Methanolic leaf extract of Gymnema sylvestre augments glucose uptake and ameliorates insulin resistance by upregulating glucose transporter-4, peroxisome proliferator-activated receptor-gamma, adiponectin, and leptin levels in vitro.

PubMed

Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V; Manjunath, Kirangadur; Viswanatha, Gollapalle L; Ashok, Godavarthi

2016-01-01

The present study was undertaken to evaluate the effect of methanolic leaf extract of Gymnema sylvestre (MLGS) on glucose transport (GLUT) and insulin resistance in vitro. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and GLUT-4 expression were assessed in L6 myotubes for concluding the GLUT activity, and adiponectin and leptin expression was studied in 3T3 L1 murine adipocyte cell line to determine the effect of MLGS (250-750 μg/ml) on insulin resistance. The findings of the experiments have demonstrated a significant and dose-dependent increase in glucose uptake in all the tested concentrations of MLGS, further the glucose uptake activity of MLGS (750 μg/ml) was at par with rosiglitazone (50 μg/ml). Concomitantly, MLGS has shown enhanced GLUT-4 and PPAR-γ gene expressions in L6 myotubes. Furthermore, cycloheximide (CHX) had completely abolished the glucose uptake activity of MLGS when co-incubated, which further confirmed that glucose uptake activity of MLGS was linked to enhanced expression of GLUT-4 and PPAR-γ. In addition, in another experimental set, MLGS showed enhanced expression of adiponectin and leptin, thus confirms the ameliorative effect of MLGS on insulin resistance. These findings suggest that MLGS has an enhanced glucose uptake activity in L6 myotubes, and ameliorate the insulin resistance in 3T3 L1 murine adipocyte cell line in vitro.

Methanolic leaf extract of Gymnema sylvestre augments glucose uptake and ameliorates insulin resistance by upregulating glucose transporter-4, peroxisome proliferator-activated receptor-gamma, adiponectin, and leptin levels in vitro

PubMed Central

Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V.; Manjunath, Kirangadur; Viswanatha, Gollapalle L.; Ashok, Godavarthi

2016-01-01

Aims: The present study was undertaken to evaluate the effect of methanolic leaf extract of Gymnema sylvestre (MLGS) on glucose transport (GLUT) and insulin resistance in vitro. Materials and Methods: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and GLUT-4 expression were assessed in L6 myotubes for concluding the GLUT activity, and adiponectin and leptin expression was studied in 3T3 L1 murine adipocyte cell line to determine the effect of MLGS (250-750 μg/ml) on insulin resistance. Results: The findings of the experiments have demonstrated a significant and dose-dependent increase in glucose uptake in all the tested concentrations of MLGS, further the glucose uptake activity of MLGS (750 μg/ml) was at par with rosiglitazone (50 μg/ml). Concomitantly, MLGS has shown enhanced GLUT-4 and PPAR-γ gene expressions in L6 myotubes. Furthermore, cycloheximide (CHX) had completely abolished the glucose uptake activity of MLGS when co-incubated, which further confirmed that glucose uptake activity of MLGS was linked to enhanced expression of GLUT-4 and PPAR-γ. In addition, in another experimental set, MLGS showed enhanced expression of adiponectin and leptin, thus confirms the ameliorative effect of MLGS on insulin resistance. Conclusion: These findings suggest that MLGS has an enhanced glucose uptake activity in L6 myotubes, and ameliorate the insulin resistance in 3T3 L1 murine adipocyte cell line in vitro. PMID:27104035

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 ...

Ideal discrimination of discrete clinical endpoints using multilocus genotypes.

PubMed

Hahn, Lance W; Moore, Jason H

2004-01-01

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

Pneumocystis jirovecii multilocus gene sequencing: findings and implications.

PubMed

Matos, Olga; Esteves, Francisco

2010-08-01

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

Multilocus Genotyping and Molecular Phylogenetics Resolve a Novel Head Blight Pathogen within the Fusarium graminearum Species Complex from Ethiopia

USDA-ARS?s Scientific Manuscript database

A survey of Fusarium head blight (FHB)-contaminated wheat in Ethiopia recovered 31 isolates resembling members of the Fusarium graminearum species complex. Results of a multilocus genotyping (MLGT) assay for FHB species and trichothecene chemotype determination suggested that 22 of these isolates m...

Multilocus Genotypes of Relevance for Drug Metabolizing Enzymes and Therapy with Thiopurines in Patients with Acute Lymphoblastic Leukemia

PubMed Central

Stocco, Gabriele; Franca, Raffaella; Verzegnassi, Federico; Londero, Margherita; Rabusin, Marco; Decorti, Giuliana

2013-01-01

Multilocus genotypes have been shown to be of relevance for using pharmacogenomic principles to individualize drug therapy. As it relates to thiopurine therapy, genetic polymorphisms of TPMT are strongly associated with the pharmacokinetics and clinical effects of thiopurines (mercaptopurine and azathioprine), influencing their toxicity and efficacy. We have recently demonstrated that TPMT and ITPA genotypes constitute a multilocus genotype of pharmacogenetic relevance for children with acute lymphoblastic leukemia (ALL) receiving thiopurine therapy. The use of high-throughput genomic analysis allows identification of additional candidate genetic factors associated with pharmacogenetic phenotypes, such as TPMT enzymatic activity: PACSIN2 polymorphisms have been identified by a genome-wide analysis, combining evaluation of polymorphisms and gene expression, as a significant determinant of TPMT activity in the HapMap CEU cell lines and the effects of PACSIN2 on TPMT activity and mercaptopurine induced adverse effects were confirmed in children with ALL. Combination of genetic factors of relevance for thiopurine metabolizing enzyme activity, based on the growing understanding of their association with drug metabolism and efficacy, is particularly promising for patients with pediatric ALL. The knowledge basis and clinical applications for multilocus genotypes of importance for therapy with mercaptopurine in pediatric ALL is discussed in the present review. PMID:23335936

Connections between the human gut microbiome and gestational diabetes mellitus.

PubMed

Kuang, Ya-Shu; Lu, Jin-Hua; Li, Sheng-Hui; Li, Jun-Hua; Yuan, Ming-Yang; He, Jian-Rong; Chen, Nian-Nian; Xiao, Wan-Qing; Shen, Song-Ying; Qiu, Lan; Wu, Ying-Fang; Hu, Cui-Yue; Wu, Yan-Yan; Li, Wei-Dong; Chen, Qiao-Zhu; Deng, Hong-Wen; Papasian, Christopher J; Xia, Hui-Min; Qiu, Xiu

2017-08-01

The human gut microbiome can modulate metabolic health and affect insulin resistance, and it may play an important role in the etiology of gestational diabetes mellitus (GDM). Here, we compared the gut microbial composition of 43 GDM patients and 81 healthy pregnant women via whole-metagenome shotgun sequencing of their fecal samples, collected at 21-29 weeks, to explore associations between GDM and the composition of microbial taxonomic units and functional genes. A metagenome-wide association study identified 154 837 genes, which clustered into 129 metagenome linkage groups (MLGs) for species description, with significant relative abundance differences between the 2 cohorts. Parabacteroides distasonis, Klebsiella variicola, etc., were enriched in GDM patients, whereas Methanobrevibacter smithii, Alistipes spp., Bifidobacterium spp., and Eubacterium spp. were enriched in controls. The ratios of the gross abundances of GDM-enriched MLGs to control-enriched MLGs were positively correlated with blood glucose levels. A random forest model shows that fecal MLGs have excellent discriminatory power to predict GDM status. Our study discovered novel relationships between the gut microbiome and GDM status and suggests that changes in microbial composition may potentially be used to identify individuals at risk for GDM. © The Author 2017. Published by Oxford University Press.

Multicentric epidemiological study of Aspergillus fumigatus isolates by multilocus enzyme electrophoresis.

PubMed Central

Rodriguez, E; De Meeüs, T; Mallie, M; Renaud, F; Symoens, F; Mondon, P; Piens, M A; Lebeau, B; Viviani, M A; Grillot, R; Nolard, N; Chapuis, F; Tortorano, A M; Bastide, J M

1996-01-01

The genotypes of 63 isolates of Aspergillus fumigatus obtained from three hospitals in different geographical areas and of eight culture collection strains were determined by multilocus enzyme electrophoresis. Twelve of the 17 enzymatic loci studied were polymorphic, giving rise to 48 different electrophoretic types. The existence of fixed multilocus genotypes, significant heterozygote deficits and excesses at the different loci, and linkage disequilibria within subpopulations strongly suggests a clonal reproduction mode for A. fumigatus. Numerical analysis of the comparison and disposition of the different electrophoretic types demonstrates a significant genetic differentiation between the three sampling sites. However, no correlation could be found between geographical distances and genetic differentiation. On account of the multiple discriminatory markers, multilocus enzyme electrophoresis typing seems to be a very powerful tool for epidemiological and reproductive mode studies of A. fumigatus. PMID:8880520

Molecular Epidemiology of Human Oral Chagas Disease Outbreaks in Colombia

PubMed Central

Ramírez, Juan David; Montilla, Marleny; Cucunubá, Zulma M.; Floréz, Astrid Carolina; Zambrano, Pilar; Guhl, Felipe

2013-01-01

Background Trypanosoma cruzi, the causative agent of Chagas disease, displays significant genetic variability revealed by six Discrete Typing Units (TcI-TcVI). In this pathology, oral transmission represents an emerging epidemiological scenario where different outbreaks associated to food/beverages consumption have been reported in Argentina, Bolivia, Brazil, Ecuador and Venezuela. In Colombia, six human oral outbreaks have been reported corroborating the importance of this transmission route. Molecular epidemiology of oral outbreaks is barely known observing the incrimination of TcI, TcII, TcIV and TcV genotypes. Methodology and Principal Findings High-throughput molecular characterization was conducted performing MLMT (Multilocus Microsatellite Typing) and mtMLST (mitochondrial Multilocus Sequence Typing) strategies on 50 clones from ten isolates. Results allowed observing the occurrence of TcI, TcIV and mixed infection of distinct TcI genotypes. Thus, a majority of specific mitochondrial haplotypes and allelic multilocus genotypes associated to the sylvatic cycle of transmission were detected in the dataset with the foreseen presence of mitochondrial haplotypes and allelic multilocus genotypes associated to the domestic cycle of transmission. Conclusions These findings suggest the incrimination of sylvatic genotypes in the oral outbreaks occurred in Colombia. We observed patterns of super-infection and/or co-infection with a tailored association with the severe forms of myocarditis in the acute phase of the disease. The transmission dynamics of this infection route based on molecular epidemiology evidence was unraveled and the clinical and biological implications are discussed. PMID:23437405

Population sub-structuring among Trypanosoma evansi stocks.

PubMed

Njiru, Z K; Constantine, C C

2007-10-01

To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei's genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (IA=0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed Fst=0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei's genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.

Genotypic diversity of european Phytophthora ramorum isolates based on SSR analysis

Treesearch

Kris Van Poucke; Annelies Vercauteren; Martine Maes; Sabine Werres; Kurt Heungens

2013-01-01

in Scotland were genotyped using seven microsatellite markers as described by Vercauteren et al. (2010). Thirty multilocus genotypes were identified within the Scottish population, with 51 percent of the isolates belonging to the main European genotype EU1MG1 and 13 unique detected genotypes. Ten of those genotypes were site specific, often represented by...

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 ...

Genetic diversity of Phytophthora ramorum in nursery trade and managed environment in Scotland

Treesearch

Alexandra Schlenzig; David Cook

2013-01-01

in Scotland were genotyped using seven microsatellite markers as described by Vercauteren et al. (2010). Thirty multilocus genotypes were identified within the Scottish population, with 51 percent of the isolates belonging to the main European genotype EU1MG1 and 13 unique detected genotypes. Ten of those genotypes were site specific, often represented by...

Cryptosporidium genotyping in Europe: The current status and processes for a harmonised multi-locus genotyping scheme.

PubMed

Chalmers, Rachel M; Pérez-Cordón, Gregorio; Cacció, Simone M; Klotz, Christian; Robertson, Lucy J

2018-06-13

Due to the occurrence of genetic recombination, a reliable and discriminatory method to genotype Cryptosporidium isolates at the intra-species level requires the analysis of multiple loci, but a standardised scheme is not currently available. A workshop was held at the Robert Koch Institute, Berlin in 2016 that gathered 23 scientists with appropriate expertise (in either Cryptosporidium genotyping and/or surveillance, epidemiology or outbreaks) to discuss the processes for the development of a robust, standardised, multi-locus genotyping (MLG) scheme and propose an approach. The background evidence and main conclusions were outlined in a previously published report; the objectives of this further report are to describe 1) the current use of Cryptosporidium genotyping, 2) the elicitation and synthesis of the participants' opinions, and 3) the agreed processes and criteria for the development, evaluation and validation of a standardised MLG scheme for Cryptosporidium surveillance and outbreak investigations. Cryptosporidium was characterised to the species level in 7/12 (58%) participating European countries, mostly for human outbreak investigations. Further genotyping was mostly by sequencing the gp60 gene. A ranking exercise of performance and convenience criteria found that portability, biological robustness, typeability, and discriminatory power were considered by participants as the most important attributes in developing a multilocus scheme. The major barrier to implementation was lack of funding. A structured process for marker identification, evaluation, validation, implementation, and maintenance was proposed and outlined for application to Cryptosporidium, with prioritisation of Cryptosporidium parvum to support investigation of transmission in Europe. Copyright © 2018 Elsevier Inc. All rights reserved.

Highly Discriminatory Variable-Number Tandem-Repeat Markers for Genotyping of Trichophyton interdigitale Strains

PubMed Central

Drira, Ines; Hadrich, Ines; Neji, Sourour; Mahfouth, Nedia; Trabelsi, Houaida; Sellami, Hayet; Makni, Fattouma

2014-01-01

Trichophyton interdigitale is the second most frequent cause of superficial fungal infections of various parts of the human body. Studying the population structure and genotype differentiation of T. interdigitale strains may lead to significant improvements in clinical practice. The present study aimed to develop and select suitable variable-number tandem-repeat (VNTR) markers for 92 clinical strains of T. interdigitale. On the basis of an analysis of four VNTR markers, four to eight distinct alleles were detected for each marker. The marker with the highest discriminatory power had eight alleles and a D value of 0.802. The combination of all four markers yielded a D value of 0.969 with 29 distinct multilocus genotypes. VNTR typing revealed the genetic diversity of the strains, identifying three populations according to their colonization sites. A correlation between phenotypic characteristics and multilocus genotypes was observed. Seven patients harbored T. interdigitale strains with different genotypes. Typing of clinical T. interdigitale samples by VNTR markers displayed excellent discriminatory power and 100% reproducibility. PMID:24989614

[Multilocus genotyping of polymorphous STR-loci of chromosomal DNA in individual cells: technical difficulties].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu; Kobylianskiĭ, A G; Dziubenko, E V

2013-01-01

This study was designed to estimate the effectiveness of special technical procedures for the enhancement of sensitivity of multiplex analysis of DNA, such as the use of low-plexity PCR systems and the whole genome preamplification technology, and the possibility of their application for the purpose of forensic medical genotyping of polymorphous STR-loci of chromosomal DNA in individual cells. The authors refused to use the imitation model (equivalent DNA dilutions) for the sake of obtaining the maximally informative data and chose to work with real preparations of solitary buccal epithelial cells isolated by the laser microdissection technique. It was shown that neither the use of the low-plexity multilocus PCR systems nor the whole genome pre-amplification technology makes possible reliable genotyping of STR-loci of chromosomal DNA in individual cells. The proposed techniques allow for DNA genotyping in preparations consisting of 10 diploid cells whereas the methods for reliable genotyping of STR-loci of chromosomal DNA in individual cells remains to be developed.

Detecting genotypic changes associated with selective mortality at sea in Atlantic salmon: polygenic multilocus analysis surpasses genome scan.

PubMed

Bourret, Vincent; Dionne, Mélanie; Bernatchez, Louis

2014-09-01

Wild populations of Atlantic salmon have declined worldwide. While the causes for this decline may be complex and numerous, increased mortality at sea is predicted to be one of the major contributing factors. Examining the potential changes occurring in the genome-wide composition of populations during this migration has the potential to tease apart some of the factors influencing marine mortality. Here, we genotyped 5568 SNPs in Atlantic salmon populations representing two distinct regional genetic groups and across two cohorts to test for differential allelic and genotypic frequencies between juveniles (smolts) migrating to sea and adults (grilses) returning to freshwater after 1 year at sea. Given the complexity of the traits potentially associated with sea mortality, we contrasted the outcomes of a single-locus F(ST) based genome scan method with a new multilocus framework to test for genetically based differential mortality at sea. While numerous outliers were identified by the single-locus analysis, no evidence for parallel, temporally repeated selection was found. In contrast, the multilocus approach detected repeated patterns of selection for a multilocus group of 34 covarying SNPs in one of the two populations. No significant pattern of selective mortality was detected in the other population, suggesting different causes of mortality among populations. These results first support the hypothesis that selection mainly causes small changes in allele frequencies among many covarying loci rather than a small number of changes in loci with large effects. They also point out that moving away from the a strict 'selective sweep paradigm' towards a multilocus genetics framework may be a more useful approach for studying the genomic signatures of natural selection on complex traits in wild populations. © 2014 John Wiley & Sons Ltd.

Multi-locus variable number tandem repeat analysis for Escherichia coli causing extraintestinal infections.

PubMed

Manges, Amee R; Tellis, Patricia A; Vincent, Caroline; Lifeso, Kimberley; Geneau, Geneviève; Reid-Smith, Richard J; Boerlin, Patrick

2009-11-01

Discriminatory genotyping methods for the analysis of Escherichia coli other than O157:H7 are necessary for public health-related activities. A new multi-locus variable number tandem repeat analysis protocol is presented; this method achieves an index of discrimination of 99.5% and is reproducible and valid when tested on a collection of 836 diverse E. coli.

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

PubMed Central

2009-01-01

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

Genotypic Diversity of Methicillin-Resistant Staphylococcus aureus Isolates in Korean Hospitals

PubMed Central

Soo Ko, Kwan; Kim, Yeon-Sook; Song, Jae-Hoon; Yeom, Joon-Sup; Lee, Hyuck; Jung, Sook-In; Jeong, Doo-Ryun; Kim, Shin-Woo; Chang, Hyun-Ha; Ki, Hyun Kyun; Moon, Chisook; Oh, Won Sup; Peck, Kyong Ran; Lee, Nam Yong

2005-01-01

Ninety-six methicillin-resistant Staphylococcus aureus (MRSA) isolates from eight Korean hospitals were analyzed by multilocus sequence typing, SCCmec typing, and spa typing. The predominant genotype was ST5-MRSA-II of clonal complex 5, which was found in 36 isolates from six hospitals, but ST239-MRSA-III was also common. Overall, results showed a notable genotypic diversity of MRSA strains circulating in Korean hospitals. PMID:16048991

Genotypic diversity of methicillin-resistant Staphylococcus aureus isolates in Korean hospitals.

PubMed

Soo Ko, Kwan; Kim, Yeon-Sook; Song, Jae-Hoon; Yeom, Joon-Sup; Lee, Hyuck; Jung, Sook-In; Jeong, Doo-Ryun; Kim, Shin-Woo; Chang, Hyun-Ha; Ki, Hyun Kyun; Moon, Chisook; Oh, Won Sup; Peck, Kyong Ran; Lee, Nam Yong

2005-08-01

Ninety-six methicillin-resistant Staphylococcus aureus (MRSA) isolates from eight Korean hospitals were analyzed by multilocus sequence typing, SCCmec typing, and spa typing. The predominant genotype was ST5-MRSA-II of clonal complex 5, which was found in 36 isolates from six hospitals, but ST239-MRSA-III was also common. Overall, results showed a notable genotypic diversity of MRSA strains circulating in Korean hospitals.

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

PubMed

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

2012-01-01

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

Molecular appraisal of intestinal parasitic infection in transplant recipients

PubMed Central

Yadav, Pooja; Khalil, Shehla; Mirdha, Bijay Ranjan

2016-01-01

Background & objectives: Diarrhoea is the main clinical manifestation caused by intestinal parasitic infections in patients, with special reference to transplant recipients who require careful consideration to reduce morbidity and mortality. Further, molecular characterization of some important parasites is necessary to delineate the different modes of transmission to consider appropriate management strategies. We undertook this study to investigate the intestinal parasitic infections in transplant recipients with or without diarrhoea, and the genotypes of the isolated parasites were also determined. Methods: Stool samples from 38 transplant recipients comprising 29 post-renal, two liver and seven bone marrow transplant (BMT) recipients presenting with diarrhoea and 50 transplant recipients (42 post-renal transplant, eight BMT) without diarrhoea were examined for the presence of intestinal parasites by light microscopy using wet mount, modified Ziehl–Neelsen staining for intestinal coccidia and modified trichrome staining for microsporidia. Genotypes of Cryptosporidium species were determined by multilocus genotyping using small subunit ribosomal (SSUrRNA), Cryptosporidium oocyst wall protein (COWP) and dihydrofolate reductase (DHFR) as the target genes. Assemblage study for Giardia lamblia was performed using triose phosphate isomerase (TPI) as the target gene. Samples were also screened for bacterial, fungal and viral pathogens. Results: The parasites that were detected included Cryptosporidium species (21%, 8/38), Cystoisospora (Isospora) belli (8%, 3), Cyclospora cayetanensis (5%, 2), G. lamblia (11%, 4), Hymenolepis nana (11%, 4), Strongyloides stercoralis (3%, 1) and Blastocystis hominis (3%, 1). Multilocus genotyping of Cryptosporidium species at SSUrRNA, COWP and DHFR loci could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. All the C. hominis isolates were detected in adult post-renal transplant (PRT) recipients, whereas the C. parvum isolates included a child with BMT and an adult with PRT. Clostridium difficle, cytomegalovirus and Candida albicans were found in 2, 3 and 2 patients, respectively. Interpretation & conclusions: In the present study, C. hominis was observed as an important parasite causing intestinal infections in transplant recipients. Multilocus genotyping of Cryptosporidium species could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. Genotyping of G. lamblia revealed that assemblage B was most common. PMID:27934806

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...

Multilocus sequence analysis of Anaplasma phagocytophilum reveals three distinct lineages with different host ranges in clinically ill French cattle.

PubMed

Chastagner, Amélie; Dugat, Thibaud; Vourc'h, Gwenaël; Verheyden, Hélène; Legrand, Loïc; Bachy, Véronique; Chabanne, Luc; Joncour, Guy; Maillard, Renaud; Boulouis, Henri-Jean; Haddad, Nadia; Bailly, Xavier; Leblond, Agnès

2014-12-09

Molecular epidemiology represents a powerful approach to elucidate the complex epidemiological cycles of multi-host pathogens, such as Anaplasma phagocytophilum. A. phagocytophilum is a tick-borne bacterium that affects a wide range of wild and domesticated animals. Here, we characterized its genetic diversity in populations of French cattle; we then compared the observed genotypes with those found in horses, dogs, and roe deer to determine whether genotypes of A. phagocytophilum are shared among different hosts. We sampled 120 domesticated animals (104 cattle, 13 horses, and 3 dogs) and 40 wild animals (roe deer) and used multilocus sequence analysis on nine loci (ankA, msp4, groESL, typA, pled, gyrA, recG, polA, and an intergenic region) to characterize the genotypes of A. phagocytophilum present. Phylogenic analysis revealed three genetic clusters of bacterial variants in domesticated animals. The two principal clusters included 98% of the bacterial genotypes found in cattle, which were only distantly related to those in roe deer. One cluster comprised only cattle genotypes, while the second contained genotypes from cattle, horses, and dogs. The third contained all roe deer genotypes and three cattle genotypes. Geographical factors could not explain this clustering pattern. These results suggest that roe deer do not contribute to the spread of A. phagocytophilum in cattle in France. Further studies should explore if these different clusters are associated with differing disease severity in domesticated hosts. Additionally, it remains to be seen if the three clusters of A. phagocytophilum genotypes in cattle correspond to distinct epidemiological cycles, potentially involving different reservoir hosts.

Diversity among French Bacillus anthracis isolates.

PubMed

Fouet, Agnès; Smith, Kimothy L; Keys, Chris; Vaissaire, Josée; Le Doujet, Claudine; Lévy, Martine; Mock, Michèle; Keim, Paul

2002-12-01

While outbreaks of animal anthrax zoonoses still regularly occur in France, little is known about the epidemiology links between them. We have used the eight-locus multilocus variable-number tandem repeat analysis typing technique against a collection of 50 Bacillus anthracis isolates from France. There were eight distinct genotypes belonging to two dissimilar genetic clusters. Regional strain patterns were observed, with the B2 genotypes prevalent in southern France and the A1a genotypes found only in northern France.

Multilocus Sequence Typing of Serially Collected Isolates of Cryptococcus from HIV-Infected Patients in South Africa

PubMed Central

Van Wyk, Marelize; Govender, Nelesh P.; Litvintseva, Anastasia P.

2014-01-01

Patients with cryptococcal meningitis in sub-Saharan Africa frequently relapse following treatment. The natural history and etiology of these recurrent episodes warrant investigation. Here, we used multilocus sequence typing (MLST) to compare the molecular genotypes of strains of Cryptococcus neoformans and Cryptococcus gattii isolated from serial episodes of cryptococcal meningitis that were separated by at least 110 days. The most common MLST genotypes among the isolates were the dominant global clinical genotypes (M5 and M4) of molecular type VNI, as well as the VNI genotypes apparently restricted to southern Africa. In addition, there was considerable genetic diversity among these South African isolates, as 15% of the patients had unique genotypes. Eleven percent of the patients were reinfected with a genetically different strain following their initial diagnosis and treatment. However, the majority of serial episodes (89%) were caused by strains with the same genotype as the original strain. These results indicate that serial episodes of cryptococcosis in South Africa are frequently associated with persistence or relapse of the original infection. Using a reference broth microdilution method, we found that the serial isolates of 11% of the patients infected with strains of C. neoformans var. grubii with identical genotypes exhibited ≥4-fold increases in the MICs to fluconazole. Therefore, these recurrent episodes may have been precipitated by inadequate induction or consolidation of antifungal treatment and occasionally may have been due to increased resistance to fluconazole, which may have developed during the chronic infection. PMID:24648562

Molecular typing of avian pathogenic Escherichia coli colonies originating from outbreaks of E. coli peritonitis syndrome in chicken flocks.

PubMed

Landman, W J M; Buter, G J; Dijkman, R; van Eck, J H H

2014-01-01

Escherichia coli colonies isolated from the bone marrow of fresh dead hens of laying flocks with the E. coli peritonitis syndrome (EPS) were genotyped using pulsed-field gel electrophoresis (PFGE). Typing is important from an epidemiological point of view and also if the use of autogenous (auto)vaccines is considered. Birds with EPS originated from one house of each of three layer farms and one broiler breeder farm. Farms were considered as separate epidemiological units. In total, six flocks were examined including two successive flocks of one layer farm and the broiler breeder farm. E. coli colonies (one per bird) from nine to 16 hens of each flock were genotyped. The clonality of E. coli within birds was studied using five colonies of each of nine to 14 birds per flock. E. coli genotypes, which totalled 15, differed between farms and flocks except for two successive layer flocks that shared three genotypes. One to five genotypes were found per flock with one or two genotypes dominating each outbreak. Within hens, E. coli bacteria were always clonal. Colonies of the same PFGE type always had the same multilocus sequence type. However, four PFGE types shared sequence type 95. Neither PFGE types nor multilocus sequence types were unambiguously related to avian pathogenic E. coli from EPS. In cases where persistence of E. coli strains associated with EPS is found to occur frequently, routine genotyping to select strains for autovaccines should be considered.

Statistical genetics and evolution of quantitative traits

NASA Astrophysics Data System (ADS)

Neher, Richard A.; Shraiman, Boris I.

2011-10-01

The distribution and heritability of many traits depends on numerous loci in the genome. In general, the astronomical number of possible genotypes makes the system with large numbers of loci difficult to describe. Multilocus evolution, however, greatly simplifies in the limit of weak selection and frequent recombination. In this limit, populations rapidly reach quasilinkage equilibrium (QLE) in which the dynamics of the full genotype distribution, including correlations between alleles at different loci, can be parametrized by the allele frequencies. This review provides a simplified exposition of the concept and mathematics of QLE which is central to the statistical description of genotypes in sexual populations. Key results of quantitative genetics such as the generalized Fisher’s “fundamental theorem,” along with Wright’s adaptive landscape, are shown to emerge within QLE from the dynamics of the genotype distribution. This is followed by a discussion under what circumstances QLE is applicable, and what the breakdown of QLE implies for the population structure and the dynamics of selection. Understanding the fundamental aspects of multilocus evolution obtained through simplified models may be helpful in providing conceptual and computational tools to address the challenges arising in the studies of complex quantitative phenotypes of practical interest.

Population size estimation in Yellowstone wolves with error-prone noninvasive microsatellite genotypes.

PubMed

Creel, Scott; Spong, Goran; Sands, Jennifer L; Rotella, Jay; Zeigle, Janet; Joe, Lawrence; Murphy, Kerry M; Smith, Douglas

2003-07-01

Determining population sizes can be difficult, but is essential for conservation. By counting distinct microsatellite genotypes, DNA from noninvasive samples (hair, faeces) allows estimation of population size. Problems arise because genotypes from noninvasive samples are error-prone, but genotyping errors can be reduced by multiple polymerase chain reaction (PCR). For faecal genotypes from wolves in Yellowstone National Park, error rates varied substantially among samples, often above the 'worst-case threshold' suggested by simulation. Consequently, a substantial proportion of multilocus genotypes held one or more errors, despite multiple PCR. These genotyping errors created several genotypes per individual and caused overestimation (up to 5.5-fold) of population size. We propose a 'matching approach' to eliminate this overestimation bias.

Population genetic analysis of Enterocytozoon bieneusi in humans.

PubMed

Li, Wei; Cama, Vitaliano; Feng, Yaoyu; Gilman, Robert H; Bern, Caryn; Zhang, Xichen; Xiao, Lihua

2012-01-01

Genotyping based on sequence analysis of the ribosomal internal transcribed spacer has revealed significant genetic diversity in Enterocytozoonbieneusi. Thus far, the population genetics of E. bieneusi and its significance in the epidemiology of microsporidiosis have not been examined. In this study, a multilocus sequence typing of E. bieneusi in AIDS patients in Lima, Peru was conducted, using 72 specimens previously genotyped as A, D, IV, EbpC, WL11, Peru7, Peru8, Peru10 and Peru11 at the internal transcribed spacer locus. Altogether, 39 multilocus genotypes were identified among the 72 specimens. The observation of strong intragenic linkage disequilibria and limited genetic recombination among markers were indicative of an overall clonal population structure of E. bieneusi. Measures of pair-wise intergenic linkage disequilibria and a standardised index of association (IAS) based on allelic profile data further supported this conclusion. Both sequence-based and allelic profile-based phylogenetic analyses showed the presence of two genetically isolated groups in the study population, one (group 1) containing isolates of the anthroponotic internal transcribed spacer genotype A, and the other (group 2) containing isolates of multiple internal transcribed spacer genotypes (mainly genotypes D and IV) with zoonotic potential. The measurement of linkage disequilibria and recombination indicated group 2 had a clonal population structure, whereas group 1 had an epidemic population structure. The formation of the two sub-populations was confirmed by STRUCTURE and Wright's fixation index (FST) analyses. The data highlight the power of MLST in understanding the epidemiology of E. bieneusi. Published by Elsevier Ltd.

[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.

Genomic Sequencing of Bordetella pertussis for Epidemiology and Global Surveillance of Whooping Cough.

PubMed

Bouchez, Valérie; Guglielmini, Julien; Dazas, Mélody; Landier, Annie; Toubiana, Julie; Guillot, Sophie; Criscuolo, Alexis; Brisse, Sylvain

2018-06-01

Bordetella pertussis causes whooping cough, a highly contagious respiratory disease that is reemerging in many world regions. The spread of antigen-deficient strains may threaten acellular vaccine efficacy. Dynamics of strain transmission are poorly defined because of shortcomings in current strain genotyping methods. Our objective was to develop a whole-genome genotyping strategy with sufficient resolution for local epidemiologic questions and sufficient reproducibility to enable international comparisons of clinical isolates. We defined a core genome multilocus sequence typing scheme comprising 2,038 loci and demonstrated its congruence with whole-genome single-nucleotide polymorphism variation. Most cases of intrafamilial groups of isolates or of multiple isolates recovered from the same patient were distinguished from temporally and geographically cocirculating isolates. However, epidemiologically unrelated isolates were sometimes nearly undistinguishable. We set up a publicly accessible core genome multilocus sequence typing database to enable global comparisons of B. pertussis isolates, opening the way for internationally coordinated surveillance.

Molecular Investigation of Tularemia Outbreaks, Spain, 1997–2008

PubMed Central

Ariza-Miguel, Jaime; Johansson, Anders; Fernández-Natal, María Isabel; Martínez-Nistal, Carmen; Orduña, Antonio; Rodríguez-Ferri, Elías F.; Hernández, Marta

2014-01-01

Tularemia outbreaks occurred in northwestern Spain in 1997–1998 and 2007–2008 and affected >1,000 persons. We assessed isolates involved in these outbreaks by using pulsed-field gel electrophoresis with 2 restriction enzymes and multilocus variable number tandem repeat analysis of 16 genomic loci of Francisella tularensis, the cause of this disease. Isolates were divided into 3 pulsotypes by pulsed-field gel electrophoresis and 8 allelic profiles by multilocus variable number tandem repeat analysis. Isolates obtained from the second tularemia outbreak had the same genotypes as isolates obtained from the first outbreak. Both outbreaks were caused by genotypes of genetic subclade B.Br:FTNF002–00, which is widely distributed in countries in central and western Europe. Thus, reemergence of tularemia in Spain was not caused by the reintroduction of exotic strains, but probably by persistence of local reservoirs of infection. PMID:24750848

Three Divergent Subpopulations of the Malaria Parasite Plasmodium knowlesi

PubMed Central

Lin, Lee C.; Rovie-Ryan, Jeffrine J.; Kadir, Khamisah A.; Anderios, Fread; Hisam, Shamilah; Sharma, Reuben S.K.; Singh, Balbir; Conway, David J.

2017-01-01

Multilocus microsatellite genotyping of Plasmodium knowlesi isolates previously indicated 2 divergent parasite subpopulations in humans on the island of Borneo, each associated with a different macaque reservoir host species. Geographic divergence was also apparent, and independent sequence data have indicated particularly deep divergence between parasites from mainland Southeast Asia and Borneo. To resolve the overall population structure, multilocus microsatellite genotyping was conducted on a new sample of 182 P. knowlesi infections (obtained from 134 humans and 48 wild macaques) from diverse areas of Malaysia, first analyzed separately and then in combination with previous data. All analyses confirmed 2 divergent clusters of human cases in Malaysian Borneo, associated with long-tailed macaques and pig-tailed macaques, and a third cluster in humans and most macaques in peninsular Malaysia. High levels of pairwise divergence between each of these sympatric and allopatric subpopulations have implications for the epidemiology and control of this zoonotic species. PMID:28322705

Clonal Expansion of the Pseudogymnoascus destructans Genotype in North America Is Accompanied by Significant Variation in Phenotypic Expression

PubMed Central

Khankhet, Jordan; Vanderwolf, Karen J.; McAlpine, Donald F.; McBurney, Scott; Overy, David P.; Slavic, Durda; Xu, Jianping

2014-01-01

Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition - dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America. PMID:25122221

Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression.

PubMed

Khankhet, Jordan; Vanderwolf, Karen J; McAlpine, Donald F; McBurney, Scott; Overy, David P; Slavic, Durda; Xu, Jianping

2014-01-01

Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition--dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

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

Variations in the Phytophthora infestans Population in Nepal as Revealed by Nuclear and Mitochondrial DNA Polymorphisms.

PubMed

Ghimire, S R; Hyde, K D; Hodgkiss, I J; Shaw, D S; Liew, E C Y

2003-02-01

ABSTRACT Phytophthora infestans isolates collected from potato and tomato crops from various parts of Nepal during the 1999 and 2000 crop seasons were characterized for nuclear and mitochondrial DNA polymorphisms using restriction fragment length polymorphism markers. The nuclear DNA probe RG57 detected 11 multilocus genotypes among 280 isolates. Three genotypes were detected 21 times or more, constituting 94% of the total population, whereas frequencies of other genotypes ranged from 0.004 to 0.014. The overall genotypic diversity as estimated by the Gleason index was 1.78. Most of the overall diversity was present at the highest level (i.e., interregional, 46%), indicating limited gene flow among regions. Cluster analysis of multilocus genotypes derived from RG57 and mating type data for Nepalese isolates and representative isolates worldwide showed Nepalese isolates grouping into four clusters. Characterization of 67 isolates for mitochondrial DNA polymorphisms revealed the presence of two mt-haplotypes, Ia and Ib with the proportions of 0.88 and 0.12, respectively. Polymorphisms in nuclear and mitochondrial DNA revealed a moderate level of diversity in this population. Genotype NP3 had an identical RG57 fingerprint to US1 and had mt-haplotype Ib, confirming the presence of an old population in Nepal. Most of the genotypes had a different RG57 fingerprint than that of US1 and mt-haplotype Ia, the common characteristics of new populations. The presence of a new population at high proportions in Nepal was consistent with the global trend of mt-haplotype distribution, and suggests the displacement of old populations. This study indicates at least three possible introductions of P. infestans to Nepal.

Genetic Population Structure of Cacao Plantings within a Young Production Area in Nicaragua

PubMed Central

Trognitz, Bodo; Scheldeman, Xavier; Hansel-Hohl, Karin; Kuant, Aldo; Grebe, Hans; Hermann, Michael

2011-01-01

Significant cocoa production in the municipality of Waslala, Nicaragua, began in 1961. Since the 1980s, its economic importance to rural smallholders increased, and the region now contributes more than 50% of national cocoa bean production. This research aimed to assist local farmers to develop production of high-value cocoa based on optimal use of cacao biodiversity. Using microsatellite markers, the allelic composition and genetic structure of cacao was assessed from 44 representative plantings and two unmanaged trees. The population at Waslala consists of only three putative founder genotype spectra (lineages). Two (B and R) were introduced during the past 50 years and occur in >95% of all trees sampled, indicating high rates of outcrossing. Based on intermediate allelic diversity, there was large farm-to-farm multilocus genotypic variation. GIS analysis revealed unequal distribution of the genotype spectra, with R being frequent within a 2 km corridor along roads, and B at more remote sites with lower precipitation. The third lineage, Y, was detected in the two forest trees. For explaining the spatial stratification of the genotype spectra, both human intervention and a combination of management and selection driven by environmental conditions, appear responsible. Genotypes of individual trees were highly diverse across plantings, thus enabling selection for farm-specific qualities. On-farm populations can currently be most clearly recognized by the degree of the contribution of the three genotype spectra. Of two possible strategies for future development of cacao in Waslala, i.e. introducing more unrelated germplasm, or working with existing on-site diversity, the latter seems most appropriate. Superior genotypes could be selected by their specific composite genotype spectra as soon as associations with desired quality traits are established, and clonally multiplied. The two Y trees from the forest share a single multilocus genotype, possibly representing the Mayan, ‘ancient Criollo’ cacao. PMID:21264251

Genetic population structure of cacao plantings within a young production area in Nicaragua.

PubMed

Trognitz, Bodo; Scheldeman, Xavier; Hansel-Hohl, Karin; Kuant, Aldo; Grebe, Hans; Hermann, Michael

2011-01-14

Significant cocoa production in the municipality of Waslala, Nicaragua, began in 1961. Since the 1980s, its economic importance to rural smallholders increased, and the region now contributes more than 50% of national cocoa bean production. This research aimed to assist local farmers to develop production of high-value cocoa based on optimal use of cacao biodiversity. Using microsatellite markers, the allelic composition and genetic structure of cacao was assessed from 44 representative plantings and two unmanaged trees. The population at Waslala consists of only three putative founder genotype spectra (lineages). Two (B and R) were introduced during the past 50 years and occur in >95% of all trees sampled, indicating high rates of outcrossing. Based on intermediate allelic diversity, there was large farm-to-farm multilocus genotypic variation. GIS analysis revealed unequal distribution of the genotype spectra, with R being frequent within a 2 km corridor along roads, and B at more remote sites with lower precipitation. The third lineage, Y, was detected in the two forest trees. For explaining the spatial stratification of the genotype spectra, both human intervention and a combination of management and selection driven by environmental conditions, appear responsible. Genotypes of individual trees were highly diverse across plantings, thus enabling selection for farm-specific qualities. On-farm populations can currently be most clearly recognized by the degree of the contribution of the three genotype spectra. Of two possible strategies for future development of cacao in Waslala, i.e. introducing more unrelated germplasm, or working with existing on-site diversity, the latter seems most appropriate. Superior genotypes could be selected by their specific composite genotype spectra as soon as associations with desired quality traits are established, and clonally multiplied. The two Y trees from the forest share a single multilocus genotype, possibly representing the Mayan, 'ancient Criollo' cacao.

Seroprevalence and first multilocus microsatellite genotyping of Neospora caninum in dairy cattle in Henan, central China.

PubMed

Qian, Weifeng; Wang, Tianqi; Yan, Wenchao; Zhang, Min; Han, Lifang; Xue, Rui; Song, Shaofu; Lv, Chaochao

2017-09-15

Neospora caninum is one of the important causes of abortion in cattle worldwide, and losses due to neosporosis to the cattle industry are considerable. However, the knowledge of genetic characterization of this parasite is limited. The aim of the present study is to determine the prevalence and genetic characterization of N. caninum from dairy cows in Henan Province, central China. A total of 510 blood samples and 7 aborted fetuses were collected from 8 dairy farms in Henan Province. Serum antibodies to N. caninum were examined by ELISA using a recombinant tNcSRS2 protein as the coating antigen. The overall seroprevalence of N. caninum in dairy cows was 41.2% (210/510). The seropositivity rate of N. caninum in aborting cows (49.3%) was statistically significant higher than that (29.3%) in non-aborting cows (p<0.05) with an odds ratio of 2.44 (95% CI, 1.61-3.41). Statistical association was also found between farm type and the seropositivity rate of N. caninum infection in cows (p<0.01).N. caninum DNA was detected from 6 of 396 blood samples (1.5%) and 4 of 7 aborted fetuses by nested PCR based on NC5 gene, and the 10N. caninum positive DNA samples were further analyzed by multilocus microsatellite (MS) genotyping for MS4, MS5, MS6A, MS7, MS8, MS10, and MS12. Only 2 samples were successfully genotyped at all genetic loci, and two unique profiles including two novel allelic patterns were identified. To our knowledge, this study is the first report of genetic characterization of N. caninum isolates from naturally infected dairy cows based on multilocus microsatellites (more than 2 loci) in China. Copyright © 2017 Elsevier B.V. All rights reserved.

The long-term evolution of multilocus traits under frequency-dependent disruptive selection.

PubMed

van Doorn, G Sander; Dieckmann, Ulf

2006-11-01

Frequency-dependent disruptive selection is widely recognized as an important source of genetic variation. Its evolutionary consequences have been extensively studied using phenotypic evolutionary models, based on quantitative genetics, game theory, or adaptive dynamics. However, the genetic assumptions underlying these approaches are highly idealized and, even worse, predict different consequences of frequency-dependent disruptive selection. Population genetic models, by contrast, enable genotypic evolutionary models, but traditionally assume constant fitness values. Only a minority of these models thus addresses frequency-dependent selection, and only a few of these do so in a multilocus context. An inherent limitation of these remaining studies is that they only investigate the short-term maintenance of genetic variation. Consequently, the long-term evolution of multilocus characters under frequency-dependent disruptive selection remains poorly understood. We aim to bridge this gap between phenotypic and genotypic models by studying a multilocus version of Levene's soft-selection model. Individual-based simulations and deterministic approximations based on adaptive dynamics theory provide insights into the underlying evolutionary dynamics. Our analysis uncovers a general pattern of polymorphism formation and collapse, likely to apply to a wide variety of genetic systems: after convergence to a fitness minimum and the subsequent establishment of genetic polymorphism at multiple loci, genetic variation becomes increasingly concentrated on a few loci, until eventually only a single polymorphic locus remains. This evolutionary process combines features observed in quantitative genetics and adaptive dynamics models, and it can be explained as a consequence of changes in the selection regime that are inherent to frequency-dependent disruptive selection. Our findings demonstrate that the potential of frequency-dependent disruptive selection to maintain polygenic variation is considerably smaller than previously expected.

Population Structure and History in Developing Core Sets in Wild Germplasm

USDA-ARS?s Scientific Manuscript database

Accurate inference of genetic discontinuities between populations is an essential component in studies of intraspecific biodiversity and evolution, as well as associative genetics. Multi-locus genotypes were amplified from 949 individuals representing seedling trees from 88 half-sib families from ei...

Population Structure And History In Developing Core Sets In Wild Germplasm.

USDA-ARS?s Scientific Manuscript database

Accurate inference of genetic discontinuities between populations is an essential component in studies of intraspecific biodiversity and evolution, as well as associative genetics. Multi-locus genotypes were amplified from 949 individuals representing seedling trees from 88 half-sib families from ei...

Age-dependent, negative heterozygosity-fitness correlations and local effects in an endangered Caribbean reptile, Iguana delicatissima.

PubMed

Judson, Jessica L Martin; Knapp, Charles R; Welch, Mark E

2018-02-01

Inbreeding depression can have alarming impacts on threatened species with small population sizes. Assessing inbreeding has therefore become an important focus of conservation research. In this study, heterozygosity-fitness correlations (HFCs) were measured by genotyping 7 loci in 83 adult and 184 hatchling Lesser Antillean Iguanas, Iguana delicatissima, at a communal nesting site in Dominica to assess the role of inbreeding depression on hatchling fitness and recruitment to the adult population in this endangered species. We found insignificant correlations between multilocus heterozygosity and multiple fitness proxies in hatchlings and adults. Further, multilocus heterozygosity did not differ significantly between hatchlings and adults, which suggests that the survivorship of homozygous hatchlings does not differ markedly from that of their heterozygous counterparts. However, genotypes at two individual loci were correlated with hatching date, a finding consistent with the linkage between specific marker loci and segregating deleterious recessive alleles. These results provide only modest evidence that inbreeding depression influences the population dynamics of I. delicatissima on Dominica.

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

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/.

Epidemiological study of erythromycin-resistant Streptococcus pyogenes from Korea and Japan by emm genotyping and multilocus sequence typing.

PubMed

Takahashi, Takashi; Arai, Kazuaki; Lee, Dong Hyun; Koh, Eun Ha; Yoshida, Haruno; Yano, Hisakazu; Kaku, Mitsuo; Kim, Sunjoo

2016-01-01

We determined the epidemiological characteristics of erythromycin (EM)-resistant Streptococcus pyogenes (group A streptococci, GAS) strains isolated from Korea and Japan, using emm genotyping and multilocus sequence typing (MLST). Clinical isolates of GAS had been collected from 1992 to 2012 in Korea and from 2004 to 2009 in Japan. EM resistance was determined by the microdilution method, and resistance genotypes were assessed by PCR. The emm genotyping and MLST were performed by DNA sequencing. The emm genotypes and sequence types (STs) were concordant in 143 (85.1%) of 168 EM-resistant GAS strains from Korea. ST36/emm12 (35.1%), ST52/emm28 (22.6%), and ST49/emm75 (16.1%) were the most common types. Most of the ST36 (93.9%) and ST52 (95.8%) strains harbored erm(B), whereas strains ST49, ST42, and ST15 contained mef(A). The concordance between emm genotypes and STs was 41 (93.2%) among 44 EM-resistant GAS strains from Japan. ST36/emm12 (34.1%), ST49/emm75 (18.2%), and ST28/emm1 (15.9%) were the major types. ST36 isolates harbored either erm(B) (56.3%) or mef(A) (37.5%), whereas isolates ST28, ST49, and ST38 carried only mef(A). The proportion of erm(B) and mef(A) was 66.1% and 33.3% in Korea and 22.7% and 68.2% in Japan, respectively. The common STs in Korea and Japan were ST36 and ST49, whereas ST52 was present only in Korea and ST28 only in Japan. Genotype erm(B) was predominant in Korea, whereas mef(A) was frequent in Japan. There were differences between Korea and Japan regarding the frequencies of emm genotypes, STs, and EM resistance genes among the EM-resistant GAS.

Microsatellite DNA fingerprinting, differentiation, and genetic relationships of clones, cultivars, and varieties of six poplar species from three sections of the genus Populus.

PubMed

Rahman, Muhammad H; Rajora, Om P

2002-12-01

Accurate identification of Populus clones and cultivars is essential for effective selection, breeding, and genetic resource management programs. The unit of cultivation and breeding in poplars is a clone, and individual cultivars are normally represented by a single clone. Microsatellite DNA markers of 10 simple sequence repeat loci were used for genetic fingerprinting and differentiation of 96 clones/cultivars and varieties belonging to six Populus species (P. deltoides, P. nigra, P. balsamifera, P. trichocarpa, P. grandidentata, and P maximowiczii) from three sections of the genus. All 96 clones/cultivars could be uniquely fingerprinted based on their single- or multilocus microsatellite genotypes. The five P. grandidentata clones could be differentiated based on their single-locus genotypes, while six clones of P. trichocarpa and 11 clones of P. maximowiczii could be identified by their two-locus genotypes. Twenty clones of P. deltoides and 25 clones of P. nigra could be differentiated by their multilocus genotypes employing three loci, and 29 clones of P. balsamifera required the use of multilocus genotypes at five loci for their genetic fingerprinting and differentiation. The loci PTR3, PTR5, and PTR7 were found to be the most informative for genetic fingerprinting and differentiation of the clones. The mean number of alleles per locus ranged from 2.9 in P. trichocarpa or P. grandidentata to 6.0 in P. balsamifera and 11.2 in 96 clones of the six species. The mean number of observed genotypes per locus ranged from 2.4 in P. grandidentata to 7.4 in P. balsamifera and 19.6 in 96 clones of the six species. The mean number of unique genotypes per locus ranged from 1.3 in P. grandidentata to 3.9 in P. deltoides and 8.8 in 96 clones of the six species. The power of discrimination of the microsatellite DNA markers in the 96 clones ranged from 0.726 for PTR4 to 0.939 for PTR7, with a mean of 0.832 over the 10 simple sequence repeat loci. Clones/cultivars from the same species showed higher microsatellite DNA similarities than the clones from the different species. A UPGMA cluster plot constructed from the microsatellite genotypic similarities separated the 96 clones into six major groups corresponding to their species. Populus nigra var. italica clones were genetically differentiated from the P. nigra var. nigra clones. Microsatellite DNA markers could be useful in genetic fingerprinting, identification, classification, certification, and registration of clones, clultivars, and varieties as well as genetic resource management and protection of plant breeders' rights in Populus.

Genotypic and phenotypic characterization of Cryptococcus flavescens, beneficial biocontrol agents for controlling Fusarium head blight of wheat

USDA-ARS?s Scientific Manuscript database

Cryptococcus flavescens strain OH182.9_3C (3C) previously displayed significant biological control activity against Fusarium head blight, a globally important disease of wheat; however, the diversity within C. flavescens has not been previously characterized. Multilocus sequence typing was performed...

Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high hetrogeneity

USDA-ARS?s Scientific Manuscript database

Fecal specimens from 120 lambs in Valencia (Spain) were analyzed for Giardia duodenalis by IFA and nested-PCR using the beta giardin, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and small subunit ribosomal RNA (ssurRNA) genes. The highest prevalence was obtained using the ssurRN...

Multilocus sequence typing of Mycoplasma bovis reveals host-specific genotypes in cattle versus bison

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and is the bacterium isolated most frequently from the polymicrobial syndrome known as bovine respiratory disease complex (BRDC). Recently, M. bovis has emerged as a significant health problem in bison, causing necro...

Multi-locus mixed model analysis of stem rust resistance in a worldwide collection of winter wheat

USDA-ARS?s Scientific Manuscript database

Genome-wide association mapping is a powerful tool for dissecting the relationship between phenotypes and genetic variants in diverse populations. With improved cost efficiency of high-throughput genotyping platforms, association mapping is a desirable method to mine populations for favorable allele...

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

PubMed

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

2011-02-01

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

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

PubMed

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

2017-12-01

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

Relationships between functional genes in Lactobacillus delbrueckii ssp. bulgaricus isolates and phenotypic characteristics associated with fermentation time and flavor production in yogurt elucidated using multilocus sequence typing.

PubMed

Liu, Wenjun; Yu, Jie; Sun, Zhihong; Song, Yuqin; Wang, Xueni; Wang, Hongmei; Wuren, Tuoya; Zha, Musu; Menghe, Bilige; Heping, Zhang

2016-01-01

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is well known for its worldwide application in yogurt production. Flavor production and acid producing are considered as the most important characteristics for starter culture screening. To our knowledge this is the first study applying functional gene sequence multilocus sequence typing technology to predict the fermentation and flavor-producing characteristics of yogurt-producing bacteria. In the present study, phenotypic characteristics of 35 L. bulgaricus strains were quantified during the fermentation of milk to yogurt and during its subsequent storage; these included fermentation time, acidification rate, pH, titratable acidity, and flavor characteristics (acetaldehyde concentration). Furthermore, multilocus sequence typing analysis of 7 functional genes associated with fermentation time, acid production, and flavor formation was done to elucidate the phylogeny and genetic evolution of the same L. bulgaricus isolates. The results showed that strains significantly differed in fermentation time, acidification rate, and acetaldehyde production. Combining functional gene sequence analysis with phenotypic characteristics demonstrated that groups of strains established using genotype data were consistent with groups identified based on their phenotypic traits. This study has established an efficient and rapid molecular genotyping method to identify strains with good fermentation traits; this has the potential to replace time-consuming conventional methods based on direct measurement of phenotypic traits. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

Multilocus genotyping of a ‘Candidatus Phytoplasma aurantifolia’-related strain associated with cauliflower phyllody disease in China

USDA-ARS?s Scientific Manuscript database

A new cauliflower disease characterized by formation of leaf-like inflorescences and malformed flowers occurred in a seed production filed located in Yunnan, a southwest province of China. In the diseased plants, floral organs in three inner whorls (petals, carpels, and stamens) were under-develope...

Clonality, recombination, and hybridization in the plumbing-inhabiting human pathogen Fusarium keratoplasticum inferred from multilocus sequence typing

USDA-ARS?s Scientific Manuscript database

Recent work has shown that Fusarium species and genotypes most commonly associated with human infections, particularly of the cornea (mycotic keratitis), are the same as those most commonly isolated from plumbing systems. The species most dominant in plumbing biofilms is Fusarium keratoplasticum, a ...

Composition and toxigenic potential of the Fusarium graminearum species complex from maize ears, stalks and stubble in Brazil

USDA-ARS?s Scientific Manuscript database

Detailed knowledge of the composition and toxigenic potential of the Fusarium graminearum species complex affecting maize crops in Brazil is lacking. A multilocus genotype approach was used to identify 539 isolates from three sub-collections: 1) maize kernels (n= 110) from five states spanning sout...

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 genotyping identifies a highly homogeneous phytoplasma lineage associated with sweet cherry virescence disease in China and its carriage by an erythroneurine leafhopper

USDA-ARS?s Scientific Manuscript database

Phytoplasmas are a diverse group of insect-transmitted, cell wall-less bacteria that colonize plant phloem sieve elements and cause numerous diseases in economically important crops. Living a trans-kingdom parasitic life, phytoplasmas possess dynamic genomes and evolve rapidly toward formation of di...

Multilocus genotypes indicate differentiation among Puccinia psidii populations from South America and Hawaii

Treesearch

R. N. Graca; A. C. Alfenas; A. L. Ross-Davis; Ned Klopfenstein; M. -S. Kim; T. L. Peever; P. G. Cannon; J. Y. Uchida; C. Y. Kadooka; R. D. Hauff

2011-01-01

Puccinia psidii is the cause of rust disease of many host species in the Myrtaceae family, including guava (Psidium spp.), eucalypt (Eucalyptus spp.), rose apple (Syzygium jambos), and 'ohi'a (Metrosideros polymorpha). First reported in 1884 on guava in Brazil, the rust has since been detected in South America (Argentina, Brazil, Colombia, Paraguay, Uruguay,...

Genetic structuring of Coues white-tailed deer in the southwestern United States

Treesearch

Roy G. Lopez

2006-01-01

The manuscripts in this thesis examine different aspects of white-tailed deer. In the first manuscript I used microsatellite DNA markers in the form of multilocus genotype data and microsatellite allele frequencies to examine spatial patterns of genetic relatedness for Coues white-tailed deer (Odocoileus virginianus couesi) in Arizona and New Mexico...

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

PubMed

Tellapragada, Chaitanya; Kamthan, Aayushi; Shaw, Tushar; Ke, Vandana; Kumar, Subodh; Bhat, Vinod; Mukhopadhyay, Chiranjay

2016-01-01

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006-2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as "outliers" on the eBURST "Population snapshot", suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

Lymphogranuloma venereum rates increased and Chlamydia trachomatis genotypes changed among men who have sex with men in Sweden 2004-2016.

PubMed

Isaksson, Jenny; Carlsson, Ola; Airell, Åsa; Strömdahl, Susanne; Bratt, Göran; Herrmann, Björn

2017-11-01

This study aimed to determine the incidence of lymphogranuloma venereum (LGV) in Sweden since 2004 and to study in detail a consecutive number of Chlamydia trachomatis cases in men who have sex with men (MSM) during a 10 month period (September 2014 to July 2015). LGV increased from sporadic import cases in 2004 to comprise a spread within Sweden in 2016. Initially, only the L2b ompA genotype was detected, but in 2015 half of the genotyped LGV cases were L2 genotype. The changing genotype distribution in Sweden is linked to increased LGV spread in Europe. High-resolution multilocus sequence typing of 168 C. trachomatis cases from MSM in 2015 resulted in 29 sequence types, of which 3 accounted for 49 % of cases. The increased rates and different genotypes of LGV indicate that more concern for high-risk taking MSM is needed to avoid further spread of this invasive infection.

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

PubMed

Giannoutsou, E; Sotiriou, P; Apostolakos, P; Galatis, B

2013-10-01

The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.

Clonal Population Structure of Pseudomonas stutzeri, a Species with Exceptional Genetic Diversity

PubMed Central

Rius, Núria; Fusté, M. Carme; Guasp, Caterina; Lalucat, Jorge; Lorén, José G.

