithm. Furthermore, an efficient symmetric searchable encryption (SSE) scheme is constructed based on our QSHE scheme. SSE is important in the cloud storage, which allows users to offload search queries to the untrusted cloud. Then the cloud is responsible for returning encrypted files that match search queries (also encrypted), which protects users' privacy.

A Quantum Multi-proxy Blind Signature Scheme Based on Genuine Four-Qubit Entangled State

NASA Astrophysics Data System (ADS)

Tian, Juan-Hong; Zhang, Jian-Zhong; Li, Yan-Ping

2016-02-01

In this paper, we propose a multi-proxy blind signature scheme based on controlled teleportation. Genuine four-qubit entangled state functions as quantum channel. The scheme uses the physical characteristics of quantum mechanics to implement delegation, signature and verification. The security analysis shows the scheme satisfies the security features of multi-proxy signature, unforgeability, undeniability, blindness and unconditional security.

A multihop key agreement scheme for wireless ad hoc networks based on channel characteristics.

PubMed

Hao, Zhuo; Zhong, Sheng; Yu, Nenghai

2013-01-01

A number of key agreement schemes based on wireless channel characteristics have been proposed recently. However, previous key agreement schemes require that two nodes which need to agree on a key are within the communication range of each other. Hence, they are not suitable for multihop wireless networks, in which nodes do not always have direct connections with each other. In this paper, we first propose a basic multihop key agreement scheme for wireless ad hoc networks. The proposed basic scheme is resistant to external eavesdroppers. Nevertheless, this basic scheme is not secure when there exist internal eavesdroppers or Man-in-the-Middle (MITM) adversaries. In order to cope with these adversaries, we propose an improved multihop key agreement scheme. We show that the improved scheme is secure against internal eavesdroppers and MITM adversaries in a single path. Both performance analysis and simulation results demonstrate that the improved scheme is efficient. Consequently, the improved key agreement scheme is suitable for multihop wireless ad hoc networks.

A Multihop Key Agreement Scheme for Wireless Ad Hoc Networks Based on Channel Characteristics

PubMed Central

Yu, Nenghai

2013-01-01

A number of key agreement schemes based on wireless channel characteristics have been proposed recently. However, previous key agreement schemes require that two nodes which need to agree on a key are within the communication range of each other. Hence, they are not suitable for multihop wireless networks, in which nodes do not always have direct connections with each other. In this paper, we first propose a basic multihop key agreement scheme for wireless ad hoc networks. The proposed basic scheme is resistant to external eavesdroppers. Nevertheless, this basic scheme is not secure when there exist internal eavesdroppers or Man-in-the-Middle (MITM) adversaries. In order to cope with these adversaries, we propose an improved multihop key agreement scheme. We show that the improved scheme is secure against internal eavesdroppers and MITM adversaries in a single path. Both performance analysis and simulation results demonstrate that the improved scheme is efficient. Consequently, the improved key agreement scheme is suitable for multihop wireless ad hoc networks. PMID:23766725

Comparison of two SVD-based color image compression schemes.

PubMed

Li, Ying; Wei, Musheng; Zhang, Fengxia; Zhao, Jianli

2017-01-01

Color image compression is a commonly used process to represent image data as few bits as possible, which removes redundancy in the data while maintaining an appropriate level of quality for the user. Color image compression algorithms based on quaternion are very common in recent years. In this paper, we propose a color image compression scheme, based on the real SVD, named real compression scheme. First, we form a new real rectangular matrix C according to the red, green and blue components of the original color image and perform the real SVD for C. Then we select several largest singular values and the corresponding vectors in the left and right unitary matrices to compress the color image. We compare the real compression scheme with quaternion compression scheme by performing quaternion SVD using the real structure-preserving algorithm. We compare the two schemes in terms of operation amount, assignment number, operation speed, PSNR and CR. The experimental results show that with the same numbers of selected singular values, the real compression scheme offers higher CR, much less operation time, but a little bit smaller PSNR than the quaternion compression scheme. When these two schemes have the same CR, the real compression scheme shows more prominent advantages both on the operation time and PSNR.