2001-01-01

Genetic diversity and genetic relationships among 42 Pseudomonas stutzeri strains belonging to several genomovars and isolated from different sources were investigated in an examination of 20 metabolic enzymes by multilocus enzyme electrophoresis analysis. Forty-two distinct allele profiles were identified, indicating that all multilocus genotypes were represented by a single strain. All 20 loci were exceptionally polymorphic, with an average of 15.9 alleles per locus. To the best of our knowledge, this P. stutzeri sample exhibited the highest mean genetic diversity (H = 0.876) found to date in all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles was identified. The index of association (IA) for the P. stutzeri strains analyzed was 1.10. The IA values were always significantly different from zero for all subgroups studied, including clinical and environmental isolates and strains classified as genomovar 1. These results suggest that the population structure of P. stutzeri is strongly clonal, indicating that there is no significant level of assortative recombination that might destroy linkage disequilibrium. PMID:11133969

Gene-gene interactions of CYP2A6 and MAOA polymorphisms on smoking behavior in Chinese male population.

PubMed

Tang, Xun; Guo, Song; Sun, Hongqiang; Song, Xuemei; Jiang, Zuonin; Sheng, Lixiang; Zhou, Dongfeng; Hu, Yonghua; Chen, Dafang

2009-05-01

Nicotine is the major psychoactive ingredient in tobacco, and is responsible for dependence through the nicotine-stimulated reward pathway mediated by the central dopaminergic system. Consequently, genetic polymorphisms in both nicotine metabolism and dopamine catabolism genes may influence smoking behavior, and interact with each other resulting in risk modulation. In this study, we investigated the association and multilocus gene-gene interactions of cytochrome P450 2A6 (CYP2A6), dopamine beta-hydroxylase (DBH), catechol O-methyl transferase (COMT), and monoamine oxidase A (MAOA) polymorphisms with smoking behavior in a community-based Chinese male population. The polymorphisms were genotyped in 203 current smokers, 66 former smokers, and 102 never smokers. Multivariate logistic regression models and the multifactor dimensionality reduction method were used to analyze the association and multilocus gene-gene interactions. Statistically significant trends were shown for increased risk of smoking initiation in participants with CYP2A6*1B/CYP2A6*1B genotypes compared with those with CYP2A6*1A/CYP2A6*1A genotypes [odds ratio (OR)=3.5, 95% confidence interval (CI)= 1.5-8.1], and participants with CYP2A6*1/CYP2A6*1 genotypes were at higher risk of smoking initiation (OR=2.4, 95% CI=1.2-4.5) and smoking persistence (OR=4.0, 95% CI=1.5-10.3) than those who have CYP2A6*4C genotypes. Moreover, the best model involved a gene-gene interaction between MAOA and CYP2A6 was characterized by the multifactor dimensionality reduction method (64.11% accuracy, P<0.001), and indicated that carriers of the combined 1460 T/O genotype for MAOA EcoRV and CYP2A6*1/CYP2A6*1 genotypes were at higher risk of smoking (OR=15.4, 95% CI=4.5-52.5). These findings suggested a substantial influence of CYP2A6 polymorphism as well as the interaction with MAOA resulting in risk modulation on smoking behavior in Chinese male population.

Genetic epidemiology of the Sudden Oak Death pathogen Phytophthora ramorum in California

Treesearch

S. Mascheretti; P.J.P. Croucher; M. Kozanitas; L. Baker; M. Garbelotto

2009-01-01

A total of 669 isolates of Phytophthora ramorum, the pathogen responsible for Sudden Oak Death, were collected from 34 Californian forests and from the ornamental plant-trade. Seven microsatellite markers revealed 82 multilocus genotypes (MGs) of which only three were abundant (>10%). Iteratively collapsing based upon minimum ΦST, yielded five meta-samples and five...

Isolation of Neospora caninum from kidney and brain of a bovine foetus and molecular characterization in Brazil.

PubMed

Locatelli Dittrich, Rosangela; Regidor-Cerrillo, Javier; Ortega-Mora, Luis Miguel; Oliveira Koch, Marília de; Busch, Ana Paula B; Gonçalves, Kamila Alcalá; Cruz, Amilcar A

2018-02-01

Bovine neosporosis has become a disease of international concern as it is among the main causes of abortion in cattle. Viable N. caninum has been isolated from brains of fetuses and neonatal calves, and there is no report of isolation of tachyzoites from kidney. Also, detailed information about the genetic diversity of N. caninum is scarce. N. caninum tachyzoites were isolated from the kidney and the brain of an aborted 4-month-old bovine foetus. The parasite was confirmed to be N. caninum by PCR. The tachyzoites of the new isolate, named BNC-PR4, were propagated in Vero cell cultures. Pathogenicity of the parasite was examined in BALB/c mice. Mice inoculated intraperitoneally with BNC-PR4 failed to yield clinical signs of disease and did not induce severe brain lesions, suggesting a bovine isolate with low virulence. The N. caninum-positive DNA sample was further analyzed by multilocus microsatellite (MS) genotyping for MS4, MS5, MS6A, MS6B, MS7, MS8, MS10, MS12, and MS21. Multilocus-microsatellite genotyping revealed a unique genetic profile that differed from previously reported isolates. Published by Elsevier Inc.

Genotypic analysis of Mucor from the platypus in Australia.

PubMed

Connolly, J H; Stodart, B J; Ash, G J

2010-01-01

Mucor amphibiorum is the only pathogen known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads (Bufo marinus) and green tree frogs (Litoria caerulea) from mainland Australia but has not been confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of 21 Mucor isolates representing isolates from the platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of most of the isolates. Representative isolates from infected platypuses formed a clade containing the reference isolates of M. amphibiorum from the Centraal Bureau voor Schimmelcultures repository. The M. amphibiorum isolates showed a close sequence identity with Mucor indicus and consisted of two haplotypes, differentiated by single nucleotide polymorphisms within the ITS1 and ITS2 regions. With the exception of isolate 96-4049, all isolates from platypuses were in one haplotype. Multilocus fingerprinting via the use of intersimple sequence repeats polymerase chain reaction identified 19 genotypes. Two major clusters were evident: 1) M. amphibiorum and Mucor racemosus; and 2) Mucor circinelloides, Mucor ramosissimus, and Mucor fragilis. Seven M. amphibiorum isolates from platypuses were present in two subclusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate subcluster, distinct from other Mucor spp. The combination of sequencing and multilocus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functional diversity will provide critical information for its management in Tasmanian river systems.

Significant spread of extensively drug-resistant Acinetobacter baumannii genotypes of clonal complex 92 among intensive care unit patients in a university hospital in southern Iran.

PubMed

Saffari, Fereshteh; Monsen, Tor; Karmostaji, Afsaneh; Azimabad, Fahimeh Bahadori; Widerström, Micael

2017-11-01

Infections associated with Acinetobacter baumannii represent an increasing threat in healthcare settings. Therefore, we investigated the epidemiological relationship between clinical isolates of A. baumannii obtained from patients in a university hospital in Bandar Abbas in southern Iran. Sixty-four consecutive non-duplicate clinical isolates collected during 2014-2015 were subjected to susceptibility testing, clonal relationship analysis using PFGE, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST), and examined for the presence of carbapenemases and integrons. Almost all A. baumannii isolates were extensively drug-resistant (XDR; 98 %) and carried an OXA carbapenemase gene (blaOXA-23-like; 98 %) and class 1 integrons (48 %). PFGE and MLST analysis identified three major genotypes, all belonging to clonal complex 92 (CC92): sequence type 848 (ST848) (n=23), ST451 (n=16) and ST195 (n=8). CC92 has previously been documented in the hospital setting in northern Iran, and ST195 has been reported in Arab States of the Persian Gulf. These data suggest national and global transmission of A. baumannii CC92. This report demonstrates the occurrence and potential spread of closely related XDR genotypes of A. baumannii CC92 within a university hospital in southern Iran. These genotypes were found in the majority of the investigated isolates, showed high prevalence of blaOXA-23 and integron class 1, and were associated with stay in the intensive care unit. Very few treatment options remain for healthcare-adapted XDR A. baumannii, and hence effective measures are desperately needed to reduce the spread of these strains and resultant infections in the healthcare setting.

Particular Candida albicans Strains in the Digestive Tract of Dyspeptic Patients, Identified by Multilocus Sequence Typing

PubMed Central

Gong, Yan-Bing; Zheng, Jian-Ling; Jin, Bo; Zhuo, De-Xiang; Huang, Zhu-Qing; Qi, He; Zhang, Wei; Duan, Wei; Fu, Ji-Ting; Wang, Chui-Jie; Mao, Ze-Bin

2012-01-01

Background Candida albicans is a human commensal that is also responsible for chronic gastritis and peptic ulcerous disease. Little is known about the genetic profiles of the C. albicans strains in the digestive tract of dyspeptic patients. The aim of this study was to evaluate the prevalence, diversity, and genetic profiles among C. albicans isolates recovered from natural colonization of the digestive tract in the dyspeptic patients. Methods and Findings Oral swab samples (n = 111) and gastric mucosa samples (n = 102) were obtained from a group of patients who presented dyspeptic symptoms or ulcer complaints. Oral swab samples (n = 162) were also obtained from healthy volunteers. C. albicans isolates were characterized and analyzed by multilocus sequence typing. The prevalence of Candida spp. in the oral samples was not significantly different between the dyspeptic group and the healthy group (36.0%, 40/111 vs. 29.6%, 48/162; P > 0.05). However, there were significant differences between the groups in the distribution of species isolated and the genotypes of the C. albicans isolates. C. albicans was isolated from 97.8% of the Candida-positive subjects in the dyspeptic group, but from only 56.3% in the healthy group (P < 0.001). DST1593 was the dominant C. albicans genotype from the digestive tract of the dyspeptic group (60%, 27/45), but not the healthy group (14.8%, 4/27) (P < 0.001). Conclusions Our data suggest a possible link between particular C. albicans strain genotypes and the host microenvironment. Positivity for particular C. albicans genotypes could signify susceptibility to dyspepsia. PMID:22536371

Genetic diversity in Trichomonas vaginalis.

PubMed

Meade, John C; Carlton, Jane M

2013-09-01

Recent advances in genetic characterisation of Trichomonas vaginalis isolates show that the extensive clinical variability in trichomoniasis and its disease sequelae are matched by significant genetic diversity in the organism itself, suggesting a connection between the genetic identity of isolates and their clinical manifestations. Indeed, a high degree of genetic heterogeneity in T vaginalis isolates has been observed using multiple genotyping techniques. A unique two-type population structure that is both local and global in distribution has been identified, and there is evidence of recombination within each group, although sexual recombination between the groups appears to be constrained. There is conflicting evidence in these studies for correlations between T vaginalis genetic identity and clinical presentation, metronidazole susceptibility, and the presence of T vaginalis virus, underscoring the need for adoption of a common standard for genotyping the parasite. Moving forward, microsatellite genotyping and multilocus sequence typing are the most robust techniques for future investigations of T vaginalis genotype-phenotype associations.

Incidence, Antimicrobial Resistance, and Molecular Characteristics of Nontyphoidal Salmonella Including Extended-Spectrum β-Lactamase Producers in Retail Chicken Meat.

PubMed

Choi, Dasom; Chon, Jung-Whan; Kim, Hong-Seok; Kim, Dong-Hyeon; Lim, Jong-Soo; Yim, Jin-Hyeok; Seo, Kun-Ho

2015-11-01

The present study was undertaken to determine the prevalence of Salmonella in 100 chicken carcass samples from five integrated broiler operation brands in Korea. Serotypes, antibiotic resistance patterns, extended-spectrum β-lactamase (ESBL) genotype, and clonal divergence using multilocus sequence typing of the isolated strains were analyzed. A total of 42 chicken samples were contaminated with nontyphoidal Salmonella (NTS) isolates: 16 isolates (38%) were Salmonella Virchow, 9 (21%) were Salmonella Bareilly, and 8 (19%) were Salmonella Infantis. A multidrug resistance (MDR; resistant to more than three classes of antibiotics) phenotype was observed in 29% of the isolates, which were resistant to five or more classes of antibiotics. The dominant MDR type was resistance to classes of penicillin, cephalosporins, aminoglycosides, quinolones, and tetracyclines. All the MDR isolates were positive for ESBL producers, and all but one (with the CTX-M-1 genotype) had the CTX-M-15 genotype. Multilocus sequence typing of the isolates revealed ST16 as the dominant sequence type; Salmonella Virchow, Salmonella Infantis, and Salmonella Richmond were all ST16, indicating a close genetic relationship between these serovars. This is the first study in Korea showing the CTX-M-1 type of NTS and the prevalence of ESBL-producing strains among NTS isolated from retail chicken meat. Our findings suggest that MDR Salmonella contamination is widely prevalent in retail chicken meat, and consumption of inadequately cooked products could lead to dissemination of NTS, which is hazardous to human health.

Tracing the first steps of American sturgeon pioneers in Europe

USGS Publications Warehouse

Ludwig, A.; Arndt, U.; Lippold, S.; Benecke, N.; Debus, L.; King, T.L.; Matsumura, S.

2008-01-01

Background. A Baltic population of Atlantic sturgeon was founded ???1,200 years ago by migrants from North America, but after centuries of persistence, the population was extirpated in the 1960s, mainly as a result of over-harvest and habitat alterations. As there are four genetically distinct groups of Atlantic sturgeon inhabiting North American rivers today, we investigated the genetic provenance of the historic Baltic population by ancient DNA analyses using mitochondrial and nuclear markers. Results. The phylogeographic signal obtained from multilocus microsatellite DNA genotypes and mitochondrial DNA control region haplotypes, when compared to existing baseline datasets from extant populations, allowed for the identification of the region-of-origin of the North American Atlantic sturgeon founders. Moreover, statistical and simulation analyses of the multilocus genotypes allowed for the calculation of the effective number of individuals that originally founded the European population of Atlantic sturgeon. Our findings suggest that the Baltic population of A. oxyrinchus descended from a relatively small number of founders originating from the northern extent of the species' range in North America. Conclusion. These results demonstrate that the most northerly distributed North American A. oxyrinchus colonized the Baltic Sea ???1,200 years ago, suggesting that Canadian specimens should be the primary source of broodstock used for restoration in Baltic rivers. This study illustrates the great potential of patterns obtained from ancient DNA to identify population-of-origin to investigate historic genotype structure of extinct populations. ?? 2008 Ludwig et al; licensee BioMed Central Ltd.

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms.

PubMed

Tong, Steven Y C; Xie, Shirley; Richardson, Leisha J; Ballard, Susan A; Dakh, Farshid; Grabsch, Elizabeth A; Grayson, M Lindsay; Howden, Benjamin P; Johnson, Paul D R; Giffard, Philip M

2011-01-01

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.

Giardia duodenalis Infections in Humans and Other Animals in China

PubMed Central

Li, Junqiang; Wang, Haiyan; Wang, Rongjun; Zhang, Longxian

2017-01-01

Giardia duodenalis is an important zoonotic pathogen in both public and veterinary health, and has been genotyped into at least eight assemblages (A–H), each with a distinct host range. In recent years, this intestinal protozoan parasite has been identified widely in humans and various other animals, and has even been recorded in environmental contaminants. Along with whole genome sequencing of G. duodenalis, multilocus sequence typing is increasingly being used to characterize G. duodenalis isolates. Here, we review the epidemiology, genotyping, and subtyping of G. duodenalis from humans and a wide range of other animals, as well as from wastewater, in China. PMID:29081771

Unraveling the etiology of North American grapevine yellows (NAGY): multilocus genotyping and structural analysis of secY proteins distinguish NAGYIII phytoplasma strains from strains causing X-disease

USDA-ARS?s Scientific Manuscript database

North American grapevine yellows (NAGY) disease has sometimes been ascribed to infection of Vitis vinifera L. by X-disease phytoplasma, but the accuracy of this attribution has remained open to question. In the present study of NAGY etiology, the disease was discovered in Maryland, Pennsylvania, Oh...

Multi-Locus Candidate Gene Analyses of Lipid Levels in a Pediatric Turkish Cohort: Lessons Learned on LPL, CETP, LIPC, ABCA1, and SHBG

PubMed Central

Eren, Fatih; Agirbasli, Deniz; White, Marquitta J.; Williams, Scott M

2013-01-01

Abstract Cardiovascular risk factors and atherosclerosis precursors were examined in 365 Turkish children and adolescents. Study participants were recruited at five different state schools. We tested single and multi-locus effects of six polymorphisms from five candidate genes, chosen based on prior known association with lipid levels in adults, for association with low (≤10th percentile) high density lipoprotein cholesterol (HDL-C) and high (≥90th percentile) triglycerides (TG), and the related continuous outcomes. We observed an association between CETP variant rs708272 and low HDL-C (allelic p=0.020, genotypic p=0.046), which was supported by an independent analysis, PRAT (PRAT control p=0.027). Sex-stratified logistic regression analysis showed that the B2 allele of rs708272 decreased odds of being in the lower tenth percentile of HDL-C measurements (OR=0.36, p=0.02) in girls; this direction of effect was also seen in boys but was not significant (OR=0.64, p=0.21). Logistic regression analysis also revealed that the T allele of rs6257 (SHBG) decreased odds of being in the top tenth percentile of TG measurements in boys (OR=0.43, p=0.03). Analysis of lipid levels as a continuous trait revealed a significant association between rs708272 (CETP) and LDL-C levels in males (p=0.02) with the B2B2 genotype group having the lowest mean LDL-C; the same direction of effect was also seen in females (p=0.05). An effect was also seen between rs708272 and HDL-C levels in girls (p=0.01), with the B2B2 genotype having the highest mean HDL-C levels. Multi-locus analysis, using quantitative multifactor dimensionality reduction (qMDR) identified the previously mentioned CETP variant as the best single locus model, and overall model, for predicting HDL-C levels in children. This study provides evidence for association between CETP and low HDL-C phenotype in children, but the results appear to be weaker in children than previous results in adults and may also be subject to gender effects. PMID:23988150

Relationship between Trypanosoma brucei rhodesiense genetic diversity and clinical spectrum among sleeping sickness patients in Uganda.

PubMed

Kato, Charles D; Mugasa, Claire M; Nanteza, Ann; Matovu, Enock; Alibu, Vincent P

2017-10-27

Human African trypanosomiasis (HAT) due to Trypanosoma brucei rhodesiense in East and southern Africa is reported to be clinically diverse. We tested the hypothesis that this clinical diversity is associated with a variation in trypanosome genotypes. Trypanosome DNA isolated from HAT patients was genotyped using 7 microsatellite markers directly from blood spotted FTA cards following a whole genome amplification. All markers were polymorphic and identified 17 multi-locus genotypes with 56% of the isolates having replicate genotypes. We did not observe any significant clustering between isolates and bootstrap values across major tree nodes were insignificant. When genotypes were compared among patients with varying clinical presentation or outcome, replicate genotypes were observed at both extremes showing no significant association between genetic diversity and clinical outcome. Our study shows that T. b. rhodesiense isolates are homogeneous within a focus and that observed clinical diversity may not be associated with parasite genetic diversity. Other factors like host genetics and environmental factors might be involved in determining clinical diversity. Our study may be important in designing appropriate control measures that target the parasite.

How many genetic markers to tag an individual? An empirical assessment of false matching rates among close relatives.

PubMed

Rew, Mary Beth; Robbins, Jooke; Mattila, David; Palsbøll, Per J; Bérube, Martine

2011-04-01

Genetic identification of individuals is now commonplace, enabling the application of tagging methods to elusive species or species that cannot be tagged by traditional methods. A key aspect is determining the number of loci required to ensure that different individuals have non-matching multi-locus genotypes. Closely related individuals are of particular concern because of elevated matching probabilities caused by their recent co-ancestry. This issue may be addressed by increasing the number of loci to a level where full siblings (the relatedness category with the highest matching probability) are expected to have non-matching multi-locus genotypes. However, increasing the number of loci to meet this "full-sib criterion" greatly increases the laboratory effort, which in turn may increase the genotyping error rate resulting in an upward-biased mark-recapture estimate of abundance as recaptures are missed due to genotyping errors. We assessed the contribution of false matches from close relatives among 425 maternally related humpback whales, each genotyped at 20 microsatellite loci. We observed a very low (0.5-4%) contribution to falsely matching samples from pairs of first-order relatives (i.e., parent and offspring or full siblings). The main contribution to falsely matching individuals from close relatives originated from second-order relatives (e.g., half siblings), which was estimated at 9%. In our study, the total number of observed matches agreed well with expectations based upon the matching probability estimated for unrelated individuals, suggesting that the full-sib criterion is overly conservative, and would have required a 280% relative increase in effort. We suggest that, under most circumstances, the overall contribution to falsely matching samples from close relatives is likely to be low, and hence applying the full-sib criterion is unnecessary. In those cases where close relatives may present a significant issue, such as unrepresentative sampling, we propose three different genotyping strategies requiring only a modest increase in effort, which will greatly reduce the number of false matches due to the presence of related individuals.

Development of a High-Resolution Multi-Locus Microsatellite Typing Method for Colletotrichum gloeosporioides.

PubMed

Mehta, Nikita; Hagen, Ferry; Aamir, Sadaf; Singh, Sanjay K; Baghela, Abhishek

2017-12-01

Colletotrichum gloeosporioides is an economically important fungal pathogen causing substantial yield losses indifferent host plants. To understand the genetic diversity and molecular epidemiology of this fungus, we have developed a novel, high-resolution multi-locus microsatellite typing (MLMT) method. Bioinformatic analysis of C. gloeosporioides unannotated genome sequence yielded eight potential microsatellite loci, of which five, CG1 (GT) n , CG2 (GT1) n , CG3 (TC) n , CG4 (CT) n , and CG5 (CT1) n were selected for further study based on their universal amplification potential, reproducibility, and repeat number polymorphism. The selected microsatellites were used to analyze 31 strains of C. gloeosporioides isolated from 20 different host plants from India. All microsatellite loci were found to be polymorphic, and the approximate fragment sizes of microsatellite loci CG1, CG2, CG3, CG4, and CG5 were in ranges of 213-241, 197-227, 231-265, 209-275, and 132-188, respectively. Among the 31 isolates, 55 different genotypes were identified. The Simpson's index of diversity (D) values for the individual locus ranged from 0.79 to 0.92, with the D value of all combined five microsatellite loci being 0.99. Microsatellite data analysis revealed that isolates from Ocimum sanctum , Capsicum annuum (chili pepper), and Mangifera indica (mango) formed distinct clusters, therefore exhibited some level of correlation between certain genotypes and host. The developed MLMT method would be a powerful tool for studying the genetic diversity and any possible genotype-host correlation in C. gloeosporioides .

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.

Isolation and characterization of microsatellite loci in Alasmidonta heterodon (Bivalvia: Unionidae)

USGS Publications Warehouse

Shaw, K.M.; King, T.L.; Lellis, W.A.; Eackles, M.S.

2006-01-01

We developed 13 species-specific microsatellite markers for the federally endangered Atlantic slope unionid Alasmidonta heterodon. Four to 18 alleles per locus were observed among 30 individuals. Observed heterozygosity throughout the loci ranged from 26.9 to 86.2% and averaged 63.6%. Estimates of individual pairwise genetic distances indicated that levels of genetic diversity among loci were sufficient to produce unique multilocus genotypes for all animals surveyed. Randomization tests showed that genotypes for this collection were consistent with Hardy-Weinberg expectations, and no significant linkage disequilibrium was observed between loci. These loci therefore appear suitable for population surveys, kinship assessment and other such applications. ?? 2006 Blackwell Publishing Ltd.

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu

2012-01-01

The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

Molecular Characteristics and Drug Susceptibility of Mycobacterium tuberculosis Isolates from Patients Co-infected with Human Immunodeficiency Virus in Beijing, China.

PubMed

Liu, Jie; Wang, Hui Zhu; Lian, Lu Lu; Yu, Yan Hua; Zhao, Xiu Qin; Guo, Cai Ping; Liu, Hai Can; Liu, Shu Mei; Zhao, Hui; Zeng, Zhao Ying; Zhao, Xiu Ying; Wan, Kang Lin

2015-03-01

70 clinical Mycobacterium tuberculosis strains isolated from AIDS patients in two HIV/AIDS referral hospitals in Beijing were used in this study. M. tuberculosis and non-tuberculosis mycobacterium (NTM) were identified by using multi-locus PCR. M. tuberculosis was genotyped by using 15-locus MIRU-VNTR technique and spoligotyping afterwards. Meanwhile, the drug susceptibilities of the strains to the four first-line anti TB drugs (rifampin, isoniazid, streptomycin, and ethambutol) and the four second-line anti-TB drugs (capreomycin, kanamycin, ofloxacin, and ethionanide) were tested with proportional method. In this study, M. tuberculosis and NTM strains isolated from AIDS patients with TB-like symptoms were identified and genotyping analysis indicated that Beijing genotype was the predominant genotype. In addition, the prevalence of drug-resistant TB, especially the prevalence of XDR-TB, was higher than that in TB patients without HIV infection. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Diverse Genotypes of Yersinia pestis Caused Plague in Madagascar in 2007.

PubMed

Riehm, Julia M; Projahn, Michaela; Vogler, Amy J; Rajerison, Minoaerisoa; Andersen, Genevieve; Hall, Carina M; Zimmermann, Thomas; Soanandrasana, Rahelinirina; Andrianaivoarimanana, Voahangy; Straubinger, Reinhard K; Nottingham, Roxanne; Keim, Paul; Wagner, David M; Scholz, Holger C

2015-06-01

Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging. DNA was extracted directly from 93 clinical samples from patients with a clinical diagnosis of plague in Madagascar in 2007. The extracted DNAs were then genotyped using three molecular genotyping methods, including, single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. These methods provided increasing resolution, respectively. The results of these analyses revealed that, in 2007, ten molecular groups, two newly described here and eight previously identified, were responsible for causing human plague in geographically distinct areas of Madagascar. Plague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks.

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries.

PubMed

Ko, Kwan Soo; Oh, Won Sup; Peck, Kyong Ran; Lee, Jang Ho; Lee, Nam Yong; Song, Jae-Hoon

2005-07-01

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.

Novel genotype of Tritrichomonas foetus from cattle in Southern Africa.

PubMed

Casteriano, Andrea; Molini, Umberto; Kandjumbwa, Kornelia; Khaiseb, Siegfried; Frey, Caroline F; Šlapeta, Jan

2016-12-01

Bovine trichomonosis caused by Tritrichomonas foetus is a significant reproductive disease of cattle. Preputial samples were collected using sheath washing technique in bulls in Namibia. Thirty-six trichomonad cultures were characterized using the TaqMan-probe commercial real-time polymerase chain reaction (PCR) diagnostic assay (VetMAX™-Gold Trich Detection Kit) and CYBR real-time PCR assay based on TFR3/4 primers. Diagnostic real-time PCRs and DNA sequencing of the internal transcribed region confirmed presence of T. foetus in 35 out of 36 samples. Multilocus genotyping using cysteine proteases (CP1, CP2, CP4, CP5, CP6, CP7, CP8, CP9) and malate dehydrogenase (MDH1) gene sequences demonstrate that the T. foetus in Namibia are genetically distinct from those characterized elsewhere. We report the discovery of a novel genotype of T. foetus in Namibian cattle, distinct from other T. foetus genotypes in Europe, South and North America and Australia. We suggest recognition of a 'Southern African' genotype of T. foetus. Identification of the new genotype of T. foetus demonstrates the need for wider global sampling to fully understand the diversity and origin of T. foetus causing disease in cattle or cats.

Assessing the Organization and Capabilities of 4th Marine Logistics Group

DTIC Science & Technology

2010-05-14

Logistics Group (MLG) should adopt further organizational changes within specific parameters to complement the structure and capabilities of the Active...In order to enhance operational effectiveness and continue its mandated responsibility to augment and reinforce, 4th MLG should adopt further...of the Deputy Commandant, Installations and Logistics (DC, I&L), Marine Forces Reserve ( MFR ), and the MLGs. The paper also relies heavily on the

A Flexible and Highly Sensitive Pressure Sensor Based on a PDMS Foam Coated with Graphene Nanoplatelets

PubMed Central

Rinaldi, Andrea; Tamburrano, Alessio; Fortunato, Marco; Sarto, Maria Sabrina

2016-01-01

The demand for high performance multifunctional wearable devices is more and more pushing towards the development of novel low-cost, soft and flexible sensors with high sensitivity. In the present work, we describe the fabrication process and the properties of new polydimethylsiloxane (PDMS) foams loaded with multilayer graphene nanoplatelets (MLGs) for application as high sensitive piezoresistive pressure sensors. The effective DC conductivity of the produced foams is measured as a function of MLG loading. The piezoresistive response of the MLG-PDMS foam-based sensor at different strain rates is assessed through quasi-static pressure tests. The results of the experimental investigations demonstrated that sensor loaded with 0.96 wt.% of MLGs is characterized by a highly repeatable pressure-dependent conductance after a few stabilization cycles and it is suitable for detecting compressive stresses as low as 10 kPa, with a sensitivity of 0.23 kPa−1, corresponding to an applied pressure of 70 kPa. Moreover, it is estimated that the sensor is able to detect pressure variations of ~1 Pa. Therefore, the new graphene-PDMS composite foam is a lightweight cost-effective material, suitable for sensing applications in the subtle or low and medium pressure ranges. PMID:27999251

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.

Origin, Migration Routes and Worldwide Population Genetic Structure of the Wheat Yellow Rust Pathogen Puccinia striiformis f.sp. tritici

PubMed Central

Ali, Sajid; Gladieux, Pierre; Leconte, Marc; Gautier, Angélique; Justesen, Annemarie F.; Hovmøller, Mogens S.; Enjalbert, Jérôme; de Vallavieille-Pope, Claude

2014-01-01

Analyses of large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. This is particularly important for long-distance migrating fungal pathogens such as Puccinia striiformis f.sp. tritici (PST), capable of rapid spread to new regions and crop varieties. Although a range of recent PST invasions at continental scales are well documented, the worldwide population structure and the center of origin of the pathogen were still unknown. In this study, we used multilocus microsatellite genotyping to infer worldwide population structure of PST and the origin of new invasions based on 409 isolates representative of distribution of the fungus on six continents. Bayesian and multivariate clustering methods partitioned the set of multilocus genotypes into six distinct genetic groups associated with their geographical origin. Analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan regions (China) and a predominant clonal population structure in other regions. The higher genotypic diversity, recombinant population structure and high sexual reproduction ability in the Himalayan and neighboring regions suggests this area as the putative center of origin of PST. We used clustering methods and approximate Bayesian computation (ABC) to compare different competing scenarios describing ancestral relationship among ancestral populations and more recently founded populations. Our analyses confirmed the Middle East-East Africa as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. Although most geographic populations are not markedly affected by recent dispersal events, this study emphasizes the influence of human activities on recent long-distance spread of the pathogen. PMID:24465211

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

PubMed

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

2008-04-01

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

In silico genomic analyses reveal three distinct lineages of Escherichia coli O157:H7, one of which is associated with hyper-virulence.

PubMed

Laing, Chad R; Buchanan, Cody; Taboada, Eduardo N; Zhang, Yongxiang; Karmali, Mohamed A; Thomas, James E; Gannon, Victor Pj

2009-06-29

Many approaches have been used to study the evolution, population structure and genetic diversity of Escherichia coli O157:H7; however, observations made with different genotyping systems are not easily relatable to each other. Three genetic lineages of E. coli O157:H7 designated I, II and I/II have been identified using octamer-based genome scanning and microarray comparative genomic hybridization (mCGH). Each lineage contains significant phenotypic differences, with lineage I strains being the most commonly associated with human infections. Similarly, a clade of hyper-virulent O157:H7 strains implicated in the 2006 spinach and lettuce outbreaks has been defined using single-nucleotide polymorphism (SNP) typing. In this study an in silico comparison of six different genotyping approaches was performed on 19 E. coli genome sequences from 17 O157:H7 strains and single O145:NM and K12 MG1655 strains to provide an overall picture of diversity of the E. coli O157:H7 population, and to compare genotyping methods for O157:H7 strains. In silico determination of lineage, Shiga-toxin bacteriophage integration site, comparative genomic fingerprint, mCGH profile, novel region distribution profile, SNP type and multi-locus variable number tandem repeat analysis type was performed and a supernetwork based on the combination of these methods was produced. This supernetwork showed three distinct clusters of strains that were O157:H7 lineage-specific, with the SNP-based hyper-virulent clade 8 synonymous with O157:H7 lineage I/II. Lineage I/II/clade 8 strains clustered closest on the supernetwork to E. coli K12 and E. coli O55:H7, O145:NM and sorbitol-fermenting O157 strains. The results of this study highlight the similarities in relationships derived from multi-locus genome sampling methods and suggest a "common genotyping language" may be devised for population genetics and epidemiological studies. Future genotyping methods should provide data that can be stored centrally and accessed locally in an easily transferable, informative and extensible format based on comparative genomic analyses.

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China.

PubMed

Liu, Xuehan; He, Tingmei; Zhong, Zhijun; Zhang, Hemin; Wang, Rongjun; Dong, Haiju; Wang, Chengdong; Li, Desheng; Deng, Jiabo; Peng, Guangneng; Zhang, Longxian

2013-10-01

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60×3.99 μm (n=50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype. © 2013 Elsevier Ireland Ltd. All rights reserved.

Inference on the Strength of Balancing Selection for Epistatically Interacting Loci

PubMed Central

Buzbas, Erkan Ozge; Joyce, Paul; Rosenberg, Noah A.

2011-01-01

Existing inference methods for estimating the strength of balancing selection in multi-locus genotypes rely on the assumption that there are no epistatic interactions between loci. Complex systems in which balancing selection is prevalent, such as sets of human immune system genes, are known to contain components that interact epistatically. Therefore, current methods may not produce reliable inference on the strength of selection at these loci. In this paper, we address this problem by presenting statistical methods that can account for epistatic interactions in making inference about balancing selection. A theoretical result due to Fearnhead (2006) is used to build a multi-locus Wright-Fisher model of balancing selection, allowing for epistatic interactions among loci. Antagonistic and synergistic types of interactions are examined. The joint posterior distribution of the selection and mutation parameters is sampled by Markov chain Monte Carlo methods, and the plausibility of models is assessed via Bayes factors. As a component of the inference process, an algorithm to generate multi-locus allele frequencies under balancing selection models with epistasis is also presented. Recent evidence on interactions among a set of human immune system genes is introduced as a motivating biological system for the epistatic model, and data on these genes are used to demonstrate the methods. PMID:21277883

Detection of Epistasis for Flowering Time Using Bayesian Multilocus Estimation in a Barley MAGIC Population

PubMed Central

Mathew, Boby; Léon, Jens; Sannemann, Wiebke; Sillanpää, Mikko J.

2018-01-01

Gene-by-gene interactions, also known as epistasis, regulate many complex traits in different species. With the availability of low-cost genotyping it is now possible to study epistasis on a genome-wide scale. However, identifying genome-wide epistasis is a high-dimensional multiple regression problem and needs the application of dimensionality reduction techniques. Flowering Time (FT) in crops is a complex trait that is known to be influenced by many interacting genes and pathways in various crops. In this study, we successfully apply Sure Independence Screening (SIS) for dimensionality reduction to identify two-way and three-way epistasis for the FT trait in a Multiparent Advanced Generation Inter-Cross (MAGIC) barley population using the Bayesian multilocus model. The MAGIC barley population was generated from intercrossing among eight parental lines and thus, offered greater genetic diversity to detect higher-order epistatic interactions. Our results suggest that SIS is an efficient dimensionality reduction approach to detect high-order interactions in a Bayesian multilocus model. We also observe that many of our findings (genomic regions with main or higher-order epistatic effects) overlap with known candidate genes that have been already reported in barley and closely related species for the FT trait. PMID:29254994

Prenatal maternal depression and child serotonin transporter linked polymorphic region (5-HTTLPR) and dopamine receptor D4 (DRD4) genotype predict negative emotionality from 3 to 36 months.

PubMed

Green, Cathryn Gordon; Babineau, Vanessa; Jolicoeur-Martineau, Alexia; Bouvette-Turcot, Andrée-Anne; Minde, Klaus; Sassi, Roberto; St-André, Martin; Carrey, Normand; Atkinson, Leslie; Kennedy, James L; Steiner, Meir; Lydon, John; Gaudreau, Helene; Burack, Jacob A; Levitan, Robert; Meaney, Michael J; Wazana, Ashley

2017-08-01

Prenatal maternal depression and a multilocus genetic profile of two susceptibility genes implicated in the stress response were examined in an interaction model predicting negative emotionality in the first 3 years. In 179 mother-infant dyads from the Maternal Adversity, Vulnerability, and Neurodevelopment cohort, prenatal depression (Center for Epidemiologic Studies Depressions Scale) was assessed at 24 to 36 weeks. The multilocus genetic profile score consisted of the number of susceptibility alleles from the serotonin transporter linked polymorphic region gene (5-HTTLPR): no long-rs25531(A) (LA: short/short, short/long-rs25531(G) [LG], or LG/LG] vs. any LA) and the dopamine receptor D4 gene (six to eight repeats vs. two to five repeats). Negative emotionality was extracted from the Infant Behaviour Questionnaire-Revised at 3 and 6 months and the Early Child Behavior Questionnaire at 18 and 36 months. Mixed and confirmatory regression analyses indicated that prenatal depression and the multilocus genetic profile interacted to predict negative emotionality from 3 to 36 months. The results were characterized by a differential susceptibility model at 3 and 6 months and by a diathesis-stress model at 36 months.

Phenotypic Diversification Is Associated with Host-Induced Transposon Derepression in the Sudden Oak Death Pathogen Phytophthora ramorum

PubMed Central

Kasuga, Takao; Kozanitas, Melina; Bui, Mai; Hüberli, Daniel; Rizzo, David M.; Garbelotto, Matteo

2012-01-01

The oomycete pathogen Phytophthora ramorum is responsible for sudden oak death (SOD) in California coastal forests. P. ramorum is a generalist pathogen with over 100 known host species. Three or four closely related genotypes of P. ramorum (from a single lineage) were originally introduced in California forests and the pathogen reproduces clonally. Because of this the genetic diversity of P. ramorum is extremely low in Californian forests. However, P. ramorum shows diverse phenotypic variation in colony morphology, colony senescence, and virulence. In this study, we show that phenotypic variation among isolates is associated with the host species from which the microbe was originally cultured. Microarray global mRNA profiling detected derepression of transposable elements (TEs) and down-regulation of crinkler effector homologs (CRNs) in the majority of isolates originating from coast live oak (Quercus agrifolia), but this expression pattern was not observed in isolates from California bay laurel (Umbellularia californica). In some instances, oak and bay laurel isolates originating from the same geographic location had identical genotypes based on multilocus simples sequence repeat (SSR) marker analysis but had different phenotypes. Expression levels of the two marker genes analyzed by quantitative reverse transcription PCR were correlated with originating host species, but not with multilocus genotypes. Because oak is a nontransmissive dead-end host for P. ramorum, our observations are congruent with an epi-transposon hypothesis; that is, physiological stress is triggered on P. ramorum while colonizing oak stems and disrupts epigenetic silencing of TEs. This then results in TE reactivation and possibly genome diversification without significant epidemiological consequences. We propose the P. ramorum-oak host system in California forests as an ad hoc model for epi-transposon mediated diversification. PMID:22529930

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.

Mouse-virulent Toxoplasma gondii isolated from feral cats on Mona Island, Puerto Rico.

PubMed

Dubey, J P; López-Torres, H Y; Sundar, N; Velmurugan, G V; Ajzenberg, D; Kwok, O C H; Hill, R; Dardé, M L; Su, C

2007-12-01

Cats are essential in the life cycle of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts. Samples of serum, feces, and tissues from cats from Mona, a remote island off the coast of Puerto Rico, were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test and found in 16 of 19 (84.2%) of cats, with titers of 1:10 in 2, 1:80 in 1, 1:160 in 4, 1:320 in 3, and 1:1,280 or higher in 6. Tissues of 19 of the 20 cats were bioassayed in mice for T. gondii infection. Toxoplasma gondii was isolated from tissues of 12 cats: from the hearts of 9, skeletal muscle of 10, and brain of 1 cat. All infected mice from 10 of 12 isolates died of acute toxoplasmosis during primary infection. Genotyping of these 12 T. gondii isolates (designated (TgCatPr 1-12) by 10 multilocus PCR-RFLP markers, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico, and the 6 multilocus microsatellite markers TUB2, W35, TgM-A, B18, B17, and M33, revealed 7 genotypes; 5 isolates had Type I alleles at all loci except at 1 microsatellite locus, and the remainder were atypical. The latter isolates of T. gondii were different biologically and phenotypically from the feline isolates from the rest of the Americas. One isolate (TgCatPr 12) was a mixed infection with 2 genotypes.

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.

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

PubMed Central

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

2007-01-01

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

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

PubMed

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

2007-11-01

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

The EmsB Tandemly Repeated Multilocus Microsatellite: a New Tool To Investigate Genetic Diversity of Echinococcus granulosus Sensu Lato▿

PubMed Central

Maillard, S.; Gottstein, B.; Haag, K. L.; Ma, S.; Colovic, I.; Benchikh-Elfegoun, M. C.; Knapp, J.; Piarroux, R.

2009-01-01

Cystic echinococcosis (CE) is a widespread and severe zoonotic disease caused by infection with the larval stage of the eucestode Echinococcus granulosus sensu lato. The polymorphism exhibited by nuclear and mitochondrial markers conventionally used for the genotyping of different parasite species and strains does not reach the level necessary for the identification of genetic variants linked to restricted geographical areas. EmsB is a tandemly repeated multilocus microsatellite that proved its usefulness for the study of genetic polymorphisms within the species E. multilocularis, the causative agent of alveolar echinococcosis. In the present study, EmsB was used to characterize E. granulosus sensu lato samples collected from different host species (sheep, cattle, dromedaries, dogs, and human patients) originating from six different countries (Algeria, Mauritania, Romania, Serbia, Brazil, and the People's Republic of China). The conventional mitochondrial cox1 and nad1 markers identified genotypes G1, G3, G5, G6, and G7, which are clustered into three groups corresponding to the species E. granulosus sensu stricto, E. ortleppi, and E. canadensis. With the same samples, EmsB provided a higher degree of genetic discrimination and identified variations that correlated with the relatively small-scale geographic origins of the samples. In addition, one of the Brazilian single hydatid cysts presented a hybrid genotypic profile that suggested genetic exchanges between E. granulosus sensu stricto and E. ortleppi. In summary, the EmsB microsatellite exhibits an interesting potential for the elaboration of a detailed map of the distribution of genetic variants and therefore for the determination and tracking of the source of CE. PMID:19741078

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.

Evolution and polymorphism in the multilocus Levene model with no or weak epistasis.

PubMed

Bürger, Reinhard

2010-09-01

Evolution and the maintenance of polymorphism under the multilocus Levene model with soft selection are studied. The number of loci and alleles, the number of demes, the linkage map, and the degree of dominance are arbitrary, but epistasis is absent or weak. We prove that, without epistasis and under mild, generic conditions, every trajectory converges to a stationary point in linkage equilibrium. Consequently, the equilibrium and stability structure can be determined by investigating the much simpler gene-frequency dynamics on the linkage-equilibrium manifold. For a haploid species an analogous result is shown. For weak epistasis, global convergence to quasi-linkage equilibrium is established. As an application, the maintenance of multilocus polymorphism is explored if the degree of dominance is intermediate at every locus and epistasis is absent or weak. If there are at least two demes, then arbitrarily many multiallelic loci can be maintained polymorphic at a globally asymptotically stable equilibrium. Because this holds for an open set of parameters, such equilibria are structurally stable. If the degree of dominance is not only intermediate but also deme independent, and loci are diallelic, an open set of parameters yielding an internal equilibrium exists only if the number of loci is strictly less than the number of demes. Otherwise, a fully polymorphic equilibrium exists only nongenerically, and if it exists, it consists of a manifold of equilibria. Its dimension is determined. In the absence of genotype-by-environment interaction, however, a manifold of equilibria occurs for an open set of parameters. In this case, the equilibrium structure is not robust to small deviations from no genotype-by-environment interaction. In a quantitative-genetic setting, the assumptions of no epistasis and intermediate dominance are equivalent to assuming that in every deme directional selection acts on a trait that is determined additively, i.e., by nonepistatic loci with dominance. Some of our results are exemplified in this quantitative-genetic context. Copyright 2010 Elsevier Inc. All rights reserved.

Microsatellite DNA markers for delineating population structure and kinship among the endangered Kirtland's warbler (Dendroica kirtlandii)

USGS Publications Warehouse

King, T.L.; Eackles, M.S.; Henderson, A.P.; Bocetti, Carol I.; Currie, D.; Wunderle, J.M.

2005-01-01

We document the isolation and characterization of 23 microsatellite DNA markers for the endangered Kirtland's warbler (Dendroica kirtlandii), a Nearctic/Neotropical migrant passerine. This suite of markers revealed moderate to high levels of allelic diversity (averaging 7.7 alleles per locus) and heterozygosity (averaging 72%). Genotypic frequencies at 22 of 23 (95%) markers conformed to Hardy-Weinberg equilibrium expectations, and no linkage disequilibrium was observed in blood samples taken from 14 warblers found on the wintering grounds in the Bahamas archipelago. Multilocus genotypes resulting from this suite of markers should reduce the amount of resources required for initiating new genetic studies assessing breeding structure, parentage, demographics, and individual-level ecological interactions for D. kirtlandii. ?? 2005 Blackwell Publishing Ltd.

Colonisation with toxigenic Corynebacterium diphtheriae in a Scottish burns patient, June 2015.

PubMed

Deshpande, Ashutosh; Inkster, Teresa; Hamilton, Kate; Litt, David; Fry, Norman; Kennedy, Iain T R; Shookhye-Dickson, Jacqueline; Hill, Robert L R

2015-01-01

On 12 June 2015, Corynebacterium diphtheriae was identified in a skin swab from a burns patient in Scotland. The isolate was confirmed to be genotypically and phenotypically toxigenic. Multilocus sequence typing of three patient isolates yielded sequence type ST 125. The patient was clinically well. We summarise findings of this case, and results of close contact identification and screening: 12 family and close contacts and 32 hospital staff have been found negative for C. diphtheriae.

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.

Inference of Antibiotic Resistance and Virulence Among Diverse Group A Streptococcus Strains Using emm Sequencing and Multilocus Genotyping Methods

DTIC Science & Technology

2009-09-04

apparent GAS-associated conditions were sampled by oropharyn- geal swab. Swabs were streaked on blood agar plates using Table 3. Isolate properties by...testing, samples were re-streaked on blood agar plates (5% sheep blood in TSA base) (Hardy Diagnostics, Santa Maria, CA), and incubated at 35–37uC with 5–10...sensitivity (A-disk method, Hardy Diagnostics) and positive GAS latex agglutination reaction (Hardy Diagnostics). Confirmed GAS isolates were then

Isolation of Cryptococcus gattii from a Castanopsis argyrophylla tree hollow (Mai-Kaw), Chiang Mai, Thailand.

PubMed

Khayhan, Kantarawee; Hagen, Ferry; Norkaew, Treepradab; Puengchan, Tanpalang; Boekhout, Teun; Sriburee, Pojana

2017-04-01

The pathogenic yeast Cryptococcus gattii was isolated from a tree hollow of a Castanopsis argyrophylla King ex Hook.f. (Fagaceae) in Chiang Mai, Thailand. Molecular characterization with amplified fragment length polymorphism analysis and multi-locus sequence typing showed that this isolate belonged to genotype AFLP4/VGI representing C. gattii sensu stricto. Subsequent comparison of the environmental isolate with those from clinical samples from Thailand showed that they grouped closely together in a single cluster.

Deepwater Program: Exploration and Research of Northern Gulf of Mexico Deepwater Natural and Artificial Hard Bottom Habitats with Emphasis on Coral Communities: Reefs, Rigs and Wrecks

DTIC Science & Technology

2011-01-01

this analysis. They coordinate with Dr. Christina A. Kellogg with USGS for other microbiology aspects during the project. Dr. Bernie Bernard, TDI...of Mexico OCS Region, New Orleans, LA. OCS Study MMS 2008-018. Church, Robert and Lynn Samuel. 2004. Archaeological, Engineering and Hazard Study...methods using multilocus genotypes from 20 chicken breeds. Genetics 159, 699-713. Ross, S. W. and A. M. Quattrini. 2007. The fish fauna

Genetic Predisposition to Ischemic Stroke

PubMed Central

Kamatani, Yoichiro; Takahashi, Atsushi; Hata, Jun; Furukawa, Ryohei; Shiwa, Yuh; Yamaji, Taiki; Hara, Megumi; Tanno, Kozo; Ohmomo, Hideki; Ono, Kanako; Takashima, Naoyuki; Matsuda, Koichi; Wakai, Kenji; Sawada, Norie; Iwasaki, Motoki; Yamagishi, Kazumasa; Ago, Tetsuro; Ninomiya, Toshiharu; Fukushima, Akimune; Hozawa, Atsushi; Minegishi, Naoko; Satoh, Mamoru; Endo, Ryujin; Sasaki, Makoto; Sakata, Kiyomi; Kobayashi, Seiichiro; Ogasawara, Kuniaki; Nakamura, Motoyuki; Hitomi, Jiro; Kita, Yoshikuni; Tanaka, Keitaro; Iso, Hiroyasu; Kitazono, Takanari; Kubo, Michiaki; Tanaka, Hideo; Tsugane, Shoichiro; Kiyohara, Yutaka; Yamamoto, Masayuki; Sobue, Kenji; Shimizu, Atsushi

2017-01-01

Background and Purpose— The prediction of genetic predispositions to ischemic stroke (IS) may allow the identification of individuals at elevated risk and thereby prevent IS in clinical practice. Previously developed weighted multilocus genetic risk scores showed limited predictive ability for IS. Here, we investigated the predictive ability of a newer method, polygenic risk score (polyGRS), based on the idea that a few strong signals, as well as several weaker signals, can be collectively informative to determine IS risk. Methods— We genotyped 13 214 Japanese individuals with IS and 26 470 controls (derivation samples) and generated both multilocus genetic risk scores and polyGRS, using the same derivation data set. The predictive abilities of each scoring system were then assessed using 2 independent sets of Japanese samples (KyushuU and JPJM data sets). Results— In both validation data sets, polyGRS was shown to be significantly associated with IS, but weighted multilocus genetic risk scores was not. Comparing the highest with the lowest polyGRS quintile, the odds ratios for IS were 1.75 (95% confidence interval, 1.33–2.31) and 1.99 (95% confidence interval, 1.19–3.33) in the KyushuU and JPJM samples, respectively. Using the KyushuU samples, the addition of polyGRS to a nongenetic risk model resulted in a significant improvement of the predictive ability (net reclassification improvement=0.151; P<0.001). Conclusions— The polyGRS was shown to be superior to weighted multilocus genetic risk scores as an IS prediction model. Thus, together with the nongenetic risk factors, polyGRS will provide valuable information for individual risk assessment and management of modifiable risk factors. PMID:28034966

SINEs of progress: Mobile element applications to molecular ecology.

PubMed

Ray, David A

2007-01-01

Mobile elements represent a unique and under-utilized set of tools for molecular ecologists. They are essentially homoplasy-free characters with the ability to be genotyped in a simple and efficient manner. Interpretation of the data generated using mobile elements can be simple compared to other genetic markers. They exist in a wide variety of taxa and are useful over a wide selection of temporal ranges within those taxa. Furthermore, their mode of evolution instills them with another advantage over other types of multilocus genotype data: the ability to determine loci applicable to a range of time spans in the history of a taxon. In this review, I discuss the application of mobile element markers, especially short interspersed elements (SINEs), to phylogenetic and population data, with an emphasis on potential applications to molecular ecology.

Examination of tetrahydrobiopterin pathway genes in autism.

PubMed

Schnetz-Boutaud, N C; Anderson, B M; Brown, K D; Wright, H H; Abramson, R K; Cuccaro, M L; Gilbert, J R; Pericak-Vance, M A; Haines, J L

2009-11-01

Autism is a complex disorder with a high degree of heritability and significant phenotypic and genotypic heterogeneity. Although candidate gene studies and genome-wide screens have failed to identify major causal loci associated with autism, numerous studies have proposed association with several variations in genes in the dopaminergic and serotonergic pathways. Because tetrahydrobiopterin (BH4) is the essential cofactor in the synthesis of these two neurotransmitters, we genotyped 25 SNPs in nine genes of the BH4 pathway in a total of 403 families. Significant nominal association was detected in the gene for 6-pyruvoyl-tetrahydropterin synthase, PTS (chromosome 11), with P = 0.009; this result was not restricted to an affected male-only subset. Multilocus interaction was detected in the BH4 pathway alone, but not across the serotonin, dopamine and BH4 pathways.

Microsatellite markers identify three lineages of Phytophthora ramorum in US nurseries, yet single lineages in US forest and European nursery populations.

PubMed

Ivors, K; Garbelotto, M; Vries, I D E; Ruyter-Spira, C; Te Hekkert, B; Rosenzweig, N; Bonants, P

2006-05-01

Analysis of 12 polymorphic simple sequence repeats identified in the genome sequence of Phytophthora ramorum, causal agent of 'sudden oak death', revealed genotypic diversity to be significantly higher in nurseries (91% of total) than in forests (18% of total). Our analysis identified only two closely related genotypes in US forests, while the genetic structure of populations from European nurseries was of intermediate complexity, including multiple, closely related genotypes. Multilocus analysis determined populations in US forests reproduce clonally and are likely descendants of a single introduced individual. The 151 isolates analysed clustered in three clades. US forest and European nursery isolates clustered into two distinct clades, while one isolate from a US nursery belonged to a third novel clade. The combined microsatellite, sequencing and morphological analyses suggest the three clades represent distinct evolutionary lineages. All three clades were identified in some US nurseries, emphasizing the role of commercial plant trade in the movement of this pathogen.

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.

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.

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

PubMed

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

2011-12-15

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

Genetic diversity of Brucella ovis isolates from Rio Grande do Sul, Brazil, by MLVA16

PubMed Central

2014-01-01

Background Ovine epididymitis is predominantly associated with Brucella ovis infection. Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats (VNTR) analyses (MLVA) have proved to be a powerful tool for epidemiological trace-back studies. Thus, the aim of this study was to evaluate the genetic diversity of Brucella ovis isolates from Rio Grande do Sul State, Brazil, by MLVA16. Findings MLVA16 genotyping identified thirteen distinct genotypes and a Hunter-Gaston diversity index of 0.989 among the fourteen B. ovis genotyped strains. All B. ovis MLVA16 genotypes observed in the present study represented non-previously described profiles. Analyses of the eight conserved loci included in panel 1 (MLVA8) showed three different genotypes, two new and one already described for B. ovis isolates. Among ten B. ovis isolates from same herd only two strains had identical pattern, whereas the four isolates with no epidemiologic information exhibited a single MLVA16 pattern each. Analysis of minimal spanning tree, constructed using the fourteen B. ovis strains typed in this study together with all nineteen B. ovis MLVA16 genotypes available in the MLVAbank 2014, revealed the existence of two clearly distinct major clonal complexes. Conclusions In conclusion, the results of the present study showed a high genetic diversity among B. ovis field isolates from Rio Grande do Sul State, Brazil, by MLVA16. PMID:25015223

Genetic diversity of Brucella ovis isolates from Rio Grande do Sul, Brazil, by MLVA16.

PubMed

Dorneles, Elaine M S; Freire, Guilherme N; Dasso, Maurício G; Poester, Fernando P; Lage, Andrey P

2014-07-12

Ovine epididymitis is predominantly associated with Brucella ovis infection. Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats (VNTR) analyses (MLVA) have proved to be a powerful tool for epidemiological trace-back studies. Thus, the aim of this study was to evaluate the genetic diversity of Brucella ovis isolates from Rio Grande do Sul State, Brazil, by MLVA16. MLVA16 genotyping identified thirteen distinct genotypes and a Hunter-Gaston diversity index of 0.989 among the fourteen B. ovis genotyped strains. All B. ovis MLVA16 genotypes observed in the present study represented non-previously described profiles. Analyses of the eight conserved loci included in panel 1 (MLVA8) showed three different genotypes, two new and one already described for B. ovis isolates. Among ten B. ovis isolates from same herd only two strains had identical pattern, whereas the four isolates with no epidemiologic information exhibited a single MLVA16 pattern each. Analysis of minimal spanning tree, constructed using the fourteen B. ovis strains typed in this study together with all nineteen B. ovis MLVA16 genotypes available in the MLVAbank 2014, revealed the existence of two clearly distinct major clonal complexes. In conclusion, the results of the present study showed a high genetic diversity among B. ovis field isolates from Rio Grande do Sul State, Brazil, by MLVA16.

Zoonotic Potential and Molecular Epidemiology of Giardia Species and Giardiasis†

PubMed Central

Feng, Yaoyu; Xiao, Lihua

2011-01-01

Summary: Molecular diagnostic tools have been used recently in assessing the taxonomy, zoonotic potential, and transmission of Giardia species and giardiasis in humans and animals. The results of these studies have firmly established giardiasis as a zoonotic disease, although host adaptation at the genotype and subtype levels has reduced the likelihood of zoonotic transmission. These studies have also identified variations in the distribution of Giardia duodenalis genotypes among geographic areas and between domestic and wild ruminants and differences in clinical manifestations and outbreak potentials of assemblages A and B. Nevertheless, our efforts in characterizing the molecular epidemiology of giardiasis and the roles of various animals in the transmission of human giardiasis are compromised by the lack of case-control and longitudinal cohort studies and the sampling and testing of humans and animals living in the same community, the frequent occurrence of infections with mixed genotypes and subtypes, and the apparent heterozygosity at some genetic loci for some G. duodenalis genotypes. With the increased usage of multilocus genotyping tools, the development of next-generation subtyping tools, the integration of molecular analysis in epidemiological studies, and an improved understanding of the population genetics of G. duodenalis in humans and animals, we should soon have a better appreciation of the molecular epidemiology of giardiasis, the disease burden of zoonotic transmission, the taxonomy status and virulences of various G. duodenalis genotypes, and the ecology of environmental contamination. PMID:21233509

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

PubMed

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

2018-03-01

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

Microsatellite DNA markers for assessing phylogeographic and population structure in Preble's meadow jumping mice (Zapus hudsonius preblei) and cross-amplification among neighbouring taxa

USGS Publications Warehouse

King, T.L.; Eackles, M.S.; Young, C.

2006-01-01

We document the isolation and characterization of 14 tetranucleotide microsatellite DNA markers in Preble's meadow jumping mouse (Zapus hudsonius preblei). The identified markers displayed moderate levels of allelic diversity (averaging 4.9 alleles per locus) and heterozygosity (averaging 55.1%). Genotypic and allelic frequencies in a collection of 30 individuals conformed to Hardy-Weinberg equilibrium expectations and indicated no linkage disequilibrium. High levels of cross-amplification (95% overall) among neighbouring subspecies and two congeners (Zapus princeps and Zapus trinotatus) were observed. Multilocus genotypes resulting from these markers appear to provide ample genetic diversity for studies assessing individual- and population-level ecological interactions within Z. h. preblei and evolutionary relationships among neighbouring subspecies (Z. h. campestris, Z. h. intermedius, Z. h. pallidus and Z. h. luteus). ?? 2006 The Authors.

Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.

PubMed

O'Farrell, Brian; Haase, Jana K; Velayudhan, Vimalkumar; Murphy, Ronan A; Achtman, Mark

2012-01-01

Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost.

Transforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial Strains

PubMed Central

Velayudhan, Vimalkumar; Murphy, Ronan A.; Achtman, Mark

2012-01-01

Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost. PMID:23144721

Genetic Diversity and Population Structure of Leishmania infantum from Southeastern France: Evaluation Using Multi-Locus Microsatellite Typing

PubMed Central

Pomares, Christelle; Marty, Pierre; Bañuls, Anne Laure; Lemichez, Emmanuel; Pratlong, Francine; Faucher, Benoît; Jeddi, Fakhri; Moore, Sandy; Michel, Grégory; Aluru, Srikanth; Piarroux, Renaud; Hide, Mallorie

2016-01-01

In the south of France, Leishmania infantum is responsible for numerous cases of canine leishmaniasis (CanL), sporadic cases of human visceral leishmaniasis (VL) and rare cases of cutaneous and muco-cutaneous leishmaniasis (CL and MCL, respectively). Several endemic areas have been clearly identified in the south of France including the Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO). Within these endemic areas, the two cities of Nice (AM) and Marseille (P), which are located 150 km apart, and their surroundings, concentrate the greatest number of French autochthonous leishmaniasis cases. In this study, 270 L. infantum isolates from an extended time period (1978–2011) from four endemic areas, AM, P, CE and CO, were assessed using Multi-Locus Microsatellite Typing (MLMT). MLMT revealed a total of 121 different genotypes with 91 unique genotypes and 30 repeated genotypes. Substantial genetic diversity was found with a strong genetic differentiation between the Leishmania populations from AM and P. However, exchanges were observed between these two endemic areas in which it seems that strains spread from AM to P. The genetic differentiations in these areas suggest strong epidemiological structuring. A model-based analysis using STRUCTURE revealed two main populations: population A (consisting of samples primarily from the P and AM endemic areas with MON-1 and non-MON-1 strains) and population B consisting of only MON-1 strains essentially from the AM endemic area. For four patients, we observed several isolates from different biological samples which provided insight into disease relapse and re-infection. These findings shed light on the transmission dynamics of parasites in humans. However, further data are required to confirm this hypothesis based on a limited sample set. This study represents the most extensive population analysis of L. infantum strains using MLMT conducted in France. PMID:26808522

Targeted stock identification using multilocus genotype 'familyprinting'

USGS Publications Warehouse

Letcher, B.H.; King, T.L.

1999-01-01

We present an approach to stock identification of small, targeted populations that uses multilocus microsatellite genotypes of individual mating adults to uniquely identify first- and second-generation offspring in a mixture. We call the approach 'familyprinting'; unlike DNA fingerprinting where tissue samples of individuals are matched, offspring from various families are assigned to pairs of parents or sets of four grandparents with known genotypes. The basic unit of identification is the family, but families can be nested within a variety of stock units ranging from naturally reproducing groups of fish in a small tributary or pond from which mating adults can be sampled to large or small collections of families produced in hatcheries and stocked in specific locations. We show that, with as few as seven alleles per locus using four loci without error, first-generation offspring can be uniquely assigned to the correct family. For second-generation applications in a hatchery more alleles per locus (10) and loci (10) are required for correct assignment of all offspring to the correct set of grandparents. Using microsatellite DNA variation from an Atlantic salmon (Salmo solar) restoration river (Connecticut River, USA), we also show that this population contains sufficient genetic diversity in sea-run returns for 100% correct first, generation assignment and 97% correct second-generation assignment using 14 loci. We are currently using first- and second-generation familyprinting in this population with the ultimate goal of identifying stocking tributary. In addition to within-river familyprinting, there also appears to be sufficient genetic diversity within and between Atlantic salmon populations for identification of 'familyprinted' fish in a mixture of multiple populations. We also suggest that second-generation familyprinting with multiple populations may also provide a tool for examining stock structure. Familyprinting with microsatellite DNA markers is a viable method for identification of offspring of randomly mating adults from small, targeted stocks and should provide a useful addition to current mixed stock analyses with genetic markers.

Microbe-ID: an open source toolbox for microbial genotyping and species identification.

PubMed

Tabima, Javier F; Everhart, Sydney E; Larsen, Meredith M; Weisberg, Alexandra J; Kamvar, Zhian N; Tancos, Matthew A; Smart, Christine D; Chang, Jeff H; Grünwald, Niklaus J

2016-01-01

Development of tools to identify species, genotypes, or novel strains of invasive organisms is critical for monitoring emergence and implementing rapid response measures. Molecular markers, although critical to identifying species or genotypes, require bioinformatic tools for analysis. However, user-friendly analytical tools for fast identification are not readily available. To address this need, we created a web-based set of applications called Microbe-ID that allow for customizing a toolbox for rapid species identification and strain genotyping using any genetic markers of choice. Two components of Microbe-ID, named Sequence-ID and Genotype-ID, implement species and genotype identification, respectively. Sequence-ID allows identification of species by using BLAST to query sequences for any locus of interest against a custom reference sequence database. Genotype-ID allows placement of an unknown multilocus marker in either a minimum spanning network or dendrogram with bootstrap support from a user-created reference database. Microbe-ID can be used for identification of any organism based on nucleotide sequences or any molecular marker type and several examples are provided. We created a public website for demonstration purposes called Microbe-ID (microbe-id.org) and provided a working implementation for the genus Phytophthora (phytophthora-id.org). In Phytophthora-ID, the Sequence-ID application allows identification based on ITS or cox spacer sequences. Genotype-ID groups individuals into clonal lineages based on simple sequence repeat (SSR) markers for the two invasive plant pathogen species P. infestans and P. ramorum. All code is open source and available on github and CRAN. Instructions for installation and use are provided at https://github.com/grunwaldlab/Microbe-ID.

Fast genomic predictions via Bayesian G-BLUP and multilocus models of threshold traits including censored Gaussian data.

PubMed

Kärkkäinen, Hanni P; Sillanpää, Mikko J

2013-09-04

Because of the increased availability of genome-wide sets of molecular markers along with reduced cost of genotyping large samples of individuals, genomic estimated breeding values have become an essential resource in plant and animal breeding. Bayesian methods for breeding value estimation have proven to be accurate and efficient; however, the ever-increasing data sets are placing heavy demands on the parameter estimation algorithms. Although a commendable number of fast estimation algorithms are available for Bayesian models of continuous Gaussian traits, there is a shortage for corresponding models of discrete or censored phenotypes. In this work, we consider a threshold approach of binary, ordinal, and censored Gaussian observations for Bayesian multilocus association models and Bayesian genomic best linear unbiased prediction and present a high-speed generalized expectation maximization algorithm for parameter estimation under these models. We demonstrate our method with simulated and real data. Our example analyses suggest that the use of the extra information present in an ordered categorical or censored Gaussian data set, instead of dichotomizing the data into case-control observations, increases the accuracy of genomic breeding values predicted by Bayesian multilocus association models or by Bayesian genomic best linear unbiased prediction. Furthermore, the example analyses indicate that the correct threshold model is more accurate than the directly used Gaussian model with a censored Gaussian data, while with a binary or an ordinal data the superiority of the threshold model could not be confirmed.

Fast Genomic Predictions via Bayesian G-BLUP and Multilocus Models of Threshold Traits Including Censored Gaussian Data

PubMed Central

Kärkkäinen, Hanni P.; Sillanpää, Mikko J.

2013-01-01

Because of the increased availability of genome-wide sets of molecular markers along with reduced cost of genotyping large samples of individuals, genomic estimated breeding values have become an essential resource in plant and animal breeding. Bayesian methods for breeding value estimation have proven to be accurate and efficient; however, the ever-increasing data sets are placing heavy demands on the parameter estimation algorithms. Although a commendable number of fast estimation algorithms are available for Bayesian models of continuous Gaussian traits, there is a shortage for corresponding models of discrete or censored phenotypes. In this work, we consider a threshold approach of binary, ordinal, and censored Gaussian observations for Bayesian multilocus association models and Bayesian genomic best linear unbiased prediction and present a high-speed generalized expectation maximization algorithm for parameter estimation under these models. We demonstrate our method with simulated and real data. Our example analyses suggest that the use of the extra information present in an ordered categorical or censored Gaussian data set, instead of dichotomizing the data into case-control observations, increases the accuracy of genomic breeding values predicted by Bayesian multilocus association models or by Bayesian genomic best linear unbiased prediction. Furthermore, the example analyses indicate that the correct threshold model is more accurate than the directly used Gaussian model with a censored Gaussian data, while with a binary or an ordinal data the superiority of the threshold model could not be confirmed. PMID:23821618

Population genetic characterization of Cyclospora cayetanensis from discrete geographical regions.

PubMed

Guo, Yaqiong; Li, Na; Ortega, Ynes R; Zhang, Longxian; Roellig, Dawn M; Feng, Yaoyu; Xiao, Lihua

2018-01-01

Cyclospora cayetanensis is an emerging pathogen that is endemic in developing countries and responsible for many large foodborne cyclosporiasis outbreaks in North America since 1990s. Because of the lack of typing targets, the genetic diversity and population genetics of C. cayetanensis have not been investigated. In this study, we undertook a population genetic analysis of multilocus sequence typing data we recently collected from 64 C. cayetanensis specimens. Despite the extensive genetic heterogeneity in the overall C. cayetanensis population, there were significant intra- and inter-genic linkage disequilibria (LD). A disappearance of LD was observed when only multilocus genotypes were included in the population genetic analysis, indicative of an epidemic nature of C. cayetanensis. Geographical segregation-associated sub-structuring was observed between specimens from China and those from Peru and the United States. The two subpopulations had reduced LD, indicating the likely occurrence of genetic exchange among isolates in endemic areas. Further analyses of specimens from other geographical regions are necessary to fully understand the population genetics of C. cayetanensis. Copyright © 2017 Elsevier Inc. All rights reserved.

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants.

PubMed

Tapia-Hernández; Bustillos-Cristales; Jiménez-Salgado; Caballero-Mellado; Fuentes-Ramírez

2000-01-01

The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed.

Molecular characterization of Shiga-toxigenic Escherichia coli isolated from diverse sources from India by multi-locus variable number tandem repeat analysis (MLVA).

PubMed

Kumar, A; Taneja, N; Sharma, R K; Sharma, H; Ramamurthy, T; Sharma, M

2014-12-01

In a first study from India, a diverse collection of 140 environmental and clinical non-O157 Shiga-toxigenic Escherichia coli strains from a large geographical area in north India was typed by multi-locus variable number tandem repeat analysis (MLVA). The distribution of major virulence genes stx1, stx2 and eae was found to be 78%, 70% and 10%, respectively; 15 isolates were enterohaemorrhagic E. coli (stx1 +/stx2 + and eae +). By MLVA analysis, 44 different alleles were obtained. Dendrogram analysis revealed 104 different genotypes and 19 MLVA-type complexes divided into two main lineages, i.e. mutton and animal stool. Human isolates presented a statistically significant greater odds ratio for clustering with mutton samples compared to animal stool isolates. Five human isolates clustered with animal stool strains suggesting that some of the human infections may be from cattle, perhaps through milk, contact or the environment. Further epidemiological studies are required to explore these sources in context with occurrence of human cases.

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

PubMed

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

2016-10-01

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

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.

One haploid parent contributes 100% of the gene pool for a widespread species in northwest North America.

PubMed

Karlin, E F; Andrus, R E; Boles, S B; Shaw, A J

2011-02-01

The monoicous peatmoss Sphagnum subnitens has a tripartite distribution that includes disjunct population systems in Europe (including the Azores), northwestern North America and New Zealand. Regional genetic diversity was highest in European S. subnitens but in northwestern North America, a single microsatellite-based multilocus haploid genotype was detected across 16 sites ranging from Coos County, Oregon, to Kavalga Island in the Western Aleutians (a distance of some 4115 km). Two multilocus haploid genotypes were detected across 14 sites on South Island, New Zealand. The microsatellite-based regional genetic diversity detected in New Zealand and North American S. subnitens is the lowest reported for any Sphagnum. The low genetic diversity detected in both of these regions most likely resulted from a founder event associated with vegetative propagation and complete selfing, with one founding haploid plant in northwest North America and two in New Zealand. Thus, one plant appears to have contributed 100% of the gene pool for the population systems of S. subnitens occurring in northwest North America, and this is arguably the most genetically uniform group of plants having a widespread distribution yet detected. Although having a distribution spanning 12.5° of latitude and 56° of longitude, there was no evidence of any genetic diversification in S. subnitens in northwest North America. No genetic structure was detected among the three regions, and it appears that European plants of S. subnitens provided the source for New Zealand and northwest North American populations. © 2010 Blackwell Publishing Ltd.

CRISPR: A Useful Genetic Feature to Follow Vaginal Carriage of Group B Streptococcus

PubMed Central

Beauruelle, Clémence; Pastuszka, Adeline; Horvath, Philippe; Perrotin, Franck; Mereghetti, Laurent; Lanotte, Philippe

2017-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) and Cas (CRISPR-associated proteins) play a critical role in adaptive immunity against mobile genetic elements, especially phages, through their ability to acquire novel spacer sequences. Polarized spacer acquisition results in spacer polymorphism and temporal organization of CRISPR loci, making them attractive epidemiological markers. Group B Streptococcus (GBS), a genital commensal for 10 to 30% of healthy women and a major neonatal pathogen, possesses a ubiquitous and functional CRISPR1 locus. Our aim was to assess the CRISPR1 locus as an epidemiological marker to follow vaginal carriage of GBS in women. This study also allowed us to observe the evolution of the CRISPR1 locus in response to probable phage infection occurring in vivo. We followed carriage of GBS among 100 women over an 11-year period, with a median duration of approximately 2 years. The CRISPR1 locus was highly conserved over time. The isolates that show the same CRISPR1 genotype were collected from 83% of women. There was an agreement between CRISPR genotyping and other typing methods [MLVA (multilocus variable number of tandem repeat Analysis) and MLST (multilocus sequence typing)] for 94% of the cases. The CRISPR1 locus of the isolates from 18 women showed modifications, four of which acquired polarized spacer, highlighting the in vivo functionality of the system. The novel spacer of one isolate had sequence similarity with phage, suggesting that phage infection occurred during carriage. These findings improve our understanding of CRISPR-Cas evolution in GBS and provide a glimpse of host-phage dynamics in vivo. PMID:29075246

A novel Sarcocystis neurona genotype XIII is associated with severe encephalitis in an unexpectedly broad range of marine mammals from the northeastern Pacific Ocean.

PubMed

Barbosa, Lorraine; Johnson, Christine K; Lambourn, Dyanna M; Gibson, Amanda K; Haman, Katherine H; Huggins, Jessica L; Sweeny, Amy R; Sundar, Natarajan; Raverty, Stephen A; Grigg, Michael E

2015-08-01

Sarcocystis neurona is an important cause of protozoal encephalitis among marine mammals in the northeastern Pacific Ocean. To characterise the genetic type of S. neurona in this region, samples from 227 stranded marine mammals, most with clinical or pathological evidence of protozoal disease, were tested for the presence of coccidian parasites using a nested PCR assay. The frequency of S. neurona infection was 60% (136/227) among pinnipeds and cetaceans, including seven marine mammal species not previously known to be susceptible to infection by this parasite. Eight S. neurona fetal infections identified this coccidian parasite as capable of being transmitted transplacentally. Thirty-seven S. neurona-positive samples were multilocus sequence genotyped using three genetic markers: SnSAG1-5-6, SnSAG3 and SnSAG4. A novel genotype, referred to as Type XIII within the S. neurona population genetic structure, has emerged recently in the northeastern Pacific Ocean and is significantly associated with an increased severity of protozoal encephalitis and mortality among multiple stranded marine mammal species. Published by Elsevier Ltd.

Distribution of Serogroups and Genotypes among Disease-Associated and Carried Isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway

PubMed Central

Yazdankhah, Siamak P.; Kriz, Paula; Tzanakaki, Georgina; Kremastinou, Jenny; Kalmusova, Jitka; Musilek, Martin; Alvestad, Torill; Jolley, Keith A.; Wilson, Daniel J.; McCarthy, Noel D.; Caugant, Dominique A.; Maiden, Martin C. J.

2004-01-01

The distribution of serogroups and multilocus sequence types (STs) in collections of disease-associated and carried meningococci from the period 1991 to 2000 in three European countries (the Czech Republic, Greece, and Norway) was investigated. A total of 314 patient isolates and 353 isolates from asymptomatic carriers were characterized. The frequency distributions of serogroups and clone complexes differed among countries and between disease and carrier isolate collections. Highly significant differentiation was seen at each housekeeping locus. A marked positive association of serogroup C with disease was evidenced. The ST-11 complex was strongly positively associated with disease; associations for other clone complexes were weaker. The genetic diversity of the clone complexes differed. A single ST dominated the ST-11 clone complex, while the ST-41/44 complex exhibited greater levels of diversity. These data robustly demonstrated differences in the distribution of meningococcal genotypes in disease and carrier isolates and among countries. Further, they indicated that differences in genotype diversity and pathogenicity exist between meningococcal clone complexes. PMID:15528708

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

Molecular epidemiological investigation of Brucella melitensis circulating in Mongolia by MLVA16.

PubMed

Kang, Sung-Il; Her, Moon; Erdenebaataar, Janchivdorj; Vanaabaatar, Batbaatar; Cho, Hyorim; Sung, So-Ra; Lee, Jin Ju; Jung, Suk Chan; Park, Yong Ho; Kim, Ji-Yeon

2017-02-01

Mongolia has a high incidence of brucellosis in human and animals due to livestock husbandry. To investigate the genetic characteristics of Mongolian B. melitensis, an MLVA (multi-locus variable-number tandem-repeat analysis)-16 assay was performed with 94 B. melitensis isolates. They were identified as B. melitensis biovar (bv.) 1 (67), 3 (10) and Rev. 1 vaccine strains (17) using a classical biotyping and multiplex PCR. In genotyping, three human isolates were grouped at 2 genotypes with sheep isolates, and it implies that B. melitensis are cross-infected between human and livestock. In the parsimony analysis, Mongolian B. melitensis isolates had high genetic similarity with Chinese strains, likely due to the geographical proximity, clustered distinctively as compared with other foreign isolates. B. melitensis Rev. 1 vaccine strains were divided into 4 genotypes with 92% similarity. In the analysis of Rev.1 strains, the risk of mutation of vaccine strain might not be overlooked. Animal quarantines should be strengthened to prevent the spread of Brucella species among adjacent countries. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel Sarcocystis neurona genotype XIII is associated with severe encephalitis in an unexpectedly broad range of marine mammals from the northeastern Pacific Ocean

PubMed Central

Barbosa, Lorraine; Johnson, Christine K.; Lambourn, Dyanna M.; Gibson, Amanda K.; Haman, Katherine H.; Huggins, Jessica L.; Sweeny, Amy R.; Sundar, Natarajan; Raverty, Stephen A.; Grigg, Michael E.

2015-01-01

Sarcocystis neurona is an important cause of protozoal encephalitis among marine mammals in the northeastern Pacific Ocean. To characterize the genetic type of S. neurona in this region, samples from 227 stranded marine mammals, most with clinical or pathological evidence of protozoal disease, were tested for the presence of coccidian parasites using a nested PCR assay. The frequency of S. neurona infection was 60% (136/227) among pinnipeds and cetaceans, including seven marine mammal species not previously known to be susceptible to infection by this parasite. Eight S. neurona fetal infections identified this coccidian parasite as capable of being transmitted transplacentally. Thirty-seven S. neurona-positive samples were multilocus sequence genotyped using three genetic markers: SnSAG1-5-6, SnSAG3 and SnSAG4. A novel genotype, referred to as Type XIII within the S. neurona population genetic structure, has emerged recently in the northeastern Pacific Ocean and is significantly associated with an increased severity of protozoal encephalitis and mortality among multiple stranded marine mammal species. PMID:25997588

The population genetics of a solitary oligolectic sweat bee, Lasioglossum (Sphecodogastra) oenotherae (Hymenoptera: Halictidae).

PubMed

Zayed, A; Packer, L

2007-10-01

Strong evidence exists for global declines in pollinator populations. Data on the population genetics of solitary bees, especially diet specialists, are generally lacking. We studied the population genetics of the oligolectic bee Lasioglossum oenotherae, a specialist on the pollen of evening primrose (Onagraceae), by genotyping 455 females from 15 populations across the bee's North American range at six hyper-variable microsatellite loci. We found significant levels of genetic differentiation between populations, even at small geographic scales, as well as significant patterns of isolation by distance. However, using multilocus genotype assignment tests, we detected 11 first-generation migrants indicating that L. oenotherae's sub-populations are experiencing ongoing gene flow. Southern populations of L. oenotherae were significantly more likely to deviate from Hardy-Weinberg equilibrium and from genotypic equilibrium, suggesting regional differences in gene flow and/or drift and inbreeding. Short-term N(e) estimated using temporal changes in allele frequencies in several populations ranged from approximately 223 to 960. We discuss our findings in terms of the conservation genetics of specialist pollinators, a group of considerable ecological importance.

Joint genotype- and ancestry-based genome-wide association studies in admixed populations.

PubMed

Szulc, Piotr; Bogdan, Malgorzata; Frommlet, Florian; Tang, Hua

2017-09-01

In genome-wide association studies (GWAS) genetic loci that influence complex traits are localized by inspecting associations between genotypes of genetic markers and the values of the trait of interest. On the other hand, admixture mapping, which is performed in case of populations consisting of a recent mix of two ancestral groups, relies on the ancestry information at each locus (locus-specific ancestry). Recently it has been proposed to jointly model genotype and locus-specific ancestry within the framework of single marker tests. Here, we extend this approach for population-based GWAS in the direction of multimarker models. A modified version of the Bayesian information criterion is developed for building a multilocus model that accounts for the differential correlation structure due to linkage disequilibrium (LD) and admixture LD. Simulation studies and a real data example illustrate the advantages of this new approach compared to single-marker analysis or modern model selection strategies based on separately analyzing genotype and ancestry data, as well as to single-marker analysis combining genotypic and ancestry information. Depending on the signal strength, our procedure automatically chooses whether genotypic or locus-specific ancestry markers are added to the model. This results in a good compromise between the power to detect causal mutations and the precision of their localization. The proposed method has been implemented in R and is available at http://www.math.uni.wroc.pl/~mbogdan/admixtures/. © 2017 WILEY PERIODICALS, INC.

Methods for MHC genotyping in non-model vertebrates.

PubMed

Babik, W

2010-03-01

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems. © 2009 Blackwell Publishing Ltd.

Highly diverse MLVA-ompA genotypes of rectal Chlamydia trachomatis among men who have sex with men in Brighton, UK and evidence for an HIV-related sexual network.

PubMed

Labiran, Clare; Marsh, Peter; Zhou, Judith; Bannister, Alan; Clarke, Ian Nicholas; Goubet, Stephanie; Soni, Suneeta

2016-06-01

In this prospective study, we aimed to determine the distribution of genotypes by multilocus variable number tandem repeat (VNTR) analysis plus analysis of the ompA gene (MLVA-ompA) of rectal Chlamydia trachomatis among men who have sex with men (MSM) attending Brighton Genitourinary Medicine (GUM) Clinic and to examine any correlations with clinical variables, including HIV status, and to isolate rectal C. trachomatis cultures maximising the possibility of obtaining complete genotyping data. Samples were assigned genotypes by PCR and sequencing of the markers of the MLVA-ompA genotyping system. Rectal C. trachomatis was isolated in cell culture using McCoy cells. Data regarding demographics, HIV status, rectal symptoms and history of sexually transmitted infections, including C. trachomatis, were collected. 1809 MSM attending the clinic between October 2011 and January 2013 took part in the study, 112 (6.2%) of whom had rectal samples that tested positive for C. trachomatis. 85/112 (75.9%) C. trachomatis-positive rectal samples were assigned 66 different genotypes. Two distinct genotype subclusters were identified: subcluster 1 consisted of more HIV-negative men than subcluster 2 (p=0.025), and the MLVA-ompA genotypes in these subclusters reflected this. Isolates were successfully cultured from 37 of the 112 specimens, from which 27 otherwise unobtainable (from direct PCR) MLVA-ompA genotypes were gained. The most prevalent genotypes were G, E and D representing some overlap with the heterosexual distribution in UK. Subcluster 1 consisted of more 'heterosexual genotypes' and significantly more HIV-negative men than subcluster 2, associated with 'MSM genotypes'. There was a higher diversity of C. trachomatis strains among MSM in Brighton than observed in other cities. 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 genetic analysis and trichothecene profiling of Fusarium graminearum from wheat in Uruguay.

PubMed

Pan, D; Mionetto, A; Calero, N; Reynoso, M M; Torres, A; Bettucci, L

2016-03-11

Fusarium graminearum sensu stricto (F. graminearum s.s.) is the major causal agent of Fusarium head blight of wheat worldwide, and contaminates grains with trichothecene mycotoxins that cause serious threats to food safety and animal health. An important aspect of managing this pathogen and reducing mycotoxin contamination of wheat is knowledge regarding its population genetics. Therefore, isolates of F. graminearum s.s. from the major wheat-growing region of Uruguay were analyzed by amplified fragment length polymorphism assays, PCR genotyping, and chemical analysis of trichothecene production. Of the 102 isolates identified as having the 15-ADON genotype via PCR genotyping, all were DON producers, but only 41 strains were also 15-ADON producers, as determined by chemical analysis. The populations were genotypically diverse but genetically similar, with significant genetic exchange occurring between them. Analysis of molecular variance indicated that most of the genetic variability resulted from differences between isolates within populations. Multilocus linkage disequilibrium analysis suggested that the isolates had a panmictic population genetic structure and that there is significant recombination occurs in F. graminearum s.s. In conclusion, tour findings provide the first detailed description of the genetic structure and trichothecene production of populations of F. graminearum s.s. from Uruguay, and expands our understanding of the agroecology of F. graminearum and of the correlation between genotypes and trichothecene chemotypes.

Microbe-ID: an open source toolbox for microbial genotyping and species identification

PubMed Central

Tabima, Javier F.; Everhart, Sydney E.; Larsen, Meredith M.; Weisberg, Alexandra J.; Kamvar, Zhian N.; Tancos, Matthew A.; Smart, Christine D.; Chang, Jeff H.

2016-01-01

Development of tools to identify species, genotypes, or novel strains of invasive organisms is critical for monitoring emergence and implementing rapid response measures. Molecular markers, although critical to identifying species or genotypes, require bioinformatic tools for analysis. However, user-friendly analytical tools for fast identification are not readily available. To address this need, we created a web-based set of applications called Microbe-ID that allow for customizing a toolbox for rapid species identification and strain genotyping using any genetic markers of choice. Two components of Microbe-ID, named Sequence-ID and Genotype-ID, implement species and genotype identification, respectively. Sequence-ID allows identification of species by using BLAST to query sequences for any locus of interest against a custom reference sequence database. Genotype-ID allows placement of an unknown multilocus marker in either a minimum spanning network or dendrogram with bootstrap support from a user-created reference database. Microbe-ID can be used for identification of any organism based on nucleotide sequences or any molecular marker type and several examples are provided. We created a public website for demonstration purposes called Microbe-ID (microbe-id.org) and provided a working implementation for the genus Phytophthora (phytophthora-id.org). In Phytophthora-ID, the Sequence-ID application allows identification based on ITS or cox spacer sequences. Genotype-ID groups individuals into clonal lineages based on simple sequence repeat (SSR) markers for the two invasive plant pathogen species P. infestans and P. ramorum. All code is open source and available on github and CRAN. Instructions for installation and use are provided at https://github.com/grunwaldlab/Microbe-ID. PMID:27602267

The coding region of the UFGT gene is a source of diagnostic SNP markers that allow single-locus DNA genotyping for the assessment of cultivar identity and ancestry in grapevine (Vitis vinifera L.)

PubMed Central

2013-01-01

Background Vitis vinifera L. is one of society’s most important agricultural crops with a broad genetic variability. The difficulty in recognizing grapevine genotypes based on ampelographic traits and secondary metabolites prompted the development of molecular markers suitable for achieving variety genetic identification. Findings Here, we propose a comparison between a multi-locus barcoding approach based on six chloroplast markers and a single-copy nuclear gene sequencing method using five coding regions combined with a character-based system with the aim of reconstructing cultivar-specific haplotypes and genotypes to be exploited for the molecular characterization of 157 V. vinifera accessions. The analysis of the chloroplast target regions proved the inadequacy of the DNA barcoding approach at the subspecies level, and hence further DNA genotyping analyses were targeted on the sequences of five nuclear single-copy genes amplified across all of the accessions. The sequencing of the coding region of the UFGT nuclear gene (UDP-glucose: flavonoid 3-0-glucosyltransferase, the key enzyme for the accumulation of anthocyanins in berry skins) enabled the discovery of discriminant SNPs (1/34 bp) and the reconstruction of 130 V. vinifera distinct genotypes. Most of the genotypes proved to be cultivar-specific, and only few genotypes were shared by more, although strictly related, cultivars. Conclusion On the whole, this technique was successful for inferring SNP-based genotypes of grapevine accessions suitable for assessing the genetic identity and ancestry of international cultivars and also useful for corroborating some hypotheses regarding the origin of local varieties, suggesting several issues of misidentification (synonymy/homonymy). PMID:24298902

Emergence of Cryptosporidium hominis Monkey Genotype II and Novel Subtype Family Ik in the Squirrel Monkey (Saimiri sciureus) in China.

PubMed

Liu, Xuehan; Xie, Na; Li, Wei; Zhou, Ziyao; Zhong, Zhijun; Shen, Liuhong; Cao, Suizhong; Yu, Xingming; Hu, Yanchuan; Chen, Weigang; Peng, Gangneng

2015-01-01

A single Cryptosporidium isolate from a squirrel monkey with no clinical symptoms was obtained from a zoo in Ya'an city, China, and was genotyped by PCR amplification and DNA sequencing of the small-subunit ribosomal RNA (SSU rRNA), 70-kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein, and actin genes. This multilocus genetic characterization determined that the isolate was Cryptosporidium hominis, but carried 2, 10, and 6 nucleotide differences in the SSU rRNA, HSP70, and actin loci, respectively, which is comparable to the variations at these loci between C. hominis and the previously reported monkey genotype (2, 3, and 3 nucleotide differences). Phylogenetic studies, based on neighbor-joining and maximum likelihood methods, showed that the isolate identified in the current study had a distinctly discordant taxonomic status, distinct from known C. hominis and also from the monkey genotype, with respect to the three loci. Restriction fragment length polymorphisms of the SSU rRNA gene obtained from this study were similar to those of known C. hominis but clearly differentiated from the monkey genotype. Further subtyping was performed by sequence analysis of the gene encoding the 60-kDa glycoprotein (gp60). Maximum homology of only 88.3% to C. hominis subtype IdA10G4 was observed for the current isolate, and phylogenetic analysis demonstrated that this particular isolate belonged to a novel C. hominis subtype family, IkA7G4. This study is the first to report C. hominis infection in the squirrel monkey and, based on the observed genetic characteristics, confirms a new C. hominis genotype, monkey genotype II. Thus, these results provide novel insights into genotypic variation in C. hominis.

Ontogenetic change in relative performance of allozyme genotypes influences detection of heterosis in the earthworm Eisenia andrei.

PubMed

McElroy, T C; Diehl, W J

2005-02-01

The effect of ontogeny on relationships between allozyme genotypes and fresh weight was measured weekly throughout the life history of the earthworm Eisenia andrei to test the hypothesis that there is an ontogenetic component to variation in such relationships. Two of six allozyme loci showed a significant increase in apparent heterosis with ontogeny, while one locus showed a significant decrease in apparent heterosis. Three loci showed a significant decrease in the performance of common homozygotes with ontogeny. Patterns of relative genotypic performance varied among loci, but the cumulative effect was an increase in apparent allozyme heterosis later in ontogeny coinciding with a series of positive relationships between multilocus heterozygosity and fresh weight. The results could not be used to determine whether these patterns were caused by selection acting on the loci directly or on loci tightly linked to allozyme loci. However, because the same individuals were used throughout this study and thus allele frequencies and heterozygote deficiency were constant, the presence of both ontogenetic effects and differences in such patterns among loci is not compatible with a general inbreeding effect. Examining relative genotypic performance repetitively using the same individuals through ontogeny or in different environments is a very powerful experimental design for testing the effects of inbreeding or other populational factors.

Population structure and antimicrobial susceptibility of Paenibacillus larvae isolates from American foulbrood cases in Apis mellifera in Japan.

PubMed

Ueno, Yuichi; Yoshida, Emi; Misumi, Wakako; Watando, Eri; Suzuki, Kenta; Hirai, Yuko; Okura, Masatoshi; Osaki, Makoto; Katsuda, Ken; Takamatsu, Daisuke

2018-04-01

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most destructive disease of the honey bee brood. In this study, we investigated the population structure and antimicrobial susceptibility of Japanese P. larvae using 100 isolates isolated between 1993 and 2017 in 17 prefectures. Using repetitive-element PCR and multilocus sequence typing, isolates from diverse origins were classified into six genotypes, including the novel genotype ERIC II-ST24. Among these genotypes, ERIC I-ST15 is the most common in Japan, while ERIC II-ST10 isolates were found to be increasing during the 2010s. Regardless of genotype or origin, all isolates were susceptible to the major antimicrobials used in the control of AFB, including mirosamicin and tylosin, which were approved for the prevention of AFB in Japan in 1999 and 2017 respectively. Despite nearly 20 years of use, mirosamicin is still effective against Japanese P. larvae in vitro; however, the development of AFB in honey bee colonies may not always be suppressed by this drug. The case information collected in this study provides insight into the conditions under which prophylactic medicine may not exert sufficient preventive effects in vivo. © 2018 Society for Applied Microbiology and 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.

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.

Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China.

PubMed

Sun, Mingjun; Jing, Zhigang; Di, Dongdong; Yan, Hao; Zhang, Zhicheng; Xu, Quangang; Zhang, Xiyue; Wang, Xun; Ni, Bo; Sun, Xiangxiang; Yan, Chengxu; Yang, Zhen; Tian, Lili; Li, Jinping; Fan, Weixing

2017-01-01

Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2) was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2) and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs) were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS)-single-nucleotide polymorphism (SNP)-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

Multilocus Sequence Typing Reveals a New Cluster of Closely Related Candida tropicalis Genotypes in Italian Patients With Neurological Disorders.

PubMed

Scordino, Fabio; Giuffrè, Letterio; Barberi, Giuseppina; Marino Merlo, Francesca; Orlando, Maria Grazia; Giosa, Domenico; Romeo, Orazio

2018-01-01

Candida tropicalis is a pathogenic yeast that has emerged as an important cause of candidemia especially in elderly patients with hematological malignancies. Infections caused by this species are mainly reported from Latin America and Asian-Pacific countries although recent epidemiological data revealed that C. tropicalis accounts for 6-16.4% of the Candida bloodstream infections (BSIs) in Italy by representing a relevant issue especially for patients receiving long-term hospital care. The aim of this study was to describe the genetic diversity of C. tropicalis isolates contaminating the hands of healthcare workers (HCWs) and hospital environments and/or associated with BSIs occurring in patients with different neurological disorders and without hematological disease. A total of 28 C. tropicalis isolates were genotyped using multilocus sequence typing analysis of six housekeeping ( ICL1, MDR1, SAPT2, SAPT4, XYR1 , and ZWF1 ) genes and data revealed the presence of only eight diploid sequence types (DSTs) of which 6 (75%) were completely new. Four eBURST clonal complexes (CC2, CC10, CC11, and CC33) contained all DSTs found in this study and the CC33 resulted in an exclusive, well-defined, clonal cluster from Italy. In conclusion, C. tropicalis could represent an important cause of BSIs in long-term hospitalized patients with no underlying hematological disease. The findings of this study also suggest a potential horizontal transmission of a specific C. tropicalis clone through hands of HCWs and expand our understanding of the molecular epidemiology of this pathogen whose population structure is still far from being fully elucidated as its complexity increases as different categories of patients and geographic areas are examined.

Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection

PubMed Central

Llewellyn, Martin S.; Miles, Michael A.; Carrasco, Hernan J.; Lewis, Michael D.; Yeo, Matthew; Vargas, Jorge; Torrico, Faustino; Diosque, Patricio; Valente, Vera; Valente, Sebastiao A.; Gaunt, Michael W.

2009-01-01

Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs) reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is “hidden” at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed. PMID:19412340

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

PubMed Central

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

2006-01-01

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

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia.

PubMed

Opavski, Natasa; Gajic, Ina; Borek, Anna L; Obszańska, Katarzyna; Stanojevic, Maja; Lazarevic, Ivana; Ranin, Lazar; Sitkiewicz, Izabela; Mijac, Vera

2015-07-01

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004-2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes--emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

Validation of a pXO2-A PCR Assay To Explore Diversity among Italian Isolates of Bacillus anthracis Strains Closely Related to the Live, Attenuated Carbosap Vaccine

PubMed Central

Muscillo, M.; La Rosa, G.; Sali, M.; De Carolis, E.; Adone, R.; Ciuchini, F.; Fasanella, A.

2005-01-01

Several circulating Bacillus anthracis strains isolated in Italy and belonging to the A1.a cluster, genotype 3 (A1.a-3) are genotypically indistinguishable from Carbosap, a live attenuated vaccine strain, containing both pXO1 and pXO2 plasmids. The genotype was assessed by using eight-locus multilocus variable-number tandem repeat analysis. We describe here the use of a ninth locus able to explore variability among strains that have the same genotype. It is important to be able to genotype the wild isolate of B. anthracis strains from outbreaks of anthrax in areas where Carbosap vaccination of cattle and sheep is common practice. A total of 27 representative field strains isolated in Italy and four vaccinal strains, namely, Carbosap, Sterne, Pasteur I, and Pasteur II, were characterized by a ninth marker, called pXO2-A. Twenty-three field strains were genotype 3 and therefore identical to Carbosap. The marker was in the pXO2 plasmid and is based on the polymorphism of the already-known VX2-3 locus. Detection was obtained by PCR with fluorescence-labeled forward primers in order to produce appropriate fragments for capillary electrophoresis with an ABI 310 genetic analyzer. Genetic relationships showed heterogeneity in all of the examined samples. Interestingly, with respect to genotype 3, samples grouped into eight different subtypes, A to H, and the subtype G, had only two samples indistinguishable from Carbosap. The results of the present study confirm the validity of a hierarchical progressive protocol for discrimination among closely related isolates. PMID:16145138

High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA.

PubMed

De Barba, M; Miquel, C; Lobréaux, S; Quenette, P Y; Swenson, J E; Taberlet, P

2017-05-01

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies. © 2016 John Wiley & Sons Ltd.

Multi-locus variable-number tandem repeat analysis of Chinese Brucella strains isolated from 1953 to 2013.

PubMed

Tian, Guo-Zhong; Cui, Bu-Yun; Piao, Dong-Ri; Zhao, Hong-Yan; Li, Lan-Yu; Liu, Xi; Xiao, Pei; Zhao, Zhong-Zhi; Xu, Li-Qing; Jiang, Hai; Li, Zhen-Jun

2017-05-02

Brucellosis was a common human and livestock disease caused by Brucella strains, the category B priority pathogens by the US Center for Disease Control (CDC). Identified as a priority disease in human and livestock populations, the increasing incidence in recent years in China needs urgent control measures for this disease but the molecular background important for monitoring the epidemiology of Brucella strains at the national level is still lacking. A total of 600 Brucella isolates collected during 60 years (from 1953 to 2013) in China were genotyped by multiple locus variable-number tandem repeat analysis (MLVA) and the variation degree of MLVA11 loci was calculated by the Hunter Gaston Diversity Index (HGDI) values. The charts and map were processed by Excel 2013, and cluster analysis and epidemiological distribution was performed using BioNumerics (version 5.1). The 600 representative Brucella isolates fell into 104 genotypes with 58 singleton genotypes by the MLVA11 assay, including B. melitensis biovars 2 and 3 (five main genotypes), B. abortus biovars 1 and 3 (two main genotypes), B. suis biovars 1 and 3 (three main genotypes), and B. canis (two main genotypes) respectively. While most B. suis biovar 1 and biovar 3 were respectively found in northern provinces and southern provinces, B. melitensis and B. abortus strains were dominant in China. Canine Brucellosis was only found in animals without any human cases reported. Eight Brucellosis epidemic peaks emerged during the 60 years between 1953 and 2013: 1955 - 1959, 1962 - 1969, 1971 - 1975, 1977 - 1983, 1985 - 1989, 1992 - 1997, 2000 - 2008 and 2010 - 2013 in China. Brucellosis has its unique molecular epidemiological patterns with specific spatial and temporal distribution according to MLVA. IDOP-D-16-00101.

Differences in the Fecal Concentrations and Genetic Diversities of Campylobacter jejuni Populations among Individual Cows in Two Dairy Herds

PubMed Central

Ross, Colleen M.; Pleydell, Eve J.; Muirhead, Richard W.

2012-01-01

Dairy cows have been identified as common carriers of Campylobacter jejuni, which causes many of the human gastroenteritis cases reported worldwide. To design on-farm management practices that control the human infection sourced from dairy cows, the first step is to acquire an understanding of the excretion patterns of the cow reservoir. We monitored the same 35 cows from two dairy farms for C. jejuni excretion fortnightly for up to 12 months. The objective was to examine the concentration of C. jejuni and assess the genetic relationship of the C. jejuni populations excreted by individual cows. Significant differences (P < 0.01) in C. jejuni fecal concentration were observed among the 35 cows, with median concentrations that varied by up to 3.6 log10 · g−1 feces. A total of 36 different genotypes were identified from the 514 positive samples by using enterobacterial repetitive intergenic consensus (ERIC)-PCR. Although 22 of these genotypes were excreted by more than one cow, the analysis of frequencies and distribution of the genotypes by model-based statistics revealed a high degree of individuality in the C. jejuni population in each cow. The observed variation in the frequency of excretion of a genotype among cows and the analysis by multilocus sequence typing (MLST) of these genotypes suggest that excretion of C. jejuni in high numbers is due to a successful adaptation of a particular genotype to a particular cow's gut environment, but that animal-related factors render some individual cows resistant to colonization by particular genotypes. The reasons for differences in C. jejuni colonization of animals warrant further investigation. PMID:22904055

Colonization of patients, healthcare workers, and the environment with healthcare-associated Staphylococcus epidermidis genotypes in an intensive care unit: a prospective observational cohort study.

PubMed

Widerström, Micael; Wiström, Johan; Edebro, Helén; Marklund, Elisabeth; Backman, Mattias; Lindqvist, Per; Monsen, Tor

2016-12-09

During the last decades, healthcare-associated genotypes of methicillin-resistant Staphylococcus epidermidis (HA-MRSE) have been established as important opportunistic pathogens. However, data on potential reservoirs on HA-MRSE is limited. The aim of the present study was to investigate the dynamics and to which extent HA-MRSE genotypes colonize patients, healthcare workers (HCWs) and the environment in an intensive care unit (ICU). Over 12 months in 2006-2007, swab samples were obtained from patients admitted directly from the community to the ICU and patients transferred from a referral hospital, as well as from HCWs, and the ICU environment. Patients were sampled every third day during hospitalization. Antibiotic susceptibility testing was performed according to EUCAST guidelines. Pulsed-field gel electrophoresis and multilocus sequence typing were used to determine the genetic relatedness of a subset of MRSE isolates. We identified 620 MRSE isolates from 570 cultures obtained from 37 HCWs, 14 patients, and 14 environmental surfaces in the ICU. HA-MRSE genotypes were identified at admission in only one of the nine patients admitted directly from the community, of which the majority subsequently were colonized by HA-MRSE genotypes within 3 days during hospitalization. Almost all (89%) of HCWs were nasal carriers of HA-MRSE genotypes. Similarly, a significant proportion of patients transferred from the referral hospital and fomites in the ICU were widely colonized with HA-MRSE genotypes. Patients transferred from a referral hospital, HCWs, and the hospital environment serve as important reservoirs for HA-MRSE. These observations highlight the need for implementation of effective infection prevention and control measures aiming at reducing HA-MRSE transmission in the healthcare setting.

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.

Multilocus Sequence Typing of Bartonella henselae in the United Kingdom Indicates that Only a Few, Uncommon Sequence Types Are Associated with Zoonotic Disease▿†

PubMed Central

Chaloner, Gemma L.; Harrison, Timothy G.; Coyne, Karen P.; Aanensen, David M.; Birtles, Richard J.

2011-01-01

Bartonella henselae is one of the most common zoonotic agents acquired from companion animals (cats) in industrialized countries. Nonetheless, although the prevalence of infections in cats is high, the number of human cases reported is relatively low. One hypothesis for this discrepancy is that B. henselae strains vary in their zoonotic potential. To test this hypothesis, we employed structured sampling to explore the population structure of B. henselae in the United Kingdom and to determine the distribution of strains associated with zoonotic disease within this structure. A total of 118 B. henselae strains were delineated into 12 sequence types (STs) using multilocus sequence typing. We observed that most (85%) of the zoonosis-associated strains belonged to only three genotypes, i.e., ST2, ST5, and ST8. Conversely, most (74%) of the feline isolates belonged to ST4, ST6, and ST7. The difference in host association of ST2, ST5, and ST8 (zoonosis associated) and ST6 (feline) was statistically significant (P < 0.05), indicating that a few, uncommon STs were responsible for the majority of symptomatic human infections. PMID:21471345

The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa.

PubMed

Nunney, L; Elfekih, S; Stouthamer, R

2012-05-01

Microbial identification methods have evolved rapidly over the last few decades. One such method is multilocus sequence typing (MLST). MLST is a powerful tool for understanding the evolutionary dynamics of pathogens and to gain insight into their genetic diversity. We illustrate the importance of accurate typing by reporting on three problems that have arisen in the study of a single bacterial species, the plant pathogen Xylella fastidiosa. Two of these were particularly serious since they concerned contamination of important research material that has had detrimental consequences for Xylella research: the contamination of DNA used in the sequencing of an X. fastidiosa genome (Ann-1) with DNA from another X. fastidiosa strain, and the unrecognized mislabeling of a strain (Temecula1) distributed from a culture collection (ATCC). We advocate the routine use of MLST to define strains maintained in culture collections and emphasize the importance of confirming the purity of DNA submitted for sequencing. We also present a third example that illustrates the value of MLST in guiding the choice of taxonomic types. Beyond these situations, there is a strong case for MLST whenever an isolate is used experimentally, especially where genotypic differences are suspected to influence the outcome.

Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species

PubMed Central

Cavanagh, Daniel; Casey, Aidan; Altermann, Eric; Cotter, Paul D.; Fitzgerald, Gerald F.

2015-01-01

Lactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among “wild” strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ∼14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of <0.99 in addition to ANI values of <95%, implicating that these two groups are separate species. These findings suggest the requirement for a revision of L. lactis taxonomy. PMID:25841018

Haplotypic Analysis of Wellcome Trust Case Control Consortium Data

PubMed Central

Browning, Brian L.; Browning, Sharon R.

2008-01-01

We applied a recently developed multilocus association testing method (localized haplotype clustering) to Wellcome Trust Case Control Consortium data (14,000 cases of seven common diseases and 3,000 shared controls genotyped on the Affymetrix 500K array). After rigorous data quality filtering, we identified three disease-associated loci with strong statistical support from localized haplotype cluster tests but with only marginal significance in single marker tests. These loci are chromosomes 10p15.1 with type 1 diabetes (p = 5.1 × 10-9), 12q15 with type 2 diabetes (p = 1.9 × 10-7) and 15q26.2 with hypertension (p = 2.8 × 10-8). We also detected the association of chromosome 9p21.3 with type 2 diabetes (p = 2.8 × 10-8), although this locus did not pass our stringent genotype quality filters. The association of 10p15.1 with type 1 diabetes and 9p21.3 with type 2 diabetes have both been replicated in other studies using independent data sets. Overall, localized haplotype cluster analysis had better success detecting disease associated variants than a previous single-marker analysis of imputed HapMap SNPs. We found that stringent application of quality score thresholds to genotype data substantially reduced false-positive results arising from genotype error. In addition, we demonstrate that it is possible to simultaneously phase 16,000 individuals genotyped on genome-wide data (450K markers) using the Beagle software package. PMID:18224336

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

PubMed

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

2016-03-01

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

Development of Polymorphic Microsatellite Loci for Potato Wart from Next-Generation Sequence Data.

PubMed

Gagnon, Marie-Claude; van der Lee, Theo A J; Bonants, Peter J M; Smith, Donna S; Li, Xiang; Lévesque, C André; Bilodeau, Guillaume J

2016-06-01

Synchytrium endobioticum is the fungal agent causing potato wart disease. Because of its severity and persistence, quarantine measures are enforced worldwide to avoid the spread of this disease. Molecular markers exist for species-specific detection of this pathogen, yet markers to study the intraspecific genetic diversity of S. endobioticum were not available. Whole-genome sequence data from Dutch pathotype 1 isolate MB42 of S. endobioticum were mined for perfect microsatellite motifs. Of the 62 selected microsatellites, 21 could be amplified successfully and displayed moderate levels of polymorphism in 22 S. endobioticum isolates from different countries. Nineteen multilocus genotypes were observed, with only three isolates from Canada displaying identical profiles. The majority of isolates from Canada clustered genetically. In contrast, most isolates collected in Europe show no genetic clustering associated with their geographic origin. S. endobioticum isolates with the same pathotype displayed highly variable genotypes and none of the microsatellite markers correlated with a specific pathotype. The markers developed in this study can be used to assess intraspecific genetic diversity of S. endobioticum and allow track and trace of genotypes that will generate a better understanding of the migration and spread of this important fungal pathogen and support management of this disease.

Population cage experiments with a vertebrate: The temporal demography and cytonuclear genetics of hybridization on Gambusia fishes

USGS Publications Warehouse

Scribner, Kim T.; Avise, John C.

1994-01-01

The dynamics of mitochondrial and multilocus nuclear genotypic frequencies were monitored for 2 yr in experimental populations established with equal numbers of two poeciliid fishes (Gambusia affinis and Gambusia holbrooki) that hybridize naturally in the southeastern United States. In replicated "small-pool" populations (experiment I), 1018 sampled individuals at six time periods revealed an initial flush of hybridization, followed by a rapid decline in frequencies of G. affinis nuclear and mitochondrial alleles over 64 wk. Decay of gametic and cytonuclear disequilibria differed from expectations under random mating as well as under a model of assortative mating involving empirically estimated mating propensities. In two replicate "large-pond" populations (experiment II), 841 sampled individuals across four reproductive cohorts revealed lower initial frequencies of F1 hybrids than in experiment I, but again G. holbrooki alleles achieved high frequencies over four generations (72 wk). Thus, evolution within experimental Gambusia hybrid populations can be extremely rapid, resulting in consistent loss of G. affinis nuclear and cytoplasmic alleles. Concordance in results between experiments and across genetic markers suggests strong directional selection favoring G. holbrooki genotypes. Results are interpreted in light of previous reports of genotype-specific differences in life-history traits, reproductive ecology, patterns of recruitment, and size-specific mortality, and in the context of patterns of introgression previously studied indirectly from spatial observations on cytonuclear genotypes in natural Gambusia populations.

Genetic structure and viability selection in the golden eagle (Aquila chrysaetos), a vagile raptor with a Holarctic distribution

USGS Publications Warehouse

Doyle, Jacqueline M.; Katzner, Todd E.; Roemer, Gary; Cain, James W.; Millsap, Brian; McIntyre, Carol; Sonsthagen, Sarah A.; Fernandez, Nadia B.; Wheeler, Maria; Bulut, Zafer; Bloom, Peter; DeWoody, J. Andrew

2016-01-01

Molecular markers can reveal interesting aspects of organismal ecology and evolution, especially when surveyed in rare or elusive species. Herein, we provide a preliminary assessment of golden eagle (Aquila chrysaetos) population structure in North America using novel single nucleotide polymorphisms (SNPs). These SNPs included one molecular sexing marker, two mitochondrial markers, 85 putatively neutral markers that were derived from noncoding regions within large intergenic intervals, and 74 putatively nonneutral markers found in or very near protein-coding genes. We genotyped 523 eagle samples at these 162 SNPs and quantified genotyping error rates and variability at each marker. Our samples corresponded to 344 individual golden eagles as assessed by unique multilocus genotypes. Observed heterozygosity of known adults was significantly higher than of chicks, as was the number of heterozygous loci, indicating that mean zygosity measured across all 159 autosomal markers was an indicator of fitness as it is associated with eagle survival to adulthood. Finally, we used chick samples of known provenance to test for population differentiation across portions of North America and found pronounced structure among geographic sampling sites. These data indicate that cryptic genetic population structure is likely widespread in the golden eagle gene pool, and that extensive field sampling and genotyping will be required to more clearly delineate management units within North America and elsewhere.

Epidemiological Outbreaks of Pneumocystis jirovecii Pneumonia Are Not Limited to Kidney Transplant Recipients: Genotyping Confirms Common Source of Transmission in a Liver Transplantation Unit

PubMed Central

Dominique, Manon; Morio, Florent; Thepault, Rose-Anne; Franck-Martel, Claire; Tellier, Anne-Charlotte; Ferrandière, Martine; Hennequin, Christophe; Bernard, Louis; Salamé, Ephrem; Bailly, Éric; Chandenier, Jacques

2016-01-01

Over a 5-month period, four liver transplant patients at a single hospital were diagnosed with Pneumocystis jirovecii pneumonia (PCP). This unusually high incidence was investigated using molecular genotyping. Bronchoalveolar lavage fluids (BALF) obtained from the four liver recipients diagnosed with PCP were processed for multilocus sequence typing (MLST) at three loci (SOD, mt26s, and CYB). Twenty-four other BALF samples, which were positive for P. jirovecii and collected from 24 epidemiologically unrelated patients with clinical signs of PCP, were studied in parallel by use of the same method. Pneumocystis jirovecii isolates from the four liver recipients all had the same genotype, which was different from those of the isolates from all the epidemiologically unrelated individuals studied. These findings supported the hypothesis of a common source of contamination or even cross-transmission of a single P. jirovecii clone between the four liver recipients. Hospitalization mapping showed several possible encounters between these four patients, including outpatient consultations on one particular date when they all possibly met. This study demonstrates the value of molecular genotyping of P. jirovecii isolated from clinical samples for epidemiological investigation of PCP outbreaks. It is also the first description of a common source of exposure to a single P. jirovecii clone between liver transplant recipients and highlights the importance of prophylaxis in such a population. PMID:26935726

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

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.

Genetic characterization and antimicrobial resistance of Staphylococcus aureus isolated from bovine milk in Tunisia.

PubMed

Ben Said, M; Abbassi, M S; Bianchini, V; Sghaier, S; Cremonesi, P; Romanò, A; Gualdi, V; Hassen, A; Luini, M V

2016-12-01

Staphylococcus aureus is a major agent of bovine mastitis in dairy herds, causing economic losses in dairy industry worldwide. In addition, milk and milk-products contaminated by Staph. aureus can cause harmful human diseases. The aim of this study was to characterize Staph. aureus strains isolated from dairy farms in Tunisia. Bulk tank milk (n = 32) and individual cow milk (n = 130) samples were collected during the period of 2013-2014. Forty-three Staph. aureus isolates were recovered and typed by spa typing, 16S-23S rRNA intergenic spacer (RS-PCR) and multiplex PCRs for 22 virulence genes. Antimicrobial resistance was also investigated with a disc diffusion test. A selected subsample of 22 strains was additionally genotyped by multilocus sequence typing. Seventeen spa types were recovered, and t2421 (n = 10), t521 (n = 6) and t2112 (n = 5) were the most common. Fourteen different RS-PCR genotypes grouped into 11 clusters were detected in our study, with predominance of the R VI genotype (n = 24). Eight sequence types were identified and Clonal Complex 97, corresponding to RS-PCR cluster R, was the most common (n = 10), followed by CC1 (n = 4), CC15 (n = 3) and other four accounting for one or two strains. Different combinations of virulence genes were reported, and enterotoxin genes were present in few strains (seh, n = 4; sea, n = 2; sea and seh, n = 2; sec and sel, n = 2). The majority of strains were resistant only to penicillin; only one strain was found to be multiresistant and no methicillin-resistant Staph. aureus was demonstrated. Our study reported the isolation of CC97 from bovine milk in Tunisia for the first time and confirmed the relevance of this lineage in intramammary infection in cows. This paper describes the characteristics of Staphylococcus aureus isolated from bulk tank and individual cow milk in Tunisia. All strains were genotyped by spa typing and RS-PCR, a method based on the amplification of the 16S-23S rRNA intergenic spacer region, and multiplex PCRs for 22 virulence genes. A selected subsample of strains was also genotyped by multilocus sequence typing. All strains were tested for antimicrobial resistance. Our study evidences a predominance of strains belonging to Clonal Complex 97. Methicillin-resistant strains were not detected, and overall low level of antimicrobial resistance was reported. © 2016 The Society for Applied Microbiology.

First report of Enterocytozoon bieneusi from giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China.

PubMed

Tian, Ge-Ru; Zhao, Guang-Hui; Du, Shuai-Zhi; Hu, Xiong-Feng; Wang, Hui-Bao; Zhang, Long-Xian; Yu, San-Ke

2015-08-01

Enterocytozoon bieneusi is an emerging and opportunistic enteric pathogen triggering diarrhea and enteric disease in humans and animals. Despite extensive research on this pathogen, the prevalence and genotypes of E. bieneusi infection in precious wild animals of giant and red pandas have not been reported. In the present study, 82 faecal specimens were collected from 46 giant pandas (Ailuropoda melanoleuca) and 36 red pandas (Ailurus fulgens) in the northwest of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an overall infection rate of 10.98% (9/82) was observed in pandas, with 8.70% (4/46) for giant pandas, and 13.89% (5/36) for red pandas. Two ITS genotypes were identified: the novel genotype I-like (n=4) and genotype EbpC (n=5). Multilocus sequence typing (MLST) employing three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) showed that nine, six, six and nine positive products were amplified and sequenced successfully at four respective loci. A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that the genotype EbpC fell into 1d of group 1 of zoonotic potential, and the novel genotype I-like was clustered into group 2. The present study firstly indicated the presence of E. bieneusi in giant and red pandas, and these results suggested that integrated strategies should be implemented to effectively protect pandas and humans from infecting E. bieneusi in China. Copyright © 2015 Elsevier B.V. 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

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

PubMed

Moore, Jason H

2004-11-01

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

[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.