Comparison of two SVD-based color image compression schemes

PubMed Central

Li, Ying; Wei, Musheng; Zhang, Fengxia; Zhao, Jianli

2017-01-01

Color image compression is a commonly used process to represent image data as few bits as possible, which removes redundancy in the data while maintaining an appropriate level of quality for the user. Color image compression algorithms based on quaternion are very common in recent years. In this paper, we propose a color image compression scheme, based on the real SVD, named real compression scheme. First, we form a new real rectangular matrix C according to the red, green and blue components of the original color image and perform the real SVD for C. Then we select several largest singular values and the corresponding vectors in the left and right unitary matrices to compress the color image. We compare the real compression scheme with quaternion compression scheme by performing quaternion SVD using the real structure-preserving algorithm. We compare the two schemes in terms of operation amount, assignment number, operation speed, PSNR and CR. The experimental results show that with the same numbers of selected singular values, the real compression scheme offers higher CR, much less operation time, but a little bit smaller PSNR than the quaternion compression scheme. When these two schemes have the same CR, the real compression scheme shows more prominent advantages both on the operation time and PSNR. PMID:28257451

A Target Coverage Scheduling Scheme Based on Genetic Algorithms in Directional Sensor Networks

PubMed Central

Gil, Joon-Min; Han, Youn-Hee

2011-01-01

As a promising tool for monitoring the physical world, directional sensor networks (DSNs) consisting of a large number of directional sensors are attracting increasing attention. As directional sensors in DSNs have limited battery power and restricted angles of sensing range, maximizing the network lifetime while monitoring all the targets in a given area remains a challenge. A major technique to conserve the energy of directional sensors is to use a node wake-up scheduling protocol by which some sensors remain active to provide sensing services, while the others are inactive to conserve their energy. In this paper, we first address a Maximum Set Covers for DSNs (MSCD) problem, which is known to be NP-complete, and present a greedy algorithm-based target coverage scheduling scheme that can solve this problem by heuristics. This scheme is used as a baseline for comparison. We then propose a target coverage scheduling scheme based on a genetic algorithm that can find the optimal cover sets to extend the network lifetime while monitoring all targets by the evolutionary global search technique. To verify and evaluate these schemes, we conducted simulations and showed that the schemes can contribute to extending the network lifetime. Simulation results indicated that the genetic algorithm-based scheduling scheme had better performance than the greedy algorithm-based scheme in terms of maximizing network lifetime. PMID:22319387

Could the employment-based targeting approach serve Egypt in moving towards a social health insurance model?

PubMed

Shawky, S

2010-06-01

The current health insurance system in Egypt targets the productive population through an employment-based scheme bounded by a cost ceiling and focusing on curative care. Egypt Social Contract Survey data from 2005 were used to evaluate the impact of the employment-based scheme on health system accessibility and financing. Only 22.8% of the population in the productive age range (19-59 years) benefited from any health insurance scheme. The employment-based scheme covered 39.3% of the working population and was skewed towards urban areas, older people, females and the wealthier. It did not increase service utilization, but reduced out-of-pocket expenditure. Egypt should blend all health insurance schemes and adopt an innovative approach to reach universal coverage.

Quantum attack-resistent certificateless multi-receiver signcryption scheme.

PubMed

Li, Huixian; Chen, Xubao; Pang, Liaojun; Shi, Weisong

2013-01-01

The existing certificateless signcryption schemes were designed mainly based on the traditional public key cryptography, in which the security relies on the hard problems, such as factor decomposition and discrete logarithm. However, these problems will be easily solved by the quantum computing. So the existing certificateless signcryption schemes are vulnerable to the quantum attack. Multivariate public key cryptography (MPKC), which can resist the quantum attack, is one of the alternative solutions to guarantee the security of communications in the post-quantum age. Motivated by these concerns, we proposed a new construction of the certificateless multi-receiver signcryption scheme (CLMSC) based on MPKC. The new scheme inherits the security of MPKC, which can withstand the quantum attack. Multivariate quadratic polynomial operations, which have lower computation complexity than bilinear pairing operations, are employed in signcrypting a message for a certain number of receivers in our scheme. Security analysis shows that our scheme is a secure MPKC-based scheme. We proved its security under the hardness of the Multivariate Quadratic (MQ) problem and its unforgeability under the Isomorphism of Polynomials (IP) assumption in the random oracle model. The analysis results show that our scheme also has the security properties of non-repudiation, perfect forward secrecy, perfect backward secrecy and public verifiability. Compared with the existing schemes in terms of computation complexity and ciphertext length, our scheme is more efficient, which makes it suitable for terminals with low computation capacity like smart cards.