'Food addiction' and its association with a dopaminergic multilocus genetic profile.

PubMed

Davis, Caroline; Loxton, Natalie J; Levitan, Robert D; Kaplan, Allan S; Carter, Jacqueline C; Kennedy, James L

2013-06-13

Our objective was to employ a novel genetic methodology - whereby functional variants of the dopamine pathway were aggregated to reflect a polygenic liability - in the study of food addiction. We anticipated that the composite index of elevated dopamine signaling (a multilocus genetic profile score [MLGP]) would distinguish those with a designation of food addiction (according to the Yale Food Addiction Scale [YFAS] criteria), and age and weight equivalent controls. Our second aim was to assess whether this index was positively associated with eating-related sub-phenotypes of food addiction (e.g. binge eating and food cravings). Adults (n=120) recruited from the community were solicited for an overeating/overweight study. Eating-behavior questionnaires were completed and a blood sample was taken for genotyping. The YFAS identified 21 participants with food addiction. As predicted, the MLGP score was higher in those with YFAS-diagnosed food addiction, and it correlated positively with binge eating, food cravings, and emotional overeating. We then tested a multiple-mediation model proposing that reward-driven overeating facilitates the relationship between the MLGP score and food addiction. The model was statistically significant, supporting the view that the relationship between a composite genetic index of dopamine signaling and food addiction is mediated by certain aspects of reward-responsive overeating. Copyright © 2013 Elsevier Inc. All rights reserved.

Susceptibility and molecular characterization of Candida species from patients with vulvovaginitis.

PubMed

Fornari, Gheniffer; Vicente, Vania Aparecida; Gomes, Renata Rodrigues; Muro, Marisol Dominguez; Pinheiro, Rosangela Lameira; Ferrari, Carolina; Herkert, Patricia Fernanda; Takimura, Marcos; Carvalho, Newton Sérgio de; Queiroz-Telles, Flavio

2016-01-01

Vulvovaginal candidiasis affects women of reproductive age, which represents approximately 15-25% of vaginitis cases. The present study aimed to isolate and characterize yeast from the patients irrespective of the presentation of clinical symptoms. The isolates were subjected to in vitro susceptibility profile and characterization by molecular markers, which intended to assess the distribution of species. A total of 40 isolates were obtained and identified through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr gene. Candida albicans strains were genotyped by the ABC system and the isolates were divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae isolates was confirmed by the multilocus analysis. The strains of Candida, isolated from patients with complications, were found to be resistant to nystatin but sensitive to fluconazole, amphotericin B and ketoconazole, as observed by in vitro sensitivity profile. The isolates from asymptomatic patients, i.e., the colonized group, showed a dose-dependent sensitivity to the anti-fungal agents, fluconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed the same in vitro susceptibility profile. Copyright © 2016. Published by Elsevier Editora Ltda.

Occurrence, Genotyping, and Antibiotic Susceptibility of Cronobacter spp. in Drinking Water and Food Samples from Northeast China.

PubMed

Fei, Peng; Jiang, Yichao; Gong, Shaoying; Li, Ran; Jiang, Yan; Yuan, Xiujuan; Wang, Ziyuan; Kang, Huaibin; Ali, Md Aslam

2018-02-23

Cronobacter species (formerly Enterobacter sakazakii) are emerging opportunistic bacterial pathogens that can infect both infants and adults. This study was conducted to isolate and genotype diverse Cronobacter species from drinking water, chilled fresh pork, powdered infant formula, instant noodles, cookies, fruits, vegetables, and dishes in Northeast China and to evaluate the antibiotic resistance and susceptibility of the isolates. Thirty-four Cronobacter strains were isolated and identified: 21 C. sakazakii isolates (61.8%), 10 C. malonaticus isolates (29.4%), 2 C. dublinensis isolates (5.9%), and 1 C. turicensis isolate (2.9%). These isolates were further divided into 15 sequence types (STs) by multilocus sequence typing. C. sakazakii ST4 (10 isolates, 29.4%), ST1 (3 isolates, 8.8%), and ST8 (3 isolates, 8.8%) and C. malonaticus ST7 (four isolates, 11.8%) were dominant. Antibiotic susceptibility testing indicated that all 34 Cronobacter isolates were susceptible to ampicillin-sulbactam, cefotaxime, ciprofloxacin, gentamicin, meropenem, tetracycline, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole, 88.2% were susceptible to chloramphenicol, and 67.6% were resistant to cephalothin. The results of this study enhance knowledge about genotyping and antibiotic resistance of these Cronobacter species and could be used to prevent potential hazards caused by these strains in drinking water and various food products.

Molecular epidemiology of Bordetella pertussis in Greece, 2010-2015.

PubMed

Petridou, Evangelia; Jensen, Christel Barker; Arvanitidis, Athanasios; Giannaki-Psinaki, Maria; Michos, Athanasios; Krogfelt, Karen Angeliki; Petersen, Randi Føns

2018-03-01

To determine the predominant strains of Bordetella pertussis in Greece during 2010-2015. Infants and children (n=1150) (15 days to 14 years) of Greek, Roma and immigrant origin with different vaccination statuses were hospitalized in Athens, Greece with suspected pertussis infection. IS481/IS1001 real-time PCR confirmed Bordetella spp./B. pertussis infection in 300 samples. A subset of samples (n=153) were analysed by multi-locus variable number tandem repeat analysis (MLVA) and (n=25) by sequence-based typing of the toxin promotor region (ptxP) on DNA extracted from clinical specimens.Results/Key findings. A complete MLVA profile was determined in 66 out of 153 samples; the B. pertussis MLVA type 27 (n=55) was the dominant genotype and all tested samples (n=25) expressed the ptxP3 genotype. The vaccine coverage in the Greek population was 90 %; however, the study population expressed complete coverage in 2 out of 264 infants (0-11 months) and in 20 out of 36 children (1-14 years). Roma and immigrant minorities represent 7 % of the Greek population, but make up 50 % of the study population, indicating a low vaccine coverage among these groups. The B. pertussis MT27 and ptxP3 genotype is dominant in Greek, Roma and immigrant infants and children hospitalized in Greece. Thus, the predominant MLVA genotype in Greece is similar to other countries using acellular vaccines.

Isolation and characterization of viable Toxoplasma gondii isolates revealed possible high frequency of mixed infection in feral cats ( Felis domesticus) from St Kitts, West Indies.

PubMed

Dubey, J P; Moura, L; Majumdar, D; Sundar, N; Velmurugan, G V; Kwok, O C H; Kelly, P; Krecek, R C; Su, C

2009-05-01

Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. Samples of serum, feces, and tissues from feral cats from St Kitts, West Indies were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test, and found in 71 of 96 (73.9%) of cats with titres of 1:10 in six, 1: 20 in six,1:40 in seven,1: 80 in three, 1: 160 in 10, 1:320 in 13, 1:640 in nine, and 1:1,280 or higher in 17. Tissues of 10 cats were bio-assayed in mice. Toxoplasma gondii was isolated from tissues of 7 cats; from hearts of 6, from tongue of 5, and brains of 3 cats. All 7 isolates were avirulent for mice. Toxoplasma gondii oocysts were not found in the feces of 51 cats. Genotyping of these 7 T. gondii isolates by 10 multi-locus PCR-RFLP markers, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico, revealed 4 genotypes, including clonal Type II, Type III and 2 unique genotypes. Five of the 7 cats had infection with 2 genotypes, indicating high frequency of mixed infection in the cat population on the St Kitts island.

Identification and Characterization of Clostridium difficile Sequence Type 37 Genotype by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

PubMed

Li, Ruxin; Xiao, Di; Yang, Jing; Sun, Suju; Kaplan, Samuel; Li, Zhirong; Niu, Yanan; Qiang, Cuixin; Zhai, Yu; Wang, Xiaoming; Zhao, Xingzhen; Zhao, Baoxin; Welker, Martin; Pincus, David H; Jin, Dazhi; Kamboj, Mini; Zheng, Guanghui; Zhang, Guojun; Zhang, Jianzhong; Tang, Yi-Wei; Zhao, Jianhong

2018-05-01

Clostridium difficile multilocus sequence type 37 (ST37), which mainly corresponds to ribotype 017, has been a dominant genotype circulating in China. In this study, we report the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 204 C. difficile clinical isolates, including 49 ST37 and 155 non-ST37 isolates collected in China and other countries. The distributions of two major protein peaks ( m/z 3,242 and 3,286) were significantly different between ST37 and non-ST37 prototype strains and clinical isolates. This difference was reproducible when analysis was performed on different colonies in different runs. This finding was repeated and confirmed by both bioMérieux Vitek MS and Bruker Microflex LT systems on isolates recovered from a variety of geographic regions worldwide. The combination of the two peaks was present in 47 of 49 ST37 isolates, resulting in a sensitivity of 95.9%. In contrast, the peak combination was absent in 153 of 155 non-ST37 isolates, resulting in a specificity of 98.7%. Our results suggest that MALDI-TOF MS is a rapid and reliable tool to identify C. difficile genotype ST37. Work is in progress to characterize the two molecules having peaks at m/z 3,242 and 3,286, which appear to be specific to C. difficile genotype ST37. Copyright © 2018 American Society for Microbiology.

Epidemiological Outbreaks of Pneumocystis jirovecii Pneumonia Are Not Limited to Kidney Transplant Recipients: Genotyping Confirms Common Source of Transmission in a Liver Transplantation Unit.

PubMed

Desoubeaux, Guillaume; Dominique, Manon; Morio, Florent; Thepault, Rose-Anne; Franck-Martel, Claire; Tellier, Anne-Charlotte; Ferrandière, Martine; Hennequin, Christophe; Bernard, Louis; Salamé, Ephrem; Bailly, Éric; Chandenier, Jacques

2016-05-01

Over a 5-month period, four liver transplant patients at a single hospital were diagnosed with Pneumocystis jirovecii pneumonia (PCP). This unusually high incidence was investigated using molecular genotyping. Bronchoalveolar lavage fluids (BALF) obtained from the four liver recipients diagnosed with PCP were processed for multilocus sequence typing (MLST) at three loci (SOD, mt26s, and CYB). Twenty-four other BALF samples, which were positive for P. jirovecii and collected from 24 epidemiologically unrelated patients with clinical signs of PCP, were studied in parallel by use of the same method. Pneumocystis jirovecii isolates from the four liver recipients all had the same genotype, which was different from those of the isolates from all the epidemiologically unrelated individuals studied. These findings supported the hypothesis of a common source of contamination or even cross-transmission of a single P. jirovecii clone between the four liver recipients. Hospitalization mapping showed several possible encounters between these four patients, including outpatient consultations on one particular date when they all possibly met. This study demonstrates the value of molecular genotyping of P. jirovecii isolated from clinical samples for epidemiological investigation of PCP outbreaks. It is also the first description of a common source of exposure to a single P. jirovecii clone between liver transplant recipients and highlights the importance of prophylaxis in such a population. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Molecular epidemiology of Bordetella pertussis in Greece, 2010–2015

PubMed Central

Arvanitidis, Athanasios; Giannaki-Psinaki, Maria; Michos, Athanasios; Krogfelt, Karen Angeliki; Petersen, Randi Føns

2018-01-01

Purpose To determine the predominant strains of Bordetella pertussis in Greece during 2010–2015. Methodology Infants and children (n=1150) (15 days to 14 years) of Greek, Roma and immigrant origin with different vaccination statuses were hospitalized in Athens, Greece with suspected pertussis infection. IS481/IS1001 real-time PCR confirmed Bordetella spp./B. pertussis infection in 300 samples. A subset of samples (n=153) were analysed by multi-locus variable number tandem repeat analysis (MLVA) and (n=25) by sequence-based typing of the toxin promotor region (ptxP) on DNA extracted from clinical specimens. Results/Key findings A complete MLVA profile was determined in 66 out of 153 samples; the B. pertussis MLVA type 27 (n=55) was the dominant genotype and all tested samples (n=25) expressed the ptxP3 genotype. The vaccine coverage in the Greek population was 90 %; however, the study population expressed complete coverage in 2 out of 264 infants (0–11 months) and in 20 out of 36 children (1–14 years). Roma and immigrant minorities represent 7 % of the Greek population, but make up 50 % of the study population, indicating a low vaccine coverage among these groups. Conclusions The B. pertussis MT27 and ptxP3 genotype is dominant in Greek, Roma and immigrant infants and children hospitalized in Greece. Thus, the predominant MLVA genotype in Greece is similar to other countries using acellular vaccines. PMID:29458550

Public health significance of major genotypes of Salmonella enterica serovar Enteritidis present in both human and chicken isolates in Korea.

PubMed

Kang, Min-Su; Oh, Jae-Young; Kwon, Yong-Kuk; Lee, Deog-Yong; Jeong, Ok-Mi; Choi, Byung-Kook; Youn, So-Youn; Jeon, Byung-Woo; Lee, Hye-Jin; Lee, Hee-Soo

2017-06-01

Salmonella enterica serovar Enteritidis is one of the most common serotypes implicated in Salmonella infections in both humans and poultry worldwide. It has been reported that human salmonellosis is mainly associated with the consumption of poultry products contaminated with serovar Enteritidis. The present study was to extensively analyze the public health risk of serovar Enteritidis isolates from chickens in Korea. A total of 127 chicken isolates were collected from clinical cases, on-farm feces, and chicken meat between 1998 and 2012 and 20 human clinical isolates were obtained from patients with diarrhea between 2000 and 2006 in Korea. To characterize the isolates from chickens and humans, we compared the pulsed-field gel electrophoresis (PFGE) patterns and multilocus variable-number tandem-repeat analysis (MLVA) profiles of the isolates. We further characterized representative isolates of different genotypes using a DNA microarray. PFGE revealed 28 patterns and MLVA identified 16 allelic profiles. The DNA microarray showed high genetic variability in plasmid regions and other fimbrial subunits of the isolates although the virulence gene contents of isolates from the same source and/or of the same genotype were unrelated. PFGE and MLVA showed that major genotypes were present in both human and chicken isolates. This result suggests that chickens in Korea pose a significant risk to public health as a source of serovar Enteritidis as has been noted in other countries. Copyright © 2017 Elsevier Ltd. All rights reserved.

A linkage disequilibrium perspective on the genetic mosaic of speciation in two hybridizing Mediterranean white oaks

PubMed Central

Goicoechea, P G; Herrán, A; Durand, J; Bodénès, C; Plomion, C; Kremer, A

2015-01-01

We analyzed the genetic mosaic of speciation in two hybridizing Mediterranean white oaks from the Iberian Peninsula (Quercus faginea Lamb. and Quercus pyrenaica Willd.). The two species show ecological divergence in flowering phenology, leaf morphology and composition, and in their basic or acidic soil preferences. Ninety expressed sequence tag-simple sequence repeats (EST-SSRs) and eight nuclear SSRs were genotyped in 96 trees from each species. Genotyping was designed in two steps. First, we used 69 markers evenly distributed over the 12 linkage groups (LGs) of the oak linkage map to confirm the species genetic identity of the sampled genotypes, and searched for differentiation outliers. Then, we genotyped 29 additional markers from the chromosome bins containing the outliers and repeated the multilocus scans. We found one or two additional outliers within four saturated bins, thus confirming that outliers are organized into clusters. Linkage disequilibrium (LD) was extensive; even for loosely linked and for independent markers. Consequently, score tests for association between two-marker haplotypes and the ‘species trait' showed a broad genomic divergence, although substantial variation across the genome and within LGs was also observed. We discuss the influence of several confounding effects on neutrality tests and review the evolutionary processes leading to extensive LD. Finally, we examine how LD analyses within regions that contain outlier clusters and quantitative trait loci can help to identify regions of divergence and/or genomic hitchhiking in the light of predictions from ecological speciation theory. PMID:25515016

Improvement of Predictive Ability by Uniform Coverage of the Target Genetic Space

PubMed Central

Bustos-Korts, Daniela; Malosetti, Marcos; Chapman, Scott; Biddulph, Ben; van Eeuwijk, Fred

2016-01-01

Genome-enabled prediction provides breeders with the means to increase the number of genotypes that can be evaluated for selection. One of the major challenges in genome-enabled prediction is how to construct a training set of genotypes from a calibration set that represents the target population of genotypes, where the calibration set is composed of a training and validation set. A random sampling protocol of genotypes from the calibration set will lead to low quality coverage of the total genetic space by the training set when the calibration set contains population structure. As a consequence, predictive ability will be affected negatively, because some parts of the genotypic diversity in the target population will be under-represented in the training set, whereas other parts will be over-represented. Therefore, we propose a training set construction method that uniformly samples the genetic space spanned by the target population of genotypes, thereby increasing predictive ability. To evaluate our method, we constructed training sets alongside with the identification of corresponding genomic prediction models for four genotype panels that differed in the amount of population structure they contained (maize Flint, maize Dent, wheat, and rice). Training sets were constructed using uniform sampling, stratified-uniform sampling, stratified sampling and random sampling. We compared these methods with a method that maximizes the generalized coefficient of determination (CD). Several training set sizes were considered. We investigated four genomic prediction models: multi-locus QTL models, GBLUP models, combinations of QTL and GBLUPs, and Reproducing Kernel Hilbert Space (RKHS) models. For the maize and wheat panels, construction of the training set under uniform sampling led to a larger predictive ability than under stratified and random sampling. The results of our methods were similar to those of the CD method. For the rice panel, all training set construction methods led to similar predictive ability, a reflection of the very strong population structure in this panel. PMID:27672112

Molecular Epidemiology of Helicobacter pylori Infection in Nepal: Specific Ancestor Root

PubMed Central

Miftahussurur, Muhammad; Sharma, Rabi Prakash; Shrestha, Pradeep Krishna; Suzuki, Rumiko; Uchida, Tomohisa; Yamaoka, Yoshio

2015-01-01

Prevalence of Helicobacter pylori infection in Nepal, a low-risk country for gastric cancer, is debatable. To our knowledge, no studies have examined H. pylori virulence factors in Nepal. We determined the prevalence of H. pylori infection by using three different tests, and the genotypes of virulence factors were determined by PCR followed by sequencing. Multilocus sequence typing was used to analyze the population structure of the Nepalese strains. The prevalence of H. pylori infection in dyspeptic patients was 38.4% (56/146), and was significantly related with source of drinking water. In total, 51 strains were isolated and all were cagA-positive. Western-type-cagA (94.1%), cagA pre-EPIYA type with no deletion (92.2%), vacA s1a (74.5%), and m1c (54.9%) were the predominant genotypes. Antral mucosal atrophy levels were significantly higher in patients infected with vacA s1 than in those infected with s2 genotypes (P = 0.03). Several Nepalese strains were H. pylori recombinants with genetic features of South Asian and East Asian genotypes. These included all East-Asian-type-cagA strains, with significantly lesser activity and inflammation in the corpus than the strains of the specific South Asian genotype (P = 0.03 and P = 0.005, respectively). Although the population structure confirmed that most Nepalese strains belonged to the hpAsia2 population, some strains shared hpEurope- and Nepalese-specific components. Nepalese patients infected with strains belonging to hpEurope showed higher inflammation in the antrum than strains from the Nepalese specific population (P = 0.05). These results support that ancestor roots of Kathmandu`s people not only connected with India alone. PMID:26226153

Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps.

PubMed

Lucchini, V; Fabbri, E; Marucco, F; Ricci, S; Boitani, L; Randi, E

2002-05-01

We used noninvasive methods to obtain genetic and demographic data on the wolf packs (Canis lupus), which are now recolonizing the Alps, a century after their eradication. DNA samples, extracted from presumed wolf scats collected in the western Italian Alps (Piemonte), were genotyped to determine species and sex by sequencing parts of the mitochondrial DNA (mtDNA) control-region and ZFX/ZFY genes. Individual genotypes were identified by multilocus microsatellite analyses using a multiple tubes polymerase chain reaction (PCR). The performance of the laboratory protocols was affected by the age of samples. The quality of excremental DNA extracts was higher in samples freshly collected on snow in winter than in samples that were older or collected during summer. Preliminary mtDNA screening of all samples allowed species identification and was a good predictor of further PCR performances. Wolf, and not prey, DNA targets were preferentially amplified. Allelic dropout occurred more frequently than false alleles, but the probability of false homozygote determinations was always < 0.001. A panel of six to nine microsatellites would allow identification of individual wolf genotypes, also whether related, with a probability of identity of < 0.015. Genealogical relationships among individuals could be determined reliably if the number of candidate parents was 6-8, and most of them had been sampled and correctly genotyped. Genetic data indicate that colonizing Alpine wolves originate exclusively from the Italian source population and retain a high proportion of its genetic diversity. Spatial and temporal locations of individual genotypes, and kinship analyses, suggest that two distinct packs of closely related wolves, plus some unrelated individuals, ranged in the study areas. This is in agreement with field observations.

The effects of intraspecific competition and stabilizing selection on a polygenic trait.

PubMed Central

Bürger, Reinhard; Gimelfarb, Alexander

2004-01-01

The equilibrium properties of an additive multilocus model of a quantitative trait under frequency- and density-dependent selection are investigated. Two opposing evolutionary forces are assumed to act: (i) stabilizing selection on the trait, which favors genotypes with an intermediate phenotype, and (ii) intraspecific competition mediated by that trait, which favors genotypes whose effect on the trait deviates most from that of the prevailing genotypes. Accordingly, fitnesses of genotypes have a frequency-independent component describing stabilizing selection and a frequency- and density-dependent component modeling competition. We study how the equilibrium structure, in particular, number, degree of polymorphism, and genetic variance of stable equilibria, is affected by the strength of frequency dependence, and what role the number of loci, the amount of recombination, and the demographic parameters play. To this end, we employ a statistical and numerical approach, complemented by analytical results, and explore how the equilibrium properties averaged over a large number of genetic systems with a given number of loci and average amount of recombination depend on the ecological and demographic parameters. We identify two parameter regions with a transitory region in between, in which the equilibrium properties of genetic systems are distinctively different. These regions depend on the strength of frequency dependence relative to pure stabilizing selection and on the demographic parameters, but not on the number of loci or the amount of recombination. We further study the shape of the fitness function observed at equilibrium and the extent to which the dynamics in this model are adaptive, and we present examples of equilibrium distributions of genotypic values under strong frequency dependence. Consequences for the maintenance of genetic variation, the detection of disruptive selection, and models of sympatric speciation are discussed. PMID:15280253

Population genetic variation in the tree fern Alsophila spinulosa (Cyatheaceae): effects of reproductive strategy.

PubMed

Wang, Ting; Su, Yingjuan; Li, Yuan

2012-01-01

Essentially all ferns can perform both sexual and asexual reproduction. Their populations represent suitable study objects to test the population genetic effects of different reproductive systems. Using the diploid homosporous fern Alsophila spinulosa as an example species, the main purpose of this study was to assess the relative impact of sexual and asexual reproduction on the level and structure of population genetic variation. Inter-simple sequence repeats analysis was conducted on 140 individuals collected from seven populations (HSG, LCH, BPC, MPG, GX, LD, and ZHG) in China. Seventy-four polymorphic bands discriminated a total of 127 multilocus genotypes. Character compatibility analysis revealed that 50.0 to 70.0% of the genotypes had to be deleted in order to obtain a tree-like structure in the data set from populations HSG, LCH, MPG, BPC, GX, and LD; and there was a gradual decrease of conflict in the data set when genotypes with the highest incompatibility counts were successively deleted. In contrast, in population ZHG, only 33.3% of genotypes had to be removed to achieve complete compatibility in the data set, which showed a sharp decline in incompatibility upon the deletion of those genotypes. All populations examined possessed similar levels of genetic variation. Population ZHG was not found to be more differentiated than the other populations. Sexual recombination is the predominant source of genetic variation in most of the examined populations of A. spinulosa. However, somatic mutation contributes most to the genetic variation in population ZHG. This change of the primary mode of reproduction does not cause a significant difference in the population genetic composition. Character compatibility analysis represents an effective approach to separate the role of sexual and asexual components in shaping the genetic pattern of fern populations.

Impact of human population history on distributions of individual-level genetic distance

PubMed Central

2005-01-01

Summaries of human genomic variation shed light on human evolution and provide a framework for biomedical research. Variation is often summarised in terms of one or a few statistics (eg FST and gene diversity). Now that multilocus genotypes for hundreds of autosomal loci are available for thousands of individuals, new approaches are applicable. Recently, trees of individuals and other clustering approaches have demonstrated the power of an individual-focused analysis. We propose analysing the distributions of genetic distances between individuals. Each distribution, or common ancestry profile (CAP), is unique to an individual, and does not require a priori assignment of individuals to populations. Here, we consider a range of models of population history and, using coalescent simulation, reveal the potential insights gained from a set of CAPs. Information lies in the shapes of individual profiles -- sometimes captured by variance of individual CAPs -- and the variation across profiles. Analysis of short tandem repeat genotype data for over 1,000 individuals from 52 populations is consistent with dramatic differences in population histories across human groups. PMID:15814064

Scarce events of mitochondrial introgression in Trypanosoma cruzi: new case with a Bolivian strain.

PubMed

Barnabé, Christian; Brenière, Simone Frédérique

2012-12-01

Trypanosoma cruzi, the agent of Chagas disease, presents a predominantly clonal structure that has been shaped by recombination events leading to six genetic groups (DTUs, discrete typing units, TcI-TcVI). Several conventional and unconventional genetic exchange events have been described, including hybridization and mitochondrial introgression, which is explored here among Bolivian and Peruvian strains belonging to TcI because recombination events have been previously suspected by means of the MLMT method (multilocus microsatellite typing). We analyzed the variation of one nuclear (Gpi) and one mitochondrial (Nd1) gene among 60 TcI strains and 15 reference strains belonging to the six DTUs. The results clearly showed that one strain isolated from Triatoma infestans in the Cochabamba department (Bolivia) presented a genotype TcI for Gpi and a mitochondrial Nd1 genotype common to the DTUs TcIII, IV, V, and VI; this can be interpreted as a mitochondrial introgression event between distant DTUs. These kinds of events, although probably scarce, may have played an important role in the adaptive evolution of the species. Copyright © 2012 Elsevier B.V. All rights reserved.

Geographic parthenogenesis in a consumer-resource model for sexual reproduction.

PubMed

Song, Yixian; Scheu, Stefan; Drossel, Barbara

2011-03-21

The phenomenon of geographic parthenogenesis is closely tied to the question of why sexual reproduction is the dominant mode of reproduction in animals and plants. Geographic parthenogenesis describes the fact that many species reproduce asexually at the boundaries of their range. We present a mathematical model that derives the dominance of sexuals at the center and the dominance of asexuals at the boundary of a species' range from exactly the same mechanism. Our model is based on a set of resources that regrow slowly and that can be consumed only by those individuals that have a suitable genotype. Genotype is implemented by a multilocus model with two alleles at each locus, and with free recombination during production of sexual offspring. The model is tailored to seasonal species with intermittent mixis and low survival of offspring, such as Daphnia and aphids. Several patches of resources are arranged in a row, with a gradient of those parameters that typically vary through the range of species. By letting sexually and asexually reproducing populations compete, we obtain the typical patterns of geographic parthenogenesis. Copyright © 2010 Elsevier Ltd. All rights reserved.

Multilocus Variable-Number Tandem Repeat Typing of Mycobacterium ulcerans

PubMed Central

Ablordey, Anthony; Swings, Jean; Hubans, Christine; Chemlal, Karim; Locht, Camille; Portaels, Françoise; Supply, Philip

2005-01-01

The apparent genetic homogeneity of Mycobacterium ulcerans contributes to the poorly understood epidemiology of M. ulcerans infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related M. marinum genome sequence were screened in a collection of 23 M. ulcerans isolates, one Mycobacterium species referred to here as an intermediate species, and five M. marinum strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight M. ulcerans and five M. marinum genotypes. The results from the VNTR analysis corroborated the genetic relationships of M. ulcerans isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within M. ulcerans and M. marinum. PMID:15814964

Molecular characterization of environmental Cryptococcus neoformans VNII isolates in Jos, Plateau State, Nigeria.

PubMed

Nnadi, N E; Enweani, I B; Cogliati, M; Ayanbimpe, G M; Okolo, M O; Kim, E; Sabitu, M Z; Criseo, G; Romeo, O; Scordino, F

2016-12-01

Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeasts able to cause fatal neurological infections in both human and other mammals. Cryptococcosis is the most common fungal infection of the central nervous system and has a huge burden in sub-Saharan Africa and South East Asia. Bird excreta are considered an environmental reservoir for C. neoformans in urban areas, therefore a study aimed at isolating and characterizing this yeast is important in disease management. In this study, one hundred samples of pigeon droppings were collected in Jos, Plateau State, Nigeria. C. neoformans was isolated from three samples and initially identified using standard phenotypic and biochemical tests. Molecular analysis revealed that all three isolates belonged to C. neoformans genotype VNII, mating type α and were assigned to the sequence type ST43 by multilocus sequence typing analysis. This study reports, for the first time, the molecular characterization of C. neoformans in Nigeria, where little is still known about the environmental distribution of the genotypes, serotypes and mating types of this important human pathogen. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A tale of swinger insects: Signatures of past sexuality between divergent lineages of a parthenogenetic weevil revealed by ribosomal intraindividual variation.

PubMed

Rodriguero, Marcela S; Wirth, Sonia A; Alberghina, Josefina S; Lanteri, Analía A; Confalonieri, Viviana A

2018-01-01

Naupactus cervinus (Boheman) (Curculionidae, Naupactini) is a parthenogenetic weevil native to the Paranaense Forest which displays high levels of genetic variation. Two divergent clades were identified, one ranging in forest areas (Forest clade), and the other in open vegetation areas (Grassland clade). Both of them have individuals with high levels of heterozygosity in ribosomal sequences. Investigation of intraindividual variation in ITS1 sequences through cloning and posterior sequencing suggested that mating between both groups most likely occurred in the Paranaense Forest after a secondary contact, which led to fixed heterozygotes as a consequence of parthenogenesis. Otherwise, sexual segregation would have disrupted multilocus genotypes. Only a small number of heterozygous genotypes of all the possible combinations are found in nature. We propose the occurrence of a hybrid zone in the Paranaense Forest. The fact that it is one of the most important biodiversity hotspots of the world, together with its key role for investigating evolutionary processes, makes it worthy of conservation. This is the first genetic evidence of bisexuality in N. cervinus.

Characterization of Toxoplasma gondii from raccoons (Procyon lotor), coyotes (Canis latrans), and striped skunks (Mephitis mephitis) in Wisconsin identified several atypical genotypes

USGS Publications Warehouse

Dubey, J.P.; Sundar, N.; Nolden, C.A.; Samuel, M.D.; Velmurugan, G.V.; Bandini, L.A.; Kwok, O.C.H.; Bodenstein, B.; Su, C.

2007-01-01

During 2005-2006, sera and tissues from raccoons (Procyon lotor), coyotes (Canis latrans), and skunks (Mephitis mephitis) from the state of Wisconsin were tested for Toxoplasma gondii infection. Antibodies to T. gondii were found in 32 of 54 (59.2%) raccoons, 18 of 35 (51.4%) coyotes, and 5 of 7 (71.4%) skunks using the modified agglutination test and a cut-off titer of 1:20. Pooled tissues (brains, hearts, and tongues) from 30 raccoons, 15 coyotes, and 1 skunk were bioassayed for T. gondii infection in mice or cats. Viable T. gondii was isolated from 5 of 30 (16.7%) raccoons, 6 of 15 (40.0%) coyotes, and the skunk. Genetic characterization of the 12 parasite isolates by multilocus PCR-RFLP markers revealed 6 different genotypes including 5 atypical and 1 archetypal II lineages. The results indicate the prevalence of T. gondii in wildlife mammals is high and that these animals may serve as an important reservoir for transmission of T. gondii. ?? American Society of Parasitologists 2007.

[Genetic diversity and mating system Pinus brutia var. Stankewiczii sukacz. in small localities of Sudak (Crimea)].

PubMed

Korshikov, I I; Kalafat, L A; Milchevskaya, Ya G

2015-01-01

A comparative analysis of genetic variation at 12 polymorphic isozyme loci, and the mating system has been carried out in mature trees and their seed progeny in three small localities of Pinus brutia var. stankewiczii Sukacz. near the town of Sudak--settlement of Novyi Svet in the Crimea. We found that embryos maintain the same allelic diversity as mother plants but their observed heterozygosity is lower on the average by 37.4%. The significant deviation of genotype distribution from the theoretically expected ratios caused by the deficiency of heterozygotes was observed at 8 out of 12 loci. Multilocus estimate of outcrossing rate (t(m)) in populations varied from 68.9 to 94.9% making on the average 80.7%.

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.

Burkholderia pseudomallei Genotype Distribution in the Northern Territory, Australia

PubMed Central

Chapple, Stephanie N. J.; Price, Erin P.; Sarovich, Derek S.; McRobb, Evan; Mayo, Mark; Kaestli, Mirjam; Spratt, Brian G.; Currie, Bart J.

2016-01-01

Melioidosis is a tropical disease of high mortality caused by the environmental bacterium, Burkholderia pseudomallei. We have collected clinical isolates from the highly endemic Northern Territory of Australia routinely since 1989, and animal and environmental B. pseudomallei isolates since 1991. Here we provide a complete record of all B. pseudomallei multilocus sequence types (STs) found in the Northern Territory to date, and distribution maps of the eight most common environmental STs. We observed surprisingly restricted geographic distributions of STs, which is contrary to previous reports suggesting widespread environmental dissemination of this bacterium. Our data suggest that B. pseudomallei from soil and water does not frequently disperse long distances following severe weather events or by migration of infected animals. PMID:26526925

Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

PubMed Central

Amro, Ahmad; Mentis, Andreas; Pratlong, Francine; Dedet, Jean-Pierre; Votypka, Jan; Volf, Petr; Ozensoy Toz, Seray; Kuhls, Katrin; Schönian, Gabriele; Soteriadou, Ketty

2012-01-01

Background New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. Methodology/Principal Findings The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. Conclusion The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region. PMID:22348162

Detectability of bovine TB using the tuberculin skin test does not vary significantly according to pathogen genotype within Northern Ireland.

PubMed

Wright, David M; Allen, Adrian R; Mallon, Thomas R; McDowell, Stanley W J; Bishop, Stephen C; Glass, Elizabeth J; Bermingham, Mairead L; Woolliams, John A; Skuce, Robin A

2013-10-01

Strains of many infectious diseases differ in parameters that influence epidemic spread, for example virulence, transmissibility, detectability and host specificity. Knowledge of inter-strain variation can be exploited to improve management and decrease disease incidence. Bovine tuberculosis (bTB) is increasingly prevalent among farmed cattle in the UK, exerting a heavy economic burden on the farming industry and government. We aimed to determine whether strains of Mycobacterium bovis (the causative agent of bTB) identified and classified using genetic markers (spoligotyping and multi-locus VNTR analysis) varied in response to the tuberculin skin test; this being the primary method of bTB detection used in the UK. Inter-strain variation in detectability of M. bovis could have important implications for disease control. The skin test is based on a differential delayed type hypersensitivity (DTH) response to intradermal injections of purified protein derivative (PPD) from M. bovis (PPD-B) and Mycobacterium avium (PPD-A). We searched for an association between skin test response (PPD-B skin rise minus PPD-A skin rise) and M. bovis genotype at the disclosing test in culture-confirmed cases using a field dataset consisting of 21,000 isolates belonging to 63 genotypes of M. bovis from cattle in Northern Ireland. We found no substantial variation among genotypes (estimated responses clustered tightly around the mean) controlling for animal sex, breed and test effects. We also estimated the ratio of skin test detected to undetected cases (i.e. cases only detected at abattoir). The skin test detection ratio varied among abattoirs with some detecting a greater proportion of cases than others but this variation was unrelated to the community composition of genotypes within each abattoir catchment. These two lines of evidence indicate that M. bovis genotypes in Northern Ireland have similar detectability using the skin test. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Genotype patterns at PICALM, CR1, BIN1, CLU, and APOE genes are associated with episodic memory

PubMed Central

Barral, S.; Bird, T.; Goate, A.; Farlow, M.R.; Diaz-Arrastia, R.; Bennett, D.A.; Graff-Radford, N.; Boeve, B.F.; Sweet, R.A.; Stern, Y.; Wilson, R.S.; Foroud, T.; Ott, J.; Green, Robert; Kowall, Neil; Farrer, Lindsay; Williamson, Jennifer; Santana, Vincent; Schmechel, Donald; Gaskel, Peter; Ghetti, Bernardino; Farlow, Martin R.; Faber, Kelley; Prentice, Heather; Horner, Kelly; Growdon, John H.; Blacker, Deborah; Tanzi, Rudolph E.; Hyman, Bradley T.; Boeve, Bradley; Kuntz, Karen; Norgaard, Lindsay; Larson, Nathan; Kistler, Dana; Parfitt, Francine; Haddow, Jenny; Silverman, Jeremy; Beeri, Michal Schnaider; Sano, Mary; Wang, Joy; Lally, Rachel; Johnson, Nancy; Mesulam, Marcel; Weintraub, Sandra; Bigio, Eileen; Kaye, Jeffery; Kramer, Patricia; Payne-Murphy, Jessica; Bennett, David; Jacobs, Holli; Chang, Jeen-Soo; Arends, Danielle; Harrell, Lindy; Bartzokis, George; Cummings, Jeffery; Lu, Po H.; Toland, Usha; Smith, Charles; Brickhouse, Alise; Trojanowski, John; Van Deerlin, Vivianna; McCarty Wood, Elisabeth; DeKosky, Steven; Sweet, Robert; Weamer, Elise; Chui, Helena; Varpetian, Arousiak; Diaz-Arrastia, Ramon; Rosenberg, Roger; Davis, Barbara; Bird, Thomas; Schellenberg, Gerard D.; Raskind, Murray; Rumbaugh, Malia; Nickel, Kate; Goate, Alison; Morris, John; Norton, Joanne; Levitch, Denise; Grant, Betsy; Coats, Mary; Levey, Allen; Rosen, Ami; Anosike, Ezinna

2012-01-01

Objective: Several genome-wide association studies (GWAS) have associated variants in late-onset Alzheimer disease (LOAD) susceptibility genes; however, these single nucleotide polymorphisms (SNPs) have very modest effects, suggesting that single SNP approaches may be inadequate to identify genetic risks. An alternative approach is the use of multilocus genotype patterns (MLGPs) that combine SNPs at different susceptibility genes. Methods: Using data from 1,365 subjects in the National Institute on Aging Late-Onset Alzheimer's Disease Family Study, we conducted a family-based association study in which we tabulated MLGPs for SNPs at CR1, BIN1, CLU, PICALM, and APOE. We used generalized estimating equations to model episodic memory as the dependent endophenotype of LOAD and the MLGPs as predictors while adjusting for sex, age, and education. Results: Several genotype patterns influenced episodic memory performance. A pattern that included PICALM and CLU was the strongest genotypic profile for lower memory performance (β = −0.32, SE = 0.19, p = 0.021). The effect was stronger after addition of APOE (p = 0.016). Two additional patterns involving PICALM, CR1, and APOE and another pattern involving PICALM, BIN1, and APOE were also associated with significantly poorer memory performance (β = −0.44, SE = 0.09, p = 0.009 and β = −0.29, SE = 0.07, p = 0.012) even after exclusion of patients with LOAD. We also identified genotype pattern involving variants in PICALM, CLU, and APOE as a predictor of better memory performance (β = 0.26, SE = 0.10, p = 0.010). Conclusions: MLGPs provide an alternative analytical approach to predict an individual's genetic risk for episodic memory performance, a surrogate indicator of LOAD. Identifying genotypic patterns contributing to the decline of an individual's cognitive performance may be a critical step along the road to preclinical detection of Alzheimer disease. PMID:22539578

Prevalence, risk factors and genetic characterization of Toxoplasma gondii in sick pigs and stray cats in Jiangsu Province, eastern China.

PubMed

Hou, Zhao-Feng; Su, Shi-Jie; Liu, Dan-Dan; Wang, Le-le; Jia, Chuan-Li; Zhao, Zhen-Xing; Ma, Yi-Fei; Li, Qiao-Qiao; Xu, Jin-Jun; Tao, Jian-Ping

2018-06-01

Toxoplasma gondii is an obligate intracellular parasitic protozoan with a worldwide distribution. The parasites in edible tissues of pigs and oocysts from cats are the major sources of T. gondii infection in humans. However, there are no data from sick pigs in veterinary clinics or from stray cats in Jiangsu Province, eastern China. In total, biological samples from 141 sick pigs and 64 stray cats were collected from this region. The rate of T. gondii infection in sick pigs was 46.81% using a polymerase chain reaction (PCR), and the overall prevalence of toxoplasmosis in stray cats was 34.38% by PCR and an enzyme-linked immunosorbent assay (ELISA). T. gondii was significantly more prevalent in lungs and heart than in liver and spleen (P < 0.05). Age and geographic region were considered to be the main risk factors associated with T. gondii infection in these pigs. The DNA samples from 17 sick pigs and seven stray cats, were successfully genotyped by multilocus PCR-restriction fragment length polymorphism (PCR-RFLP) with 10 genetic markers [SAG1, SAG2 (5'-3'SAG2, alt. SAG2), SAG3, GRA6, PK1, c22-8, c29-2, BTUB, L358 and Apico]. Six distinct genotypes were found, which were designated ToxoDB PCR-RFLP genotypes #9 (Chinese I), #10 (Type I), #213, and #89, and New 1 and New 2. Chinese I is the most prevalent T. gondii genotype in this region. The two new genotypes (designated New 1 and New 2) are reported and the ToxoDB PCR-RFLP genotype #89 is found for the first time in China. Such information will be useful for the prevention, diagnosis and treatment of porcine toxoplasmosis in Jiangsu Province, eastern China. Copyright © 2018 Elsevier B.V. All rights reserved.

Multilocus PCR-RFLP profiling in Trypanosoma cruzi I highlights an intraspecific genetic variation pattern.

PubMed

Ramírez, Juan David; Duque, María Clara; Montilla, Marleny; Cucunubá, Zulma M; Guhl, Felipe

2012-12-01

Chagas disease represents a serious problem in public health. This zoonotic pathology is caused by the kinetoplastid Trypanosoma cruzi which displays a high genetic diversity falling into six Discrete Typing Units (TcI-TcVI). In Colombia, the prevalent DTU is TcI with findings of TcII, TcIII and TcIV in low proportions. The aim of this work was to observe the genetic variability within TcI using a multilocus PCR-RFLP strategy. We analyzed 70 single-celled clones from triatomines, reservoirs and humans that were amplified and restricted via ten PCR-RFLPs targets across TcI genome, the restriction fragments were used to construct phylograms according to calculated genetic distances. We obtained five polymorphic targets (1f8, HSP60, HSP70, SAPA and H1) and the consensus tree constructed according to these regions allowed us to observe two well-defined groups with close association to the transmission cycles (domestic/peridomestic and sylvatic) of Chagas disease in Colombia. Our findings allowed us to corroborate the previous reported genotypes based on the intergenic region of mini-exon gene. More studies examining the genetic diversity among T. cruzi I populations must be conducted in order to obtain a better understanding in regions where this DTU is endemic. Copyright © 2012 Elsevier B.V. All rights reserved.

Analyses of the population structure in a global collection of Phytophthora nicotianae isolates inferred from mitochondrial and nuclear DNA sequences.

PubMed

Mammella, Marco A; Martin, Frank N; Cacciola, Santa O; Coffey, Michael D; Faedda, Roberto; Schena, Leonardo

2013-06-01

Genetic variation within the heterothallic cosmopolitan plant pathogen Phytophthora nicotianae was determined in 96 isolates from a wide range of hosts and geographic locations by characterizing four mitochondrial (10% of the genome) and three nuclear loci. In all, 52 single-nucleotide polymorphisms (SNPs) (an average of 1 every 58 bp) and 313 sites with gaps representing 5,450 bases enabled the identification of 50 different multilocus mitochondrial haplotypes. Similarly, 24 SNPs (an average of 1 every 69 bp), with heterozygosity observed at each locus, were observed in three nuclear regions (hyp, scp, and β-tub) differentiating 40 multilocus nuclear genotypes. Both mitochondrial and nuclear markers revealed a high level of dispersal of isolates and an inconsistent geographic structuring of populations. However, a specific association was observed for host of origin and genetic grouping with both nuclear and mitochondrial sequences. In particular, the majority of citrus isolates from Italy, California, Florida, Syria, Albania, and the Philippines clustered in the same mitochondrial group and shared at least one nuclear allele. A similar association was also observed for isolates recovered from Nicotiana and Solanum spp. The present study suggests an important role of nursery populations in increasing genetic recombination within the species and the existence of extensive phenomena of migration of isolates that have been likely spread worldwide with infected plant material.

Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation.

PubMed

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

2017-02-01

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

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

PubMed

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

2017-04-01

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

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.

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.

Multilocus enzyme electrophoresis on agarose gel as an aid to the identification of entomopathogenic Bacillus sphaericus strains.

PubMed

Zahner, V; Rabinovitch, L; Cavados, C F; Momen, H

1994-04-01

Sixty strains of Bacillus sphaericus, including 31 insect pathogens were studied by multilocus enzyme electrophoresis and were classified into 44 zymovars (electrophoretic types). Among the entomopathogenic strains, 11 belong to the same zymovar (Z59) indicating a widespread frequent genotype. Bands of enzyme activity were not detected among the strains for the loci GPI (E.C.5.3.1.9), G6P (E.C.1.1.1.49), 6PG (E.C.1.1.1.44) and ME (E.C.1.1.1.40). The enzymatic loci NP (E.C.2.4.2.1) and ACON (E.C.4.2.1.3) were monomorphic while the other enzymes, MDH (E.C.1.1.1.37), LeDH (E.C.1.4.1.9), ADH (E.C.1.4.1.1), EST (E.C.3.1.1.1), PEP-2 (E.C.3.4.11.1), PEP-3 (E.C.3.4.11) and PEP-D (E.C. 3.4.13.9) were polymorphic. The genetic variation in the non-insect pathogenic group seemed to be greater than in the entomopathogenic group. This latter group appears to be distinct from other strains of these species. All insect pathogens were recovered in the same phenetic cluster and a diagnostic allele is reported for the identification of entomopathogenic strains.

Shift Work in Nurses: Contribution of Phenotypes and Genotypes to Adaptation

PubMed Central

Gamble, Karen L.; Motsinger-Reif, Alison A.; Hida, Akiko; Borsetti, Hugo M.; Servick, Stein V.; Ciarleglio, Christopher M.; Robbins, Sam; Hicks, Jennifer; Carver, Krista; Hamilton, Nalo; Wells, Nancy; Summar, Marshall L.; McMahon, Douglas G.; Johnson, Carl Hirschie

2011-01-01

Background Daily cycles of sleep/wake, hormones, and physiological processes are often misaligned with behavioral patterns during shift work, leading to an increased risk of developing cardiovascular/metabolic/gastrointestinal disorders, some types of cancer, and mental disorders including depression and anxiety. It is unclear how sleep timing, chronotype, and circadian clock gene variation contribute to adaptation to shift work. Methods Newly defined sleep strategies, chronotype, and genotype for polymorphisms in circadian clock genes were assessed in 388 hospital day- and night-shift nurses. Results Night-shift nurses who used sleep deprivation as a means to switch to and from diurnal sleep on work days (∼25%) were the most poorly adapted to their work schedule. Chronotype also influenced efficacy of adaptation. In addition, polymorphisms in CLOCK, NPAS2, PER2, and PER3 were significantly associated with outcomes such as alcohol/caffeine consumption and sleepiness, as well as sleep phase, inertia and duration in both single- and multi-locus models. Many of these results were specific to shift type suggesting an interaction between genotype and environment (in this case, shift work). Conclusions Sleep strategy, chronotype, and genotype contribute to the adaptation of the circadian system to an environment that switches frequently and/or irregularly between different schedules of the light-dark cycle and social/workplace time. This study of shift work nurses illustrates how an environmental “stress” to the temporal organization of physiology and metabolism can have behavioral and health-related consequences. Because nurses are a key component of health care, these findings could have important implications for health-care policy. PMID:21533241

Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections in stray and pet cats (Felis catus) in northwest China: co-infections and risk factors.

PubMed

Cong, Wei; Meng, Qing-Feng; Blaga, Radu; Villena, Isabelle; Zhu, Xing-Quan; Qian, Ai-Dong

2016-01-01

This study was conducted to estimate the prevalence of Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections among stray and pet cats in Lanzhou, northwest China, and to identify the influence of age, gender, and regions on seropositivity. T. gondii antibodies were examined in cat sera by the modified agglutination test (MAT). The circulating antigens of D. immitis and FeLV and specific antibodies to FIV were examined using kits commercially available. The overall prevalence of T. gondii, FIV, FeLV, and D. immitis was 19.34, 9.12, 11.33, and 3.04 %, respectively. For the genetic characterization of T. gondii genotypes in cats, genomic DNA was extracted from the seropositive cats and the T. gondii B1 gene was amplified using a semi-nested PCR. DNA samples giving positive B1 amplification were then genotyped using multilocus PCR-RFLP. Two T. gondii genotypes (ToxoDB#9 and ToxoDB#1) were identified. Results of the multivariate logistic regression analysis showed that older cats are more likely to be seropositive than juveniles for T. gondii, FIV, FeLV, and D. immitis. This is the first report of T. gondii genotypes in cats in northwest China. Moreover, the present study is the first study of retrovirus and D. immitis seroprevalence in cats in China. The results revealed that T. gondii, FIV, and FeLV infections are common in stray and pet cats in northwest China.

Outbreak of Pneumocystis Pneumonia in Renal and Liver Transplant Patients Caused by Genotypically Distinct Strains of Pneumocystis jirovecii1

PubMed Central

Rostved, Andreas A.; Sassi, Monica; Kurtzhals, Jørgen A. L.; Sørensen, Søren Schwartz; Rasmussen, Allan; Ross, Christian; Gogineni, Emile; Huber, Charles; Kutty, Geetha; Kovacs, Joseph A.; Helweg-Larsen, Jannik

2013-01-01

Background An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. Methods P. jirovecii isolates from 22 transplant-cases, 2 colonized RTRs and 19 Pneumocystis-control samples were genotyped by restriction fragment length polymorphism and multi-locus sequence typing analysis. Contact tracing were used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. Results Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs and a colonized RTR in 3 distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis-control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only CMV viremia was consistently associated with PCP (P = 0.03; P = 0.009). Mycophenolate mofetile was associated with PCP risk only in the RTR population (P = 0.04). Conclusion We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by inter-human transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months post-transplantation. Since patients at risk cannot be identified clinically and outbreaks cannot be predicted, six months of PCP chemoprophylaxis should be considered for all renal and liver transplant recipients. PMID:23903011

Outbreak of pneumocystis pneumonia in renal and liver transplant patients caused by genotypically distinct strains of Pneumocystis jirovecii.

PubMed

Rostved, Andreas A; Sassi, Monica; Kurtzhals, Jørgen A L; Sørensen, Søren Schwartz; Rasmussen, Allan; Ross, Christian; Gogineni, Emile; Huber, Charles; Kutty, Geetha; Kovacs, Joseph A; Helweg-Larsen, Jannik

2013-11-15

An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. P. jirovecii isolates from 22 transplant cases, 2 colonized RTRs, and 19 Pneumocystis control samples were genotyped by restriction fragment length polymorphism and multilocus sequence typing analysis. Contact tracing was used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs, and a colonized RTR in three distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only cytomegalovirus viremia was consistently associated with PCP (P=0.03; P=0.009). Mycophenolate mofetil was associated with PCP risk only in the RTR population (P=0.04). We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by interhuman transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months after transplantation. Because patients at risk cannot be identified clinically and outbreaks cannot be predicted, 6 months of PCP chemoprophylaxis should be considered for all RTRs and LTRs.

CoCoa: a software tool for estimating the coefficient of coancestry from multilocus genotype data.

PubMed

Maenhout, Steven; De Baets, Bernard; Haesaert, Geert

2009-10-15

Phenotypic data collected in breeding programs and marker-trait association studies are often analyzed by means of linear mixed models. In these models, the covariance between the genetic background effects of all genotypes under study is modeled by means of pairwise coefficients of coancestry. Several marker-based coancestry estimation procedures allow to estimate this covariance matrix, but generally introduce a certain amount of bias when the examined genotypes are part of a breeding program. CoCoa implements the most commonly used marker-based coancestry estimation procedures and as such, allows to select the best fitting covariance structure for the phenotypic data at hand. This better model fit translates into an increased power and improved type I error control in association studies and an improved accuracy in phenotypic prediction studies. The presented software package also provides an implementation of the new Weighted Alikeness in State (WAIS) estimator for use in hybrid breeding programs. Besides several matrix manipulation tools, CoCoa implements two different bending heuristics, in case the inverse of an ill-conditioned coancestry matrix estimate is needed. The software package CoCoa is freely available at http://webs.hogent.be/cocoa. Source code, manual, binaries for 32 and 64-bit Linux systems and an installer for Microsoft Windows are provided. The core components of CoCoa are written in C++, while the graphical user interface is written in Java.

Molecular detection and genetic characterization of Toxoplasma gondii infection in sika deer (Cervus nippon) in China.

PubMed

Cong, Wei; Qin, Si-Yuan; Meng, Qing-Feng; Zou, Feng-Cai; Qian, Ai-Dong; Zhu, Xing-Quan

2016-04-01

The objective of the present study was to investigate the prevalence and genetic characterization of Toxoplasma gondii infection in sika deer in China. During August 2014 to November 2014, a total of 450 tissue samples coming from 150 sika deer were collected to detect the T. gondii B1 gene using a nested PCR, and the positive samples were genotyped at 11 genetic markers (SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and Apico) using multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. Seventeen of 150 sika deer (11.33%) were tested positive by nested PCR. Six DNA samples from the 17 positive samples were completely typed, in which 4 samples from lung tissues, and 2 from muscular tissues, were identified as ToxoDB Genotype #9 (http://toxodb.org/toxo/). The results of the present study revealed the existence of T. gondii infection in sika deer in China, which provided the information of T. gondii genetic diversity in this host species. This study also indicated that ToxoDB Genotype #9 has a wide distribution in sika deer that could be potential reservoirs for T. gondii transmission, which may pose a threat to human health. Copyright © 2016 Elsevier B.V. All rights reserved.

Segregation distortion causes large-scale differences between male and female genomes in hybrid ants.

PubMed

Kulmuni, Jonna; Seifert, Bernhard; Pamilo, Pekka

2010-04-20

Hybridization in isolated populations can lead either to hybrid breakdown and extinction or in some cases to speciation. The basis of hybrid breakdown lies in genetic incompatibilities between diverged genomes. In social Hymenoptera, the consequences of hybridization can differ from those in other animals because of haplodiploidy and sociality. Selection pressures differ between sexes because males are haploid and females are diploid. Furthermore, sociality and group living may allow survival of hybrid genotypes. We show that hybridization in Formica ants has resulted in a stable situation in which the males form two highly divergent gene pools whereas all the females are hybrids. This causes an exceptional situation with large-scale differences between male and female genomes. The genotype differences indicate strong transmission ratio distortion depending on offspring sex, whereby the mother transmits some alleles exclusively to her daughters and other alleles exclusively to her sons. The genetic differences between the sexes and the apparent lack of multilocus hybrid genotypes in males can be explained by recessive incompatibilities which cause the elimination of hybrid males because of their haploid genome. Alternatively, differentiation between sexes could be created by prezygotic segregation into male-forming and female-forming gametes in diploid females. Differentiation between sexes is stable and maintained throughout generations. The present study shows a unique outcome of hybridization and demonstrates that hybridization has the potential of generating evolutionary novelties in animals.

Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease

PubMed Central

Eshoo, Mark W.; Crowder, Christopher C.; Rebman, Alison W.; Rounds, Megan A.; Matthews, Heather E.; Picuri, John M.; Soloski, Mark J.; Ecker, David J.; Schutzer, Steven E.; Aucott, John N.

2012-01-01

Direct molecular tests in blood for early Lyme disease can be insensitive due to low amount of circulating Borrelia burgdorferi DNA. To address this challenge, we have developed a sensitive strategy to both detect and genotype B. burgdorferi directly from whole blood collected during the initial patient visit. This strategy improved sensitivity by employing 1.25 mL of whole blood, a novel pre-enrichment of the entire specimen extract for Borrelia DNA prior to a multi-locus PCR and electrospray ionization mass spectrometry detection assay. We evaluated the assay on blood collected at the initial presentation from 21 endemic area patients who had both physician-diagnosed erythema migrans (EM) and positive two-tiered serology either at the initial visit or at a follow-up visit after three weeks of antibiotic therapy. Results of this DNA analysis showed detection of B. burgdorferi in 13 of 21 patients (62%). In most cases the new assay also provided the B. burgdorferi genotype. The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion. PMID:22590620

Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

PubMed Central

Yang, Yongchun; Liu, Yinglong; Ding, Yunlei; Yi, Li; Ma, Zhe; Fan, Hongjie; Lu, Chengping

2013-01-01

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae. PMID:23874442

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.

A Longitudinal 6-Year Study of the Molecular Epidemiology of Clinical Campylobacter Isolates in Oxfordshire, United Kingdom

PubMed Central

McCarthy, Noel M.; Wimalarathna, Helen L.; Colles, Frances M.; Clark, Lorraine; Bowler, Ian C. J. W.; Maiden, Martin C. J.; Dingle, Kate E.

2012-01-01

Temporal and seasonal trends in Campylobacter genotypes causing human gastroenteritis were investigated in a 6-year study of 3,300 recent isolates from Oxfordshire, United Kingdom. Genotypes (sequence types [ST]) were defined using multilocus sequence typing and assigned to a clonal complex (a cluster of related strains that share four or more identical alleles with a previously defined central genotype). A previously undescribed clonal complex (ST-464) was identified which, together with ST-42, ST-45, and ST-52 complexes, showed increasing incidence. Concurrently, the incidence of ST-574, ST-607, and ST-658 complexes declined. The relative frequencies of three clonal complexes (ST-45, ST-283, and ST-42) peaked during summer and those of two (ST-353 and ST-403) peaked during winter. Nine clonal complexes (ST-22, ST-45, ST-48, ST-61, ST-257, ST-283, ST-403, ST-658, and ST-677) were significantly associated with ciprofloxacin sensitivity (P < 0.05). Seven clonal complexes (ST-49, ST-206, ST-354, ST-446, ST-460, ST-464, and ST-607) were associated with ciprofloxacin resistance (P < 0.05). Clonal complexes exhibited changing incidence and differences in seasonality and antibiotic resistance phenotype. These data also demonstrated that detailed surveillance at a single site captures information which reflects that observed nationally. PMID:22814466

Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis

PubMed Central

Harrabi, Myriam; Bettaieb, Jihène; Ghawar, Wissem; Toumi, Amine; Zaâtour, Amor; Yazidi, Rihab; Chaâbane, Sana; Chalghaf, Bilel; Hide, Mallorie; Bañuls, Anne-Laure; Ben Salah, Afif

2015-01-01

In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991–1992 and 2008–2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991–1992 and 2008–2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008–2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations. PMID:26302440

Genotypes of Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis in dogs and cats in Shanghai, China.

PubMed

Xu, Hailing; Jin, Yue; Wu, Wenxian; Li, Pei; Wang, Lin; Li, Na; Feng, Yaoyu; Xiao, Lihua

2016-03-01

Controversies exist on the potential role of companion animals in the transmission of enteric pathogens in humans. This study was conducted to examine the genotype distribution of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in companion animals in Shanghai, China, and to assess their zoonotic potential. Fecal specimens from 485 dogs and 160 cats were examined for the occurrence and genotype distribution of the three pathogens by PCR. PCR products were sequenced to determine the species and genotypes. The χ(2) test was used to compare differences in infection rates between living conditions or age groups. Cryptosporidium spp., E. bieneusi and G. duodenalis were found in 39 (8.0 %), 29 (6.0 %) and 127 (26.2 %) of dogs, and 6 (3.8 %), 9 (5.6 %) and 21 (13.1 %) of cats, respectively. Infection rates of the pathogens in dogs from pet shops and a clinic were higher than those in household dogs, and higher in cats from one animal shelter than from pet shops. No significant differences in infection rates were detected among age groups. Cryptosporidium canis and C. felis were the only Cryptosporidium species found in dogs and cats, respectively. Enterocytozoon bieneusi genotype PtEb IX was the dominant genotype in dogs, whereas Type IV and D were the most common ones in cats. Multi-locus sequence typing at the glutamate dehydrogenase, β-giardin, and triosephosphate isomerase loci revealed the presence of G. duodenalis assemblages A (n = 23), B (n = 1), C (n = 26), and D (n = 58) in dogs (only A in household dogs) and assemblages A (n = 2), B (n = 6), C (n = 2), D (n = 1), and F (n = 7) in cats. Co-infection was detected in 24 dogs and 5 cats, especially those living in crowded conditions. Living condition is a major risk factor affecting the occurrence of enteric protists in companion animals in China, and although dogs and cats can be potential sources of human infections, the different distribution of pathogen species and genotypes between dogs and cats suggests that inter-species transmission of these pathogens is probably rare in the study area.

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.

Bifiguratus adelaidae, gen. et sp. nov., a new member of Mucoromycotina in endophytic and soil-dwelling habitats

DOE PAGES

Torres-Cruz, Terry J.; Billingsley Tobias, Terri L.; Almatruk, Maryam; ...

2017-08-08

Illumina amplicon sequencing of soil in a temperate pine forest in the southeastern United States detected an abundant, nitrogen (N)-responsive fungal genotype of unknown phylogenetic affiliation. Two isolates with ribosomal sequences consistent with that genotype were subsequently obtained. Examination of records in GenBank revealed that a genetically similar fungus had been isolated previously as an endophyte of moss in a pine forest in the southwestern United States. The three isolates were characterized using morphological, genomic, and multilocus molecular data (18S, internal transcribed spacer [ITS], and 28S rRNA sequences). Phylogenetic and maximum likelihood phylogenomic reconstructions revealed that the taxon represents amore » novel lineage in Mucoromycotina, only preceded by Calcarisporiella, the earliest diverging lineage in the subphylum. Sequences for the novel taxon are frequently detected in environmental sequencing studies, and it is currently part of UNITE’s dynamic list of most wanted fungi. The fungus is dimorphic, grows best at room temperature, and is associated with a wide variety of bacteria. In this paper, a new monotypic genus, Bifiguratus, is proposed, typified by Bifiguratus adelaidae.« less

Multiple introductions of divergent genetic lineages in an invasive fungal pathogen, Cryphonectria parasitica, in France.

PubMed

Dutech, C; Fabreguettes, O; Capdevielle, X; Robin, C

2010-08-01

The occurrence of multiple introductions may be a crucial factor in the successful establishment of invasive species, but few studies focus on the introduction of fungal pathogens, despite their significant effect on invaded habitats. Although Cryphonectria parasitica, the chestnut blight fungus introduced in North America and Europe from Asia during the 20th century, caused dramatic changes in its new range, the history of its introduction is not well retraced in Europe. Using 10 microsatellite loci, we investigated the genetic diversity of 583 isolates in France, where several introductions have been hypothesized. Our analyses showed that the seven most frequent multilocus genotypes belonged to three genetic lineages, which had a different and geographically limited distribution. These results suggest that different introduction events occurred in France. Genetic recombination was low among these lineages, despite the presence of the two mating types in each chestnut stand analysed. The spatial distribution of lineages suggests that the history of introductions in France associated with the slow expansion of the disease has contributed to the low observed rate of recombination among the divergent lineages. However, we discuss the possibility that environmental conditions or viral interactions could locally reduce recombination among genotypes.

Genetic diversity, kinship analysis, and broodstock management of captive Atlantic sturgeon for population restoration

USGS Publications Warehouse

Henderson, A.P.; Spidle, A.P.; King, T.L.

2005-01-01

Captive Atlantic sturgeon Acipenser oxyrinchus considered for use as broodstock in a restoration program were genotyped using nuclear DNA microsatellites and compared to wild collections from the Hudson River, New York (source of parents of the captive sturgeon) and from Albemarle Sound, North Carolina. Because the potential broodfish were the progeny of a small number of parents, maintaining genetic diversity and minimizing inbreeding is essential to a successful breeding and supplementation program. The microsatellite loci used in this analysis generated unique multilocus genotypes for each of 136 Atlantic sturgeon. Analyses indicated significant genetic separation between the New York and North Carolina collections and correctly identified the potential broodstock as a subset of the Hudson River population. Pairwise genetic distance (-In proportion of shared alleles) between half and full siblings in the potential broodfish was as great as 1.386, a value exceeded by only 36% of the sampled broodfish pairs available for mating. Because the current broodstock population does not seem to have deviated far from their ancestral population in the Hudson River, progeny from that broodstock, or the parents themselves, would seem to be genetically suitable for release back into the Hudson River.

Genetic differentiation of methicillin-resistant Staphylococcus aureus strains from Korea and Japan.

PubMed

Soo Ko, Kwan; Peck, Kyong Ran; Sup Oh, Won; Lee, Nam Yong; Hiramatsu, Keiichi; Song, Jae-Hoon

2005-01-01

In this study, we evaluated genetic differentiation between methicillin-resistant Staphylococcus aureus (MRSA) strains from Korea and Japan. Seventy-five MRSA strains, including 25 h VISA strains, were analyzed by molecular typing methods, including multilocus sequence typing (MLST), SCC mec typing, and spa typing. The most prevalent genotype of MRSA strains, in both Korea and Japan, was ST 5-MRSA-II with the DMGMK spa motif, characteristic of the New York/Japan MRSA clone. In spite of these common features in MRSA strains from Korea and Japan, we also observed some genotypic divergence in MRSA from the two countries. Several spa types might be differentiated from a prevalent prototype (TJMBMDMGMK) that is shared by the two countries, revealing a unique geographic distribution. SCC mec type II lacking pUB110, designated type IIA, was found more frequently in Korea than in Japan. The rate of gentamicin resistance was also dramatically different between the two countries: 87.2% (Korea) vs. 28.6% (Japan). These preliminary findings suggested that MRSA strains from Korea and Japan might have originated from a common ancestor, but then clearly differentiated according to locality. A further comprehensive study should be performed to document the hypotheses from this study.

Bifiguratus adelaidae, gen. et sp. nov., a new member of Mucoromycotina in endophytic and soil-dwelling habitats

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

Torres-Cruz, Terry J.; Billingsley Tobias, Terri L.; Almatruk, Maryam

Illumina amplicon sequencing of soil in a temperate pine forest in the southeastern United States detected an abundant, nitrogen (N)-responsive fungal genotype of unknown phylogenetic affiliation. Two isolates with ribosomal sequences consistent with that genotype were subsequently obtained. Examination of records in GenBank revealed that a genetically similar fungus had been isolated previously as an endophyte of moss in a pine forest in the southwestern United States. The three isolates were characterized using morphological, genomic, and multilocus molecular data (18S, internal transcribed spacer [ITS], and 28S rRNA sequences). Phylogenetic and maximum likelihood phylogenomic reconstructions revealed that the taxon represents amore » novel lineage in Mucoromycotina, only preceded by Calcarisporiella, the earliest diverging lineage in the subphylum. Sequences for the novel taxon are frequently detected in environmental sequencing studies, and it is currently part of UNITE’s dynamic list of most wanted fungi. The fungus is dimorphic, grows best at room temperature, and is associated with a wide variety of bacteria. In this paper, a new monotypic genus, Bifiguratus, is proposed, typified by Bifiguratus adelaidae.« less

Isolation and Genetic Characterization of Toxoplasma gondii from Black Bears (Ursus americanus), Bobcats (Lynx rufus), and Feral Cats (Felis catus) from Pennsylvania.

PubMed

Dubey, Jitender P; Verma, Shiv K; Calero-Bernal, Rafael; Cassinelli, Ana B; Kwok, Oliver C H; Van Why, Kyle; Su, Chunlei; Humphreys, Jan G

2015-01-01

Toxoplasma gondii infects virtually all warm-blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear (Ursus americanus), bobcat (Lynx rufus), and feral cat (Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture-derived tachyzoites of these isolates was characterized using multilocus PCR-RFLP markers. Three genotypes were revealed, including ToxoDB PCR-RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Epidemiology of invasive group A streptococcal infections in Norway 2010-2014: A retrospective cohort study.

PubMed

Naseer, U; Steinbakk, M; Blystad, H; Caugant, D A

2016-10-01

Streptococcus pyogenes or group A streptococcus (GAS) causes mild to severe infections in humans. GAS genotype emm1 is the leading cause of invasive disease worldwide. In the Nordic countries emm28 has been the dominant type since the 1980s. Recently, a resurgence of genotype emm1 was reported from Sweden. Here we present the epidemiology of invasive GAS (iGAS) infections and their association with emm-types in Norway from 2010-2014. We retrospectively collected surveillance data on antimicrobial susceptibility, multilocus sequence type and emm-type, and linked them with demographic and clinical manifestation data to calculate age and sex distributions, major emm- and sequence types and prevalence ratios (PR) on associations between emm-types and clinical manifestations. We analysed 756 iGAS cases and corresponding isolates, with overall incidence of 3.0 per 100000, median age of 59 years (range, 0-102), and male 56 %. Most frequent clinical manifestation was sepsis (49 %) followed by necrotizing fasciitis (9 %). Fifty-two different emm-types and 67 sequence types were identified, distributed into five evolutionary clusters. The most prevalent genotype was emm1 (ST28) in all years (range, 20-33 %) followed by 15 % emm28 in 2014. All isolates were susceptible to penicillin, 15 % resistant to tetracycline and <4 % resistant to erythromycin. A PR of 4.5 (95 % CI, 2.3-8.9) was calculated for emm2 and necrotizing fasciitis. All emm22 isolates were resistant to tetracycline PR 7.5 (95 % CI, 5.8-9.9). This study documented the dominance of emm1, emergence of emm89 and probable import of tetracycline resistant emm112.2 into Norway (2010-2014). Genotype fluctuations between years suggested a mutual exclusive dominance of evolutionary clades.

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

Prevalence of Toxoplasma gondii infection in HIV-infected patients and food animals and direct genotyping of T. gondii isolates, Southern Ghana.

PubMed

Pappoe, Faustina; Cheng, Weisheng; Wang, Lin; Li, Yuanling; Obiri-Yeboah, Dorcas; Nuvor, Samuel Victor; Ambachew, Henock; Hu, Xiaodong; Luo, Qingli; Chu, Deyong; Xu, Yuanhong; Shen, Jilong

2017-06-01

Toxoplasma gondii is of public health and veterinary importance causing severe diseases in immunocompromised individuals including HIV/AIDS patients and in congenital cases and animals. There is limited information on the epidemiology of T. gondii infection in humans, particularly HIV patients and food animals and the parasite genotypes in Ghana. A total of 394 HIV-infected patients from three hospitals were screened for T. gondii anti-IgG and IgM using ELISA. DNAs from blood samples of seropositve participants and 95 brain tissues of food animals were PCR assayed to detect Toxoplasma gra6. DNA positive samples were genotyped using multilocus nested polymerase chain reaction restriction fragment length polymorphism at 10 loci: sag1, alt.sag2, sag3, btub, gra6, l358, c22-8, c29-2, pk1, and apico. The overall seroprevalence was 74.37% (293/394). Toxoplasma DNAs were detected in 3.07% of the seropositive participants and 9.47% of the animals. Six of the human DNA positive samples were partly typed at sag3: 33.33, 50, and 16.67% isolates had type I, II, and III alleles, respectively. All nine isolates from food animals typed at nine loci except apico were atypical: six isolates were identical to ToxoDB #41 and #145, and one was identical to TgCkBrRj2 all identified in Brazil. The genotype of two isolates has not been reported previously and was named as TgCtGh1. T. gondii seroprevalence is high among the HIV-infected individuals with T. gondii circulating in Ghana being genetically diverse.

Phenotypic and molecular characterizations of Yersinia pestis isolates from Kazakhstan and adjacent regions.

PubMed

Lowell, Jennifer L; Zhansarina, Aigul; Yockey, Brook; Meka-Mechenko, Tatyana; Stybayeva, Gulnaz; Atshabar, Bakyt; Nekrassova, Larissa; Tashmetov, Rinat; Kenghebaeva, Kuralai; Chu, May C; Kosoy, Michael; Antolin, Michael F; Gage, Kenneth L

2007-01-01

Recent interest in characterizing infectious agents associated with bioterrorism has resulted in the development of effective pathogen genotyping systems, but this information is rarely combined with phenotypic data. Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales using multi-locus variable number tandem repeat analysis (MLVA), with emphasis on evolutionary patterns using old isolate collections from countries where Y. pestis has existed the longest. Worldwide MLVA studies are largely based on isolates that have been in long-term laboratory culture and storage, or on field material from parts of the world where Y. pestis has potentially circulated in nature for thousands of years. Diversity in these isolates suggests that they may no longer represent the wild-type organism phenotypically, including the possibility of altered pathogenicity. This study focused on the phenotypic and genotypic properties of 48 Y. pestis isolates collected from 10 plague foci in and bordering Kazakhstan. Phenotypic characterization was based on diagnostic tests typically performed in reference laboratories working with Y. pestis. MLVA was used to define the genotypic relationships between the central-Asian isolates and a group of North American isolates, and to examine Kazakh Y. pestis diversity according to predefined plague foci and on an intermediate geographical scale. Phenotypic properties revealed that a large portion of this collection lacks one or more plasmids necessary to complete the blocked flea/mammal transmission cycle, has lost Congo red binding capabilities (Pgm-), or both. MLVA analysis classified isolates into previously identified biovars, and in some cases groups of isolates collected within the same plague focus formed a clade. Overall, MLVA did not distinguish unique phylogeographical groups of Y. pestis isolates as defined by plague foci and indicated higher genetic diversity among older biovars.

Population diversity and multiplicity of infection in Theileria annulata

PubMed Central

Weir, William; Karagenç, Tülin; Gharbi, Mohamed; Simuunza, Martin; Aypak, Suleyman; Aysul, Nuran; Darghouth, Mohamed Aziz; Shiels, Brian; Tait, Andrew

2011-01-01

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n = 305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced. PMID:20833170

Integration of least angle regression with empirical Bayes for multi-locus genome-wide association studies

USDA-ARS?s Scientific Manuscript database

Multi-locus genome-wide association studies has become the state-of-the-art procedure to identify quantitative trait loci (QTL) associated with traits simultaneously. However, implementation of multi-locus model is still difficult. In this study, we integrated least angle regression with empirical B...

Reproduction at the extremes: pseudovivipary, hybridization and genetic mosaicism in Posidonia australis (Posidoniaceae).

PubMed

Sinclair, Elizabeth A; Statton, John; Hovey, Renae; Anthony, Janet M; Dixon, Kingsley W; Kendrick, Gary A

2016-02-01

Organisms occupying the edges of natural geographical ranges usually survive at the extreme limits of their innate physiological tolerances. Extreme and prolonged fluctuations in environmental conditions, often associated with climate change and exacerbated at species' geographical range edges, are known to trigger alternative responses in reproduction. This study reports the first observations of adventitious inflorescence-derived plantlet formation in the marine angiosperm Posidonia australis, growing at the northern range edge (upper thermal and salinity tolerance) in Shark Bay, Western Australia. These novel plantlets are described and a combination of microsatellite DNA markers and flow cytometry is used to determine their origin. Polymorphic microsatellite DNA markers were used to generate multilocus genotypes to determine the origin of the adventitious inflorescence-derived plantlets. Ploidy and genome size were estimated using flow cytometry. All adventitious plantlets were genetically identical to the maternal plant and were therefore the product of a novel pseudoviviparous reproductive event. It was found that 87 % of the multilocus genotypes contained three alleles in at least one locus. Ploidy was identical in all sampled plants. The genome size (2 C value) for samples from Shark Bay and from a separate site much further south was not significantly different, implying they are the same ploidy level and ruling out a complete genome duplication (polyploidy). Survival at range edges often sees the development of novel responses in the struggle for survival and reproduction. This study documents a physiological response at the trailing edge, whereby reproductive strategy can adapt to fluctuating conditions and suggests that the lower-than-usual water temperature triggered unfertilized inflorescences to 'switch' to growing plantlets that were adventitious clones of their maternal parent. This may have important long-term implications as both genetic and ecological constraints may limit the ability to adapt or range-shift; this seagrass meadow in Shark Bay already has low genetic diversity, no sexual reproduction and no seedling recruitment. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Temperature-Dependent Growth Modeling of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes

PubMed Central

Sharaby, Yehonatan; Rodríguez-Martínez, Sarah; Oks, Olga; Pecellin, Marina; Mizrahi, Hila; Peretz, Avi; Brettar, Ingrid; Höfle, Manfred G.

2017-01-01

ABSTRACT Legionella pneumophila causes waterborne infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA). Recently, we found that different MLVA genotypes of L. pneumophila dominated different sites in a small drinking-water network, with a genotype-related temperature and abundance regime. The present study focuses on understanding the temperature-dependent growth kinetics of the genotypes that dominated the water network. Our aim was to model mathematically the influence of temperature on the growth kinetics of different environmental and clinical L. pneumophila genotypes and to compare it with the influence of their ecological niches. Environmental strains showed a distinct temperature preference, with significant differences among the growth kinetics of the three studied genotypes (Gt4, Gt6, and Gt15). Gt4 strains exhibited superior growth at lower temperatures (25 and 30°C), while Gt15 strains appeared to be best adapted to relatively higher temperatures (42 and 45°C). The temperature-dependent growth traits of the environmental genotypes were consistent with their distribution and temperature preferences in the water network. Clinical isolates exhibited significantly higher growth rates and reached higher maximal cell densities at 37°C and 42°C than the environmental strains. Further research on the growth preferences of L. pneumophila clinical and environmental genotypes will result in a better understanding of their ecological niches in drinking-water systems as well as in the human body. IMPORTANCE Legionella pneumophila is a waterborne pathogen that threatens humans in developed countries. The bacteria inhabit natural and man-made freshwater environments. Here we demonstrate that different environmental L. pneumophila genotypes have different temperature-dependent growth kinetics. Moreover, Legionella strains that belong to the same species but were isolated from environmental and clinical sources possess adaptations for growth at different temperatures. These growth preferences may influence the bacterial colonization at specific ecological niches within the drinking-water network. Adaptations for growth at human body temperatures may facilitate the abilities of some L. pneumophila strains to infect and cause illness in humans. Our findings may be used as a tool to improve Legionella monitoring in drinking-water networks. Risk assessment models for predicting the risk of legionellosis should take into account not only Legionella concentrations but also the temperature-dependent growth kinetics of the isolates. PMID:28159784

Clinical and microbiological characterization of Clostridium difficile infection in a tertiary care hospital in Shanghai, China.

PubMed

Dong, Danfeng; Peng, Yibing; Zhang, Lihua; Jiang, Cen; Wang, Xuefeng; Mao, Enqiang

2014-01-01

Over the last decade, Clostridium difficile infection (CDI) has emerged as a significant nosocomial infection, yet little has been reported from China. This study aimed to characterize the clinical and microbiological features of CDI from a hospital in Shanghai. Patients with CDI seen between December 2010 and March 2013 were included in this study, of which clinical data were retrospectively collected. The microbiological features of corresponding isolates were analyzed including genotype by multi-locus sequence typing (MLST), antimicrobial susceptibility, toxin production, sporulation capacity, biofilm formation, and motility. Ninety-four cases of CDI were included during this study period, 12 of whom were severe cases. By reviewing the clinical data, all patients were treated empirically with proton pump inhibitor or antibiotics or both, and they were distributed widely across various wards, most frequently to the digestive ward (28/94, 29.79%). Comparing the severe with mild cases, no significant differences were found in the basic epidemiological data or the microbiological features. Among the 94 isolates, 31 were toxin A-negative toxin B-positive all genotyped as ST37. They generated fewer toxins and spores, as well as similar amounts of biofilm and motility percentages, but exhibited highest drug resistance to cephalosporins, quinolones, macrolide-lincosamide and streptogramin (MLSB), and tetracycline. No specific clinical genotype or microbiological features were found in severe cases; antimicrobial resistance could be the primary reason for epidemic strains leading to the dissemination and persistence of CDI.

Molecular characterization of high performance inbred lines of Brazilian common beans.

PubMed

Cardoso, P C B; Veiga, M M; de Menezes, I P P; Valdisser, P A M R; Borba, T C O; Melo, L C; Del Peloso, M J; Brondani, C; Vianello, R P

2013-02-06

The identification of germplasm genetic variability in breeding programs of the common bean (Phaseolus vulgaris) is essential for determining the potential of each combination of parent plants to obtain superior genotypes. The present study aimed to estimated the extent of genetic diversity in 172 lineages and cultivars of the common bean by integrating five tests of value for cultivation and use (VCU) that were conducted over the last eight years by the breeding program of Embrapa Arroz e Feijão in Brazil. Nine multilocus genotyping systems composed of 36 fluorescent microsatellite markers distributed across 11 different chromosomes of the common bean were used, of which 24 were polymorphic in all trials. One hundred and eighty-seven alleles were identified, with an average of 7.79 alleles per locus and an average gene diversity of 0.65. The combined probability of identity for all loci was 1.32 x 10(-16). Lineages that are more genetically divergent between the selection cycles were identified, allowing the breeding program to develop a crossbreed between elite genotypes with a low degree of genetic relatedness. HE values ranged from 0.31 to 0.63, with a large reduction in the genetic base over successive selection cycles. The test showed a significant degree of differentiation (FST = 0.159). Private alleles (26%) were identified and can be directly incorporated into the gene pool of cultivated germplasm, thereby contributing effectively to the expansion of genetic diversity in this bean-breeding program.

Heterogeneous Vancomycin-Intermediate Susceptibility Phenotype in Bloodstream Methicillin-Resistant Staphylococcus aureus Isolates from an International Cohort of Patients with Infective Endocarditis: Prevalence, Genotype, and Clinical Significance

PubMed Central

Bae, In-Gyu; Federspiel, Jerome J.; Miro, Jose M.; Woods, Christopher W.; Park, Lawrence; Rybak, Michael J.; Rude, Thomas H.; Bradley, Suzanne; Bukovski, Suzana; de la Maria, Cristina Garcia; Kanj, Souha S.; Korman, Tony; Marco, Francesc; Murdoch, David R.; Plesiat, Patrick; Rodriguez-Creixems, Marta; Ryan, Suzanne; Steed, Lisa; Tattevin, Pierre; Tripodi, Marie-Françoise; Newton, Karly L.; Corey, G. Ralph; Fowler, Vance G.

2013-01-01

Background The significance of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is unknown. Using a multinational collection of isolates from methicillin-resistant S. aureus (MRSA) infective endocarditis (IE), we characterized IE patients with and without hVISA, and genotyped the infecting strains. Methods MRSA bloodstream isolates from 65 patients with definite IE from 8 countries underwent PCR for 31 virulence genes, pulsed-field gel electrophoresis, and multilocus sequence typing. hVISA was defined using population analysis profiling (PAP). Results Nineteen (29.2%) of 65 MRSA IE isolates exhibited hVISA by PAP. Isolates from Oceania and Europe were more likely to exhibit hVISA than isolates from the United States (77.8% vs. 35.0% vs. 13.9%; P < .001). The prevalence of hVISA was higher among isolates with a vancomycin minimum inhibitory concentration of 2 mg/L (P = .026). hVISA-infected patients were more likely to have persistent bacteremia (68.4% vs. 37.0%; P = .029) and heart failure (47.4% vs. 19.6%; P = .033). Mortality of hVISA- and non-hVISA-infected patients did not differ (42.1% vs. 34.8%, P = .586). hVISA and non-hVISA isolates were genotypically similar. Conclusions In these analyses, hVISA occurred in over one-quarter of MRSA IE isolates, was associated with certain IE complications, and varied in frequency by geographic region. PMID:19811099

Universality Classes of Interaction Structures for NK Fitness Landscapes

NASA Astrophysics Data System (ADS)

Hwang, Sungmin; Schmiegelt, Benjamin; Ferretti, Luca; Krug, Joachim

2018-07-01

Kauffman's NK-model is a paradigmatic example of a class of stochastic models of genotypic fitness landscapes that aim to capture generic features of epistatic interactions in multilocus systems. Genotypes are represented as sequences of L binary loci. The fitness assigned to a genotype is a sum of contributions, each of which is a random function defined on a subset of k ≤ L loci. These subsets or neighborhoods determine the genetic interactions of the model. Whereas earlier work on the NK model suggested that most of its properties are robust with regard to the choice of neighborhoods, recent work has revealed an important and sometimes counter-intuitive influence of the interaction structure on the properties of NK fitness landscapes. Here we review these developments and present new results concerning the number of local fitness maxima and the statistics of selectively accessible (that is, fitness-monotonic) mutational pathways. In particular, we develop a unified framework for computing the exponential growth rate of the expected number of local fitness maxima as a function of L, and identify two different universality classes of interaction structures that display different asymptotics of this quantity for large k. Moreover, we show that the probability that the fitness landscape can be traversed along an accessible path decreases exponentially in L for a large class of interaction structures that we characterize as locally bounded. Finally, we discuss the impact of the NK interaction structures on the dynamics of evolution using adaptive walk models.

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.

High variation in clonal vs. sexual reproduction in populations of the wild strawberry, Fragaria virginiana (Rosaceae)

PubMed Central

Wilk, John A.; Kramer, Andrea T.; Ashley, Mary V.

2009-01-01

Background and Aims Many plants reproduce both clonally and sexually, and the balance between the two modes of reproduction will vary among populations. Clonal reproduction was characterized in three populations of the wild strawberry, Fragaria virginiana, to determine the extent that reproductive mode varied locally between sites. The study sites were fragmented woodlands in Cook County, Illinois, USA. Methods A total of 95 strawberry ramets were sampled from the three sites via transects. Ramets were mapped and genotyped at five variable microsatellite loci. The variability at these five loci was sufficient to assign plants to clones with high confidence, and the spatial pattern of genets was mapped at each site. Key Results A total of 27 distinct multilocus genotypes were identified. Of these, 18 genotypes were detected only once, with the remaining nine detected in multiple ramets. The largest clone was identified in 16 ramets. No genets were shared between sites, and each site exhibited markedly different clonal and sexual recruitment patterns, ranging from two non-overlapping and widespread genets to 19 distinct genets. Only one flowering genet was female; the remainder were hermaphrodites. Conclusions Local population history or fine-scale ecological differences can result in dramatically different reproductive patterns at small spatial scales. This finding may be fairly widespread among clonal plant species, and studies that aim to characterize reproductive modes in species capable of asexual reproduction need to evaluate reproductive modes in multiple populations and sites. PMID:19797422

Universality Classes of Interaction Structures for NK Fitness Landscapes

NASA Astrophysics Data System (ADS)

Hwang, Sungmin; Schmiegelt, Benjamin; Ferretti, Luca; Krug, Joachim

2018-02-01

Kauffman's NK-model is a paradigmatic example of a class of stochastic models of genotypic fitness landscapes that aim to capture generic features of epistatic interactions in multilocus systems. Genotypes are represented as sequences of L binary loci. The fitness assigned to a genotype is a sum of contributions, each of which is a random function defined on a subset of k ≤ L loci. These subsets or neighborhoods determine the genetic interactions of the model. Whereas earlier work on the NK model suggested that most of its properties are robust with regard to the choice of neighborhoods, recent work has revealed an important and sometimes counter-intuitive influence of the interaction structure on the properties of NK fitness landscapes. Here we review these developments and present new results concerning the number of local fitness maxima and the statistics of selectively accessible (that is, fitness-monotonic) mutational pathways. In particular, we develop a unified framework for computing the exponential growth rate of the expected number of local fitness maxima as a function of L, and identify two different universality classes of interaction structures that display different asymptotics of this quantity for large k. Moreover, we show that the probability that the fitness landscape can be traversed along an accessible path decreases exponentially in L for a large class of interaction structures that we characterize as locally bounded. Finally, we discuss the impact of the NK interaction structures on the dynamics of evolution using adaptive walk models.

Heterogeneous vancomycin-intermediate susceptibility phenotype in bloodstream methicillin-resistant Staphylococcus aureus isolates from an international cohort of patients with infective endocarditis: prevalence, genotype, and clinical significance.

PubMed

Bae, In-Gyu; Federspiel, Jerome J; Miró, José M; Woods, Christopher W; Park, Lawrence; Rybak, Michael J; Rude, Thomas H; Bradley, Suzanne; Bukovski, Suzana; de la Maria, Cristina Garcia; Kanj, Souha S; Korman, Tony M; Marco, Francesc; Murdoch, David R; Plesiat, Patrick; Rodriguez-Creixems, Marta; Reinbott, Porl; Steed, Lisa; Tattevin, Pierre; Tripodi, Marie-Françoise; Newton, Karly L; Corey, G Ralph; Fowler, Vance G

2009-11-01

The significance of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is unknown. Using a multinational collection of isolates from methicillin-resistant S. aureus (MRSA) infective endocarditis (IE), we characterized patients with IE with and without hVISA, and we genotyped the infecting strains. MRSA bloodstream isolates from 65 patients with definite IE from 8 countries underwent polymerase chain reaction (PCR) for 31 virulence genes, pulsed-field gel electrophoresis, and multilocus sequence typing. hVISA was defined using population analysis profiling. Nineteen (29.2%) of 65 MRSA IE isolates exhibited the hVISA phenotype by population analysis profiling. Isolates from Oceania and Europe were more likely to exhibit the hVISA phenotype than isolates from the United States (77.8% and 35.0% vs 13.9%; P < .001). The prevalence of hVISA was higher among isolates with a vancomycin minimum inhibitory concentration of 2 mg/L (P = .026). hVISA-infected patients were more likely to have persistent bacteremia (68.4% vs 37.0%; P = .029) and heart failure (47.4% vs 19.6%; P = .033). Mortality did not differ between hVISA- and non-hVISA-infected patients (42.1% vs 34.8%, P = .586). hVISA and non-hVISA isolates were genotypically similar. In these analyses, the hVISA phenotype occurred in more than one-quarter of MRSA IE isolates, was associated with certain IE complications, and varied in frequency by geographic region.

Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

PubMed

Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc

2015-06-25

Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex analysis. Using this combined multiplex approach also substantially reduced the production of waste material. These genotyping assays appear therefore to be biologically, economically and ecologically highly relevant, being worth to be integrated in genetic-based predictive strategies for better selecting patients at risk for prostate cancer. In addition, the same approach could now equally be transposed to other clinical/research applications relying on the computation of MGRS based on multi-SNP genotyping.

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

PubMed

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

2011-01-01

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

Typing Candida albicans oral isolates from healthy brazilian schoolchildren using multilocus enzyme electrophoresis reveals two highly polymorphic taxa

PubMed Central

Boriollo, Marcelo Fabiano Gomes; Spolidorio, Denise Madalena Palomari; Barros, Letizia Monteiro; Bassi, Rodrigo Carlos; Garcia, José Antonio Dias; Costa, Ana Maria Duarte Dias; Rosa, Edvaldo Antonio Ribeiro; Höfling, José Francisco

2011-01-01

The genetic diversity of C. albicans oral isolates from 75 healthy schoolchildren from eight schools located in different geographic areas of Piracicaba city, São Paulo state, Brazil, was established using isoenzymes marker (Multilocus Enzyme Electrophoresis – MLEE) and cluster analysis. Patterns of monoclonal and polyclonal oral colonization by C. albicans within and between groups of schoolchildren were identified. However, significant divergence between the observed and the expected genotypic frequencies (Hardy-Weinberg equilibrium test) was not detected in the geographically adjacent groups, suggesting the hypothesis that populations of healthy schoolchildren do not correspond to the selection factor (differential survival) of strains. Two highly polymorphic and distantly genetically related taxa (A and B) were identified within the total population of yeasts, each contained subgroups (A1, A2, A3, A4, B1 and B2) and clusters of moderately related strains (from I to X), suggesting the existence of strains restricted or not to certain groups of geographically limited, healthy students. However, the coexistence of identical strains in healthy schoolchildren from the same school (geographically related) reinforces the hypothesis of oral transmission, where the sources of propagation could be explored. Furthermore, this could also be used in current and retrospective analyses of C. albicans isolated from immunocompetent and immunocompromised people, in order to detect commensal or potentially pathogenic yeast groups, predominantly in candidiasis, and in the development of strategies to prevent transmission or human propagation. PMID:24031720

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 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

Multi-Locus Sequence Typing of Bartonella henselae Isolates from Three Continents Reveals Hypervirulent and Feline-Associated Clones

PubMed Central

Arvand, Mardjan; Feil, Edward J.; Giladi, Michael; Boulouis, Henri-Jean; Viezens, Juliane

2007-01-01

Bartonella henselae is a zoonotic pathogen and the causative agent of cat scratch disease and a variety of other disease manifestations in humans. Previous investigations have suggested that a limited subset of B. henselae isolates may be associated with human disease. In the present study, 182 human and feline B. henselae isolates from Europe, North America and Australia were analysed by multi-locus sequence typing (MLST) to detect any associations between sequence type (ST), host species and geographical distribution of the isolates. A total of 14 sequence types were detected, but over 66% (16/24) of the isolates recovered from human disease corresponded to a single genotype, ST1, and this type was detected in all three continents. In contrast, 27.2% (43/158) of the feline isolates corresponded to ST7, but this ST was not recovered from humans and was restricted to Europe. The difference in host association of STs 1 (human) and 7 (feline) was statistically significant (P≤0.001). eBURST analysis assigned the 14 STs to three clonal lineages, which contained two or more STs, and a singleton comprising ST7. These groups were broadly consistent with a neighbour-joining tree, although splits decomposition analysis was indicative of a history of recombination. These data indicate that B. henselae lineages differ in their virulence properties for humans and contribute to a better understanding of the population structure of B. henselae. PMID:18094753

Combined use of a new SNP-based assay and multilocus SSR markers to assess genetic diversity of Xylella fastidiosa subsp. pauca infecting citrus and coffee plants.

PubMed

Montes-Borrego, Miguel; Lopes, Joao R S; Jiménez-Díaz, Rafael M; Landa, Blanca B

2015-03-01

Two haplotypes of Xylella fastidiosa subsp. pauca (Xfp) that correlated with their host of origin were identified in a collection of 90 isolates infecting citrus and coffee plants in Brazil, based on a single-nucleotide polymorphism in the gyrB sequence. A new single-nucleotide primer extension (SNuPE) protocol was designed for rapid identification of Xfp according to the host source. The protocol proved to be robust for the prediction of the Xfp host source in blind tests using DNA from cultures of the bacterium, infected plants, and insect vectors allowed to feed on Xfp-infected citrus plants. AMOVA and STRUCTURE analyses of microsatellite data separated most Xfp populations on the basis of their host source, indicating that they were genetically distinct. The combined use of the SNaPshot protocol and three previously developed multilocus SSR markers showed that two haplotypes and distinct isolates of Xfp infect citrus and coffee in Brazil and that multiple, genetically different isolates can be present in a single orchard or infect a single tree. This combined approach will be very useful in studies of the epidemiology of Xfp-induced diseases, host specificity of bacterial genotypes, the occurrence of Xfp host jumping, vector feeding habits, etc., in economically important cultivated plants or weed host reservoirs of Xfp in Brazil and elsewhere. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Multilocus adaptation associated with heat resistance in reef-building corals.

PubMed

Bay, Rachael A; Palumbi, Stephen R

2014-12-15

The evolution of tolerance to future climate change depends on the standing stock of genetic variation for resistance to climate-related impacts, but genes contributing to climate tolerance in wild populations are poorly described in number and effect. Physiology and gene expression patterns have shown that corals living in naturally high-temperature microclimates are more resistant to bleaching because of both acclimation and fixed effects, including adaptation. To search for potential genetic correlates of these fixed effects, we genotyped 15,399 single nucleotide polymorphisms (SNPs) in 23 individual tabletop corals, Acropora hyacinthus, within a natural temperature mosaic in backreef lagoons on Ofu Island, American Samoa. Despite overall lack of population substructure, we identified 114 highly divergent SNPs as candidates for environmental selection, via multiple stringent outlier tests, and correlations with temperature. Corals from the warmest reef location had higher minor allele frequencies across these candidate SNPs, a pattern not seen for noncandidate loci. Furthermore, within backreef pools, colonies in the warmest microclimates had a higher number and frequency of alternative alleles at candidate loci. These data suggest mild selection for alternate alleles at many loci in these corals during high heat episodes and possible maintenance of extensive polymorphism through multilocus balancing selection in a heterogeneous environment. In this case, a natural population harbors a reservoir of alleles preadapted to high temperatures, suggesting potential for future evolutionary response to climate change. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clinical and epidemiological analysis of Campylobacter fetus subsp. fetus infections in humans and comparative genetic analysis with strains isolated from cattle.

PubMed

Escher, Robert; Brunner, Colette; von Steiger, Niklaus; Brodard, Isabelle; Droz, Sara; Abril, Carlos; Kuhnert, Peter

2016-05-14

Campylobacter fetus subspecies fetus (CFF) is an important pathogen for both cattle and humans. We performed a systematic epidemiological and clinical study of patients and evaluated the genetic relatedness of 17 human and 17 bovine CFF isolates by using different genotyping methods. In addition, the serotype, the dissemination of the genomic island containing a type IV secretion system (T4SS) and resistance determinants for tetracycline and streptomycin were also evaluated. The isolates from patients diagnosed with CFF infection as well as those from faecal samples of healthy calves were genotyped using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), as well as single locus sequence typing (SLST) targeting cmp1 and cmp2 genes encoding two major outer membrane proteins in CFF. The presence of the genomic island and identification of serotype was determined by PCRs targeting genes of the T4SS and the sap locus, respectively. Tetracycline and streptomycin resistance phenotypes were determined by minimal inhibitory concentration. Clinical data obtained from medical records and laboratory data were supplemented by data obtained via telephone interviews with the patients and treating physicians. PFGE analysis defined two major clusters; cluster A containing 16 bovine (80 %) isolates and cluster B containing 13 human (92 %) isolates, suggesting a host preference. Further genotypic analysis using MLST, SLST as well as sap and T4SS PCR showed the presence of genotypically identical isolates in cattle and humans. The low diversity observed within the cmp alleles of CFF corroborates the clonal nature of this pathogen. The genomic island containing the tetracycline and streptomycin resistance determinants was found in 55 % of the isolates in cluster A and correlated with phenotypic antibiotic resistance. Most human and bovine isolates were separated on two phylogenetic clusters. However, several human and bovine isolates were identical by diverse genotyping methods, indicating a possible link between strains from these two hosts.

SNP Design from 454 Sequencing of Podosphaera plantaginis Transcriptome Reveals a Genetically Diverse Pathogen Metapopulation with High Levels of Mixed-Genotype Infection

PubMed Central

Tollenaere, Charlotte; Susi, Hanna; Nokso-Koivisto, Jussi; Koskinen, Patrik; Tack, Ayco; Auvinen, Petri; Paulin, Lars; Frilander, Mikko J.; Lehtonen, Rainer; Laine, Anna-Liisa

2012-01-01

Background Molecular tools may greatly improve our understanding of pathogen evolution and epidemiology but technical constraints have hindered the development of genetic resources for parasites compared to free-living organisms. This study aims at developing molecular tools for Podosphaera plantaginis, an obligate fungal pathogen of Plantago lanceolata. This interaction has been intensively studied in the Åland archipelago of Finland with epidemiological data collected from over 4,000 host populations annually since year 2001. Principal Findings A cDNA library of a pooled sample of fungal conidia was sequenced on the 454 GS-FLX platform. Over 549,411 reads were obtained and annotated into 45,245 contigs. Annotation data was acquired for 65.2% of the assembled sequences. The transcriptome assembly was screened for SNP loci, as well as for functionally important genes (mating-type genes and potential effector proteins). A genotyping assay of 27 SNP loci was designed and tested on 380 infected leaf samples from 80 populations within the Åland archipelago. With this panel we identified 85 multilocus genotypes (MLG) with uneven frequencies across the pathogen metapopulation. Approximately half of the sampled populations contain polymorphism. Our genotyping protocol revealed mixed-genotype infection within a single host leaf to be common. Mixed infection has been proposed as one of the main drivers of pathogen evolution, and hence may be an important process in this pathosystem. Significance The developed SNP panel offers exciting research perspectives for future studies in this well-characterized pathosystem. Also, the transcriptome provides an invaluable novel genomic resource for powdery mildews, which cause significant yield losses on commercially important crops annually. Furthermore, the features that render genetic studies in this system a challenge are shared with the majority of obligate parasitic species, and hence our results provide methodological insights from SNP calling to field sampling protocols for a wide range of biological systems. PMID:23300684

pLARmEB: integration of least angle regression with empirical Bayes for multilocus genome-wide association studies.

PubMed

Zhang, J; Feng, J-Y; Ni, Y-L; Wen, Y-J; Niu, Y; Tamba, C L; Yue, C; Song, Q; Zhang, Y-M

2017-06-01

Multilocus genome-wide association studies (GWAS) have become the state-of-the-art procedure to identify quantitative trait nucleotides (QTNs) associated with complex traits. However, implementation of multilocus model in GWAS is still difficult. In this study, we integrated least angle regression with empirical Bayes to perform multilocus GWAS under polygenic background control. We used an algorithm of model transformation that whitened the covariance matrix of the polygenic matrix K and environmental noise. Markers on one chromosome were included simultaneously in a multilocus model and least angle regression was used to select the most potentially associated single-nucleotide polymorphisms (SNPs), whereas the markers on the other chromosomes were used to calculate kinship matrix as polygenic background control. The selected SNPs in multilocus model were further detected for their association with the trait by empirical Bayes and likelihood ratio test. We herein refer to this method as the pLARmEB (polygenic-background-control-based least angle regression plus empirical Bayes). Results from simulation studies showed that pLARmEB was more powerful in QTN detection and more accurate in QTN effect estimation, had less false positive rate and required less computing time than Bayesian hierarchical generalized linear model, efficient mixed model association (EMMA) and least angle regression plus empirical Bayes. pLARmEB, multilocus random-SNP-effect mixed linear model and fast multilocus random-SNP-effect EMMA methods had almost equal power of QTN detection in simulation experiments. However, only pLARmEB identified 48 previously reported genes for 7 flowering time-related traits in Arabidopsis thaliana.

Isolation and characterization of atypical Listeria monocytogenes associated with a canine urinary tract infection.

PubMed

Palerme, Jean-Sébastien; Pan, Po Ching; Parsons, Cameron T; Kathariou, Sophia; Ward, Todd J; Jacob, Megan E

2016-09-01

Listeria monocytogenes, a well-described cause of encephalitis and abortion in ruminants and of food-borne illness in humans, is rarely associated with disease in companion animals. A case of urinary tract infection associated with an atypical, weakly hemolytic L. monocytogenes strain is described in a diabetic dog. The serotype of the L. monocytogenes isolate was determined to be 1/2a (3a), with the multilocus genotyping pattern 2.72_1/2a. A nucleotide substitution (Gly145Asp) was detected at residue 145 in the promoter prfA region. This residue is within the critical helix-turn-helix motif of PrfA. The source of the L. monocytogenes strain remains unknown, and the dog recovered after a 4-week course of cephalexin (30 mg/kg orally twice daily). © 2016 The Author(s).

Metallo-β-lactamase-producing Pseudomonas aeruginosa in the Netherlands: the nationwide emergence of a single sequence type.

PubMed

Van der Bij, A K; Van der Zwan, D; Peirano, G; Severin, J A; Pitout, J D D; Van Westreenen, M; Goessens, W H F

2012-09-01

Recently, the first outbreak of clonally related VIM-2 metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in a Dutch tertiary-care centre was described. Subsequently, a nationwide surveillance study was performed in 2010-2011, which identified the presence of VIM-2 MBL-producing P. aeruginosa in 11 different hospitals. Genotyping by multiple-locus variable-number tandem-repeat analysis (MLVA) showed that the majority of the 82 MBL-producing isolates found belonged to a single MLVA type (n = 70, 85%), identified as ST111 by multilocus sequence typing (MLST). As MBL-producing isolates cause serious infections that are difficult to treat, the presence of clonally related isolates in various hospitals throughout the Netherlands is of nationwide concern. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Fatal Toxoplasma gondii infection in the giant panda.

PubMed

Ma, Hongyu; Wang, Zedong; Wang, Chengdong; Li, Caiwu; Wei, Feng; Liu, Quan

2015-01-01

Toxoplasma gondii can infect nearly all warm-blooded animals. We report an acute fatal T. gondii infection in the endangered giant panda (Ailuropoda melanoleuca) in a zoo in China, characterized by acute gastroenteritis and respiratory symptoms. T. gondii infection was confirmed by immunological and molecular methods. Multilocus nested PCR-RFLP revealed clonal type I at the SAG1 and c29-2 loci, clonal type II at the SAG2, BTUB, GRA6, c22-8, and L358 loci, and clonal type III at the alternative SAG2 and SAG3 loci, thus, a potential new genotype of T. gondii in the giant panda. Other possible pathogens were not detected. To our knowledge, this is the first report of clinical toxoplasmosis in a giant panda. © H. Ma et al., published by EDP Sciences, 2015.

Development of eighteen microsatellite loci in walleye (Sander vitreus)

USGS Publications Warehouse

Coykendall, D. Katharine; Morrison, Cheryl L.; Stott, Wendylee; Springmann, Marcus J.

2014-01-01

A suite of tri- and tetra-nucleotide microsatellite loci were developed for walleye (Sander vitreus) from 454 pyrosequencing data. Eighteen of the 50 primer sets tested amplified consistently in 35 walleye from two lakes on Isle Royale, Lake Superior: Chickenbone Lake and Whittlesey Lake. The loci displayed moderate levels of allelic diversity (average 5.5 alleles/locus) and heterozygosity (average 35.8 %). Levels of genetic diversity were sufficient to produce unique multi-locus genotypes and detect phylogeographic structuring as individuals assigned back to their population of origin. Cross-species amplification within S. canadensis(sauger) was successful for 15 loci, and 11 loci were diagnostic to species. The loci characterized here will be useful for detecting fine-scale spatial structuring, resolving the taxonomic status of Sander species and sub-species, and detecting walleye/sauger hybrids.

Molecular Epidemiology of Clostridium difficile Infection in Hospitalized Patients in Eastern China.

PubMed

Jin, Dazhi; Luo, Yun; Huang, Chen; Cai, Jian; Ye, Julian; Zheng, Yi; Wang, Liqian; Zhao, Peng; Liu, Anbing; Fang, Weijia; Wang, Xianjun; Xia, Shichang; Jiang, Jianmin; Tang, Yi-Wei

2017-03-01

Few studies on risk factors for and transmission of Clostridium difficile infection (CDI) in China have been reported. A cross-sectional study was conducted for 3 years in eastern China. Consecutive stool specimens from hospitalized patients with diarrhea were cultured for C. difficile. C. difficile isolates from these patients then were analyzed for toxin genes, genotypes, and antimicrobial resistance. A severity score for the CDI in each patient was determined by a blinded review of the medical record, and these scores ranged from 1 to 6. A total of 397 out of 3,953 patients (10.0%) with diarrhea were found to have CDI. Severity of CDI was mild to moderate, and the average (± standard deviation) severity score was 2.61 ± 1.01. C. difficile was isolated from stool specimens in 432 (10.9%) of all the patients who had diarrhea. C. difficile genotypes were determined by multilocus sequence analysis and PCR ribotyping; sequence type 37 (ST37)/ribotype 017 (RT017) ( n = 68, 16.5%) was the dominant genotype. Eleven patients (16.2%) with this genotype had a CDI severity score of 5. Overall, three RTs and four STs were predominant; these genotypes were associated with significantly different antimicrobial resistance patterns in comparison to all genotypes (χ 2 = 79.56 to 97.76; P < 0.001). Independent risk factors associated with CDI included age greater than 55 years (odds ratio [95% confidence interval], 26.80 [18.76 to 38.29]), previous hospitalization (12.42 [8.85 to 17.43]), previous antimicrobial treatment within 8 weeks (150.56 [73.11 to 310.06]), hospital stay more than 3 days before sampling (2.34 [1.71 to 3.22]), undergoing chemotherapy (3.31 [2.22 to 4.92]), and undergoing abdominal surgery (4.82 [3.54 to 6.55]). CDI is clearly a problem in eastern China and has a prevalence of 10.0% in hospitalized patients. Among risk factors for CDI, the advanced age threshold was younger for Chinese patients than that reported for patients in developed countries. Copyright © 2017 American Society for Microbiology.

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.

Multilocus genetics to reconstruct aeromonad evolution

PubMed Central

2012-01-01

Background Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. Results The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. Conclusions Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or “disease specialized” while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters. PMID:22545815

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.

Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer.

PubMed

Nooshadokht, Maryam; Kalantari-Khandani, Behjat; Sharifi, Iraj; Kamyabi, Hossein; Liyanage, Namal P M; Lagenaur, Laurel A; Kagnoff, Martin F; Singer, Steven M; Babaei, Zahra; Solaymani-Mohammadi, Shahram

2017-10-01

Human infection with the protozoan parasite Giardia duodenalis is one the most common parasitic diseases worldwide. Higher incidence rates of giardiasis have been reported from human subjects with multiple debilitating chronic conditions, including hypogammaglobulinemia and common variable immunodeficiency (CVID). In the current study, stool specimens were collected from 199 individuals diagnosed with HIV or cancer and immunocompetent subjects. The sensitivity of microscopy-based detection on fresh stool preparations, trichrome staining and stool antigen immunodetection for the diagnosis of G. duodenalis were 36%, 45.5% and 100%, respectively when compared with a highly sensitive stool-based PCR method as the gold standard. Further multilocus molecular analyses using glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci demonstrated that the AI genotype of G. duodenalis was the most prevalent, followed by the AII genotype and mixed (AI+B) infections. We concluded that stool antigen immunodetection-based immunoassays and stool-based PCR amplification had comparable sensitivity and specificity for the diagnosis of G. duodenalis infections in these populations. Stool antigen detection-based diagnostic modalities are rapid and accurate and may offer alternatives to conventional microscopy and PCR-based diagnostic methods for the diagnosis of G. duodenalis in human subjects living with HIV or cancer. Copyright © 2017. Published by Elsevier B.V.

Human Birth Weight and Reproductive Immunology: Testing for Interactions between Maternal and Offspring KIR and HLA-C Genes.

PubMed

Clark, Michelle M; Chazara, Olympe; Sobel, Eric M; Gjessing, Håkon K; Magnus, Per; Moffett, Ashley; Sinsheimer, Janet S

2016-01-01

Maternal and offspring cell contact at the site of placentation presents a plausible setting for maternal-fetal genotype (MFG) interactions affecting fetal growth. We test hypotheses regarding killer cell immunoglobulin-like receptor (KIR) and HLA-C MFG effects on human birth weight by extending the quantitative MFG (QMFG) test. Until recently, association testing for MFG interactions had limited applications. To improve the ability to test for these interactions, we developed the extended QMFG test, a linear mixed-effect model that can use multi-locus genotype data from families. We demonstrate the extended QMFG test's statistical properties. We also show that if an offspring-only model is fit when MFG effects exist, associations can be missed or misattributed. Furthermore, imprecisely modeling the effects of both KIR and HLA-C could result in a failure to replicate if these loci's allele frequencies differ among populations. To further illustrate the extended QMFG test's advantages, we apply the extended QMFG test to a UK cohort study and the Norwegian Mother and Child Cohort (MoBa) study. We find a significant KIR-HLA-C interaction effect on birth weight. More generally, the QMFG test can detect genetic associations that may be missed by standard genome-wide association studies for quantitative traits. © 2017 S. Karger AG, Basel.

The effects of quantitative fecundity in the haploid stage on reproductive success and diploid fitness in the aquatic peat moss Sphagnum macrophyllum

PubMed Central

Johnson, M G; Shaw, A J

2016-01-01

A major question in evolutionary biology is how mating patterns affect the fitness of offspring. However, in animals and seed plants it is virtually impossible to investigate the effects of specific gamete genotypes. In bryophytes, haploid gametophytes grow via clonal propagation and produce millions of genetically identical gametes throughout a population. The main goal of this research was to test whether gamete identity has an effect on the fitness of their diploid offspring in a population of the aquatic peat moss Sphagnum macrophyllum. We observed a heavily male-biased sex ratio in gametophyte plants (ramets) and in multilocus microsatellite genotypes (genets). There was a steeper relationship between mating success (number of different haploid mates) and fecundity (number of diploid offspring) for male genets compared with female genets. At the sporophyte level, we observed a weak effect of inbreeding on offspring fitness, but no effect of brood size (number of sporophytes per maternal ramet). Instead, the identities of the haploid male and haploid female parents were significant contributors to variance in fitness of sporophyte offspring in the population. Our results suggest that intrasexual gametophyte/gamete competition may play a role in determining mating success in this population. PMID:26905464

The effects of quantitative fecundity in the haploid stage on reproductive success and diploid fitness in the aquatic peat moss Sphagnum macrophyllum.

PubMed

Johnson, M G; Shaw, A J

2016-06-01

A major question in evolutionary biology is how mating patterns affect the fitness of offspring. However, in animals and seed plants it is virtually impossible to investigate the effects of specific gamete genotypes. In bryophytes, haploid gametophytes grow via clonal propagation and produce millions of genetically identical gametes throughout a population. The main goal of this research was to test whether gamete identity has an effect on the fitness of their diploid offspring in a population of the aquatic peat moss Sphagnum macrophyllum. We observed a heavily male-biased sex ratio in gametophyte plants (ramets) and in multilocus microsatellite genotypes (genets). There was a steeper relationship between mating success (number of different haploid mates) and fecundity (number of diploid offspring) for male genets compared with female genets. At the sporophyte level, we observed a weak effect of inbreeding on offspring fitness, but no effect of brood size (number of sporophytes per maternal ramet). Instead, the identities of the haploid male and haploid female parents were significant contributors to variance in fitness of sporophyte offspring in the population. Our results suggest that intrasexual gametophyte/gamete competition may play a role in determining mating success in this population.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe.

PubMed

Cvetnić, Željko; Duvnjak, Sanja; Đuras, Martina; Gomerčić, Tomislav; Reil, Irena; Zdelar-Tuk, Maja; Špičić, Silvio

2016-11-30

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health. Copyright © 2016 Elsevier B.V. All rights reserved.

A likelihood-based approach for assessment of extra-pair paternity and conspecific brood parasitism in natural populations

USGS Publications Warehouse

Lemons, Patrick R.; Marshall, T.C.; McCloskey, Sarah E.; Sethi, S.A.; Schmutz, Joel A.; Sedinger, James S.

2015-01-01

Genotypes are frequently used to assess alternative reproductive strategies such as extra-pair paternity and conspecific brood parasitism in wild populations. However, such analyses are vulnerable to genotyping error or molecular artifacts that can bias results. For example, when using multilocus microsatellite data, a mismatch at a single locus, suggesting the offspring was not directly related to its putative parents, can occur quite commonly even when the offspring is truly related. Some recent studies have advocated an ad-hoc rule that offspring must differ at more than one locus in order to conclude that they are not directly related. While this reduces the frequency with which true offspring are identified as not directly related young, it also introduces bias in the opposite direction, wherein not directly related young are categorized as true offspring. More importantly, it ignores the additional information on allele frequencies which would reduce overall bias. In this study, we present a novel technique for assessing extra-pair paternity and conspecific brood parasitism using a likelihood-based approach in a new version of program cervus. We test the suitability of the technique by applying it to a simulated data set and then present an example to demonstrate its influence on the estimation of alternative reproductive strategies.

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.

Identification of a high-virulence clone of type III Streptococcus agalactiae (group B Streptococcus) causing invasive neonatal disease.

PubMed

Musser, J M; Mattingly, S J; Quentin, R; Goudeau, A; Selander, R K

1989-06-01

Chromosomal genotypes of 128 isolates of six serotypes (Ia, Ib, Ic, II, Ic/II, and III) of Streptococcus agalactiae (group B Streptococcus) recovered predominantly from human infants in the United States were characterized by an analysis of electrophoretically demonstrable allelic profiles at 11 metabolic enzyme loci. Nineteen distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Mean genetic diversity per locus among ETs of isolates of the same serotype was, on average, nearly equal to that in all 19 ETs. Cluster analysis of the ETs revealed two primary phylogenetic divisions at a genetic distance of 0.65. A single clone (ET 1) represented by 40 isolates expressing type III antigen formed division I. Division II was composed of 18 ETs in three major lineages diverging from one another at distances greater than 0.35 and included strains of all six antigenic classes. The type III organisms in division I produce more extracellular neuraminidase and apparently are more virulent than the type III strains in division II, which are related to strains of other serotypes that cause disease much less frequently. The existence of this unusually virulent clone accounts, in major part, for the high morbidity and mortality associated with infection by type III organisms.

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

Genetic diversity and antibiotic susceptibility of Staphylococcus aureus isolates from wild boars.