A splitting scheme based on the space-time CE/SE method for solving multi-dimensional hydrodynamical models of semiconductor devices

NASA Astrophysics Data System (ADS)

Nisar, Ubaid Ahmed; Ashraf, Waqas; Qamar, Shamsul

2016-08-01

Numerical solutions of the hydrodynamical model of semiconductor devices are presented in one and two-space dimension. The model describes the charge transport in semiconductor devices. Mathematically, the models can be written as a convection-diffusion type system with a right hand side describing the relaxation effects and interaction with a self consistent electric field. The proposed numerical scheme is a splitting scheme based on the conservation element and solution element (CE/SE) method for hyperbolic step, and a semi-implicit scheme for the relaxation step. The numerical results of the suggested scheme are compared with the splitting scheme based on Nessyahu-Tadmor (NT) central scheme for convection step and the same semi-implicit scheme for the relaxation step. The effects of various parameters such as low field mobility, device length, lattice temperature and voltages for one-space dimensional hydrodynamic model are explored to further validate the generic applicability of the CE/SE method for the current model equations. A two dimensional simulation is also performed by CE/SE method for a MESFET device, producing results in good agreement with those obtained by NT-central scheme.

An enhanced biometric-based authentication scheme for telecare medicine information systems using elliptic curve cryptosystem.

PubMed

Lu, Yanrong; Li, Lixiang; Peng, Haipeng; Yang, Yixian

2015-03-01

The telecare medical information systems (TMISs) enable patients to conveniently enjoy telecare services at home. The protection of patient's privacy is a key issue due to the openness of communication environment. Authentication as a typical approach is adopted to guarantee confidential and authorized interaction between the patient and remote server. In order to achieve the goals, numerous remote authentication schemes based on cryptography have been presented. Recently, Arshad et al. (J Med Syst 38(12): 2014) presented a secure and efficient three-factor authenticated key exchange scheme to remedy the weaknesses of Tan et al.'s scheme (J Med Syst 38(3): 2014). In this paper, we found that once a successful off-line password attack that results in an adversary could impersonate any user of the system in Arshad et al.'s scheme. In order to thwart these security attacks, an enhanced biometric and smart card based remote authentication scheme for TMISs is proposed. In addition, the BAN logic is applied to demonstrate the completeness of the enhanced scheme. Security and performance analyses show that our enhanced scheme satisfies more security properties and less computational cost compared with previously proposed schemes.

Cryptanalysis of Chatterjee-Sarkar Hierarchical Identity-Based Encryption Scheme at PKC 06

NASA Astrophysics Data System (ADS)

Park, Jong Hwan; Lee, Dong Hoon

In 2006, Chatterjee and Sarkar proposed a hierarchical identity-based encryption (HIBE) scheme which can support an unbounded number of identity levels. This property is particularly useful in providing forward secrecy by embedding time components within hierarchical identities. In this paper we show that their scheme does not provide the claimed property. Our analysis shows that if the number of identity levels becomes larger than the value of a fixed public parameter, an unintended receiver can reconstruct a new valid ciphertext and decrypt the ciphertext using his or her own private key. The analysis is similarly applied to a multi-receiver identity-based encryption scheme presented as an application of Chatterjee and Sarkar's HIBE scheme.

Algorithms for adaptive stochastic control for a class of linear systems

NASA Technical Reports Server (NTRS)

Toda, M.; Patel, R. V.

1977-01-01

Control of linear, discrete time, stochastic systems with unknown control gain parameters is discussed. Two suboptimal adaptive control schemes are derived: one is based on underestimating future control and the other is based on overestimating future control. Both schemes require little on-line computation and incorporate in their control laws some information on estimation errors. The performance of these laws is studied by Monte Carlo simulations on a computer. Two single input, third order systems are considered, one stable and the other unstable, and the performance of the two adaptive control schemes is compared with that of the scheme based on enforced certainty equivalence and the scheme where the control gain parameters are known.