PubMed

Seinige, D; Von Altrock, A; Kehrenberg, C

2017-10-01

We here report the occurrence of S. aureus in wild boars and characterize isolates genotypically and phenotypically in order to get knowledge about the occurrence of clonal lineages and genotypes in free-living wild animals. Forty-one S. aureus isolates obtained from 111 wild boars hunted in Lower Saxony, Germany, were investigated and compared to human and livestock isolates. The S. aureus belonged to multilocus sequence types ST1, ST7, ST30, ST133, ST425, ST804, ST890 and to the new ST3237, ST3238, ST3255 and ST3369. The livestock associated CC398-MRSA lineage, however, was not found. In addition to well-known spa types, the new types t14999, t15000, t15001 and t15002 were detected. Macrorestriction analysis revealed a variety of different SmaI fragment patterns. Most isolates were susceptible to all antimicrobials tested, including methicillin, and resistance was detected only to ampicillin, penicillin and erythromycin. PCR analysis confirmed the presence of staphylococcal enterotoxin genes (seh) in all t127-ST1 isolates. A high degree of genetic diversity was detected with many spa types and clonal lineages previously reported in humans and livestock animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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

2012-09-01

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

The Host Genotype and Environment Affect Strain Types of Bifidobacterium longum subsp. longum Inhabiting the Intestinal Tracts of Twins.

PubMed

Zhang, Min; Hang, Xiaomin; Tan, Jing; Yang, Hong

2015-07-01

To investigate the influences of host genotype and environment on Bifidobacterium longum subsp. longum inhabiting human intestines at the strain level, six pairs of twins, divided into two groups (children and adults), were recruited. Each group consisted of two monozygotic (MZ) twin pairs and one dizygotic (DZ) twin pair. Child twins had been living together from birth, while adult twins had been living separately for 5 to 10 years. A total of 345 B. longum subsp. longum isolates obtained from 60 fecal samples from these twins were analyzed by multilocus sequence typing (MLST), and 35 sequence types (STs) were finally acquired. Comparison of strains within and between the twin pairs showed that no strains with identical STs were observed between unrelated individuals or within adult DZ twin pairs. Eight STs were found to be monophyletic, existing within MZ twins and child DZ twins. The similarity of strain types within child cotwins was significantly higher than that within adult cotwins, which indicated that environment was one of the important determinants in B. longum subsp. longum strain types inhabiting human intestines. However, although these differences between MZ and DZ twins were observed, it is still difficult to reach an exact conclusion about the impact of host genotype. This is mainly because of the limited number of subjects tested in the present study and the lack of strain types tracing in the same twin pairs from birth until adulthood. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Seed dispersal at alpine treeline: long distance dispersal maintains alpine treelines

NASA Astrophysics Data System (ADS)

Johnson, J. S.; Gaddis, K. D.; Cairns, D. M.; Krutovsky, K.

2016-12-01

Alpine treelines are expected to advance to higher elevations in conjunction with global warming. Nevertheless, the importance of reproductive method and seed dispersal distances at the alpine treeline ecotone remains unresolved. We address two research questions at mountain hemlock treelines on the Kenai Peninsula, Alaska: (1) What is the primary mode of reproduction, and (2) are recruits derived from local treeline populations or are they arriving from more distant seed sources? We addressed our research questions by exhaustively sampling mountain hemlock individuals along a single mountain slope and then genotyped DNA single nucleotide polymorphisms using a genotyping by sequencing approach (ddRAD Seq). First we assessed mode of reproduction by determining the proportion of sampled individuals with identical multilocus genotypes that are the product of clonal reproduction. Second, we used a categorical allocation based parentage analysis to identify parent-offspring pairs, so that the proportion of treeline reproduction events could be quantified spatially and dispersal distance measured. We identified sexual reproduction as the primary mode of reproduction at our study site. Seedling establishment was characterized by extensive cryptic seed dispersal and gene flow into the ecotone. The average dispersal distance was 73 meters with long distance dispersal identified as dispersal occurring at distances greater than 450 meters. We show that production of seeds within the alpine treeline ecotone is not a necessary requirement for treelines to advance to higher elevations in response to climate change. The extensive cryptic seed dispersal and gene flow into the alpine treeline ecotone is likely sufficient to propel the ecotone higher under more favorable climate.

Genome-Wide Association Study on Resistance to Stalk Rot Diseases in Grain Sorghum

PubMed Central

Adeyanju, Adedayo; Little, Christopher; Yu, Jianming; Tesso, Tesfaye

2015-01-01

Stalk rots are important biotic constraints to sorghum production worldwide. Several pathogens may be associated with the disease, but Macrophomina phaseolina and Fusarium thapsinum are recognized as the major causal organisms. The diseases become more aggressive when drought and high-temperature stress occur during grain filling. Progress in genetic improvement efforts has been slow due to lack of effective phenotyping protocol and the strong environmental effect on disease incidence and severity. Deployment of modern molecular tools is expected to accelerate efforts to develop resistant hybrids. This study was aimed at identifying genomic regions associated with resistance to both causal organisms. A sorghum diversity panel consisting of 300 genotypes assembled from different parts of the world was evaluated for response to infection by both pathogens. Community resources of 79,132 single nucleotide polymorphic (SNP) markers developed on the panel were used in association studies using a multi-locus mixed model to map loci associated with stalk rot resistance. Adequate genetic variation was observed for resistance to both pathogens. Structure analysis grouped the genotypes into five subpopulations primarily based on the racial category of the genotypes. Fourteen loci and a set of candidate genes appear to be involved in connected functions controlling plant defense response. However, each associated SNP had relatively small effect on the traits, accounting for 19–30% of phenotypic variation. Linkage disequilibrium analyses suggest that significant SNPs are genetically independent. Estimation of frequencies of associated alleles revealed that durra and caudatum subpopulations were enriched for resistant alleles, but the results suggest complex molecular mechanisms underlying resistance to both pathogens. PMID:25882062

Change in genotype of methicillin-resistant Staphylococcus aureus (MRSA) affects the antibiogram of hospital-acquired MRSA.

PubMed

Harada, Dai; Nakaminami, Hidemasa; Miyajima, Eri; Sugiyama, Taku; Sasai, Nao; Kitamura, Yoshinobu; Tamura, Taku; Kawakubo, Takashi; Noguchi, Norihisa

2018-07-01

Recently, the dissemination of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) into hospitals has frequently been reported worldwide. Hospital-acquired MRSA (HA-MRSA) strains exhibit high-level resistance to multiple antimicrobial agents, whereas CA-MRSA strains are usually susceptible to non-β-lactams. Thus, it is predicted that the antibiogram of the HA-MRSA population would change along with the change in genotype of MRSA. Here, we investigated the changes in the MRSA population along with the MRSA antibiogram in a hospital between 2010 and 2016. Staphylococcal cassette chromosome (SCC) mec typing showed that the predominant HA-MRSA strains in the hospital dramatically changed from SCCmec type II, which is the major type of HA-MRSA, to SCCmec type IV, which is the major type of CA-MRSA. Multilocus sequence typing revealed that the predominant SCCmec type IV strain was a clonal complex (CC) 8 clone, which is mainly found among CA-MRSA. Furthermore, the CC1-SCCmec type IV (CC1-IV) clone significantly increased. Both the CC8-IV and CC1-IV clones exhibited high antimicrobial susceptibility. The antibiogram change of the HA-MRSA population was consistent with the antimicrobial susceptibilities and increased prevalence of the CC8-IV and CC1-IV clones. Our data reveal that the change in the genotypes of MRSA strains could impact the antibiogram of HA-MRSA population. Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

USA300-related methicillin-resistant Staphylococcus aureus clone is the predominant cause of community and hospital MRSA infections in Colombian children.

PubMed

Márquez-Ortiz, Ricaurte Alejandro; Álvarez-Olmos, Martha I; Escobar Pérez, Javier Antonio; Leal, Aura Lucia; Castro, Betsy Esperanza; Mariño, Ana Cristina; Barrero, Esther Rocio; Mujica, Sandra Celina; Gaines, Sebastián; Vanegas, Natasha

2014-08-01

Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) isolates are known to be more virulent and clinically aggressive in children. The goal of the present study was characterize the molecular epidemiology of MRSA isolates causing infections in Colombian children. An observational and prospective study was conducted between April 2009 and June 2011 at 15 hospitals in Bogotá, Colombia. A detailed epidemiological profile was made of 162 children infected with MRSA. The isolates were subjected to antimicrobial susceptibility testing, molecular characterization including 21 virulence genes, SCCmec, spa and agr typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Among all isolates included in the study, 85.8% were obtained from patients whose infectious process was initiated in the community; of these, 69,8% occurred in patients without healthcare-associated risk factors. The molecular characterization of the isolates showed a high proportion (95.1%) containing a community-genotype profile with a high prevalence of SCCmec type IV, PVL-positives, and also related to CC8. Most CG-MRSA isolates (143, 92.9%) were genetically related to the pandemic clone USA300, differing by the presence of SCCmec IVc and the absence of the arginine catabolic mobile element (ACME). An increase in the frequency of CG-MRSA infections has been reported worldwide. In this study we found that almost all MRSA infections in our pediatric population were caused by community-genotype isolates, supporting the success of the CG-MRSA clones. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Spatial Statistical Model for Landscape Genetics

PubMed Central

Guillot, Gilles; Estoup, Arnaud; Mortier, Frédéric; Cosson, Jean François

2005-01-01

Landscape genetics is a new discipline that aims to provide information on how landscape and environmental features influence population genetic structure. The first key step of landscape genetics is the spatial detection and location of genetic discontinuities between populations. However, efficient methods for achieving this task are lacking. In this article, we first clarify what is conceptually involved in the spatial modeling of genetic data. Then we describe a Bayesian model implemented in a Markov chain Monte Carlo scheme that allows inference of the location of such genetic discontinuities from individual geo-referenced multilocus genotypes, without a priori knowledge on populational units and limits. In this method, the global set of sampled individuals is modeled as a spatial mixture of panmictic populations, and the spatial organization of populations is modeled through the colored Voronoi tessellation. In addition to spatially locating genetic discontinuities, the method quantifies the amount of spatial dependence in the data set, estimates the number of populations in the studied area, assigns individuals to their population of origin, and detects individual migrants between populations, while taking into account uncertainty on the location of sampled individuals. The performance of the method is evaluated through the analysis of simulated data sets. Results show good performances for standard data sets (e.g., 100 individuals genotyped at 10 loci with 10 alleles per locus), with high but also low levels of population differentiation (e.g., FST < 0.05). The method is then applied to a set of 88 individuals of wolverines (Gulo gulo) sampled in the northwestern United States and genotyped at 10 microsatellites. PMID:15520263

Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries

PubMed Central

Menegon, Michela; Bardají, Azucena; Martínez-Espinosa, Flor; Bôtto-Menezes, Camila; Ome-Kaius, Maria; Mueller, Ivo; Betuela, Inoni; Arévalo-Herrera, Myriam; Kochar, Swati; Kochar, Sanjay K.; Jaju, Puneet; Hans, Dhiraj; Chitnis, Chetan; Padilla, Norma; Castellanos, María Eugenia; Ortiz, Lucía; Sanz, Sergi; Piqueras, Mireia; Desai, Meghna; Mayor, Alfredo; del Portillo, Hernando; Menéndez, Clara; Severini, Carlo

2016-01-01

Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied. PMID:27011010

A detailed view on Model-Based Multifactor Dimensionality Reduction for detecting gene-gene interactions in case-control data in the absence and presence of noise

PubMed Central

CATTAERT, TOM; CALLE, M. LUZ; DUDEK, SCOTT M.; MAHACHIE JOHN, JESTINAH M.; VAN LISHOUT, FRANÇOIS; URREA, VICTOR; RITCHIE, MARYLYN D.; VAN STEEN, KRISTEL

2010-01-01

SUMMARY Analyzing the combined effects of genes and/or environmental factors on the development of complex diseases is a great challenge from both the statistical and computational perspective, even using a relatively small number of genetic and non-genetic exposures. Several data mining methods have been proposed for interaction analysis, among them, the Multifactor Dimensionality Reduction Method (MDR), which has proven its utility in a variety of theoretical and practical settings. Model-Based Multifactor Dimensionality Reduction (MB-MDR), a relatively new MDR-based technique that is able to unify the best of both non-parametric and parametric worlds, was developed to address some of the remaining concerns that go along with an MDR-analysis. These include the restriction to univariate, dichotomous traits, the absence of flexible ways to adjust for lower-order effects and important confounders, and the difficulty to highlight epistasis effects when too many multi-locus genotype cells are pooled into two new genotype groups. Whereas the true value of MB-MDR can only reveal itself by extensive applications of the method in a variety of real-life scenarios, here we investigate the empirical power of MB-MDR to detect gene-gene interactions in the absence of any noise and in the presence of genotyping error, missing data, phenocopy, and genetic heterogeneity. For the considered simulation settings, we show that the power is generally higher for MB-MDR than for MDR, in particular in the presence of genetic heterogeneity, phenocopy, or low minor allele frequencies. PMID:21158747

Genetic diversity of wild germplasm of "yerba mate" (Ilex paraguariensis St. Hil.) from Uruguay.

PubMed

Cascales, Jimena; Bracco, Mariana; Poggio, Lidia; Gottlieb, Alexandra Marina

2014-12-01

The "yerba mate" tree, Ilex paraguariensis St. Hil., is a crop native to subtropical South America, marketed for the elaboration of the highly popular "mate" beverage. The Uruguayan germplasm occupies the southernmost area of the species distribution range and carries adaptations to environments that considerably differ from the current production area. We characterized the genetic variability of the germplasm from this unexplored area by jointly analyzing individuals from the diversification center (ABP, Argentina, Brazil and Paraguay) with 19 nuclear and 11 plastidic microsatellite markers. For the Uruguayan germplasm, we registered 55 alleles (18 % private), and 80 genotypes (44 % exclusive), whereas 63 alleles (28.6 % private) and 81 genotypes (42 % exclusive) were recorded for individuals from ABP. Only two plastidic haplotypes were detected. Distance-based and multilocus genotype analyses showed that individuals from ABP intermingle and that the Uruguayan germplasm is differentiated in three gene-pools. Significant positive correlations between genetic and geographic distances were detected. Our results concur in that ABP individuals harbor greater genetic variation than those from the tail of the distribution, as to the number of alleles (1.15-fold), He (1.19-fold), Rs (1.39-fold), and the between-group genetic distances (1.16-fold). Also the shape of the genetic landscape interpolation analysis suggests that the genetic variation decays southward towards the Uruguayan territory. We showed that Uruguayan germplasm hosts a combination of nuclear alleles not present in the central region, constituting a valuable breeding resource. Future conservation efforts should concentrate in collecting numerous individuals of "yerba mate" per site to gather the existent variation.

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.

Preliminary evidence that negative symptom severity relates to multilocus genetic profile for dopamine signaling capacity and D2 receptor binding in healthy controls and in schizophrenia.

PubMed

Eisenstein, Sarah A; Bogdan, Ryan; Chen, Ling; Moerlein, Stephen M; Black, Kevin J; Perlmutter, Joel S; Hershey, Tamara; Barch, Deanna M

2017-03-01

Deficits in central, subcortical dopamine (DA) signaling may underlie negative symptom severity, particularly anhedonia, in healthy individuals and in schizophrenia. To investigate these relationships, we assessed negative symptoms with the Schedule for the Assessment of Negative Symptoms and the Brief Negative Symptom Scale (BNSS) and self-reported anhedonia with the Scales for Physical and Social Anhedonia (SPSA), Temporal Experience of Pleasure Scale, and Snaith-Hamilton Pleasure Scale in 36 healthy controls (HC), 27 siblings (SIB) of individuals with schizophrenia, and 66 individuals with schizophrenia or schizoaffective disorder (SCZ). A subset of participants (N = 124) were genotyped for DA-related polymorphisms in genes for DRD4, DRD2/ANKK1, DAT1, and COMT, which were used to construct biologically-informed multi-locus genetic profile (MGP) scores reflective of subcortical dopaminergic signaling. DA receptor type 2 (D2R) binding was assessed among a second subset of participants (N = 23) using PET scans with the D2R-selective, non-displaceable radioligand (N-[ 11 C]methyl)benperidol. Higher MGP scores, reflecting elevated subcortical dopaminergic signaling capacity, were associated with less negative symptom severity, as measured by the BNSS, across all participants. In addition, higher striatal D2R binding was associated with less physical and social anhedonia, as measured by the SPSA, across HC, SIB, and SCZ. The current preliminary findings support the hypothesis that subcortical DA function may contribute to negative symptom severity and self-reported anhedonia, independent of diagnostic status. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Recurrent hybridization and recent origin obscure phylogenetic relationships within the ‘white-headed’ gull (Larus sp.) complex

USGS Publications Warehouse

Sonsthagen, Sarah A.; Wilson, Robert E.; Chesser, Terry; Pons, Jean-Marc; Crochet, Pierre-Andre; Driscoll, Amy; Dove, Carla

2016-01-01

Species complexes that have undergone recent radiations are often characterized by extensive allele sharing due to recent ancestry and (or) introgressive hybridization. This can result in discordant evolutionary histories of genes and heterogeneous genomes, making delineating species limits difficult. Here we examine the phylogenetic relationships among a complex group of birds, the white-headed gulls (Aves: Laridae), which offer a unique window into the speciation process due to their recent evolutionary history and propensity to hybridize. Relationships were examined among 17 species (61 populations) using a multilocus approach, including mitochondrial and nuclear intron DNA sequences and microsatellite genotype information. Analyses of microsatellite and intron data resulted in some species-based groupings, although most species were not represented by a single cluster. Considerable allele and haplotype sharing among white-headed gull species was observed; no locus contained a species-specific clade. Despite this, our multilocus approach provided better resolution among some species than previous studies. Interestingly, most clades appear to correspond to geographic locality: our BEAST analysis recovered strong support for a northern European/Icelandic clade, a southern European/Russian clade, and a western North American/canus clade, with weak evidence for a high latitude clade spanning North America and northwestern Europe. This geographical structuring is concordant with behavioral observations of pervasive hybridization in areas of secondary contact. The extent of allele and haplotype sharing indicates that ecological and sexual selection are likely not strong enough to complete reproductive isolation within several species in the white-headed gull complex. This suggests that just a few genes are driving the speciation process.

Preliminary Evidence that Negative Symptom Severity Relates to Multilocus Genetic Profile for Dopamine Signaling Capacity and D2 Receptor Binding in Healthy Controls and in Schizophrenia

PubMed Central

Eisenstein, Sarah A.; Bogdan, Ryan; Chen, Ling; Moerlein, Stephen M.; Black, Kevin J.; Perlmutter, Joel S.; Hershey, Tamara; Barch, Deanna M.

2017-01-01

Deficits in central, subcortical dopamine (DA) signaling may underlie negative symptom severity, particularly anhedonia, in healthy individuals and in schizophrenia. To investigate these relationships, we assessed negative symptoms with the Schedule for the Assessment of Negative Symptoms and the Brief Negative Symptom Scale (BNSS) and self-reported anhedonia with the Scales for Physical and Social Anhedonia (SPSA), Temporal Experience of Pleasure Scale, and Snaith-Hamilton Pleasure Scale in 36 healthy controls (HC), 27 siblings (SIB) of individuals with schizophrenia, and 66 individuals with schizophrenia or schizoaffective disorder (SCZ). A subset of participants (N = 124) were genotyped for DA-related polymorphisms in genes for DRD4, DRD2/ANKK1, DAT1, and COMT, which were used to construct biologically-informed multi-locus genetic profile (MGP) scores reflective of subcortical dopaminergic signaling. DA receptor type 2 (D2R) binding was assessed among a second subset of participants (N = 23) using PET scans with the D2R-selective, non-displaceable radioligand (N-[11C]methyl)benperidol. Higher MGP scores, reflecting elevated subcortical dopaminergic signaling capacity, were associated with less negative symptom severity, as measured by the BNSS, across all participants. In addition, higher striatal D2R binding was associated with less physical and social anhedonia, as measured by the SPSA, across HC, SIB, and SCZ. The current preliminary findings support the hypothesis that subcortical DA function may contribute to negative symptom severity and self-reported anhedonia, independent of diagnostic status. PMID:27886638

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

Functional Multi-Locus QTL Mapping of Temporal Trends in Scots Pine Wood Traits

PubMed Central

Li, Zitong; Hallingbäck, Henrik R.; Abrahamsson, Sara; Fries, Anders; Gull, Bengt Andersson; Sillanpää, Mikko J.; García-Gil, M. Rosario

2014-01-01

Quantitative trait loci (QTL) mapping of wood properties in conifer species has focused on single time point measurements or on trait means based on heterogeneous wood samples (e.g., increment cores), thus ignoring systematic within-tree trends. In this study, functional QTL mapping was performed for a set of important wood properties in increment cores from a 17-yr-old Scots pine (Pinus sylvestris L.) full-sib family with the aim of detecting wood trait QTL for general intercepts (means) and for linear slopes by increasing cambial age. Two multi-locus functional QTL analysis approaches were proposed and their performances were compared on trait datasets comprising 2 to 9 time points, 91 to 455 individual tree measurements and genotype datasets of amplified length polymorphisms (AFLP), and single nucleotide polymorphism (SNP) markers. The first method was a multilevel LASSO analysis whereby trend parameter estimation and QTL mapping were conducted consecutively; the second method was our Bayesian linear mixed model whereby trends and underlying genetic effects were estimated simultaneously. We also compared several different hypothesis testing methods under either the LASSO or the Bayesian framework to perform QTL inference. In total, five and four significant QTL were observed for the intercepts and slopes, respectively, across wood traits such as earlywood percentage, wood density, radial fiberwidth, and spiral grain angle. Four of these QTL were represented by candidate gene SNPs, thus providing promising targets for future research in QTL mapping and molecular function. Bayesian and LASSO methods both detected similar sets of QTL given datasets that comprised large numbers of individuals. PMID:25305041

Functional multi-locus QTL mapping of temporal trends in Scots pine wood traits.

PubMed

Li, Zitong; Hallingbäck, Henrik R; Abrahamsson, Sara; Fries, Anders; Gull, Bengt Andersson; Sillanpää, Mikko J; García-Gil, M Rosario

2014-10-09

Quantitative trait loci (QTL) mapping of wood properties in conifer species has focused on single time point measurements or on trait means based on heterogeneous wood samples (e.g., increment cores), thus ignoring systematic within-tree trends. In this study, functional QTL mapping was performed for a set of important wood properties in increment cores from a 17-yr-old Scots pine (Pinus sylvestris L.) full-sib family with the aim of detecting wood trait QTL for general intercepts (means) and for linear slopes by increasing cambial age. Two multi-locus functional QTL analysis approaches were proposed and their performances were compared on trait datasets comprising 2 to 9 time points, 91 to 455 individual tree measurements and genotype datasets of amplified length polymorphisms (AFLP), and single nucleotide polymorphism (SNP) markers. The first method was a multilevel LASSO analysis whereby trend parameter estimation and QTL mapping were conducted consecutively; the second method was our Bayesian linear mixed model whereby trends and underlying genetic effects were estimated simultaneously. We also compared several different hypothesis testing methods under either the LASSO or the Bayesian framework to perform QTL inference. In total, five and four significant QTL were observed for the intercepts and slopes, respectively, across wood traits such as earlywood percentage, wood density, radial fiberwidth, and spiral grain angle. Four of these QTL were represented by candidate gene SNPs, thus providing promising targets for future research in QTL mapping and molecular function. Bayesian and LASSO methods both detected similar sets of QTL given datasets that comprised large numbers of individuals. Copyright © 2014 Li et al.

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

PubMed

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

2001-06-19

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

Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications

PubMed Central

Silva, Claudia; Vinuesa, Pablo; Eguiarte, Luis E.; Martínez-Romero, Esperanza; Souza, Valeria

2003-01-01

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed. PMID:12571008

Neisseria meningitidis; clones, carriage, and disease.

PubMed

Read, R C

2014-05-01

Neisseria meningitidis, the cause of meningococcal disease, has been the subject of sophisticated molecular epidemiological investigation as a consequence of the significant public health threat posed by this organism. The use of multilocus sequence typing and whole genome sequencing classifies the organism into clonal complexes. Extensive phenotypic, genotypic and epidemiological information is available on the PubMLST website. The human nasopharynx is the sole ecological niche of this species, and carrier isolates show extensive genetic diversity as compared with hyperinvasive lineages. Horizontal gene exchange and recombinant events within the meningococcal genome during residence in the human nasopharynx result in antigenic diversity even within clonal complexes, so that individual clones may express, for example, more than one capsular polysaccharide (serogroup). Successful clones are capable of wide global dissemination, and may be associated with explosive epidemics of invasive disease. © 2014 The Author Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Mapping quantitative trait loci controlling milk production in dairy cattle by exploiting progeny testing

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

Georges, M.; Nielsen, D.; Mackinnon, M.

1995-02-01

We have exploited {open_quotes}progeny testing{close_quotes} to map quantitative trait loci (QTL) underlying the genetic variation of milk production in a selected dairy cattle population. A total of 1,518 sires, with progeny tests based on the milking performances of >150,000 daughters jointly, was genotyped for 159 autosomal microsatellites bracketing 1645 centimorgan or approximately two thirds of the bovine genome. Using a maximum likelihood multilocus linkage analysis accounting for variance heterogeneity of the phenotypes, we identified five chromosomes giving very strong evidence (LOD score {ge} 3) for the presence of a QTL controlling milk production: chromosomes 1, 6, 9, 10 and 20.more » These findings demonstrate that loci with considerable effects on milk production are still segregating in highly selected populations and pave the way toward marker-assisted selection in dairy cattle breeding. 44 refs., 4 figs., 3 tabs.« less

Genotypic and phenotypic diversity in tropical strains of Aspergillus spp. (section Circumdati) isolated from insects.

PubMed

Moraes, Aurea; Holanda, Veronica; Zahner, Viviane

2006-04-01

The morphology, multilocus enzyme electrophoresis (MLEE), and RAPD-PCR profiles of a panel of 63 strains of Aspergilus section Circumdati, all isoloated from Brazilian insects, were examined. When compared to the descriptions reported in the literature, differences were observed in terms of colony diameter for the representatives studies. Numerical taxonomy based on data generated by MLEE identified two distinct subgroups among the A. ochraceus isolates. In addition, phosphoglucose isomerase (GPI-1) was detected only in A. sclerotiorum, while phosphofructokinase (FK-1) and acid phosphatase (ACP-2) were present only in strains of A. sulphureus, suggesting that these alleles (bands) could be used for species-specific detection. Using RAPD-PCR, species-specific molecular markers were identified for both A. petrakii and A. sulphureus. These results are important from the taxonomic viewpoint and may also be used in the design of screening programs for the isoloation of new strains.

Estimating population sizes for elusive animals: the forest elephants of Kakum National Park, Ghana.

PubMed

Eggert, L S; Eggert, J A; Woodruff, D S

2003-06-01

African forest elephants are difficult to observe in the dense vegetation, and previous studies have relied upon indirect methods to estimate population sizes. Using multilocus genotyping of noninvasively collected samples, we performed a genetic survey of the forest elephant population at Kakum National Park, Ghana. We estimated population size, sex ratio and genetic variability from our data, then combined this information with field observations to divide the population into age groups. Our population size estimate was very close to that obtained using dung counts, the most commonly used indirect method of estimating the population sizes of forest elephant populations. As their habitat is fragmented by expanding human populations, management will be increasingly important to the persistence of forest elephant populations. The data that can be obtained from noninvasively collected samples will help managers plan for the conservation of this keystone species.

Genetic Structure of Plasmodium falciparum and Elimination of Malaria, Comoros Archipelago

PubMed Central

Rebaudet, Stanislas; Bogreau, Hervé; Silaï, Rahamatou; Lepère, Jean-François; Bertaux, Lionel; Pradines, Bruno; Delmont, Jean; Gautret, Philippe; Parola, Philippe

2010-01-01

The efficacy of malaria control and elimination on islands may depend on the intensity of new parasite inflow. On the Comoros archipelago, where falciparum malaria remains a major public health problem because of spread of drug resistance and insufficient malaria control, recent interventions for malaria elimination were planned on Moheli, 1 of 4 islands in the Comoros archipelago. To assess the relevance of such a local strategy, we performed a population genetics analysis by using multilocus microsatellite and resistance genotyping of Plasmodium falciparum sampled from each island of the archipelago. We found a contrasted population genetic structure explained by geographic isolation, human migration, malaria transmission, and drug selective pressure. Our findings suggest that malaria elimination interventions should be implemented simultaneously on the entire archipelago rather than restricted to 1 island and demonstrate the necessity for specific chemoresistance surveillance on each of the 4 Comorian islands. PMID:21029525

Development of ten microsatellite loci in the invasive giant African land snail, Achatina (=Lissachatina) fulica Bowdich, 1822

USGS Publications Warehouse

Morrison, Cheryl L.; Springmann, Marcus J.; Iwanowicz, Deborah D.; Wade, Christopher M.

2015-01-01

A suite of tetra-nucleotide microsatellite loci were developed for the invasive giant African land snail, Achatina (=Lissachatina) fulica Bowdich, 1822, from Ion Torrent next-generation sequencing data. Ten of the 96 primer sets tested amplified consistently in 30 snails from Miami, Florida, plus 12 individuals representative of their native East Africa, Indian and Pacific Ocean regions. The loci displayed moderate levels of allelic diversity (average 5.6 alleles/locus) and heterozygosity (average 42 %). Levels of genetic diversity were sufficient to produce unique multi-locus genotypes and detect phylogeographic structuring among regional samples. The invasive A. fulica can cause extensive damage to important food crops and natural resources, including native flora and fauna. The loci characterized here will be useful for determining the origins and tracking the spread of invasions, detecting fine-scale spatial structuring and estimating demographic parameters.

Development of twelve microsatellite loci in the red tree corals Primnoa resedaeformis and Primnoa pacifica

USGS Publications Warehouse

Morrison, Cheryl L.; Springmann, Marcus J.; Shroades, Kelsey; Stone, Robert P.

2015-01-01

A suite of tetra-, penta-, and hexa-nucleotide microsatellite loci were developed from Roche 454 pyrosequencing data for the cold-water octocorals Primnoa resedaeformis and P. pacifica. Twelve of 98 primer sets tested consistently amplified in 30 P. resedaeformis samples from Baltimore Canyon (western North Atlantic Ocean) and in 24 P. pacifica samples (Shutter Ridge, eastern Gulf of Alaska). The loci displayed moderate levels of allelic diversity (average 7.5 alleles/locus) and heterozygosity (average 47 %). Levels of genetic diversity were sufficient to produce unique multi-locus genotypes and to distinguish species. These common species are long-lived (hundreds of years) and provide essential fish habitat (P. pacifica), yet populations are provided little protection from human activities. These loci will be used to determine regional patterns of population connectivity to inform effective marine spatial planning and ecosystem-based fisheries management.

The epidemiology of infections with Giardia species and genotypes in well cared for dogs and cats in Germany.

PubMed

Pallant, Louise; Barutzki, Dieter; Schaper, Roland; Thompson, R C Andrew

2015-01-06

Giardia is now considered the most common enteric parasite in well cared for dogs and cats in developed countries. The ecology, epidemiology and clinical impact of infections with this parasite in such animals is still not fully understood due to variable results across different studies. Faecal samples were collected between 2009 and 2012 from privately owned cats and dogs in Germany presented to local veterinarians for a variety of reasons. Giardia positive samples were identified by microscopy and coproantigen methods. Total faecal DNA was extracted from Giardia positive samples and multilocus genotyping methods (18S rDNA, β-giardin, GDH) were applied. Relationships between host age, sex, and breed, season of presentation and the different species of Giardia detected were assessed. A total of 60 cat and 130 dog samples were identified as Giardia positive. Potentially zoonotic Giardia was identified in both animal species. Cats had a similarly high rate of infection with the G. duodenalis and G. cati. Cats less than 1 year were more likely to have G. duodenalis than cats older than 1 year. Pure breed cats demonstrated a greater proportion of zoonotic species than mixed breed cats. In samples from dogs, G. canis (C and D genotypes) were identified most commonly. Male dogs were more likely to have G. canis (genotype D) than female dogs. The 18S rDNA PCR protocol was the most successful followed by the β-giardin and GDH (amplifying from 92%, 42% and 13% of samples respectively). The potentially zoonotic species G. duodenalis and G. enterica were found in cat and dog samples, with G. duodenalis found in greater numbers; however, this may be due to the detection techniques utilised. Cats appeared to show a relationship between G. duodenalis and G. cati with age and breed, which may be explained by different housing habitats for pure and mixed breed cats. The different success rates for the three loci utilised highlights the usefulness of the 18S locus as a screening tool, as well as the importance of using multiple loci for genotyping to fully determine the level of multiple infection of Giardia present.

The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia.

PubMed

Alyamani, Essam J; Khiyami, Anamil M; Booq, Rayan Y; Majrashi, Majed A; Bahwerth, Fayez S; Rechkina, Elena

2017-01-06

The infection and prevalence of extended-spectrum β-lactamases (ESBLs) is a worldwide problem, and the presence of ESBLs varies between countries. In this study, we investigated the occurrence of plasmid-mediated ESBL/AmpC/carbapenemase/aminoglycoside resistance gene expression in Escherichia coli using phenotypic and genotypic techniques. A total of 58 E. coli isolates were collected from hospitals in the city of Makkah and screened for the production of ESBL/AmpC/carbapenemase/aminoglycoside resistance genes. All samples were subjected to phenotypic and genotypic analyses. The antibiotic susceptibility of the E. coli isolates was determined using the Vitek-2 system and the minimum inhibitory concentration (MIC) assay. Antimicrobial agents tested using the Vitek 2 system and MIC assay included the expanded-spectrum (or third-generation) cephalosporins (e.g., cefoxitin, cefepime, aztreonam, cefotaxime, ceftriaxone, and ceftazidime) and carbapenems (meropenem and imipenem). Reported positive isolates were investigated using genotyping technology (oligonucleotide microarray-based assay and PCR). The genotyping investigation was focused on ESBL variants and the AmpC, carbapenemase and aminoglycoside resistance genes. E. coli was phylogenetically grouped, and the clonality of the isolates was studied using multilocus sequence typing (MLST). Our E. coli isolates exhibited different levels of resistance to ESBL drugs, including ampicillin (96.61%), cefoxitin (15.25%), ciprofloxacin (79.66%), cefepime (75.58%), aztreonam (89.83%), cefotaxime (76.27%), ceftazidime (81.36%), meropenem (0%) and imipenem (0%). Furthermore, the distribution of ESBL-producing E. coli was consistent with the data obtained using an oligonucleotide microarray-based assay and PCR genotyping against genes associated with β-lactam resistance. ST131 was the dominant sequence type lineage of the isolates and was the most uropathogenic E. coli lineage. The E. coli isolates also carried aminoglycoside resistance genes. The evolution and prevalence of ESBL-producing E. coli may be rapidly accelerating in Saudi Arabia due to the high visitation seasons (especially to the city of Makkah). The health authority in Saudi Arabia should monitor the level of drug resistance in all general hospitals to reduce the increasing trend of microbial drug resistance and the impact on patient therapy.

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.

Insecticide use and competition shape the genetic diversity of the aphid Aphis gossypii in a cotton-growing landscape.

PubMed

Brévault, T; Carletto, J; Tribot, J; Vanlerberghe-Masutti, F

2011-08-01

Field populations of the cotton aphid, Aphis gossypii Glover, are structured into geographically widespread host races. In the cotton-producing regions of West and Central Africa (WCA), two genotypes have been repeatedly detected within the cotton host race, one of which (Burk1) is prevalent (>90%) and resistant to several insecticides, as opposed to the second one (Ivo). Here, we conducted whole plant and field cage experiments to test hypotheses for such low genetic diversity, including selection from insecticide treatments, interclonal competition and adaptation to host plant, or climatic conditions. To assess the genetic diversity of immigrant aphids, alatae were trapped and collected on cotton and relay host plants (okra and roselle) in the early cropping season. Individuals were genotyped at eight specific microsatellite loci and characterized by a multilocus genotype (MLG). When independently transferred from cotton (Gossypium hirustum L.) leaf discs to whole plants (G. hirsutum and G. arboreum, roselle and okra), Ivo and Burk1 performed equally well. When concurrently transferred from cotton leaf discs to the same plant species, Ivo performed better than Burk1, indicating that competition favoured Ivo. This was also the case on G. hirsutum growing outdoors. Conversely, Burk1 prevailed when cotton plants were sprayed with insecticides. In experiments where aphids were allowed to move to neighbouring plants, Burk1 was better represented than Ivo on low-populated plants, suggesting that dispersal may be a way to avoid competition on crowded plants. Most cotton aphids collected on cotton or relay host plants in the early cropping season were Burk1 (>90%), indicating high dispersal ability and, probably reflecting high frequency on host plants from which they dispersed. In the agricultural landscape of WCA, the use of broad-range insecticides on both cotton and relay host plants has led to the prevalence of one genotype of A. gossypii resistant to different classes of insecticides. Deployment of widespread and integrated pest management strategies are needed to restore cotton aphid control.

Genome-Wide Association Mapping of Flowering and Ripening Periods in Apple.

PubMed

Urrestarazu, Jorge; Muranty, Hélène; Denancé, Caroline; Leforestier, Diane; Ravon, Elisa; Guyader, Arnaud; Guisnel, Rémi; Feugey, Laurence; Aubourg, Sébastien; Celton, Jean-Marc; Daccord, Nicolas; Dondini, Luca; Gregori, Roberto; Lateur, Marc; Houben, Patrick; Ordidge, Matthew; Paprstein, Frantisek; Sedlak, Jiri; Nybom, Hilde; Garkava-Gustavsson, Larisa; Troggio, Michela; Bianco, Luca; Velasco, Riccardo; Poncet, Charles; Théron, Anthony; Moriya, Shigeki; Bink, Marco C A M; Laurens, François; Tartarini, Stefano; Durel, Charles-Eric

2017-01-01

Deciphering the genetic control of flowering and ripening periods in apple is essential for breeding cultivars adapted to their growing environments. We implemented a large Genome-Wide Association Study (GWAS) at the European level using an association panel of 1,168 different apple genotypes distributed over six locations and phenotyped for these phenological traits. The panel was genotyped at a high-density of SNPs using the Axiom®Apple 480 K SNP array. We ran GWAS with a multi-locus mixed model (MLMM), which handles the putatively confounding effect of significant SNPs elsewhere on the genome. Genomic regions were further investigated to reveal candidate genes responsible for the phenotypic variation. At the whole population level, GWAS retained two SNPs as cofactors on chromosome 9 for flowering period, and six for ripening period (four on chromosome 3, one on chromosome 10 and one on chromosome 16) which, together accounted for 8.9 and 17.2% of the phenotypic variance, respectively. For both traits, SNPs in weak linkage disequilibrium were detected nearby, thus suggesting the existence of allelic heterogeneity. The geographic origins and relationships of apple cultivars accounted for large parts of the phenotypic variation. Variation in genotypic frequency of the SNPs associated with the two traits was connected to the geographic origin of the genotypes (grouped as North+East, West and South Europe), and indicated differential selection in different growing environments. Genes encoding transcription factors containing either NAC or MADS domains were identified as major candidates within the small confidence intervals computed for the associated genomic regions. A strong microsynteny between apple and peach was revealed in all the four confidence interval regions. This study shows how association genetics can unravel the genetic control of important horticultural traits in apple, as well as reduce the confidence intervals of the associated regions identified by linkage mapping approaches. Our findings can be used for the improvement of apple through marker-assisted breeding strategies that take advantage of the accumulating additive effects of the identified SNPs.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans.

PubMed

Boriollo, Marcelo Fabiano Gomes; Dias, Ricardo Antunes; Fiorini, João Evangelista; Oliveira, Nelma de Mello Silva; Spolidório, Denise Madalena Palomari; de Souza, Henrique Marques Barbosa; Figueira, Antonio Vargas de Oliveira; Pizzirani-Kleiner, Aline Aparecida

2010-09-01

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE)xS(SM)(EK)xS(SM)(SSRs)). Clustering analyses showed a mean of 9+/-12.4 isolates per cluster (3.8+/-8 isolates/taxon) for MLEE, 6.2+/-4.9 isolates per cluster (4+/-4.5 isolates/taxon) for SSRs, and 4.1+/-2.3 isolates per cluster (2.6+/-2.3 isolates/taxon) for EK. A total of 45 (13%), 39 (11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (S(J)) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (c) 2010 Elsevier B.V. All rights reserved.

Genetically-Driven Enhancement of Dopaminergic Transmission Affects Moral Acceptability in Females but Not in Males: A Pilot Study

PubMed Central

Pellegrini, Silvia; Palumbo, Sara; Iofrida, Caterina; Melissari, Erika; Rota, Giuseppina; Mariotti, Veronica; Anastasio, Teresa; Manfrinati, Andrea; Rumiati, Rino; Lotto, Lorella; Sarlo, Michela; Pietrini, Pietro

2017-01-01

Moral behavior has been a key topic of debate for philosophy and psychology for a long time. In recent years, thanks to the development of novel methodologies in cognitive sciences, the question of how we make moral choices has expanded to the study of neurobiological correlates that subtend the mental processes involved in moral behavior. For instance, in vivo brain imaging studies have shown that distinct patterns of brain neural activity, associated with emotional response and cognitive processes, are involved in moral judgment. Moreover, while it is well-known that responses to the same moral dilemmas differ across individuals, to what extent this variability may be rooted in genetics still remains to be understood. As dopamine is a key modulator of neural processes underlying executive functions, we questioned whether genetic polymorphisms associated with decision-making and dopaminergic neurotransmission modulation would contribute to the observed variability in moral judgment. To this aim, we genotyped five genetic variants of the dopaminergic pathway [rs1800955 in the dopamine receptor D4 (DRD4) gene, DRD4 48 bp variable number of tandem repeat (VNTR), solute carrier family 6 member 3 (SLC6A3) 40 bp VNTR, rs4680 in the catechol-O-methyl transferase (COMT) gene, and rs1800497 in the ankyrin repeat and kinase domain containing 1 (ANKK1) gene] in 200 subjects, who were requested to answer 56 moral dilemmas. As these variants are all located in genes belonging to the dopaminergic pathway, they were combined in multilocus genetic profiles for the association analysis. While no individual variant showed any significant effects on moral dilemma responses, the multilocus genetic profile analysis revealed a significant gender-specific influence on human moral acceptability. Specifically, those genotype combinations that improve dopaminergic signaling selectively increased moral acceptability in females, by making their responses to moral dilemmas more similar to those provided by males. As females usually give more emotionally-based answers and engage the “emotional brain” more than males, our results, though preliminary and therefore in need of replication in independent samples, suggest that this increase in dopamine availability enhances the cognitive and reduces the emotional components of moral decision-making in females, thus favoring a more rationally-driven decision process. PMID:28900390

Genotyping Toxoplasma gondii from wildlife in Pennsylvania and identification of natural recombinants virulent to mice.

PubMed

Dubey, J P; Van Why, K; Verma, S K; Choudhary, S; Kwok, O C H; Khan, A; Behinke, M S; Sibley, L D; Ferreira, L R; Oliveira, S; Weaver, M; Stewart, R; Su, C

2014-02-24

Recent studies indicated the predominance of Toxoplasma gondii haplogroup 12 in wildlife in the USA. However, still little is known of the genetic diversity of this parasite circulating in wildlife. In the present study, we tested coyotes (Canis latrans), red foxes (Vulpes vulpes), white-tailed deer (Odocoileus virginianus), and geese (Branta canadensis) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 160 of 367 animals, including 92 (34.5%) of 266 coyotes, 49 (62.0%) of 79 white-tailed deer, 17 (85.0%) of 20 red fox, and two of two Canada geese tested by the modified agglutination test (cut off titer 1:25). Tissues from 105 seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 29 animals, including 10 of 53 coyotes, 11 of 16 foxes, 7 of 49 deer, and one of one goose. DNA isolated from culture-derived tachyzoites of these isolates was characterized initially using multilocus PCR-RFLP markers. Nine genotypes were revealed, including ToxoDB PCR-RFLP #1 (4 isolates), #2 (2 isolates), #3 (4 isolates), #4 (6 isolates), #5 (4 isolates), #54 (1 isolate), #141 (1 isolate), #143 (1 isolate), and #216 (6 isolates), indicating high genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of six T. gondii isolates (5 of #216 and #141) was determined in outbred Swiss Webster mice. Three of #216 and the #141 isolates were acute virulent to mice, and the other 2 #216 isolates were intermediate virulent. To determine the extent of genetic variation of these as well as a few recently reported virulent isolates from wildlife in North America, intron sequences were generated. Analysis of intron sequences and PCR-RFLP genotyping results indicated that the #216 isolates are likely derived from recombination of the clonal type I and III lineages. To determine if T. gondii virulence can be predicted by typing, we genotyped a collection of strains using PCR-RFLP markers for polymorphic genes ROP5, ROP16, ROP18 and GRA15, which are known to interact with host immune response. The results showed that there is an association of genotypes of ROP5 and ROP18 with mouse-virulence, however, additional gene(s) may also contribute to virulence in distinct T. gondii genotypes. Published by Elsevier B.V.

Molecular typing and epidemiological investigation of clinical populations of Pseudomonas aeruginosa using an oligonucleotide-microarray

PubMed Central

2012-01-01

Background Pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis (CF) patients, who are easily affected by chronic lung infections. For epidemiological purposes, discriminating P.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. A collection of 182 P. aeruginosa clinical strains isolated within Italian hospitals from patients with chronic infections, i.e. cystic fibrosis (CF) patients, and with acute infections were genotyped. Molecular typing was performed with the ArrayTube (AT) multimarker microarray (Alere Technologies GmbH, Jena, Germany), a cost-effective, time-saving and standardized method, which addresses genes from both the core and accessory P.aeruginosa genome. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were employed as reference genotyping techniques to estimate the ArrayTube resolution power. Results 41 AT-genotypes were identified within our collection, among which 14 were novel and 27 had been previously described in publicly available AT-databases. Almost 30% of the genotypes belonged to a main cluster of clones. 4B9A, EC2A, 3C2A were mostly associated to CF-patients whereas F469, 2C1A, 6C22 to non CF. An investigation on co-infections events revealed that almost 40% of CF patients were colonized by more than one genotype, whereas less than 4% were observed in non CF patients. The presence of the exoU gene correlated with non-CF patients within the intensive care unit (ICU) whereas the pKLC102-like island appeared to be prevalent in the CF centre. The congruence between the ArrayTube typing and PFGE or MLST was 0.077 and 0.559 (Adjusted Rand coefficient), respectively. AT typing of this Italian collection could be easily integrated with the global P. aeruginosa AT-typed population, uncovering that most AT-genotypes identified (> 80%) belonged to two large clonal clusters, and included 12 among the most abundant clones of the global population. Conclusions The ArrayTube (AT) multimarker array represented a robust and portable alternative to reference techniques for performing P. aeruginosa molecular typing, and allowed us to draw conclusions especially suitable for epidemiologists on an Italian clinical collection from chronic and acute infections. PMID:22840192

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.

Phenotypic and Genotypic Diversity of Thermophilic Campylobacter spp. in Commercial Turkey Flocks: A Longitudinal Study

PubMed Central

Kashoma, Isaac P.; Kumar, Anand; Sanad, Yasser M.; Gebreyes, Wondwossen; Kazwala, Rudovick R.; Garabed, Rebecca

2014-01-01

Abstract Poultry are recognized as a main reservoir of Campylobacter spp. However, longitudinal studies investigating the persistence of Campylobacter on commercial meat turkeys are rare. The objectives of this study were to determine the prevalence, antimicrobial susceptibility, and persistence of genotypically related strains of Campylobacter spp. recovered from three commercial turkey farms in Ohio belonging to a single producer. Eight hundred ten samples were collected from birds aged 1 week to slaughter, consisting of 750 fecal droppings and 60 ceca at slaughter. Overall Campylobacter prevalence was 55.9%. Multiplex polymerase chain reaction (PCR) confirmed 72.3% of all isolates as C. coli, 5.3% as C. jejuni, 10.6% as both, and 11.9% as other Campylobacter spp. PCR restriction fragment length polymorphism of the flaA gene subtyping detected 70 types—62 for C. coli and 8 for C. jejuni isolates—with most (80%) of flaA-types constituting farm homogeneous groups. Multilocus sequence typing of 99 selected Campylobacter isolates resulted in 23 sequence types (STs), consisting of 8 STs for C. jejuni and 15 STs for C. coli isolates. Six novel STs—four for C. jejuni and two—for C. coli, were detected. In a subset of isolates (n=98) tested for antimicrobial resistance, the most common resistance was to tetracycline (95%), followed by azithromycin (43%), while 42% and 18% of the isolates were resistant to ciprofloxacin and erythromycin, respectively. All isolates were susceptible to florfenicol. C. coli isolates displayed a higher proportion of resistance than C. jejuni to most antimicrobials. This study highlights the high prevalence, genotypic diversity, and antimicrobial resistance of Campylobacter spp. in commercial turkey from farm to slaughter. PMID:25184688

Clonal Differences between Non-Typhoidal Salmonella (NTS) Recovered from Children and Animals Living in Close Contact in The Gambia

PubMed Central

Dione, Michel M.; Ikumapayi, Usman N.; Saha, Debasish; Mohammed, Nuredin I.; Geerts, Stanny; Ieven, Margareta; Adegbola, Richard A.; Antonio, Martin

2011-01-01

Background Non-Typhoidal Salmonella (NTS) is an important cause of invasive bacterial disease and associated with mortality in Africa. However, little is known about the environmental reservoirs and predominant modes of transmission. Our study aimed to study the role of domestic animals in the transmission of NTS to humans in rural area of The Gambia. Methodology Human NTS isolates were obtained through an active population-based case-control surveillance study designated to determine the aetiology and epidemiology of enteric infections covering 27,567 Gambian children less than five years of age in the surveillance area. Fourteen children infected with NTS were traced back to their family compounds and anal swabs collected from 210 domestic animals present in their households. Identified NTSs were serotyped and genotyped by multi-locus sequencing typing. Principal Findings NTS was identified from 21/210 animal sources in the households of the 14 infected children. Chickens carried NTS more frequently than sheep and goats; 66.6%, 28.6% and 4.8% respectively. The most common NTS serovars were S. Colindale in humans (21.42%) and S. Poona in animals (14.28%). MLST on the 35 NTS revealed four new alleles and 24 sequence types (ST) of which 18 (75%) STs were novel. There was no overlap in serovars or genotypes of NTS recovered from humans or animal sources in the same household. Conclusion Our results do not support the hypothesis that humans and animals in close contact in the same household carry genotypically similar Salmonella serovars. These findings form an important baseline for future studies of transmission of NTS in humans and animals in Africa. PMID:21655353

Laboratory-acquired infections of Salmonella enterica serotype Typhi in South Africa: phenotypic and genotypic analysis of isolates.

PubMed

Smith, Anthony Marius; Smouse, Shannon Lucrecia; Tau, Nomsa Pauline; Bamford, Colleen; Moodley, Vineshree Mischka; Jacobs, Charlene; McCarthy, Kerrigan Mary; Lourens, Adré; Keddy, Karen Helena

2017-09-29

Workers in clinical microbiology laboratories are exposed to a variety of pathogenic microorganisms. Salmonella species is among the most commonly reported bacterial causes of laboratory-acquired infections. We report on three cases of laboratory-acquired Salmonella enterica serotype Typhi (Salmonella Typhi) infection which occurred over the period 2012 to 2016 in South Africa. Laboratory investigation included phenotypic and genotypic characterization of isolates. Phenotypic analysis included standard microbiological identification techniques, serotyping and antimicrobial susceptibility testing. Genotypic analysis included the molecular subtyping methodologies of pulsed-field gel electrophoresis analysis, multilocus sequence typing and whole-genome sequencing (WGS); with WGS data analysis including phylogenetic analysis based upon comparison of single nucleotide polymorphism profiles of isolates. All cases of laboratory-acquired infection were most likely the result of lapses in good laboratory practice and laboratory safety. The following critical issues were highlighted. There was misdiagnosis and misreporting of Salmonella Typhi as nontyphoidal Salmonella by a diagnostic laboratory, with associated public health implications. We highlight issues concerning the importance of accurate fluoroquinolone susceptibility testing and interpretation of results according to updated guidelines. We describe potential shortcomings of a single disk susceptibility screening test for fluoroquinolone susceptibility and suggest that confirmatory minimum inhibitory concentration testing should always be performed in cases of invasive Salmonella infections. These antimicrobial susceptibility testing issues resulted in inappropriate ciprofloxacin therapy which may have been responsible for failure in clearance of pathogen from patients. Salmonella Typhi capsular polysaccharide vaccine was not protective in one case, possibly secondarily to a faulty vaccine. Molecular subtyping of isolates proved effective to investigate the genetic relatedness of isolates. Molecular subtyping data interpreted together with epidemiological data allowed us to pinpoint the most likely sources for our cases of laboratory-acquired infection.

Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory.

PubMed

Herrera, C M; Bazaga, P

2011-04-01

Individual variation in ecologically important features of organisms is a crucial element in ecology and evolution, yet disentangling its underlying causes is difficult in natural populations. We applied a genomic scan approach using amplified fragment length polymorphism (AFLP) markers to quantify the genetic basis of long-term individual differences in herbivory by mammals at a wild population of the violet Viola cazorlensis monitored for two decades. In addition, methylation-sensitive amplified polymorphism (MSAP) analyses were used to investigate the association between browsing damage and epigenetic characteristics of individuals, an aspect that has been not previously explored for any wild plant. Structural equation modelling was used to identify likely causal structures linking genotypes, epigenotypes and herbivory. Individuals of V. cazorlensis differed widely in the incidence of browsing mammals over the 20-year study period. Six AFLP markers (1.6% of total) were significantly related to herbivory, accounting altogether for 44% of population-wide variance in herbivory levels. MSAP analyses revealed considerable epigenetic variation among individuals, and differential browsing damage was significantly related to variation in multilocus epigenotypes. In addition, variation across plants in epigenetic characteristics was related to variation in several herbivory-related AFLP markers. Statistical comparison of alternative causal models suggested that individual differences in herbivory are the outcome of a complex causal structure where genotypes and epigenotypes are interconnected and have direct and indirect effects on herbivory. Insofar as methylation states of MSAP markers influential on herbivory are transgenerationally heritable, herbivore-driven evolutionary changes at the study population will involve correlated changes in genotypic and epigenotypic distributions. © 2011 Blackwell Publishing Ltd.

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

Species identification and molecular typing of human Brucella isolates from Kuwait.

PubMed

Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

Species identification and molecular typing of human Brucella isolates from Kuwait

PubMed Central

Osman, Amr; Shaheed, Faraz; Khan, Mohd W.

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90–99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region. PMID:28800594

Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus).

PubMed

Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori

2014-05-01

Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals. Copyright © 2014 Elsevier B.V. All rights reserved.

Fine-Scale Genetic Structure of Monilinia fructicola During Brown Rot Epidemics Within Individual Peach Tree Canopies.

PubMed

Everhart, S E; Scherm, H

2015-04-01

The purpose of this study was to determine the fine-scale genetic structure of populations of the brown rot pathogen Monilinia fructicola within individual peach tree canopies to better understand within-tree plant pathogen diversity and to complement previous work on spatiotemporal development of brown rot disease at the canopy level. Across 3 years in a total of six trees, we monitored disease development, collected isolates from every M. fructicola symptom during the course of the season, and created high-resolution three-dimensional maps of all symptom and isolate locations within individual canopies using an electromagnetic digitizer. Each canopy population (65 to 173 isolates per tree) was characterized using a set of 13 microsatellite markers and analyzed for evidence of spatial genetic autocorrelation among isolates during the epidemic phase of the disease. Results showed high genetic diversity (average uh=0.529) and high genotypic diversity (average D=0.928) within canopies. The percentage of unique multilocus genotypes within trees was greater for blossom blight isolates (78.2%) than for fruit rot isolates (51.3%), indicating a greater contribution of clonal reproduction during the preharvest epidemic. For fruit rot isolates, between 54.2 and 81.7% of isolates were contained in one to four dominant clonal genotypes per tree having at least 10 members. All six fruit rot populations showed positive and significant spatial genetic autocorrelation for distance classes between 0.37 and 1.48 m. Despite high levels of within-tree pathogen diversity, the contribution of locally available inoculum combined with short-distance dispersal is likely the main factor generating clonal population foci and associated spatial genetic clustering within trees.

Development of Seven Microsatellite Markers Using Next Generation Sequencing for the Conservation on the Korean Population of Dorcus hopei (E. Saunders, 1854) (Coleoptera, Lucanidae)

PubMed Central

Kang, Tae Hwa; Han, Sang Hoon; Park, Sun Jae

2015-01-01

We developed microsatellite markers for genetic structural analyses of Dorcus hopei, a stag beetle species, using next generation sequencing and polymerase chain reaction (PCR)-based genotyping for regional populations. A total of 407,070,351 base pairs of genomic DNA containing >4000 microsatellite loci except AT repeats were sequenced. From 76 loci selected for primer design, 27 were polymorphic. Of these 27 markers, 10 were tested on three regional populations: two Chinese (Shichuan and Guangxi) and one Korean (Wanju). Three markers were excluded due to inconsistent amplification, genotyping errors, and Hardy-Weinberg equilibrium (HWE). By multi-locus genotyping, the allele number, observed heterozygosity and polymorphism information content of seven microsatellite loci were ranged 2‒10, 0.1333‒1.0000, and 0.1228‒0.8509, respectively. In an analysis on the genetic differentiation among regional populations including one Japanese population and one cross-breeding population, the individual colored bar-plots showed that both Chinese populations were closer to each other than to the Far East Asian populations. In Far East Asian populations, Wanju and Nirasaki populations could not be distinguished from each other because the frequency of genetic contents was very similar in some individuals of two populations. Moreover, the cross-breeding population contained all patterns of genetic contents shown in Chinese, Korean, and Japanese populations, compared with the genetic content frequency of each regional population. As a result, we examined whether the cross-breeding population might be a hybrid population, and might contain a possibility of interbreeding with Chinese populations in parental generations. Therefore, these markers will be useful for analyses of genetic diversity in populations, genetic relationships between regional populations, genetic structure analyses, and origin tests. PMID:26370965

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

PubMed Central

Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M.; Scholz, Holger C.; Splettstoesser, Wolf D.; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

2011-01-01

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse. PMID:22012011

Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

PubMed

Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M; Scholz, Holger C; Splettstoesser, Wolf D; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

2011-12-01

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

Genetic diversity and clonal characteristics of ciprofloxacin-resistant Campylobacter jejuni isolated from Chilean patients with gastroenteritis.