Efficient Fair Exchange from Identity-Based Signature

NASA Astrophysics Data System (ADS)

Yum, Dae Hyun; Lee, Pil Joong

A fair exchange scheme is a protocol by which two parties Alice and Bob exchange items or services without allowing either party to gain advantages by quitting prematurely or otherwise misbehaving. To this end, modern cryptographic solutions use a semi-trusted arbitrator who involves only in cases where one party attempts to cheat or simply crashes. We call such a fair exchange scheme optimistic. When no registration is required between the signer and the arbitrator, we say that the fair exchange scheme is setup free. To date, the setup-free optimist fair exchange scheme under the standard RSA assumption was only possible from the generic construction of [12], which uses ring signatures. In this paper, we introduce a new setup-free optimistic fair exchange scheme under the standard RSA assumption. Our scheme uses the GQ identity-based signature and is more efficient than [12]. The construction can also be generalized by using various identity-based signature schemes. Our main technique is to allow each user to choose his (or her) own “random” public key in the identitybased signature scheme.

Genetic and economic evaluation of Japanese Black (Wagyu) cattle breeding schemes.

PubMed

Kahi, A K; Hirooka, H

2005-09-01

Deterministic simulation was used to evaluate 10 breeding schemes for genetic gain and profitability and in the context of maximizing returns from investment in Japanese Black cattle breeding. A breeding objective that integrated the cow-calf and feedlot segments was considered. Ten breeding schemes that differed in the records available for use as selection criteria were defined. The schemes ranged from one that used carcass traits currently available to Japanese Black cattle breeders (Scheme 1) to one that also included linear measurements and male and female reproduction traits (Scheme 10). The latter scheme represented the highest level of performance recording. In all breeding schemes, sires were chosen from the proportion selected during the first selection stage (performance testing), modeling a two-stage selection process. The effect on genetic gain and profitability of varying test capacity and number of progeny per sire and of ultrasound scanning of live animals was examined for all breeding schemes. Breeding schemes that selected young bulls during performance testing based on additional individual traits and information on carcass traits from their relatives generated additional genetic gain and profitability. Increasing test capacity resulted in an increase in genetic gain in all schemes. Profitability was optimal in Scheme 2 (a scheme similar to Scheme 1, but selection of young bulls also was based on information on carcass traits from their relatives) to 10 when 900 to 1,000 places were available for performance testing. Similarly, as the number of progeny used in the selection of sires increased, genetic gain first increased sharply and then gradually in all schemes. Profit was optimal across all breeding schemes when sires were selected based on information from 150 to 200 progeny. Additional genetic gain and profitability were generated in each breeding scheme with ultrasound scanning of live animals for carcass traits. Ultrasound scanning of live animals was more important than the addition of any other traits in the selection criteria. These results may be used to provide guidance to Japanese Black cattle breeders.

A secure and robust password-based remote user authentication scheme using smart cards for the integrated EPR information system.

PubMed

Das, Ashok Kumar

2015-03-01

An integrated EPR (Electronic Patient Record) information system of all the patients provides the medical institutions and the academia with most of the patients' information in details for them to make corrective decisions and clinical decisions in order to maintain and analyze patients' health. In such system, the illegal access must be restricted and the information from theft during transmission over the insecure Internet must be prevented. Lee et al. proposed an efficient password-based remote user authentication scheme using smart card for the integrated EPR information system. Their scheme is very efficient due to usage of one-way hash function and bitwise exclusive-or (XOR) operations. However, in this paper, we show that though their scheme is very efficient, their scheme has three security weaknesses such as (1) it has design flaws in password change phase, (2) it fails to protect privileged insider attack and (3) it lacks the formal security verification. We also find that another recently proposed Wen's scheme has the same security drawbacks as in Lee at al.'s scheme. In order to remedy these security weaknesses found in Lee et al.'s scheme and Wen's scheme, we propose a secure and efficient password-based remote user authentication scheme using smart cards for the integrated EPR information system. We show that our scheme is also efficient as compared to Lee et al.'s scheme and Wen's scheme as our scheme only uses one-way hash function and bitwise exclusive-or (XOR) operations. Through the security analysis, we show that our scheme is secure against possible known attacks. Furthermore, we simulate our scheme for the formal security verification using the widely-accepted AVISPA (Automated Validation of Internet Security Protocols and Applications) tool and show that our scheme is secure against passive and active attacks.