PubMed

Collado, Luis; Muñoz, Nataly; Porte, Lorena; Ochoa, Sofía; Varela, Carmen; Muñoz, Ivo

2018-03-01

Campylobacter jejuni is a major cause of acute gastroenteritis worldwide. However, it has also been associated with other diseases such as bacteremia and with several post-infection sequelae. Although campylobacteriosis is usually a self-limited infection, antibiotics are indicated for severe and chronic conditions. Unfortunately, several industrialised nations have reported a substantial increase in antibiotic resistance of C. jejuni. However, there is still a lack of knowledge about the epidemiology of resistance developed by this pathogen in the developing world. For this reason, our objective was to determine the resistance of clinical C. jejuni strains to ciprofloxacin and erythromycin in Chile and their associated genotypes. Fifty C. jejuni isolates recovered from fecal samples of people with acute gastroenteritis, in central and southern Chile between 2006 and 2015, were analysed. Resistance to erythromycin and ciprofloxacin was assessed by disk diffusion and agar dilution methods. Furthermore, these strains were genotyped by Multilocus Sequence Typing (MLST). Only one of the isolates was resistant to erythromycin. However, 48% of them were resistant to ciprofloxacin. The minimal inhibitory concentration of these ciprofloxacin-resistant isolates was in the range between 4 and 32 μg/ml. Moreover, MLST analyses showed that most ciprofloxacin-resistant strains were grouped into three dominant clonal complexes (ST-21, ST-48 and ST-353), while the unique strain resistant to both antibiotics belonged to the ST-45 complex. Our results evidence a high ciprofloxacin resistance and suggest that there is a dissemination of resistant clonal lineages responsible for cases of campylobacteriosis in Chile. Further studies should elucidate the origin of these resistant genotypes. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular characterization and antifungal susceptibility testing of Cryptococcus neoformans sensu stricto from southern Brazil.

PubMed

Herkert, Patricia Fernanda; Meis, Jacques F; Lucca de Oliveira Salvador, Gabriel; Rodrigues Gomes, Renata; Aparecida Vicente, Vania; Dominguez Muro, Marisol; Lameira Pinheiro, Rosangela; Lopes Colombo, Arnaldo; Vargas Schwarzbold, Alexandre; Sakuma de Oliveira, Carla; Simão Ferreira, Marcelo; Queiroz-Telles, Flávio; Hagen, Ferry

2018-04-01

Cryptococcosis is acquired from the environment by the inhalation of Cryptococcus cells and may establish from an asymptomatic latent infection into pneumonia or meningoencephalitis. The genetic diversity of a Cryptococcus neoformans species complex has been investigated by several molecular tools, such as multi-locus sequence typing, amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism and microsatellite analysis. This study aimed to investigate the genotype distributions and antifungal susceptibility profiles of C. neoformans sensu lato isolates from southern Brazil. We studied 219 C. neoformans sensu lato isolates with mating- and serotyping, AFLP fingerprinting, microsatellite typing and antifungal susceptibility testing.Results/Key findings. Among the isolates, 136 (69 %) were from HIV-positive patients. Only C. neoformans mating-type α and serotype A were observed. AFLP fingerprinting analysis divided the isolates into AFLP1/VNI (n=172; 78.5 %), AFLP1A/VNII (n=19; 8.7 %), AFLP1B/VNII (n=4; 1.8 %) and a new AFLP pattern AFLP1C (n=23; 10.5 %). All isolates were susceptible to tested antifungals and no correlation between antifungal susceptibility and genotypes was observed. Through microsatellite analysis, most isolates clustered in a major microsatellite complex and Simpson's diversity index of this population was D=0.9856. The majority of C. neoformans sensu stricto infections occurred in HIV-positive patients. C. neoformans AFLP1/VNI was the most frequent genotype and all antifungal drugs had high in vitro activity against this species. Microsatellite analyses showed a high genetic diversity within the regional C. neoformans sensu stricto population, and correlation between environmental and clinical isolates, as well as a temporal and geographic relationship.

Streptococcus pneumoniae Carriage Prevalence in Nepal: Evaluation of a Method for Delayed Transport of Samples from Remote Regions and Implications for Vaccine Implementation

PubMed Central

Hanieh, Sarah; Hamaluba, Mainga; Kelly, Dominic F.; Metz, Jane A.; Wyres, Kelly L.; Fisher, Roberta; Pradhan, Rahul; Shakya, Disuja; Shrestha, Lochan; Shrestha, Amrita; Joshi, Anip; Habens, Jocelyn; Maharjan, Bishnu D.; Thorson, Stephen; Bohler, Erik; Yu, Ly-Mee; Kelly, Sarah; Plested, Emma; John, Tessa; Werno, Anja M.; Adhikari, Neelam; Murdoch, David R.; Brueggemann, Angela B.; Pollard, Andrew J.

2014-01-01

Background Pneumococcal disease is a significant cause of morbidity and mortality in young children in Nepal, and currently available pneumococcal conjugate vaccines offer moderate coverage of invasive disease isolates. Methods A prevalence study of children aged 1.5 to 24 months in urban and rural Nepal was conducted. In the urban group, nasopharyngeal swabs (NPS) were transported using silica desiccant packages (SDP) with delayed processing (2 weeks), or skim-milk-tryptone-glucose-glycerin (STGG) with immediate processing (within 8 hours). Pneumococcal nasopharyngeal carriage prevalence, serogroup/type distribution and isolate genotypes (as defined by multilocus sequence typing) were determined. Results 1101 children were enrolled into the study: 574 in the urban group and 527 in the rural group. Overall carriage prevalence based on culture from specimens transported and stored in STGG was 58.7% (337/574), compared to 40.9% (235/574) in SDP. There was concordance of detection of pneumococcus in 67% of samples. Using the SDP method, pneumococcal carriage prevalence was higher in the rural population (69.2%; 364/526) compared to the urban population (40.9%; 235/574). Serogroup/type distribution varied with geographical location. Over half of the genotypes identified in both the urban and rural pneumococcal populations were novel. Conclusion The combination of delayed culture and transport using SDP underestimates the prevalence of pneumococcal carriage; however, in remote areas, this method could still provide a useful estimate of carriage prevalence and serogroup/type distribution. Vaccine impact is unpredictable in a setting with novel genotypes and limited serotype coverage as described here. Consequently, continued surveillance of pneumococcal isolates from carriage and disease in Nepali children following the planned introduction of pneumococcal conjugate vaccines introduction will be essential. PMID:24905574

A MLVA Genotyping Scheme for Global Surveillance of the Citrus Pathogen Xanthomonas citri pv. citri Suggests a Worldwide Geographical Expansion of a Single Genetic Lineage

PubMed Central

Boyer, Karine; Leduc, Alice; Tourterel, Christophe; Drevet, Christine; Ravigné, Virginie; Gagnevin, Lionel; Guérin, Fabien; Chiroleu, Frédéric; Koebnik, Ralf; Verdier, Valérie; Vernière, Christian

2014-01-01

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/). PMID:24897119

CONTRIBUTIONS OF SEXUAL AND ASEXUAL REPRODUCTION TO POPULATION STRUCTURE IN THE CLONAL SOFT CORAL, ALCYONIUM RUDYI.

PubMed

McFadden, Catherine S

1997-02-01

Numerous studies of population structure in sessile clonal marine invertebrates have demonstrated low genotypic diversity and nonequilibrium genotype frequencies within local populations that are monopolized by relatively few, highly replicated genets. All of the species studied to date produce planktonic sexual propagules capable of dispersing long distances; despite local genotypic disequilibria, populations are often panmictic over large geographic areas. The population structure paradigm these species represent may not be typical of the majority of clonal invertebrate groups, however, which are believed to produce highly philopatric sexual propagules. I used allozyme variation to examine the population structure of the temperate soft coral, Alcyonium rudyi, a typical clonal species whose sexually produced larvae and asexually produced ramets both have very low dispersal capabilities. Like other clonal plants and invertebrates, the local population dynamics of A. rudyi are dominated by asexual reproduction, and recruitment of new sexually produced genets occurs infrequently. As expected from its philopatric larval stage, estimates of genetic differentiation among populations of A. rudyi were highly significant at all spatial scales examined (mean θ = 0.300 among 20 populations spanning a 1100-km range), suggesting that genetic exchange seldom occurs among populations separated by as little as a few hundred meters. Mapping of multilocus allozyme genotypes within a dense aggregation of A. rudyi ramets confirmed that dispersal of asexual propagules is also very limited: members of the same genet usually remain within < 50 cm of one another on the same rock surface. Unlike most previously studied clonal invertebrates, populations of A. rudyi do not appear to be dominated by a few widespread genets: estimates of genotypic diversity (G o ) within 20 geographically distinct populations did not differ from expectations for outcrossing, sexual populations. Despite theoretical suggestions that philopatric dispersal combined with typically small effective population sizes should promote inbreeding in clonal species, inbreeding does not appear to contribute significantly to the population structure of A. rudyi. Genet genotype frequencies conformed to Hardy-Weinberg expectations in all populations, and inbreeding coefficients (f) were close to zero. In general, the population structure of A. rudyi did not differ significantly from that observed among outcrossing sexual species with philopatric larval dispersal. Age estimates suggest, however, that genets of A. rudyi live for many decades. Genet longevity may promote high genotypic diversity within A. rudyi populations and may be the most important evolutionary consequence of clonal reproduction in this species and the many others that share its dispersal characteristics. © 1997 The Society for the Study of Evolution.

Genetic Diversity of Human Pathogenic Members of the Fusarium oxysporum Complex Inferred from Multilocus DNA Sequence Data and Amplified Fragment Length Polymorphism Analyses: Evidence for the Recent Dispersion of a Geographically Widespread Clonal Lineage and Nosocomial Origin

PubMed Central

O'Donnell, Kerry; Sutton, Deanna A.; Rinaldi, Michael G.; Magnon, Karen C.; Cox, Patricia A.; Revankar, Sanjay G.; Sanche, Stephen; Geiser, David M.; Juba, Jean H.; van Burik, Jo-Anne H.; Padhye, Arvind; Anaissie, Elias J.; Francesconi, Andrea; Walsh, Thomas J.; Robinson, Jody S.

2004-01-01

Fusarium oxysporum is a phylogenetically diverse monophyletic complex of filamentous ascomycetous fungi that are responsible for localized and disseminated life-threatening opportunistic infections in immunocompetent and severely neutropenic patients, respectively. Although members of this complex were isolated from patients during a pseudoepidemic in San Antonio, Tex., and from patients and the water system in a Houston, Tex., hospital during the 1990s, little is known about their genetic relatedness and population structure. This study was conducted to investigate the global genetic diversity and population biology of a comprehensive set of clinically important members of the F. oxysporum complex, focusing on the 33 isolates from patients at the San Antonio hospital and on strains isolated in the United States from the water systems of geographically distant hospitals in Texas, Maryland, and Washington, which were suspected as reservoirs of nosocomial fusariosis. In all, 18 environmental isolates and 88 isolates from patients spanning four continents were genotyped. The major finding of this study, based on concordant results from phylogenetic analyses of multilocus DNA sequence data and amplified fragment length polymorphisms, is that a recently dispersed, geographically widespread clonal lineage is responsible for over 70% of all clinical isolates investigated, including all of those associated with the pseudoepidemic in San Antonio. Moreover, strains of the clonal lineage recovered from patients were conclusively shown to genetically match those isolated from the hospital water systems of three U.S. hospitals, providing support for the hypothesis that hospitals may serve as a reservoir for nosocomial fusarial infections. PMID:15528703

The doublesex gene integrates multi-locus complementary sex determination signals in the Japanese ant, Vollenhovia emeryi.

PubMed

Miyakawa, Misato Okamoto; Tsuchida, Koji; Miyakawa, Hitoshi

2018-03-01

A female diploid, male haploid sex determination system (haplodiploidy) is found in hymenopteran taxa, such as ants, wasps, bees and sawflies. In this system, a single, complementary sex-determination (sl-CSD) locus functions as the primary sex-determination signal. In the taxa that has evolved this system, females and males are heterozygous and hemi/homozygous at the CSD locus, respectively. While the sl-CSD system enables females to alter sex ratios in the nest, it carries a high cost in terms of inbreeding, as individuals that are homozygous at the CSD locus become sterile diploid males. To counter this risk, some of hymenopteran species have evolved a multi-locus CSD (ml-CSD) system, which effectively reduces the proportion of sterile males. However, the mechanism by which these multiple primary signals are integrated and how they affect the terminal sex-differentiation signal of the molecular cascade have not yet been clarified. To resolve these questions, we examined the molecular cascade in the Japanese ant Vollenhovia emeryi, which we previously confirmed has two CSD loci. Here, we showed that the sex-determination gene, doublesex (dsx), which is highly conserved among phylogenetically distant taxa, is responsible for integrating two CSD signals in V. emeryi. After identifying and characterizing dsx, genotypes containing two CSD loci and splicing patterns of dsx were found to correspond to the sexual phenotype, suggesting that two primary signals are integrated into dsx. These findings will facilitate future molecular and functional studies of the sex determination cascade in V. emeryi, and shed light on the evolution and diversification of sex determination systems in insects. Copyright © 2018 Elsevier Ltd. All rights reserved.

Match probabilities in a finite, subdivided population

PubMed Central

Malaspinas, Anna-Sapfo; Slatkin, Montgomery; Song, Yun S.

2011-01-01

We generalize a recently introduced graphical framework to compute the probability that haplotypes or genotypes of two individuals drawn from a finite, subdivided population match. As in the previous work, we assume an infinite-alleles model. We focus on the case of a population divided into two subpopulations, but the underlying framework can be applied to a general model of population subdivision. We examine the effect of population subdivision on the match probabilities and the accuracy of the product rule which approximates multi-locus match probabilities as a product of one-locus match probabilities. We quantify the deviation from predictions of the product rule by R, the ratio of the multi-locus match probability to the product of the one-locus match probabilities.We carry out the computation for two loci and find that ignoring subdivision can lead to underestimation of the match probabilities if the population under consideration actually has subdivision structure and the individuals originate from the same subpopulation. On the other hand, under a given model of population subdivision, we find that the ratio R for two loci is only slightly greater than 1 for a large range of symmetric and asymmetric migration rates. Keeping in mind that the infinite-alleles model is not the appropriate mutation model for STR loci, we conclude that, for two loci and biologically reasonable parameter values, population subdivision may lead to results that disfavor innocent suspects because of an increase in identity-by-descent in finite populations. On the other hand, for the same range of parameters, population subdivision does not lead to a substantial increase in linkage disequilibrium between loci. Those results are consistent with established practice. PMID:21266180

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2015-01-01

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

Characterization of Staphylococcus aureus isolates from retail chicken carcasses and pet workers in Northwest Arkansas.

PubMed

Hanning, Irene; Gilmore, David; Pendleton, Sean; Fleck, Scott; Clement, Ashley; Park, Si Hong; Scott, Erin; Ricke, Steven C

2012-01-01

Staphylococcus aureus can be carried on the skin and nasal passages of humans and animals as a commensal. A case of human methicillin-resistant S. aureus infection resulting from contact with pork has been reported. Poultry carcasses are sold at retail with the skin intact, but pork and beef typically are not. Thus, the risk of methicillin-resistant S. aureus human infection from whole raw poultry carcasses may be greater than that of exposure from pork or beef. The objective of this study was to isolate and characterize S. aureus from whole retail poultry carcasses and compare the isolates to S. aureus isolates from humans. A total of 25 S. aureus isolates were collected from 222 whole poultry carcasses. The isolates were characterized phenotypically with antibiotic resistance disc diffusion assays and genotypically using multilocus sequence typing. A total of 17 S. aureus isolates obtained from healthy humans were included and characterized in the same way as the poultry isolates. Staphylococcus spp. were recovered from all poultry carcasses. Only 25 poultry carcasses (11.2%) were contaminated with S. aureus. Of these 25 isolates, 36% were resistant to at least one of the antibiotics tested and 20% were resistant to two or more antibiotics tested. However, 100% of the human isolates were resistant to at least one of the antibiotics and 94% were resistant to two or more antibiotics. The results of the multilocus sequence typing indicate that most of the isolates grouped according to source. These results indicate a low prevalence of S. aureus present in poultry, and the isolates were not phenotypically similar to human isolates. The low number of S. aureus isolates from this study indicates that chicken carcasses would appear to not be a significant source of this bacterium.

Haplotypes in CCR5-CCR2, CCL3 and CCL5 are associated with natural resistance to HIV-1 infection in a Colombian cohort.

PubMed

Vega, Jorge A; Villegas-Ospina, Simón; Aguilar-Jiménez, Wbeimar; Rugeles, María T; Bedoya, Gabriel; Zapata, Wildeman

2017-06-01

Variants in genes encoding for HIV-1 co-receptors and their natural ligands have been individually associated to natural resistance to HIV-1 infection. However, the simultaneous presence of these variants has been poorly studied. To evaluate the association of single and multilocus haplotypes in genes coding for the viral co-receptors CCR5 and CCR2, and their ligands CCL3 and CCL5, with resistance or susceptibility to HIV-1 infection. Nine variants in CCR5-CCR2, two SNPs in CCL3 and two in CCL5 were genotyped by PCR-RFLP in 35 seropositive (cases) and 49 HIV-1-exposed seronegative Colombian individuals (controls). Haplotypes were inferred using the Arlequin software, and their frequency in individual or combined loci was compared between cases and controls by the chi-square test. A p' value ;0.05 after Bonferroni correction was considered significant. Homozygosis of the human haplogroup (HH) E was absent in controls and frequent in cases, showing a tendency to susceptibility. The haplotypes C-C and T-T in CCL3 were associated with susceptibility (p'=0.016) and resistance (p';0.0001) to HIV-1 infection, respectively. Finally, in multilocus analysis, the haplotype combinations formed by HHC in CCR5-CCR2, T-T in CCL3 and G-C in CCL5 were associated with resistance (p'=0.006). Our results suggest that specific combinations of variants in genes from the same signaling pathway can define an HIV-1 resistant phenotype. Despite our small sample size, our statistically significant associations suggest strong effects; however, these results should be further validated in larger cohorts.

Human Infections Attributable to the d-Tartrate-Fermenting Variant of Salmonella enterica Serovar Paratyphi B in Germany Originate in Reptiles and, on Rare Occasions, Poultry

PubMed Central

Toboldt, Anne; Tietze, Erhard; Helmuth, Reiner; Fruth, Angelika; Junker, Ernst

2012-01-01

In this study, the population structure, incidence, and potential sources of human infection caused by the d-tartrate-fermenting variant of Salmonella enterica serovar Paratyphi B [S. Paratyphi B (dT+)] was investigated. In Germany, the serovar is frequently isolated from broilers. Therefore, a selection of 108 epidemiologically unrelated S. enterica serovar Paratyphi B (dT+) strains isolated in Germany between 2002 and 2010 especially from humans, poultry/poultry meat, and reptiles was investigated by phenotypic and genotypic methods. Strains isolated from poultry and products thereof were strongly associated with multilocus sequence type ST28 and showed antimicrobial multiresistance profiles. Pulsed-field gel electrophoresis XbaI profiles were highly homogeneous, with only a few minor XbaI profile variants. All strains isolated from reptiles, except one, were strongly associated with ST88, another distantly related type. Most of the strains were susceptible to antimicrobial agents, and XbaI profiles were heterogeneous. Strains isolated from humans yielded seven sequence types (STs) clustering in three distantly related lineages. The first lineage, comprising five STs, represented mainly strains belonging to ST43 and ST149. The other two lineages were represented only by one ST each, ST28 and ST88. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was mostly in agreement with the multilocus sequence type. Because ST28 was frequently isolated from poultry but rarely in humans over the 9-year period investigated, overall, this study indicates that in Germany S. enterica serovar Paratyphi B (dT+) poses a health risk preferentially by contact with reptiles and, to a less extent, by exposure to poultry or poultry meat. PMID:22885742

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

PubMed Central

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

2009-01-01

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

Genetic Dissection of Maize Embryonic Callus Regenerative Capacity Using Multi-Locus Genome-Wide Association Studies

PubMed Central

Ma, Langlang; Liu, Min; Yan, Yuanyuan; Qing, Chunyan; Zhang, Xiaoling; Zhang, Yanling; Long, Yun; Wang, Lei; Pan, Lang; Zou, Chaoying; Li, Zhaoling; Wang, Yanli; Peng, Huanwei; Pan, Guangtang; Jiang, Zhou; Shen, Yaou

2018-01-01

The regenerative capacity of the embryonic callus, a complex quantitative trait, is one of the main limiting factors for maize transformation. This trait was decomposed into five traits, namely, green callus rate (GCR), callus differentiating rate (CDR), callus plantlet number (CPN), callus rooting rate (CRR), and callus browning rate (CBR). To dissect the genetic foundation of maize transformation, in this study multi-locus genome-wide association studies (GWAS) for the five traits were performed in a population of 144 inbred lines genotyped with 43,427 SNPs. Using the phenotypic values in three environments and best linear unbiased prediction (BLUP) values, as a result, a total of 127, 56, 160, and 130 significant quantitative trait nucleotides (QTNs) were identified by mrMLM, FASTmrEMMA, ISIS EM-BLASSO, and pLARmEB, respectively. Of these QTNs, 63 QTNs were commonly detected, including 15 across multiple environments and 58 across multiple methods. Allele distribution analysis showed that the proportion of superior alleles for 36 QTNs was <50% in 31 elite inbred lines. Meanwhile, these superior alleles had obviously additive effect on the regenerative capacity. This indicates that the regenerative capacity-related traits can be improved by proper integration of the superior alleles using marker-assisted selection. Moreover, a total of 40 candidate genes were found based on these common QTNs. Some annotated genes were previously reported to relate with auxin transport, cell fate, seed germination, or embryo development, especially, GRMZM2G108933 (WOX2) was found to promote maize transgenic embryonic callus regeneration. These identified candidate genes will contribute to a further understanding of the genetic foundation of maize embryonic callus regeneration. PMID:29755499

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

PubMed Central

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

2016-01-01

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

Multilocus Sequence 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

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.

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.

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.

Verifying the geographic origin of mahogany (Swietenia macrophylla King) with DNA-fingerprints.

PubMed

Degen, B; Ward, S E; Lemes, M R; Navarro, C; Cavers, S; Sebbenn, A M

2013-01-01

Illegal logging is one of the main causes of ongoing worldwide deforestation and needs to be eradicated. The trade in illegal timber and wood products creates market disadvantages for products from sustainable forestry. Although various measures have been established to counter illegal logging and the subsequent trade, there is a lack of practical mechanisms for identifying the origin of timber and wood products. In this study, six nuclear microsatellites were used to generate DNA fingerprints for a genetic reference database characterising the populations of origin of a large set of mahogany (Swietenia macrophylla King, Meliaceae) samples. For the database, leaves and/or cambium from 1971 mahogany trees sampled in 31 stands from Mexico to Bolivia were genotyped. A total of 145 different alleles were found, showing strong genetic differentiation (δ(Gregorious)=0.52, F(ST)=0.18, G(ST(Hedrick))=0.65) and clear correlation between genetic and spatial distances among stands (r=0.82, P<0.05). We used the genetic reference database and Bayesian assignment testing to determine the geographic origins of two sets of mahogany wood samples, based on their multilocus genotypes. In both cases the wood samples were assigned to the correct country of origin. We discuss the overall applicability of this methodology to tropical timber trading. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

PubMed Central

Unemo, Magnus; Dillon, Jo-Anne R.

2011-01-01

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

Contrasting patterns of genetic diversity and population structure of Armillaria mellea sensu stricto in the eastern and western United States.

PubMed

Baumgartner, Kendra; Travadon, Renaud; Bruhn, Johann; Bergemann, Sarah E

2010-07-01

ABSTRACT Armillaria mellea infects hundreds of plant species in natural and managed ecosystems throughout the Northern hemisphere. Previously reported nuclear genetic divergence between eastern and western U.S. isolates is consistent with the disjunct range of A. mellea in North America, which is restricted mainly to both coasts of the United States. We investigated patterns of population structure and genetic diversity of the eastern (northern and southern Appalachians, Ozarks, and western Great Lakes) and western (Berkeley, Los Angeles, St. Helena, and San Jose, CA) regions of the United States. In total, 156 diploid isolates were genotyped using 12 microsatellite loci. Absence of genetic differentiation within either eastern subpopulations (theta(ST) = -0.002, P = 0.5 ) or western subpopulations (theta(ST) = 0.004, P = 0.3 ) suggests that spore dispersal within each region is sufficient to prevent geographic differentiation. In contrast to the western United States, our finding of more than one genetic cluster of isolates within the eastern United States (K = 3), revealed by Bayesian assignment of multilocus genotypes in STRUCTURE and confirmed by genetic multivariate analyses, suggests that eastern subpopulations are derived from multiple founder sources. The existence of amplifiable and nonamplifiable loci and contrasting patterns of genetic diversity between the two regions demonstrate that there are two geographically isolated, divergent genetic pools of A. mellea in the United States.

Significant genetic boundaries and spatial dynamics of giant pandas occupying fragmented habitat across southwest China.

PubMed

Zhu, Lifeng; Zhang, Shanning; Gu, Xiaodong; Wei, Fuwen

2011-03-01

Understanding population history and genetic structure are key drivers of ecological research. Here, we studied two highly fragmented and isolated populations (Xiaoxiangling and Daxiangling) of giant pandas (Ailuropoda melanoleuca) at the extreme southwestern edge of their distribution. This area also contains the Dadu River, national road 108 and various human infrastructure and development, providing an ideal region in which we can identify the effects of different barriers on animal movements. We used partial mitochondrial control region (mtDNA) and nine microsatellite loci (nuclear DNA) data derived from 192 faecal and one blood sample collected from the wild. We found 136 genotypes corresponding to 53 unique multilocus genotypes and eight unique control region haplotypes (653 bp). Significant genetic boundaries correlated spatially with the Dadu River (K = 2). We estimate that a major divergence took place between these populations 26,000 years bp, at around the similar time the rock surface of valley bottom formed in Dadu River. The national road has resulted in further recent population differentiation (Pairwise F(S) on mtDNA and nuclear DNA) so that in effect, four smaller sub-populations now exist. Promisingly, we identified two possible first-generation migrants and their migration paths, and recommended the immediate construction of a number of corridors. Fortunately, the Chinese government has accepted our advice and is now planning corridor construction. © 2011 Blackwell Publishing Ltd.

Treatment of feline giardiasis during an outbreak of diarrhoea in a cattery: potential effects on faecal Escherichia coli resistance patterns.

PubMed

Tysnes, Kristoffer Relling; Luyckx, Katrien; Cantas, Leon; Robertson, Lucy J

2016-08-01

An outbreak of diarrhoea involving 16 cats at a cattery in Norway was investigated. Treatment and control of the outbreak were the primary objectives, but the effects of treatment on the antimicrobial resistance profiles of Escherichia coli isolated from faeces were also investigated. Faecal samples were investigated for Giardia cysts by immunofluorescence microscopy, and multi-locus genotyping was performed to determine the Giardia genotype. Faecal E coli were assessed, before and after treatment for giardiasis, for antimicrobial resistance. The outbreak was probably caused by Giardia duodenalis, Assemblage F. Although infection was eliminated in most cats following treatment with fenbendazole, over 30% of the infected cats required a second treatment round (combined fenbendazole and metronidazole). Investigation of sensitivity to antibacterial drugs of E coli that had been isolated both prior to and following treatment demonstrated that fenbendazole treatment may select for resistant bacteria. Controlling Giardia infections in dense cat populations can be challenging, and requires strict hygiene measures. In cases where fenbendazole alone does not result in treatment success, a combination treatment with fenbendazole and metronidazole may be effective. Although this study did not include untreated controls, we suggest that the potential for changes in gut microbiota and antimicrobial resistance development should be considered when choosing antiprotozoal drugs, particularly in cases of treatment failure and where repeat treatment is required. © ISFM and AAFP 2015.

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

PubMed

Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Genetic diversity among brazilian isolates of beauveria bassiana: comparisons with non-brazilian isolates and other beauveria species

USGS Publications Warehouse

Fernandes, E.K.K.; Moraes, A.M.L.; Pacheco, R.S.; Rangel, D.E.N.; Miller, M.P.; Bittencourt, V.R.E.P.; Roberts, D.W.

2009-01-01

Aims: The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp. Methods and Results: The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp. Conclusion: Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil. Significance and Impact of the Study: This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu. The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil. ?? 2009 The Society for Applied Microbiology.

Bridging ImmunoGenomic Data Analysis Workflow Gaps (BIGDAWG): An integrated case-control analysis pipeline.

PubMed

Pappas, Derek J; Marin, Wesley; Hollenbach, Jill A; Mack, Steven J

2016-03-01

Bridging ImmunoGenomic Data-Analysis Workflow Gaps (BIGDAWG) is an integrated data-analysis pipeline designed for the standardized analysis of highly-polymorphic genetic data, specifically for the HLA and KIR genetic systems. Most modern genetic analysis programs are designed for the analysis of single nucleotide polymorphisms, but the highly polymorphic nature of HLA and KIR data require specialized methods of data analysis. BIGDAWG performs case-control data analyses of highly polymorphic genotype data characteristic of the HLA and KIR loci. BIGDAWG performs tests for Hardy-Weinberg equilibrium, calculates allele frequencies and bins low-frequency alleles for k×2 and 2×2 chi-squared tests, and calculates odds ratios, confidence intervals and p-values for each allele. When multi-locus genotype data are available, BIGDAWG estimates user-specified haplotypes and performs the same binning and statistical calculations for each haplotype. For the HLA loci, BIGDAWG performs the same analyses at the individual amino-acid level. Finally, BIGDAWG generates figures and tables for each of these comparisons. BIGDAWG obviates the error-prone reformatting needed to traffic data between multiple programs, and streamlines and standardizes the data-analysis process for case-control studies of highly polymorphic data. BIGDAWG has been implemented as the bigdawg R package and as a free web application at bigdawg.immunogenomics.org. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Ovine Mannheimia haemolytica isolates from lungs with and without pneumonic lesions belong to similar genotypes.

PubMed

García-Alvarez, Andrés; Fernández-Garayzábal, José Francisco; Chaves, Fernando; Pinto, Chris; Cid, Dolores

2018-06-01

This study investigated the genetic characteristics of 121 ovine Mannheimia haemolytica isolates from lungs with (n = 75) and without pneumonic lesions (n = 46) using multilocus sequence typing (MLST), virulence-associated gene typing and pulsed-field gel electrophoresis (PFGE). Twelve STs were identified with most isolates (81%) belonged to ST16, ST28 and ST8. Analysis of the M. haemolytica MLST Database indicate a wide distribution of these genotypes in small ruminants, never reported in bovine isolates. This could suggest the adaptation of certain genetic lineages of M. haemolytica to small ruminants. e-BURST analysis grouped most STs into three clonal complexes (CC2, CC8 and CC28), consistent with a clonal population structure of M. haemolytica. Virulence-associated gene typing identified five virulence profiles in 64% and 65.1% of the M. haemolytica isolates from lungs with and without pneumonic lesions, respectively. These data suggest that M. haemolytica isolates from the lungs with and without pneumonic lesions are genetically homogeneous. By PGFE analysis a high level of genetic diversity was observed not only within isolates from lungs without pneumonic lesions but also among isolates from pneumonic lesions (GD 0.69 and GD 0.66, respectively; P > 0.05). These results indicate that multiple strains of M. haemolytica may be associated with individual cases of pneumonia in sheep. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Joint effect of unlinked genotypes: application to type 2 diabetes in the EPIC-Potsdam case-cohort study.

PubMed

Knüppel, Sven; Meidtner, Karina; Arregui, Maria; Holzhütter, Hermann-Georg; Boeing, Heiner

2015-07-01

Analyzing multiple single nucleotide polymorphisms (SNPs) is a promising approach to finding genetic effects beyond single-locus associations. We proposed the use of multilocus stepwise regression (MSR) to screen for allele combinations as a method to model joint effects, and compared the results with the often used genetic risk score (GRS), conventional stepwise selection, and the shrinkage method LASSO. In contrast to MSR, the GRS, conventional stepwise selection, and LASSO model each genotype by the risk allele doses. We reanalyzed 20 unlinked SNPs related to type 2 diabetes (T2D) in the EPIC-Potsdam case-cohort study (760 cases, 2193 noncases). No SNP-SNP interactions and no nonlinear effects were found. Two SNP combinations selected by MSR (Nagelkerke's R² = 0.050 and 0.048) included eight SNPs with mean allele combination frequency of 2%. GRS and stepwise selection selected nearly the same SNP combinations consisting of 12 and 13 SNPs (Nagelkerke's R² ranged from 0.020 to 0.029). LASSO showed similar results. The MSR method showed the best model fit measured by Nagelkerke's R² suggesting that further improvement may render this method a useful tool in genetic research. However, our comparison suggests that the GRS is a simple way to model genetic effects since it does not consider linkage, SNP-SNP interactions, and no non-linear effects. © 2015 John Wiley & Sons Ltd/University College London.

Characterization of Streptococcus pneumoniae isolates from Austrian companion animals and horses.

PubMed

Ginders, Maximilian; Leschnik, Michael; Künzel, Frank; Kampner, Doris; Mikula, Claudia; Steindl, Georg; Eichhorn, Inga; Feßler, Andrea T; Schwarz, Stefan; Spergser, Joachim; Loncaric, Igor

2017-11-14

The aim of the present study was to investigate the genetic relatedness and the antimicrobial resistance profiles of a collection of Austrian Streptococcus pneumoniae isolates from companion animals and horses. A total of 12 non-repetitive isolates presumptively identified as S. pneumoniae were obtained during routinely diagnostic activities between March 2009 and January 2017. Isolates were confirmed as S. pneumoniae by bile solubility and optochin susceptibility testing, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry and sequence analysis of a part recA and the 16S rRNA genes. Isolates were further characterized by pneumolysin polymerase chain reaction (PCR) and genotyped by multilocus sequence typing (MLST). Antimicrobial susceptibility testing was performed and resistance genes were detected by specific PCR assays. All isolates were serotyped. Four sequence types (ST) (ST36, ST3546, ST6934 and ST6937) and four serotypes (3, 19A, 19F and 23F) were detected. Two isolates from twelve displayed a multidrug-resistance pheno- and genotype. This study represents the first comprehensive investigation on characteristics of S. pneumoniae isolates recovered from Austrian companion animals and horses. The obtained results indicate that common human sero- (23F) and sequence type (ST36) implicated in causing invasive pneumococcal disease (IPD) may circulate in dogs. Isolates obtained from other examined animals seem to be host-adapted.

Forensic and population genetic characteristics of 62 X chromosome SNPs revealed by multiplex PCR and MALDI-TOF mass spectrometry genotyping in 4 North Eurasian populations.

PubMed

Stepanov, Vadim; Vagaitseva, Ksenyia; Kharkov, Vladimir; Cherednichenko, Anastasia; Bocharova, Anna; Berezina, Galina; Svyatova, Gulnara

2016-01-01

X chromosome genetic markers are widely used in basic population genetic research as well as in forensic genetics. In this paper we analyze the genetic diversity of 62 X chromosome SNPs in 4 populations using multiplex genotyping based on multi-locus PCR and MALDI-TOF mass spectrometry, and report forensic and population genetic features of the panel of X-linked SNPs (XSNPid). Studied populations represent Siberian (Buryat and Khakas), North Asian (Khanty) and Central Asian (Kazakh) native people. Khanty, Khakas and Kazakh population demonstrate average gene diversity over 0.45. Only East Siberian Buryat population is characterized by lower average heterozygosity (0.436). AMOVA analysis of genetic structure reveals a relatively low but significant level of genetic differentiation in a group of 4 population studied (FST=0.023, p=0.0000). The XSNPid panel provides a very high discriminating power in each population. The combined probability of discrimination in females (PDf) for XSNPid panel ranged between populations from 0.99999999999999999999999982 in Khakas to 0.9999999999999999999999963 in Buryats. The combined discriminating power in males (PDm) varies from 0.999999999999999792 to 0.9999999999999999819. The developed multiplex set of X chromosome SNPs can be a useful tool for population genetic studies and for forensic identity and kinship testing. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Molecular Diversity of Seed-borne Fusarium Species Associated with Maize in India

PubMed Central

Aiyaz, Mohammed; Divakara, Shetty Thimmappa; Mudili, Venkataramana; Moore, Geromy George; Gupta, Vijai Kumar; Yli-Mattila, Tapani; Nayaka, Siddaiah Chandra; Niranjana, Siddapura Ramachandrappa

2016-01-01

A total of 106 maize seed samples were collected from different agro-climatic regions of India. Sixty-two Fusarium isolates were recovered, 90% of which were identified as Fusarium verticillioides based on morphological and molecular characters. Use of the tef-1α gene corrected/refined the morphological species identifications of 11 isolates, and confirmed those of the remaining isolates. Genetic diversity among the Fusarium isolates involved multilocus fingerprinting profiles by Inter Simple Sequence Repeats (ISSR) UPGMA and tef-1α gene phenetic analyses; for which, we observed no significant differences among the isolates based on geographic origin or fumonisin production; most of the subdivision related to species. Genotyping was performed on the F. verticillioides isolates, using 12 primer sets from the fumonisin pathway, to elucidate the molec-ular basis of fumonisin production or non-production. One fumonisin-negative isolate, UOMMF-16, was unable to amplify nine of the 12 fumonisin cluster genes tested. We also used the CD-ELISA method to confirm fumonisin production for our 62 Fusarium isolates. Only 15 isolates were found to be fumonisin-negative. Interestingly, genotypic characterization re-vealed six isolates with various gene deletion patterns that also tested positive for the production of fumonisins via CD-ELISA. Our findings confirm the importance of molecular studies for species delimitation, and for observing genetic and phenotypic diversity, among the Fusaria. PMID:27226769

Historical and biological determinants of genetic diversity in the highly endemic triploid sea lavender Limonium dufourii (Plumbaginaceae).

PubMed

Palop-Esteban, M; Segarra-Moragues, J G; González-Candelas, F

2007-09-01

Microsatellite markers were used to evaluate the genetic diversity and population genetic structure in the critically endangered Limonium dufourii (Plumbaginaceae), a highly endemic triploid species from the coasts of eastern Spain. Sixty-five alleles from 13 microsatellite regions were amplified in a sample of 122 individuals collected from the six extant populations. Microsatellite patterns were consistent with the triploid nature of L. dufourii. Alleles were unambiguously assigned to two different parental subgenomes in this hybrid species and the greater contribution of the diploid parental subgenome was confirmed. Eleven, 25 and 26 multilocus genotypes were recorded from the haploid, diploid and from the combined information of both subgenomes, respectively. Genetic diversity was mostly distributed among populations (72.06% of the total genetic variation). Genotypes from Marjal del Moro populations grouped into two highly structured clusters (88.41% of the total variance). The observed patterns of distribution of genetic diversity are interpreted to result from multiple hybridization events and isolation between populations. Threats to this species are mainly anthropogenic (urbanization and tourism pressure), although stochastic risks cannot be ignored. Therefore, in order to preserve extant genetic variation of L. dufourii, in situ strategies such as the preservation of its habitat are a high priority. Several recommendations in order to assist ex situ measures to guarantee the success of conservation strategies and maintain the relationships between individuals and populations are proposed.

Impacts of early viability selection on management of inbreeding and genetic diversity in conservation.

PubMed

Grueber, Catherine E; Hogg, Carolyn J; Ivy, Jamie A; Belov, Katherine

2015-04-01

Maintaining genetic diversity is a crucial goal of intensive management of threatened species, particularly for those populations that act as sources for translocation or re-introduction programmes. Most captive genetic management is based on pedigrees and a neutral theory of inheritance, an assumption that may be violated by selective forces operating in captivity. Here, we explore the conservation consequences of early viability selection: differential offspring survival that occurs prior to management or research observations, such as embryo deaths in utero. If early viability selection produces genotypic deviations from Mendelian predictions, it may undermine management strategies intended to minimize inbreeding and maintain genetic diversity. We use empirical examples to demonstrate that straightforward approaches, such as comparing litter sizes of inbred vs. noninbred breeding pairs, can be used to test whether early viability selection likely impacts estimates of inbreeding depression. We also show that comparing multilocus genotype data to pedigree predictions can reveal whether early viability selection drives systematic biases in genetic diversity, patterns that would not be detected using pedigree-based statistics alone. More sophisticated analysis combining genomewide molecular data with pedigree information will enable conservation scientists to test whether early viability selection drives deviations from neutrality across wide stretches of the genome, revealing whether this form of selection biases the pedigree-based statistics and inference upon which intensive management is based. © 2015 John Wiley & Sons Ltd.

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

PubMed Central

Limmathurotsakul, Direk; Max, Tamara L.; Sarovich, Derek S.; Vogler, Amy J.; Dale, Julia L.; Ginther, Jennifer L.; Leadem, Benjamin; Colman, Rebecca E.; Foster, Jeffrey T.; Tuanyok, Apichai; Wagner, David M.; Peacock, Sharon J.; Pearson, Talima; Keim, Paul

2010-01-01

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum. PMID:20090837

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

PubMed Central

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

2014-01-01

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

Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal.

PubMed

Paudel, Sarad; Mikota, Susan K; Nakajima, Chie; Gairhe, Kamal P; Maharjan, Bhagwan; Thapa, Jeewan; Poudel, Ajay; Shimozuru, Michito; Suzuki, Yasuhiko; Tsubota, Toshio

2014-05-01

Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of polysomic microsatellite markers for characterization of population structuring and phylogeography in the shortnose sturgeon (Acipenser brevirostrum)

USGS Publications Warehouse

Henderson, Anne P.; King, Tim L.

2012-01-01

Shortnose sturgeon Acipenser brevirostrum is an endangered polyploid fish species for which no nuclear DNA markers previously existed. To address this need, 86 polysomic loci were developed and characterized in 20 A. brevirostrum from five river systems and eight members (parents and six progeny) of a captive-bred family. All markers proved to be polymorphic, polysomic, and demonstrated direct inheritance when tested in a captive family. Eleven loci were included in a range-wide survey of 561 fish sampled from 17 geographic collections. Allelic diversity at these markers ranged from 7 to 24 alleles/locus and averaged 16.5 alleles/locus; sufficient diversity to produce unique multilocus genotypes. In the range-wide survey, a Mantel comparison of an ecological (1-Jaccard’s) and genetic (ΦPT; an analog to FST) distance metrics, identified a strong positive correlation (r = 0.98, P PT represents a viable metric for assessing genetic relatedness using this class of marker.

Polymorphic SSR Markers for Plasmopara obducens (Peronosporaceae), the Newly Emergent Downy Mildew Pathogen of Impatiens (Balsaminaceae)

DOE PAGES

Salgado-Salazar, Catalina; Rivera, Yazmín; Veltri, Daniel; ...

2015-11-10

Premise of the study: Simple sequence repeat (SSR) markers were developed for Plasmopara obducens, the causal agent of the newly emergent downy mildew disease of Impatiens walleriana. Methods and Results: A 202-Mb draft genome assembly was generated from P. obducens using Illumina technology and mined to identify 13,483 SSR motifs. Primers were synthesized for 62 marker candidates, of which 37 generated reliable PCR products. Testing of the 37 markers using 96 P. obducens samples showed 96% of the markers were polymorphic, with 2-6 alleles observed. Observed and expected heterozygosity ranged from 0.000-0.892 and 0.023-0.746, respectively. Just 17 markers were sufficientmore » to identify all multilocus genotypes. Conclusions: These are the first SSR markers available for this pathogen, and one of the first molecular resources. These markers will be useful in assessing variation in pathogen populations and determining the factors contributing to the emergence of destructive impatiens downy mildew disease.« less

An outbreak of East Coast Fever on the Comoros: a consequence of the import of immunised cattle from Tanzania?

PubMed

De Deken, R; Martin, V; Saido, A; Madder, M; Brandt, J; Geysen, D

2007-02-28

In 2003 and 2004, a severe epidemic decimated the cattle population on Grand Comore, the largest island of the Union of Comoros. Fatalities started soon after the import of cattle from Tanzania. Theileria parva and its vector, Rhipicephalus appendiculatus, could be identified as the main culprits of the epidemic. Characterisation by multilocus genotyping revealed that the T. parva parasites isolated on the Comoros were identical to the components of the Muguga cocktail vaccine used in Tanzania to immunise cattle. Therefore, it is believed that East Coast Fever reached the Comoros while some of the imported livestock got infected in Tanzania by ticks of which the immature stadia fed on Muguga cocktail vaccinated animals. Since the Comorian government neither has the financial means nor the competent staff to pursue an adequate epidemiosurveillance, the danger exists that without external assistance and in a context of continuing globalisation more transboundary diseases will affect the Comorian livestock sector in the future.

Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers.

PubMed

Depoorter, Eliza; Bull, Matt J; Peeters, Charlotte; Coenye, Tom; Vandamme, Peter; Mahenthiralingam, Eshwar

2016-06-01

Burkholderia is an incredibly diverse and versatile Gram-negative genus, within which over 80 species have been formally named and multiple other genotypic groups likely represent new species. Phylogenetic analysis based on the 16S rRNA gene sequence and core genome ribosomal multilocus sequence typing analysis indicates the presence of at least three major clades within the genus. Biotechnologically, Burkholderia are well-known for their bioremediation and biopesticidal properties. Within this review, we explore the ability of Burkholderia to synthesise a wide range of antimicrobial compounds ranging from historically characterised antifungals to recently described antibacterial antibiotics with activity against multiresistant clinical pathogens. The production of multiple Burkholderia antibiotics is controlled by quorum sensing and examples of quorum sensing pathways found across the genus are discussed. The capacity for antibiotic biosynthesis and secondary metabolism encoded within Burkholderia genomes is also evaluated. Overall, Burkholderia demonstrate significant biotechnological potential as a source of novel antibiotics and bioactive secondary metabolites.

Accurate determination of genetic identity for a single cacao bean, using molecular markers with a nanofluidic system, ensures cocoa authentication.

PubMed

Fang, Wanping; Meinhardt, Lyndel W; Mischke, Sue; Bellato, Cláudia M; Motilal, Lambert; Zhang, Dapeng

2014-01-15

Cacao (Theobroma cacao L.), the source of cocoa, is an economically important tropical crop. One problem with the premium cacao market is contamination with off-types adulterating raw premium material. Accurate determination of the genetic identity of single cacao beans is essential for ensuring cocoa authentication. Using nanofluidic single nucleotide polymorphism (SNP) genotyping with 48 SNP markers, we generated SNP fingerprints for small quantities of DNA extracted from the seed coat of single cacao beans. On the basis of the SNP profiles, we identified an assumed adulterant variety, which was unambiguously distinguished from the authentic beans by multilocus matching. Assignment tests based on both Bayesian clustering analysis and allele frequency clearly separated all 30 authentic samples from the non-authentic samples. Distance-based principle coordinate analysis further supported these results. The nanofluidic SNP protocol, together with forensic statistical tools, is sufficiently robust to establish authentication and to verify gourmet cacao varieties. This method shows significant potential for practical application.

Human Staphylococcus aureus lineages among Zoological Park residents in Greece

PubMed Central

Drougka, E.; Foka, A.; Posantzis, D.; Giormezis, N.; Anastassiou, E.D.; Petinaki, E.; Spiliopoulou, I.

2015-01-01

Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL) and tst (toxic shock syndrome toxin-1) were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST), spa type and Pulsed-Field Gel Electrophoresis (PFGE). Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred. PMID:26623381

Invasive Disease Due to Nontypeable Haemophilus influenzae among Children in Arkansas

PubMed Central

O'Neill, Joshua M.; St. Geme III, Joseph W.; Cutter, David; Adderson, Elisabeth E.; Anyanwu, Juliana; Jacobs, Richard F.; Schutze, Gordon E.

2003-01-01

In this study, we reviewed cases of invasive disease due to nontypeable Haemophilus influenzae among children hospitalized at Arkansas Children's Hospital from 1993 to 2001. A total of 28 cases were examined, including 21 associated with bacteremia and 4 associated with meningitis. Of the patients examined, 86% were ≤4 years of age, and 68% had underlying medical conditions. Characterization of the bacterial isolates by multilocus sequence type genotyping revealed significant overall genetic diversity, similar to the diversity in the general population structure for nontypeable H. influenzae. However, four separate pairs of isolates were closely related genetically, a relationship confirmed by pulsed-field gel electrophoresis and Southern hybridization studies using probes for the major H. influenzae adhesin genes. These results suggest that selected strains of nontypeable H. influenzae may have more invasive potential, especially in young children and patients with underlying medical conditions. At this point, the specific factors that contribute to enhanced virulence remain unclear. PMID:12843045

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

USGS Publications Warehouse

Vogler, Amy J.; Nottingham, Roxanne; Busch, Joseph D.; Sahl, Jason W.; Shuey, Megan M.; Foster, Jeffrey T.; Schupp, James M.; Smith, Susan; Rocke, Tonie E.; Klein, Paul; Wagner, David M.

2016-01-01

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

Genetic patterns across multiple introductions of the globally invasive crab genus Carcinus.

PubMed

Darling, John A; Bagley, Mark J; Roman, Joe; Tepolt, Carolyn K; Geller, Jonathan B

2008-12-01

The European green crab Carcinus maenas is one of the world's most successful aquatic invaders, having established populations on every continent with temperate shores. Here we describe patterns of genetic diversity across both the native and introduced ranges of C. maenas and its sister species, C. aestuarii, including all known non-native populations. The global data set includes sequences from the mitochondrial cytochrome c oxidase subunit I gene, as well as multilocus genotype data from nine polymorphic nuclear microsatellite loci. Combined phylogeographic and population genetic analyses clarify the global colonization history of C. maenas, providing evidence of multiple invasions to Atlantic North America and South Africa, secondary invasions to the northeastern Pacific, Tasmania, and Argentina, and a strong likelihood of C. maenas x C. aestuarii hybrids in South Africa and Japan. Successful C. maenas invasions vary broadly in the degree to which they retain genetic diversity, although populations with the least variation typically derive from secondary invasions or from introductions that occurred more than 100 years ago.

Pathogenic Potential to Humans of Bovine Escherichia coli O26, Scotland

PubMed Central

Rosser, Tracy; Allison, Lesley J.; Courcier, Emily; Evans, Judith; McKendrick, Iain J.; Pearce, Michael C.; Handel, Ian; Caprioli, Alfredo; Karch, Helge; Hanson, Mary F.; Pollock, Kevin G.J.; Locking, Mary E.; Woolhouse, Mark E.J.; Matthews, Louise; Low, J. Chris; Gally, David L.

2012-01-01

Escherichia coli O26 and O157 have similar overall prevalences in cattle in Scotland, but in humans, Shiga toxin–producing E. coli O26 infections are fewer and clinically less severe than E. coli O157 infections. To investigate this discrepancy, we genotyped E. coli O26 isolates from cattle and humans in Scotland and continental Europe. The genetic background of some strains from Scotland was closely related to that of strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type 21 strains and confirmed the role of stx2 in severe human disease. Although the prevalences of E. coli O26 and O157 on cattle farms in Scotland are equivalent, prevalence of more virulent strains is low, reducing human infection risk. However, new data on E. coli O26–associated HUS in humans highlight the need for surveillance of non-O157 enterohemorrhagic E. coli and for understanding stx2 phage acquisition. PMID:22377426

Inferring HIV Escape Rates from Multi-Locus Genotype Data

DOE PAGES

Kessinger, Taylor A.; Perelson, Alan S.; Neher, Richard A.

2013-09-03

Cytotoxic T-lymphocytes (CTLs) recognize viral protein fragments displayed by major histocompatibility complex molecules on the surface of virally infected cells and generate an anti-viral response that can kill the infected cells. Virus variants whose protein fragments are not efficiently presented on infected cells or whose fragments are presented but not recognized by CTLs therefore have a competitive advantage and spread rapidly through the population. We present a method that allows a more robust estimation of these escape rates from serially sampled sequence data. The proposed method accounts for competition between multiple escapes by explicitly modeling the accumulation of escape mutationsmore » and the stochastic effects of rare multiple mutants. Applying our method to serially sampled HIV sequence data, we estimate rates of HIV escape that are substantially larger than those previously reported. The method can be extended to complex escapes that require compensatory mutations. We expect our method to be applicable in other contexts such as cancer evolution where time series data is also available.« less

Single-Locus versus Multilocus Patterns of Local Adaptation to Climate in Eastern White Pine (Pinus strobus, Pinaceae)

PubMed Central

Zinck, John W. R.

2016-01-01

Natural plant populations are often adapted to their local climate and environmental conditions, and populations of forest trees offer some of the best examples of this pattern. However, little empirical work has focused on the relative contribution of single-locus versus multilocus effects to the genetic architecture of local adaptation in plants/forest trees. Here, we employ eastern white pine (Pinus strobus) to test the hypothesis that it is the inter-genic effects that primarily drive climate-induced local adaptation. The genetic structure of 29 range-wide natural populations of eastern white pine was determined in relation to local climatic factors using both a reference set of SSR markers, and SNPs located in candidate genes putatively involved in adaptive response to climate. Comparisons were made between marker sets using standard single-locus outlier analysis, single-locus and multilocus environment association analyses and a novel implementation of Population Graphs. Magnitudes of population structure were similar between the two marker sets. Outlier loci consistent with diversifying selection were rare for both SNPs and SSRs. However, genetic distances based on the multilocus among population covariances (cGD) were significantly more correlated to climate, even after correcting for spatial effects, for SNPs as compared to SSRs. Coalescent simulations confirmed that the differences in mutation rates between SSRs and SNPs did not affect the topologies of the Population Graphs, and hence values of cGD and their correlations with associated climate variables. We conclude that the multilocus covariances among populations primarily reflect adaptation to local climate and environment in eastern white pine. This result highlights the complexity of the genetic architecture of adaptive traits, as well as the need to consider multilocus effects in studies of local adaptation. PMID:27387485

Multilocus methylation analysis in a large cohort of 11p15-related foetal growth disorders (Russell Silver and Beckwith Wiedemann syndromes) reveals simultaneous loss of methylation at paternal and maternal imprinted loci.

PubMed

Azzi, Salah; Rossignol, Sylvie; Steunou, Virginie; Sas, Theo; Thibaud, Nathalie; Danton, Fabienne; Le Jule, Maryline; Heinrichs, Claudine; Cabrol, Sylvie; Gicquel, Christine; Le Bouc, Yves; Netchine, Irene

2009-12-15

Genomic imprinting plays an important role in mammalian development. Loss of imprinting (LOI) through loss (LOM) or gain (GOM) of methylation is involved in many human disorders and cancers. The imprinted 11p15 region is crucial for the control of foetal growth and LOI at this locus is implicated in two clinically opposite disorders: Beckwith Wiedemann syndrome (BWS) with foetal overgrowth associated with an enhanced tumour risk and Russell-Silver syndrome (RSS) with intrauterine and postnatal growth restriction. So far, only a few studies have assessed multilocus LOM in human imprinting diseases. To investigate multilocus LOI syndrome, we studied the methylation status of five maternally and two paternally methylated loci in a large series (n = 167) of patients with 11p15-related foetal growth disorders. We found that 9.5% of RSS and 24% of BWS patients showed multilocus LOM at regions other than ICR1 and ICR2 11p15, respectively. Moreover, over two third of multilocus LOM RSS patients also had LOM at a second paternally methylated locus, DLK1/GTL2 IG-DMR. No additional clinical features due to LOM of other loci were found suggesting an (epi)dominant effect of the 11p15 LOM on the clinical phenotype for this series of patients. Surprisingly, four patients displayed LOM at both ICR1 and ICR2 11p15. Three of them had a RSS and one a BWS phenotype. Our results show for the first time that multilocus LOM can also concern RSS patients. Moreover, LOM can involve both paternally and maternally methylated loci in the same patient.

Multilocus sequence analysis of phytopathogenic species of the genus Streptomyces

USDA-ARS?s Scientific Manuscript database

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

Measuring and partitioning the high-order linkage disequilibrium by multiple order Markov chains.

PubMed

Kim, Yunjung; Feng, Sheng; Zeng, Zhao-Bang

2008-05-01

A map of the background levels of disequilibrium between nearby markers can be useful for association mapping studies. In order to assess the background levels of linkage disequilibrium (LD), multilocus LD measures are more advantageous than pairwise LD measures because the combined analysis of pairwise LD measures is not adequate to detect simultaneous allele associations among multiple markers. Various multilocus LD measures based on haplotypes have been proposed. However, most of these measures provide a single index of association among multiple markers and does not reveal the complex patterns and different levels of LD structure. In this paper, we employ non-homogeneous, multiple order Markov Chain models as a statistical framework to measure and partition the LD among multiple markers into components due to different orders of marker associations. Using a sliding window of multiple markers on phased haplotype data, we compute corresponding likelihoods for different Markov Chain (MC) orders in each window. The log-likelihood difference between the lowest MC order model (MC0) and the highest MC order model in each window is used as a measure of the total LD or the overall deviation from the gametic equilibrium for the window. Then, we partition the total LD into lower order disequilibria and estimate the effects from two-, three-, and higher order disequilibria. The relationship between different orders of LD and the log-likelihood difference involving two different orders of MC models are explored. By applying our method to the phased haplotype data in the ENCODE regions of the HapMap project, we are able to identify high/low multilocus LD regions. Our results reveal that the most LD in the HapMap data is attributed to the LD between adjacent pairs of markers across the whole region. LD between adjacent pairs of markers appears to be more significant in high multilocus LD regions than in low multilocus LD regions. We also find that as the multilocus total LD increases, the effects of high-order LD tends to get weaker due to the lack of observed multilocus haplotypes. The overall estimates of first, second, third, and fourth order LD across the ENCODE regions are 64, 23, 9, and 3%.

Novel fusarium head blight pathogens from Nepal and Louisiana revealed by multilocus genealogical concordance

USDA-ARS?s Scientific Manuscript database

This study was conducted to assess evolutionary relationships, species diversity, and trichothecene toxin potential of five Fusarium graminearum complex (FGSC) isolates identified as genetically novel during prior Fusarium head blight (FHB) surveys in Nepal and Louisiana. Results of a multilocus gen...

Environmental Transport of Emerging Human-Pathogenic Cryptosporidium Species and Subtypes through Combined Sewer Overflow and Wastewater

PubMed Central

Huang, Chengchen; Hu, Yue; Wang, Lin; Wang, Yuanfei; Li, Na; Guo, Yaqiong; Xiao, Lihua

2017-01-01

ABSTRACT The environmental transport of Cryptosporidium spp. through combined sewer overflow (CSO) and the occurrence of several emerging human-pathogenic Cryptosporidium species in developing countries remain unclear. In this study, we collected 40 CSO samples and 40 raw wastewater samples from Shanghai, China, and examined them by PCR and DNA sequencing for Cryptosporidium species (targeting the small subunit rRNA gene) and Giardia duodenalis (targeting the triosephosphate isomerase, β-giardin, and glutamate dehydrogenase genes) and Enterocytozoon bieneusi (targeting the ribosomal internal transcribed spacer) genotypes. Human-pathogenic Cryptosporidium species were further subtyped by sequence analysis of the 60-kDa glycoprotein gene, with additional multilocus sequence typing on the emerging zoonotic pathogen Cryptosporidium ubiquitum. Cryptosporidium spp., G. duodenalis, and E. bieneusi were detected in 12 and 15, 33 and 32, and 37 and 40 CSO and wastewater samples, respectively, including 10 Cryptosporidium species, 3 G. duodenalis assemblages, and 8 E. bieneusi genotypes. In addition to Cryptosporidium hominis and Cryptosporidium parvum, two new pathogens identified in industrialized nations, C. ubiquitum and Cryptosporidium viatorum, were frequently detected. The two novel C. ubiquitum subtype families identified appeared to be genetic recombinants of known subtype families. Similarly, the dominant group 1 E. bieneusi genotypes and G. duodenalis subassemblage AII are known human pathogens. The similar distribution of human-pathogenic Cryptosporidium species and E. bieneusi and G. duodenalis genotypes between wastewater and CSO samples reaffirms that storm overflow is potentially a significant contamination source of pathogens in surface water. The frequent identification of C. ubiquitum and C. viatorum in urban wastewater suggests that these newly identified human pathogens may be endemic in China. IMPORTANCE Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are major waterborne pathogens. Their transport into surface water through combined sewer overflow, which remains largely untreated in developing countries, has not been examined. In addition, the identification of these pathogens to genotypes and subtypes in urban storm overflow and wastewater is necessary for rapid and accurate assessment of pathogen transmission in humans and transport in the environment. Data from this study suggest that, like untreated urban wastewater, combined sewer overflow is commonly contaminated with human-pathogenic Cryptosporidium, G. duodenalis, and E. bieneusi genotypes and subtypes, and urban storm overflow potentially plays a significant role in the contamination of drinking source water and recreational water with human pathogens. They also indicate that Cryptosporidium ubiquitum and Cryptosporidium viatorum, two newly identified human pathogens, may be common in China, and genetic recombination can lead to the emergence of novel C. ubiquitum subtype families. PMID:28600310

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

PubMed Central

Guinard, Jérémy; Latreille, Anne; Guérin, Fabien; Poussier, Stéphane

2016-01-01

ABSTRACT Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations. IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the population genetics of plant pathogen adaptation remains an open, unexplored field, especially for plant-pathogenic bacteria. MLVA has become increasingly popular for epidemiosurveillance and molecular epidemiology studies of plant pathogens. However, this method has been used mostly for genotyping and identification on a regional or global scale. In this study, we developed a new MLVA scheme, targeting phylotype I of the soilborne Ralstonia solanacearum species complex (RSSC), specifically to address the bacterial population genetics on the field scale. Such a MLVA scheme, based on fast-evolving loci, may be a tool of choice for field experimental evolution and spatial genetics studies. PMID:28003195

Genotypes and oxacillin resistance of Staphylococcus aureus from chicken and chicken meat in Poland.

PubMed

Krupa, P; Bystroń, J; Bania, J; Podkowik, M; Empel, J; Mroczkowska, A

2014-12-01

The genotypes and oxacillin resistance of 263 Staphylococcus aureus isolates cultured from chicken cloacae (n = 138) and chicken meat (n = 125) was analyzed. Fifteen spa types were determined in the studied S. aureus population. Among 5 staphylococcal protein A gene (spa) types detected in S. aureus from chicken, t002, t3478, and t13620 were the most frequent. Staphylococcus aureus isolates from meat were assigned to 14 spa types. Among them, the genotypes t002, t056, t091, t3478, and t13620 were dominant. Except for 4 chicken S. aureus isolates belonging to CC398, the remaining 134 isolates were clustered into multilocus sequence clonal complex (CC) 5. Most of meat-derived isolates were assigned to CC5, CC7, and CC15, and to the newly described spa-CC12954 complex belonging to CC1. Except for t011 (CC398), all other spa types found among chicken isolates were also present in isolates from meat. Four S. aureus isolated from chicken and one from meat were identified as methicillin-resistant S. aureus (MRSA) with oxacillin minimum inhibitory concentrations from 16 to 64 μg/mL. All MRSA were assigned to spa types belonging to ST398, and included 4 animal spa t011 SCCmecV isolates and 1 meat-derived spa t899, SCCmecIV isolate. Borderline oxacillin-resistant S. aureus (BORSA) isolates, shown to grow on plates containing 2 to 3 μg/mL of oxacillin, were found within S. aureus isolates from chicken (3 isolates) and from meat (19 isolates). The spa t091 and t084 dominated among BORSA from chicken meat, whereas t548 and t002 were found within animal BORSA. We report for the first time the presence of MRSA in chicken in Poland. We demonstrate that MRSA CC398 could be found in chicken meat indicating potential of introduction of animal-associated genotypes into the food chain. We also report for the first time the possibility of transmission of BORSA isolates from chicken to meat. ©2014 Poultry Science Association Inc.

Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China.

PubMed

Zhu, Yinchu; Dong, Wenyang; Ma, Jiale; Yuan, Lvfeng; Hejair, Hassan M A; Pan, Zihao; Liu, Guangjin; Yao, Huochun

2017-04-08

Swine extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that leads to economic and welfare costs in the swine industry worldwide, and is occurring with increasing frequency in China. By far, various virulence factors have been recognized in ExPEC. Here, we investigated the virulence genotypes and clonal structure of collected strains to improve the knowledge of phylogenetic traits of porcine ExPECs in China. We isolated 64 Chinese porcine ExPEC strains from 2013 to 14 in China. By multiplex PCR, the distribution of isolates belonging to phylogenetic groups B1, B2, A and D was 9.4%, 10.9%, 57.8% and 21.9%, respectively. Nineteen virulence-related genes were detected by PCR assay; ompA, fimH, vat, traT and iutA were highly prevalent. Virulence-related genes were remarkably more prevalent in group B2 than in groups A, B1 and D; notably, usp, cnf1, hlyD, papA and ibeA were only found in group B2 strains. Genotyping analysis was performed and four clusters of strains (named I to IV) were identified. Cluster IV contained all isolates from group B2 and Cluster IV isolates had the strongest pathogenicity in a mouse infection model. As phylogenetic group B2 and D ExPEC isolates are generally considered virulent, multilocus sequence typing (MLST) analysis was performed for these isolates to further investigate genetic relationships. Two novel sequence types, ST5170 and ST5171, were discovered. Among the nine clonal complexes identified among our group B2 and D isolates, CC12 and CC95 have been indicated to have high zoonotic pathogenicity. The distinction between group B2 and non-B2 isolates in virulence and genotype accorded with MLST analysis. This study reveals significant genetic diversity among ExPEC isolates and helps us to better understand their pathogenesis. Importantly, our data suggest group B2 (Cluster IV) strains have the highest risk of causing animal disease and illustrate the correlation between genotype and virulence.

Genome-Wide Association Mapping of Flowering and Ripening Periods in Apple

PubMed Central

Urrestarazu, Jorge; Muranty, Hélène; Denancé, Caroline; Leforestier, Diane; Ravon, Elisa; Guyader, Arnaud; Guisnel, Rémi; Feugey, Laurence; Aubourg, Sébastien; Celton, Jean-Marc; Daccord, Nicolas; Dondini, Luca; Gregori, Roberto; Lateur, Marc; Houben, Patrick; Ordidge, Matthew; Paprstein, Frantisek; Sedlak, Jiri; Nybom, Hilde; Garkava-Gustavsson, Larisa; Troggio, Michela; Bianco, Luca; Velasco, Riccardo; Poncet, Charles; Théron, Anthony; Moriya, Shigeki; Bink, Marco C. A. M.; Laurens, François; Tartarini, Stefano; Durel, Charles-Eric

2017-01-01

Deciphering the genetic control of flowering and ripening periods in apple is essential for breeding cultivars adapted to their growing environments. We implemented a large Genome-Wide Association Study (GWAS) at the European level using an association panel of 1,168 different apple genotypes distributed over six locations and phenotyped for these phenological traits. The panel was genotyped at a high-density of SNPs using the Axiom®Apple 480 K SNP array. We ran GWAS with a multi-locus mixed model (MLMM), which handles the putatively confounding effect of significant SNPs elsewhere on the genome. Genomic regions were further investigated to reveal candidate genes responsible for the phenotypic variation. At the whole population level, GWAS retained two SNPs as cofactors on chromosome 9 for flowering period, and six for ripening period (four on chromosome 3, one on chromosome 10 and one on chromosome 16) which, together accounted for 8.9 and 17.2% of the phenotypic variance, respectively. For both traits, SNPs in weak linkage disequilibrium were detected nearby, thus suggesting the existence of allelic heterogeneity. The geographic origins and relationships of apple cultivars accounted for large parts of the phenotypic variation. Variation in genotypic frequency of the SNPs associated with the two traits was connected to the geographic origin of the genotypes (grouped as North+East, West and South Europe), and indicated differential selection in different growing environments. Genes encoding transcription factors containing either NAC or MADS domains were identified as major candidates within the small confidence intervals computed for the associated genomic regions. A strong microsynteny between apple and peach was revealed in all the four confidence interval regions. This study shows how association genetics can unravel the genetic control of important horticultural traits in apple, as well as reduce the confidence intervals of the associated regions identified by linkage mapping approaches. Our findings can be used for the improvement of apple through marker-assisted breeding strategies that take advantage of the accumulating additive effects of the identified SNPs. PMID:29176988

Resilience of Zostera muelleri seagrass to small-scale disturbances: the relative importance of asexual versus sexual recovery

PubMed Central

Macreadie, Peter I; York, Paul H; Sherman, Craig DH

2014-01-01

Resilience is the ability of an ecosystem to recover from disturbance without loss of essential function. Seagrass ecosystems are key marine and estuarine habitats that are under threat from a variety of natural and anthropogenic disturbances. The ability of these ecosystems to recovery from disturbance will to a large extent depend on the internsity and scale of the disturbance, and the relative importance of sexual versus asexual reproduction within populations. Here, we investigated the resilience of Zostera muelleri seagrass (Syn. Zostera capricorni) to small-scale disturbances at four locations in Lake Macquarie – Australia's largest coastal lake – and monitored recovery over a 65-week period. Resilience of Z. muelleri varied significantly with disturbance intensity; Z. muelleri recovered rapidly (within 2 weeks) from low-intensity disturbance (shoot loss), and rates of recovery appeared related to initial shoot length. Recovery via rhizome encroachment (asexual regeneration) from high-intensity disturbance (loss of entire plant) varied among locations, ranging from 18-35 weeks, whereas the ability to recover was apparently lost (at least within the time frame of this study) when recovery depended on sexual regeneration, suggesting that seeds do not provide a mechanism of recovery against intense small-scale disturbances. The lack of sexual recruits into disturbed sites is surprising as our initial surveys of genotypic diversity (using nine polymorphic microsatellite loci) at these location indicate that populations are maintained by a mix of sexual and asexual reproduction (genotypic diversity [R] varied from 0.24 to 0.44), and populations consisted of a mosaic of genotypes with on average 3.6 unique multilocus genotypes per 300 mm diameter plot. We therefore conclude that Z. muelleri populations within Lake Macquarie rely on clonal growth to recover from small-scale disturbances and that ongoing sexual recruitment by seeds into established seagrass beds (as opposed to bare areas arising from disturbance) must be the mechanism responsible for maintaining the observed mixed genetic composition of Z. muelleri seagrass meadows. PMID:24634729

Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

PubMed Central

Flores-Páez, Luis A.; Zenteno, Juan C.; Alcántar-Curiel, María D.; Vargas-Mendoza, Carlos F.; Rodríguez-Martínez, Sandra; Cancino-Diaz, Mario E.; Jan-Roblero, Janet; Cancino-Diaz, Juan C.

2015-01-01

Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256+. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not, in almost all cases, infect the eye due to their large genotypic and phenotypic difference with the ocular infection isolates. PMID:26275056

Patients with diffuse uveitis and inactive toxoplasmic retinitis lesions test PCR positive for Toxoplasma gondii in their vitreous and blood.

PubMed

Novais, Eduardo A; Commodaro, Alessandra G; Santos, Fábio; Muccioli, Cristina; Maia, André; Nascimento, Heloisa; Moeller, Cecilia T A; Rizzo, Luiz V; Grigg, Michael E; Belfort, Rubens

2014-07-01

To determine if patients with inactive chorioretinitis lesions who experience chronic toxoplasmic uveitis test PCR positive for Toxoplasma in their ocular fluids. Two patients undergoing long-term anti-toxoplasmic treatment developed chronic uveitis and vitritis. They underwent therapeutic and diagnostic pars plana vitrectomy. Patient specimens were tested for toxoplasmosis by real-time PCR and nested PCR. Patient specimens were also tested for the presence of Toxoplasma antibodies that recognise allelic peptide motifs to determine parasite serotype. Patients tested positive for Toxoplasma by real-time PCR at the B1 gene in the vitreous and aqueous humours of patient 1, but only the vitreous of patient 2. Patients were not parasitemic by real-time PCR in plasma and blood. During surgery, only old hyperpigmented toxoplasmic scars were observed; there was no sign of active retinitis. Multilocus PCR-DNA sequence genotyping at B1, NTS2 and SAG1 loci established that two different non-archetypal Toxoplasma strains had infected patients 1 and 2. A peptide-based serotyping ELISA confirmed the molecular findings. No active lesions were observed, but both patients possessed sufficient parasite DNA in their vitreous to permit genotyping. Several hypotheses to explain the persistence of the vitritis and anterior uveitis in the absence of active retinitis are discussed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Surviving in mountain climate refugia: new insights from the genetic diversity and structure of the relict shrub Myrtus nivellei (Myrtaceae) in the Sahara Desert.

PubMed

Migliore, Jérémy; Baumel, Alex; Juin, Marianick; Fady, Bruno; Roig, Anne; Duong, Nathalie; Médail, Frédéric

2013-01-01

The identification of past glacial refugia has become a key topic for conservation under environmental change, since they contribute importantly to shaping current patterns of biodiversity. However, little attention has been paid so far to interglacial refugia despite their key role for the survival of relict species currently occurring in climate refugia. Here, we focus on the genetic consequences of range contraction on the relict populations of the evergreen shrub Myrtus nivellei, endemic in the Saharan mountains since at least the end of the last Green Sahara period, around 5.5 ka B.P. Multilocus genotypes (nuclear microsatellites and AFLP) were obtained from 215 individuals collected from 23 wadis (temporary rivers) in the three main mountain ranges in southern Algeria (the Hoggar, Tassili n'Ajjer and Tassili n'Immidir ranges). Identical genotypes were found in several plants growing far apart within the same wadis, a pattern taken as evidence of clonality. Multivariate analyses and Bayesian clustering revealed that genetic diversity was mainly structured among the mountain ranges, while low isolation by distance was observed within each mountain range. The range contraction induced by the last episode of aridification has likely increased the genetic isolation of the populations of M. nivellei, without greatly affecting the genetic diversity of the species as a whole. The pattern of genetic diversity observed here suggests that high connectivity may have prevailed during humid periods, which is consistent with recent paleoenvironmental reconstructions.

Listeria monocytogenes meningitis in the Netherlands, 1985-2014: A nationwide surveillance study.

PubMed

Koopmans, Merel M; Bijlsma, Merijn W; Brouwer, Matthijs C; van de Beek, Diederik; van der Ende, Arie

2017-07-01

Listeria monocytogenes can cause sepsis and meningitis. We report national surveillance data on L. monocytogenes meningitis in the Netherlands, describing incidence changes, genetic epidemiology and fatality rate. We analyzed data from the Netherlands Reference Laboratory of Bacterial Meningitis for cases of L. monocytogenes meningitis. Strains were assessed by serotyping and bacterial population structure by multi-locus sequence typing. A total of 375 cases of Listeria meningitis were identified between 1985 and 2014. Peak incidence rates were observed in neonates (0.61 per 100,000 live births) and older adults (peak at 87 year; 0.53 cases per 100,000 population of the same age). Neonatal listerial meningitis decreased 17-fold from 1.95 per 100,000 live births between 1985 and 1989, to 0.11 per 100,000 live births between 2010 and 2014. Overall case fatality rate was 31%, in a multivariate analysis older age and concomitant bacteremia were associated with mortality (both p < 0.01). Clonal complexes (CC) CC1, CC2 and CC3 decreased over time from respectively 32% to 12%, 33% to 9% and 10% to 2% (all p < 0.001), while CC6 increased from 2% to 26% (p < 0.001). The incidence of neonatal listerial meningitis has declined over the past 25 years. The genotype CC6 has become the predominant genotype in listerial meningitis in the Netherlands. Mortality of listeria meningitis has remained high. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Dominance of multidrug-resistant Denmark(14)-32 (ST230) clone among Streptococcus pneumoniae serotype 19A isolates causing pneumococcal disease in Bulgaria from 1992 to 2013.

PubMed

Setchanova, Lena Petrova; Alexandrova, Alexandra; Dacheva, Daniela; Mitov, Ivan; Kaneva, Radka; Mitev, Vanio

2015-02-01

A pneumococcal conjugate vaccine (PCV10) was introduced in Bulgarian national immunization program since April 2010. Clonal composition based on pulsed-field gel electrophoresis and multilocus sequence typing genotyping of 52 serotype 19A Streptococcus pneumoniae isolates was analyzed. These were invasive and respiratory isolates collected between 1992 and 2013 from both children (78.8% <5 years) and adults with pneumococcal infections. Multidrug resistance was found in 82.7% of all 19A isolates. The most prevalent genotype (63.5%) among serotype 19A pneumococcal strains was the multidrug-resistant clonal complex CC230, which is a capsular switched variant of the Denmark(14)-32 (ST230) global clone. The most frequent sequence type (ST) was ST230 (48.1%) and together with four other closely related STs (15.4%), belonging to ST1611, ST276, ST7466, and ST2013, which were single- and double-locus variants; they were included in the main CC230. The disappearance of highly drug-resistant ST663 clone and emergence of new clones as CC320 and CC199 was also observed among the rest 19A isolates. A comparison of clonal composition between invasive and noninvasive isolates did not show a great genetic diversity among both kinds of isolates. Continuous surveillance of serotype 19A population following the introduction of PCV10 is essential to evaluate the impact of the vaccine on the epidemiology of this serotype.

Population Structure of Listeria monocytogenes Serotype 4b Isolates from Sporadic Human Listeriosis Cases in the United States from 2003 to 2008

PubMed Central

Ward, Todd J.; Graves, Lewis M.; Tarr, Cheryl L.; Siletzky, Robin M.; Kathariou, Sophia

2014-01-01

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis. PMID:24705322

Population structure of Listeria monocytogenes serotype 4b isolates from sporadic human listeriosis cases in the United States from 2003 to 2008.

PubMed

Lee, Sangmi; Ward, Todd J; Graves, Lewis M; Tarr, Cheryl L; Siletzky, Robin M; Kathariou, Sophia

2014-06-01

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis.

Multilocus analysis reveals three candidate genes for Chinese migraine susceptibility.

PubMed

An, X-K; Fang, J; Yu, Z-Z; Lin, Q; Lu, C-X; Qu, H-L; Ma, Q-L

2017-08-01

Several genome-wide association studies (GWASs) in Caucasian populations have identified 12 loci that are significantly associated with migraine. More evidence suggests that serotonin receptors are also involved in migraine pathophysiology. In the present study, a case-control study was conducted in a cohort of 581 migraine cases and 533 ethnically matched controls among a Chinese population. Eighteen polymorphisms from serotonin receptors and GWASs were selected, and genotyping was performed using a Sequenom MALDI-TOF mass spectrometry iPLEX platform. The genotypic and allelic distributions of MEF2D rs2274316 and ASTN2 rs6478241 were significantly different between migraine patients and controls. Univariate and multivariate analysis revealed significant associations of polymorphisms in the MEF2D and ASTN2 genes with migraine susceptibility. MEF2D, PRDM16 and ASTN2 were also found to be associated with migraine without aura (MO) and migraine with family history. And, MEF2D and ASTN2 also served as genetic risk factors for the migraine without family history. The generalized multifactor dimensionality reduction analysis identified that MEF2D and HTR2E constituted the two-factor interaction model. Our study suggests that the MEF2D, PRDM16 and ASTN2 genes from GWAS are associated with migraine susceptibility, especially MO, among Chinese patients. It appears that there is no association with serotonin receptor related genes. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genetic structure of populations of Legionella pneumophila.

PubMed Central

Selander, R K; McKinney, R M; Whittam, T S; Bibb, W F; Brenner, D J; Nolte, F S; Pattison, P E

1985-01-01

The genetic structure of populations of Legionella pneumophila was defined by an analysis of electrophoretically demonstrable allelic variation at structural genes encoding 22 enzymes in 292 isolates from clinical and environmental sources. Nineteen of the loci were polymorphic, and 62 distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified. Principal coordinates and clustering analyses demonstrated that isolates received as L. pneumophila were a heterogeneous array of genotypes that included two previously undescribed species. For 50 ETs of L. pneumophila (strict sense), mean genetic diversity per locus was 0.312, and diversity was equivalent in ETs represented by isolates recovered from clinical sources and those collected from environmental sources. Cluster analysis revealed four major groups or lineages of ETs in L. pneumophila. Genetic diversity among ETs of the same serotype was, on average, 93% of that in the total sample of ETs. Isolates marked by particular patterns of reactivity to a panel of nine monoclonal antibodies were also genetically heterogeneous, mean diversity within patterns being about 75% of the total. Both Pontiac fever and the pneumonic form of legionellosis may be caused by isolates of the same ET. The genetic structure of L. pneumophila is clonal, and many clones apparently are worldwide in distribution. The fact that L. pneumophila is only 60% as variable as Escherichia coli raises the possibility that isolates recovered from clinical cases and man-made environments are a restricted subset of all clones in the species as a whole. PMID:4030689

Genetic Analysis of Phytophthora nicotianae Populations from Different Hosts Using Microsatellite Markers.

PubMed

Biasi, Antonio; Martin, Frank N; Cacciola, Santa O; di San Lio, Gaetano Magnano; Grünwald, Niklaus J; Schena, Leonardo

2016-09-01

In all, 231 isolates of Phytophthora nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa, and Ruta), and other plant genera were characterized using simple-sequence repeat markers. In total, 99 multilocus genotypes (MLG) were identified, revealing a strong association between genetic grouping and host of recovery, with most MLG being associated with a single host genus. Significant differences in the structure of populations were revealed but clonality prevailed in all populations. Isolates from Citrus were found to be genetically related regardless of their geographic origin and were characterized by high genetic uniformity and high inbreeding coefficients. Higher variability was observed for other populations and a significant geographical structuring was determined for isolates from Nicotiana. Detected differences were related to the propagation and cultivation systems of different crops. Isolates obtained from Citrus spp. are more likely to be distributed worldwide with infected plant material whereas Nicotiana and Lycopersicon spp. are propagated by seed, which would not contribute to the spread of the pathogen and result in a greater chance for geographic isolation of lineages. With regard to ornamental species in nurseries, the high genetic variation is likely the result of the admixture of diverse pathogen genotypes through the trade of infected plant material from various geographic origins, the presence of several hosts in the same nursery, and genetic recombination through sexual reproduction of this heterothallic species.

Genotypic diversity in root-endophytic fungi reflects efficient dispersal and environmental adaptation.

PubMed

Glynou, Kyriaki; Ali, Tahir; Kia, Sevda Haghi; Thines, Marco; Maciá-Vicente, Jose G

2017-09-01

Studying community structure and dynamics of plant-associated fungi is the basis for unravelling their interactions with hosts and ecosystem functions. A recent sampling revealed that only a few fungal groups, as defined by internal transcribed spacer region (ITS) sequence similarity, dominate culturable root endophytic communities of nonmycorrhizal Microthlaspi spp. plants across Europe. Strains of these fungi display a broad phenotypic and functional diversity, which suggests a genetic variability masked by ITS clustering into operational taxonomic units (OTUs). The aims of this study were to identify how genetic similarity patterns of these fungi change across environments and to evaluate their ability to disperse and adapt to ecological conditions. A first ITS-based haplotype analysis of ten widespread OTUs mostly showed a low to moderate genotypic differentiation, with the exception of a group identified as Cadophora sp. that was highly diverse. A multilocus phylogeny based on additional genetic loci (partial translation elongation factor 1α, beta-tubulin and actin) and amplified fragment length polymorphism profiling of 185 strains representative of the five dominant OTUs revealed a weak association of genetic differences with geography and environmental conditions, including bioclimatic and soil factors. Our findings suggest that dominant culturable root endophytic fungi have efficient dispersal capabilities, and that their distribution is little affected by environmental filtering. Other processes, such as inter- and intraspecific biotic interactions, may be more important for the local assembly of their communities. © 2017 John Wiley & Sons Ltd.

Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification.

PubMed

Houston, Rachel; Birck, Matthew; Hughes-Stamm, Sheree; Gangitano, David

2016-05-01

Marijuana (Cannabis sativa) is the most commonly used illicit substance in the USA. The development of a validated method using Cannabis short tandem repeats (STRs) could aid in the individualization of samples as well as serve as an intelligence tool to link multiple cases. For this purpose, a modified 13-loci STR multiplex method was optimized and evaluated according to ISFG and SWGDAM guidelines. A real-time PCR quantification method for C. sativa was developed and validated, and a sequenced allelic ladder was also designed to accurately genotype 199 C. sativa samples from 11 U.S. Customs and Border Protection seizures. Distinguishable DNA profiles were generated from 127 samples that yielded full STR profiles. Four duplicate genotypes within seizures were found. The combined power of discrimination of this multilocus system is 1 in 70 million. The sensitivity of the multiplex STR system is 0.25 ng of template DNA. None of the 13 STR markers cross-reacted with any of the studied species, except for Humulus lupulus (hops) which generated unspecific peaks. Phylogenetic analysis and case-to-case pairwise comparison of 11 cases using F st as genetic distance revealed the genetic association of four groups of cases. Moreover, due to their genetic similarity, a subset of samples (N = 97) was found to form a homogeneous population in Hardy-Weinberg and linkage equilibrium. The results of this research demonstrate the applicability of this 13-loci STR system in associating Cannabis cases for intelligence purposes.

Methicillin-resistant Staphylococcus aureus carriage among veterinary staff and dogs in private veterinary clinics in Hokkaido, Japan.

PubMed

Ishihara, Kanako; Saito, Mieko; Shimokubo, Natsumi; Muramatsu, Yasukazu; Maetani, Shigeki; Tamura, Yutaka

2014-03-01

To explore the prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in veterinary medical practices, MRSA carriage was tested among 96 veterinarians (Vets), 70 veterinary technicians (VTs) and 292 dogs with which they had contact at 71 private veterinary clinics (VCs) in Hokkaido, Japan. MRSA isolates were obtained from 22 Vets [22.9%] and 7 VTs [10%]. The prevalence of MRSA among Vets was as high as that found in an academic veterinary hospital in our previous study. In contrast, only two blood donor dogs and one dog with liver disease (1.0%, 3/292) yielded MRSA. All MRSA-positive dogs were reared or treated in different VCs, in each of which at least one veterinary staff member carrying MRSA worked. Sequence types (ST) identified by multilocus sequence typing, spa types, and SCCmec types for canine MRSA isolates (ST5-spa t002-SCCmec II [from two dogs] or ST30-spa t021-SCCmec IV [from a dog]) were concordant with those from veterinary staff members in the same clinics as the MRSA-positive dogs, with which they had potentially had contact. Most MRSA isolates from veterinary staff were the same genotype (SCCmec type II and spa type t002) as a major hospital-acquired MRSA clone in Japan. The remaining MRSA was the same genotypes as domestic and foreign community-associated MRSA. Measures against MRSA infection should be provided in private VCs. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Functional variation in a disease resistance gene in populations of Arabidopsis thaliana.

PubMed

Jorgensen, T H; Emerson, B C

2008-11-01

Analyses of functional genetic diversity in natural populations may provide important new insights into gene function and are necessary to understand the evolutionary processes maintaining diversity itself. The importance of including diversity within and between local populations in such studies is often ignored although many of the processes affecting genetic diversity act on this scale. Here we examine the molecular diversity in RPW8 (Recognition of Powdery Mildew), a gene conferring broad-spectrum resistance to powdery mildews in Arabidopsis thaliana stock-center accessions. Our eight UK study populations of the weedy A. thaliana were from locations judged to be subject to a minimum of anthropogenic disturbance and potentially long established. The majority of populations comprised considerable variation both in disease phenotype and RPW8 genotype. Although resistant individuals shared a major RPW8 genotype, no single allele was uniquely associated with resistance. It is concluded that RPW8 is an essential component of resistance to powdery mildews in A. thaliana, but not the only genetic factor involved in this process. No signature of selection was detected at RPW8 with a microsatellite multilocus test using an empirical null model. Unlike many previous studies of this model plant species, we found high levels of genetic diversity and relatively low differentiation (F(ST) = 0.31) between populations at 14 microsatellite markers. This is judged to be due to our sampling being aimed at potentially long established populations and highlights the importance of population choice for studies of genetic diversity within this species.

Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination.

PubMed

Perez, Federico; Endimiani, Andrea; Ray, Amy J; Decker, Brooke K; Wallace, Christopher J; Hujer, Kristine M; Ecker, David J; Adams, Mark D; Toltzis, Philip; Dul, Michael J; Windau, Anne; Bajaksouzian, Saralee; Jacobs, Michael R; Salata, Robert A; Bonomo, Robert A

2010-08-01

Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained bla(OXA-23), and four isolates possessed bla(OXA-24/40). Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing bla(KPC-2) or bla(KPC-3) were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.

Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination

PubMed Central

Perez, Federico; Endimiani, Andrea; Ray, Amy J.; Decker, Brooke K.; Wallace, Christopher J.; Hujer, Kristine M.; Ecker, David J.; Adams, Mark D.; Toltzis, Philip; Dul, Michael J.; Windau, Anne; Bajaksouzian, Saralee; Jacobs, Michael R.; Salata, Robert A.; Bonomo, Robert A.

2010-01-01

Background Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. Methods Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. Results Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained blaOXA-23, and four isolates possessed blaOXA-24/40. Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing blaKPC-2 or blaKPC-3 were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). Conclusion In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes. PMID:20513702

Tracing the source of campylobacteriosis.

PubMed

Wilson, Daniel J; Gabriel, Edith; Leatherbarrow, Andrew J H; Cheesbrough, John; Gee, Steven; Bolton, Eric; Fox, Andrew; Fearnhead, Paul; Hart, C Anthony; Diggle, Peter J

2008-09-26

Campylobacter jejuni is the leading cause of bacterial gastro-enteritis in the developed world. It is thought to infect 2-3 million people a year in the US alone, at a cost to the economy in excess of US $4 billion. C. jejuni is a widespread zoonotic pathogen that is carried by animals farmed for meat and poultry. A connection with contaminated food is recognized, but C. jejuni is also commonly found in wild animals and water sources. Phylogenetic studies have suggested that genotypes pathogenic to humans bear greatest resemblance to non-livestock isolates. Moreover, seasonal variation in campylobacteriosis bears the hallmarks of water-borne disease, and certain outbreaks have been attributed to contamination of drinking water. As a result, the relative importance of these reservoirs to human disease is controversial. We use multilocus sequence typing to genotype 1,231 cases of C. jejuni isolated from patients in Lancashire, England. By modeling the DNA sequence evolution and zoonotic transmission of C. jejuni between host species and the environment, we assign human cases probabilistically to source populations. Our novel population genetics approach reveals that the vast majority (97%) of sporadic disease can be attributed to animals farmed for meat and poultry. Chicken and cattle are the principal sources of C. jejuni pathogenic to humans, whereas wild animal and environmental sources are responsible for just 3% of disease. Our results imply that the primary transmission route is through the food chain, and suggest that incidence could be dramatically reduced by enhanced on-farm biosecurity or preventing food-borne transmission.

Severe South American Ocular Toxoplasmosis Is Associated with Decreased Ifn-γ/Il-17a and Increased Il-6/Il-13 Intraocular Levels

PubMed Central

de-la-Torre, Alejandra; Sauer, Arnaud; Pfaff, Alexander W.; Bourcier, Tristan; Brunet, Julie; Speeg-Schatz, Claude; Ballonzoli, Laurent; Villard, Odile; Ajzenberg, Daniel; Sundar, Natarajan; Grigg, Michael E.

2013-01-01

In a cross sectional study, 19 French and 23 Colombian cases of confirmed active ocular toxoplasmosis (OT) were evaluated. The objective was to compare clinical, parasitological and immunological responses and relate them to the infecting strains. A complete ocular examination was performed in each patient. The infecting strain was characterized by genotyping when intraocular Toxoplasma DNA was detectable, as well as by peptide-specific serotyping for each patient. To characterize the immune response, we assessed Toxoplasma protein recognition patterns by intraocular antibodies and the intraocular profile of cytokines, chemokines and growth factors. Significant differences were found for size of active lesions, unilateral macular involvement, unilateral visual impairment, vitreous inflammation, synechiae, and vasculitis, with higher values observed throughout for Colombian patients. Multilocus PCR-DNA sequence genotyping was only successful in three Colombian patients revealing one type I and two atypical strains. The Colombian OT patients possessed heterogeneous atypical serotypes whereas the French were uniformly reactive to type II strain peptides. The protein patterns recognized by intraocular antibodies and the cytokine patterns were strikingly different between the two populations. Intraocular IFN-γ and IL-17 expression was lower, while higher levels of IL-13 and IL-6 were detected in aqueous humor of Colombian patients. Our results are consistent with the hypothesis that South American strains may cause more severe OT due to an inhibition of the protective effect of IFN-γ. PMID:24278490

Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species

PubMed Central

Divis, Paul C. S.; Singh, Balbir; Anderios, Fread; Hisam, Shamilah; Matusop, Asmad; Kocken, Clemens H.; Assefa, Samuel A.; Duffy, Craig W.; Conway, David J.

2015-01-01

Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system. PMID:26020959

O-acetyltransferase gene neuO is segregated according to phylogenetic background and contributes to environmental desiccation resistance in Escherichia coli K1.

PubMed

Mordhorst, Ines L; Claus, Heike; Ewers, Christa; Lappann, Martin; Schoen, Christoph; Elias, Johannes; Batzilla, Julia; Dobrindt, Ulrich; Wieler, Lothar H; Bergfeld, Anne K; Mühlenhoff, Martina; Vogel, Ulrich

2009-12-01

Escherichia coli K1 causes disease in humans and birds. Its polysialic acid capsule can be O-acetylated via phase-variable expression of the acetyltransferase NeuO encoded by prophage CUS-3. The role of capsule O-acetylation in ecological adaptation or pathogenic invasion of E. coli K1 is largely unclear. A population genetics approach was performed to study the distribution of neuO among E. coli K1 isolates from human and avian sources. Multilocus sequence typing revealed 39 different sequence types (STs) among 183 E. coli K1 strains. The proportion of the ST95 complex (STC95) was 44%. NeuO was found in 98% of the STC95 strains, but only in 24% of other STs. Grouping of STs and prophage genotypes revealed a segregation of prophage types according to STs, suggesting coevolution of CUS-3 and the E. coli K1 host. Within the STC95, which is known to harbour both human and avian pathogenic isolates, CUS-3 genotypes were shared irrespective of the host species. Functional analysis of a variety of strain pairs revealed that NeuO-mediated K1 capsule O-acetylation enhanced desiccation resistance. In contrast, NeuO expression led to a reduced biofilm formation in biofilm positive E. coli K1 isolates. These findings suggest a delicate ecological balance of neuO'on'/'off' switching.

Quantitative structural organisation model for wheat endosperm cell walls: Cellulose as an important constituent.

PubMed

Gartaula, Ghanendra; Dhital, Sushil; Netzel, Gabriele; Flanagan, Bernadine M; Yakubov, Gleb E; Beahan, Cherie T; Collins, Helen M; Burton, Rachel A; Bacic, Antony; Gidley, Michael J

2018-09-15

The cell walls of cereal endosperms are a major source of fibre in many diets and of importance in seed structure and germination. Cell walls were isolated from both pure wheat endosperm and milled flour. 13 C CP/MAS NMR in conjunction with methylation analysis before and after acid hydrolysis showed that, in addition to arabinoxylan (AX) and (1, 3; 1, 4)-β-D-glucan (MLG), wheat endosperm cell walls contain a significant proportion of cellulose (ca 20%) which is tightly bound to xylans and mannans. Light microscopy showed that the cellulose was relatively evenly distributed across the grain endosperm. The cell walls contain a fibrous acid-resistant core structure laminated by matrix polysaccharides as revealed by AFM imaging. A model for endosperm cell wall structural organisation is proposed, based on a core of cellulose and interacting non-cellulosic polysaccharides which anchors AX (with very occasional diferulic acid cross-linking) that in turn retains MLGs through physical entanglement. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

Multilocus resolution of Mugilidae phylogeny (Teleostei: Mugiliformes): Implications for the family's taxonomy.

PubMed

Xia, Rong; Durand, Jean-Dominique; Fu, Cuizhang

2016-03-01

The interrelationships among mugilids (Mugiliformes: Mugilidae) remain highly debated. Using a mitochondrial gene-based phylogeny as criterion, a revised classification with 25 genera in the Mugilidae has recently been proposed. However, phylogenetic relationships of major mitochondrial lineages remain unresolved and to gain a general acceptance the classification requires confirmation based on multilocus evidence and diagnostic morphological characters. Here, we construct a species-tree using twelve nuclear and three mitochondrial loci and infer the evolution of 71 morphological characters. Our multilocus phylogeny does not agree with previous morphology-based hypotheses for the relationships within Mugilidae, confirms the revised classification with 25 genera and further resolves their phylogenetic relationships. Using the well-resolved multilocus phylogeny as the criterion, we reclassify Mugilidae genera into three new subfamilies (Myxinae, Rhinomugilinae, and Cheloninae) and one new, recombined, subfamily (Mugilinae). The Rhinomugilinae subfamily is further divided into four tribes. The revised classification of Mugilidae is supported by morpho-anatomical synapomorphies or a combination of characters. These characters are used to erect a key to the subfamilies and genera. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of Low-Fat vs Low-Carbohydrate Diet on 12-Month Weight Loss in Overweight Adults and the Association With Genotype Pattern or Insulin Secretion: The DIETFITS Randomized Clinical Trial.

PubMed

Gardner, Christopher D; Trepanowski, John F; Del Gobbo, Liana C; Hauser, Michelle E; Rigdon, Joseph; Ioannidis, John P A; Desai, Manisha; King, Abby C

2018-02-20

Dietary modification remains key to successful weight loss. Yet, no one dietary strategy is consistently superior to others for the general population. Previous research suggests genotype or insulin-glucose dynamics may modify the effects of diets. To determine the effect of a healthy low-fat (HLF) diet vs a healthy low-carbohydrate (HLC) diet on weight change and if genotype pattern or insulin secretion are related to the dietary effects on weight loss. The Diet Intervention Examining The Factors Interacting with Treatment Success (DIETFITS) randomized clinical trial included 609 adults aged 18 to 50 years without diabetes with a body mass index between 28 and 40. The trial enrollment was from January 29, 2013, through April 14, 2015; the date of final follow-up was May 16, 2016. Participants were randomized to the 12-month HLF or HLC diet. The study also tested whether 3 single-nucleotide polymorphism multilocus genotype responsiveness patterns or insulin secretion (INS-30; blood concentration of insulin 30 minutes after a glucose challenge) were associated with weight loss. Health educators delivered the behavior modification intervention to HLF (n = 305) and HLC (n = 304) participants via 22 diet-specific small group sessions administered over 12 months. The sessions focused on ways to achieve the lowest fat or carbohydrate intake that could be maintained long-term and emphasized diet quality. Primary outcome was 12-month weight change and determination of whether there were significant interactions among diet type and genotype pattern, diet and insulin secretion, and diet and weight loss. Among 609 participants randomized (mean age, 40 [SD, 7] years; 57% women; mean body mass index, 33 [SD, 3]; 244 [40%] had a low-fat genotype; 180 [30%] had a low-carbohydrate genotype; mean baseline INS-30, 93 μIU/mL), 481 (79%) completed the trial. In the HLF vs HLC diets, respectively, the mean 12-month macronutrient distributions were 48% vs 30% for carbohydrates, 29% vs 45% for fat, and 21% vs 23% for protein. Weight change at 12 months was -5.3 kg for the HLF diet vs -6.0 kg for the HLC diet (mean between-group difference, 0.7 kg [95% CI, -0.2 to 1.6 kg]). There was no significant diet-genotype pattern interaction (P = .20) or diet-insulin secretion (INS-30) interaction (P = .47) with 12-month weight loss. There were 18 adverse events or serious adverse events that were evenly distributed across the 2 diet groups. In this 12-month weight loss diet study, there was no significant difference in weight change between a healthy low-fat diet vs a healthy low-carbohydrate diet, and neither genotype pattern nor baseline insulin secretion was associated with the dietary effects on weight loss. In the context of these 2 common weight loss diet approaches, neither of the 2 hypothesized predisposing factors was helpful in identifying which diet was better for whom. clinicaltrials.gov Identifier: NCT01826591.

Multilocus phylogeny and antifungal susceptibility of Aspergillus section Circumdati from clinical samples and description of A. pseudosclerotiorum sp. nov.

USDA-ARS?s Scientific Manuscript database

A multilocus phylogenetic study was carried out to assess the species distribution in a set of 34 clinical isolates of Aspergillus section Circumdati from the USA and their in vitro antifungal susceptibility were determined against eight antifungal drugs. The genetic markers used were ITS, BenA, CaM...

Rickettsia asembonensis Characterization by Multilocus Sequence Typing of Complete Genes, Peru.

PubMed

Loyola, Steev; Flores-Mendoza, Carmen; Torre, Armando; Kocher, Claudine; Melendrez, Melanie; Luce-Fedrow, Alison; Maina, Alice N; Richards, Allen L; Leguia, Mariana

2018-05-01

While studying rickettsial infections in Peru, we detected Rickettsia asembonensis in fleas from domestic animals. We characterized 5 complete genomic regions (17kDa, gltA, ompA, ompB, and sca4) and conducted multilocus sequence typing and phylogenetic analyses. The molecular isolate from Peru is distinct from the original R. asembonensis strain from Kenya.

The association between Staphylococcus aureus nasal colonization and symptomatic infection in children in Korea where ST72 is the major genotype: A prospective observational study.

PubMed

Kang, Sunghan; Lee, Jina; Kim, Mina

2017-08-01

This study was performed to investigate the concordance in terms of molecular characteristics and antimicrobial susceptibility between colonizing and clinical Staphylococcus aureus isolates obtained from children in Korea, where ST72 is the major genotype.This was a prospective observational descriptive study of culture-confirmed S aureus infections obtained from children ≤18 years old admitted to Asan Medical Center Children's Hospital in Seoul, Korea, from March 2014 to April 2015. Molecular studies including multilocus sequence typing (MLST), SCCmec typing, polymerase chain reaction amplification of the Panton-Valentine leukocidin (PVL) genes, and antibiotic susceptibility tests were performed on S aureus isolates obtained from nares and clinical specimens.During the study period, 126 clinically significant S aureus infections were identified. Nasal swab cultures were made from 113 of the 126 children, and 46.0% (52/113) showed S aureus colonization. The overall concordance between colonizing and clinical isolates by methicillin susceptibility was 94.2% (49/52); all 3 discordant cases were HA-MSSA cases with nasal MRSA. Among the 37 pairs of colonizing and clinical S aureus isolates included in the genotyping analysis, ST72-SCCmec type IV was the most prevalent clone and the PVL genes were positive in 2 patients. Among the 31 pairs of healthcare-associated cases, concordance rates by methicillin susceptibility and sequence type (ST) were 90.3% (28/31) and 84% (26/31), respectively. For the 6 pairs of community-associated (CA) S aureus including 3 CA-MRSA cases, 100% concordance was observed by methicillin susceptibility and ST.The concordance between isolates obtained from children who required medical services was relatively high in Korean children where ST72-SCCmec type IV is the predominant clone as the colonizer and the pathogen. It is suggested that decolonization and continuous care to prevent transmission could be effective in managing and preventing both HA- and CA-SA infections in our setting.

Characterization of the Gray Whale Eschrichtius robustus Genome and a Genotyping Array Based on Single-Nucleotide Polymorphisms in Candidate Genes.

PubMed

DeWoody, J Andrew; Fernandez, Nadia B; Brüniche-Olsen, Anna; Antonides, Jennifer D; Doyle, Jacqueline M; San Miguel, Phillip; Westerman, Rick; Vertyankin, Vladimir V; Godard-Codding, Céline A J; Bickham, John W

2017-06-01

Genetic and genomic approaches have much to offer in terms of ecology, evolution, and conservation. To better understand the biology of the gray whale Eschrichtius robustus (Lilljeborg, 1861), we sequenced the genome and produced an assembly that contains ∼95% of the genes known to be highly conserved among eukaryotes. From this assembly, we annotated 22,711 genes and identified 2,057,254 single-nucleotide polymorphisms (SNPs). Using this assembly, we generated a curated list of candidate genes potentially subject to strong natural selection, including genes associated with osmoregulation, oxygen binding and delivery, and other aspects of marine life. From these candidate genes, we queried 92 autosomal protein-coding markers with a panel of 96 SNPs that also included 2 sexing and 2 mitochondrial markers. Genotyping error rates, calculated across loci and across 69 intentional replicate samples, were low (0.021%), and observed heterozygosity was 0.33 averaged over all autosomal markers. This level of variability provides substantial discriminatory power across loci (mean probability of identity of 1.6 × 10 -25 and mean probability of exclusion >0.999 with neither parent known), indicating that these markers provide a powerful means to assess parentage and relatedness in gray whales. We found 29 unique multilocus genotypes represented among our 36 biopsies (indicating that we inadvertently sampled 7 whales twice). In total, we compiled an individual data set of 28 western gray whales (WGSs) and 1 presumptive eastern gray whale (EGW). The lone EGW we sampled was no more or less related to the WGWs than expected by chance alone. The gray whale genomes reported here will enable comparative studies of natural selection in cetaceans, and the SNP markers should be highly informative for future studies of gray whale evolution, population structure, demography, and relatedness.

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.

Developing a Molecular Identification Assay of Old Landraces for the Genetic Authentication of Typical Agro-Food Products: The Case Study of the Barley ‘Agordino’

PubMed Central

Palumbo, Fabio; Galla, Giulio

2017-01-01

Summary The orzo Agordino is a very old local variety of domesticated barley (Hordeum vulgare ssp. distichum L.) that is native to the Agordo District, Province of Belluno, and is widespread in the Veneto Region, Italy. Seeds of this landrace are widely used for the preparation of very famous dishes of the dolomitic culinary tradition such as barley soup, bakery products and local beer. Understanding the genetic diversity and identity of the Agordino barley landrace is a key step to establish conservation and valorisation strategies of this local variety and also to provide molecular traceability tools useful to ascertain the authenticity of its derivatives. The gene pool of the Agordino barley landrace was reconstructed using 60 phenotypically representative individual plants and its genotypic relationships with commercial varieties were investigated using 21 pure lines widely cultivated in the Veneto Region. For genomic DNA analysis, following an initial screening of 14 mapped microsatellite (SSR) loci, seven discriminant markers were selected on the basis of their genomic position across linkage groups and polymorphic marker alleles per locus. The genetic identity of the local barley landrace was determined by analysing all SSR markers in a single multi-locus PCR assay. Extent of genotypic variation within the Agordino barley landrace and the genotypic differentiation between the landrace individuals and the commercial varieties was determined. Then, as few as four highly informative SSR loci were selected and used to develop a molecular traceability system exploitable to verify the genetic authenticity of food products deriving from the Agordino landrace. This genetic authentication assay was validated using both DNA pools from individual Agordino barley plants and DNA samples from Agordino barley food products. On the whole, our data support the usefulness and robustness of this DNA-based diagnostic tool for the orzo Agordino identification, which could be rapidly and efficiently exploited to guarantee the authenticity of local varieties and the typicality of food products. PMID:28559731

Polyparasitism Is Associated with Increased Disease Severity in Toxoplasma gondii-Infected Marine Sentinel Species

PubMed Central

Gibson, Amanda K.; Raverty, Stephen; Lambourn, Dyanna M.; Huggins, Jessica; Magargal, Spencer L.; Grigg, Michael E.

2011-01-01

In 1995, one of the largest outbreaks of human toxoplasmosis occurred in the Pacific Northwest region of North America. Genetic typing identified a novel Toxoplasma gondii strain linked to the outbreak, in which a wide spectrum of human disease was observed. For this globally-distributed, water-borne zoonosis, strain type is one variable influencing disease, but the inability of strain type to consistently explain variations in disease severity suggests that parasite genotype alone does not determine the outcome of infection. We investigated polyparasitism (infection with multiple parasite species) as a modulator of disease severity by examining the association of concomitant infection of T. gondii and the related parasite Sarcocystis neurona with protozoal disease in wild marine mammals from the Pacific Northwest. These hosts ostensibly serve as sentinels for the detection of terrestrial parasites implicated in water-borne epidemics of humans and wildlife in this endemic region. Marine mammals (151 stranded and 10 healthy individuals) sampled over 6 years were assessed for protozoal infection using multi-locus PCR-DNA sequencing directly from host tissues. Genetic analyses uncovered a high prevalence and diversity of protozoa, with 147/161 (91%) of our sampled population infected. From 2004 to 2009, the relative frequency of S. neurona infections increased dramatically, surpassing that of T. gondii. The majority of T. gondii infections were by genotypes bearing Type I lineage alleles, though strain genotype was not associated with disease severity. Significantly, polyparasitism with S. neurona and T. gondii was common (42%) and was associated with higher mortality and more severe protozoal encephalitis. Our finding of widespread polyparasitism among marine mammals indicates pervasive contamination of waterways by zoonotic agents. Furthermore, the significant association of concomitant infection with mortality and protozoal encephalitis identifies polyparasitism as an important factor contributing to disease severity in marine mammals. PMID:21629726

Discriminating micropathogen lineages and their reticulate evolution through graph theory-based network analysis: the case of Trypanosoma cruzi, the agent of Chagas disease.

PubMed

Arnaud-Haond, Sophie; Moalic, Yann; Barnabé, Christian; Ayala, Francisco José; Tibayrenc, Michel

2014-01-01

Micropathogens (viruses, bacteria, fungi, parasitic protozoa) share a common trait, which is partial clonality, with wide variance in the respective influence of clonality and sexual recombination on the dynamics and evolution of taxa. The discrimination of distinct lineages and the reconstruction of their phylogenetic history are key information to infer their biomedical properties. However, the phylogenetic picture is often clouded by occasional events of recombination across divergent lineages, limiting the relevance of classical phylogenetic analysis and dichotomic trees. We have applied a network analysis based on graph theory to illustrate the relationships among genotypes of Trypanosoma cruzi, the parasitic protozoan responsible for Chagas disease, to identify major lineages and to unravel their past history of divergence and possible recombination events. At the scale of T. cruzi subspecific diversity, graph theory-based networks applied to 22 isoenzyme loci (262 distinct Multi-Locus-Enzyme-Electrophoresis -MLEE) and 19 microsatellite loci (66 Multi-Locus-Genotypes -MLG) fully confirms the high clustering of genotypes into major lineages or "near-clades". The release of the dichotomic constraint associated with phylogenetic reconstruction usually applied to Multilocus data allows identifying putative hybrids and their parental lineages. Reticulate topology suggests a slightly different history for some of the main "near-clades", and a possibly more complex origin for the putative hybrids than hitherto proposed. Finally the sub-network of the near-clade T. cruzi I (28 MLG) shows a clustering subdivision into three differentiated lesser near-clades ("Russian doll pattern"), which confirms the hypothesis recently proposed by other investigators. The present study broadens and clarifies the hypotheses previously obtained from classical markers on the same sets of data, which demonstrates the added value of this approach. This underlines the potential of graph theory-based network analysis for describing the nature and relationships of major pathogens, thereby opening stimulating prospects to unravel the organization, dynamics and history of major micropathogen lineages.

The Changing Pattern of Population Structure of Staphylococcus aureus from Bacteremia in China from 2013 to 2016: ST239-030-MRSA Replaced by ST59-t437.

PubMed

Li, Shuguang; Sun, Shijun; Yang, Chentao; Chen, Hongbin; Yin, Yuyao; Li, Henan; Zhao, Chunjiang; Wang, Hui

2018-01-01

To investigate the epidemiology and genetic structure of Staphylococcus aureus bacteremia in China, a total of 416 isolates from 22 teaching hospitals in 12 cities from 2013 and 2016 were characterized by antibiogram analysis, multilocus sequence typing (MLST), spa typing and staphylococcal cassette chromosome mec (SCC mec ) typing. The predominant meticillin-susceptible (MSSA) genotypes in 2013 were ST188 (19.1%), ST7 (8.7%), and ST398 (7.8%), respectively, and they continued to be the main genotypes in 2016. The prevalence of meticillin-resistant S. aureus (MRSA) were 36.5% (66/181) and 36.6% (86/235) in 2013 and 2016, respectively. Interestingly, the susceptibility rates of MRSA to rifampicin and fluoroquinolones increased significantly from 2013 to 2016 ( P < 0.01), and this was associated with changes in genetic structure. ST239-t030-MRSA, the predominant genotype among all MRSAs in 2013 (34.8%), was replaced by ST59-t437-MRSA (15.1%) in 2016. Further analysis revealed that the ST239-t030-MRSA were more resistant to rifampicin, tetracycline and fluoroquinolones than ST59-t437-MRSA ( P < 0.01). To further gain insight into the mechanisms underlying the changes of genetic structure, in vitro competition and fitness measurements were performed. Importantly, ST239-t030-MRSA displayed lower growth rate and lower competitive advantage compared to ST59-t437-MRSA. Together, our findings reveal that fitness advantage of ST59-t437-MRSA over ST239-t030-MRSA may lead to changes in genetic structure and increased susceptibility of MRSA to rifampicin and fluoroquinolones in Chinese patients with S. aureus bacteremia. Our study supports temporal dynamics in MRSA clone diversities, further providing critical insights into the importance of continued monitoring of MRSA.

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.

L. monocytogenes in a cheese processing facility: Learning from contamination scenarios over three years of sampling.

PubMed

Rückerl, I; Muhterem-Uyar, M; Muri-Klinger, S; Wagner, K-H; Wagner, M; Stessl, B

2014-10-17

The aim of this study was to analyze the changing patterns of Listeria monocytogenes contamination in a cheese processing facility manufacturing a wide range of ready-to-eat products. Characterization of L. monocytogenes isolates included genotyping by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Disinfectant-susceptibility tests and the assessment of L. monocytogenes survival in fresh cheese were also conducted. During the sampling period between 2010 and 2013, a total of 1284 environmental samples were investigated. Overall occurrence rates of Listeria spp. and L. monocytogenes were 21.9% and 19.5%, respectively. Identical L. monocytogenes genotypes were found in the food processing environment (FPE), raw materials and in products. Interventions after the sampling events changed contamination scenarios substantially. The high diversity of globally, widely distributed L. monocytogenes genotypes was reduced by identifying the major sources of contamination. Although susceptible to a broad range of disinfectants and cleaners, one dominant L. monocytogenes sequence type (ST) 5 could not be eradicated from drains and floors. Significantly, intense humidity and steam could be observed in all rooms and water residues were visible on floors due to increased cleaning strategies. This could explain the high L. monocytogenes contamination of the FPE (drains, shoes and floors) throughout the study (15.8%). The outcome of a challenge experiment in fresh cheese showed that L. monocytogenes could survive after 14days of storage at insufficient cooling temperatures (8 and 16°C). All efforts to reduce L. monocytogenes environmental contamination eventually led to a transition from dynamic to stable contamination scenarios. Consequently, implementation of systematic environmental monitoring via in-house systems should either aim for total avoidance of FPE colonization, or emphasize a first reduction of L. monocytogenes to sites where contamination of the processed product is unlikely. Drying of surfaces after cleaning is highly recommended to facilitate the L. monocytogenes eradication. Copyright © 2014 Elsevier B.V. All rights reserved.

Genotypes of Ciprofloxacin-Resistant Klebsiella pneumoniae in Korea and Their Characteristics According to the Genetic Lineages.

PubMed

Park, Dong Jin; Yu, Jin Kyung; Park, Kang Gyun; Park, Yeon-Joon

2015-12-01

We investigated the molecular genotypes of ciprofloxacin-resistant Klebsiella pneumoniae and their characteristics according to the genetic lineages. For 160 K. pneumoniae collected in 2013, ciprofloxacin minimum inhibitory concentrations (MICs) were determined by agar dilution method. The genotypes of ciprofloxacin-resistant K. pneumoniae isolates were determined by multilocus sequence typing (MLST) and wzi gene typing. The presence of plasmid-mediated resistance determinants [qnrA, qnrB, qnrS, aac(6')-Ib-cr, blaCTX-M, and blaSHV] was investigated. The gyrA and parC genes were sequenced. Fifty-seven isolates showed ciprofloxacin resistance. By MLST, four major sequence types (STs) or clonal complexes (CCs), that is, ST307, CC11, CC147, and ST15, were found and the two most prevalent STs were ST307 (14/57, 24.6%) and ST11 (12/57, 21.1%). By wzi gene sequencing, 46 of the 57 isolates could be differentiated. All the ST307 isolates had an identical wzi sequence and harbored qnrB. The majority of them harbored aac(6')-Ib-cr (85.7%) and CTX-M-15 (92.9%). In contrast, 12 ST11 isolates were divided into five sublineages by wzi sequence and qnrB, qnrS, and aac(6')-Ib-cr were carried by nine, seven, and three isolates, respectively. They harbored SHV-type extended-spectrum β-lactamase more frequently than CTX-M-15 (nine and four isolates, respectively). The prevalence of CTX-M-15, qnrB1, and aac(6')-Ib-cr was significantly higher in ST307 than in ST11 (p=0.003, p=0.000, and p=0.002, respectively). Both clones had identical amino acid substitution in gyrA (S83I) and parC (S80I). K. pneumoniae ST307 and ST11 were the two most common clones, and the ST307 isolates were highly homogeneous, suggesting their recent emergence.

Resistant mechanisms and molecular epidemiology of imipenem-resistant Acinetobacter baumannii.

PubMed

Xiao, Shu-Zhen; Chu, Hai-Qing; Han, Li-Zhong; Zhang, Zhe-Min; Li, Bing; Zhao, Lan; Xu, Liyun

2016-09-01

The aim of the study was to investigate the resistant mechanisms and homology of imipenem-resistant Acinetobacter baumannii (A. baumannii). A total of 46 non-duplicate imipenem‑resistant A. baumannii clinical isolates were collected from three tertiary hospitals between July, 2011 and June, 2012. The minimal inhibitory concentrations (MICs) of antimicrobial agents were determined using the agar dilution method. Phenylalanine‑arginine β-naphthylamide was used to detect the presence of the efflux pump-mediated resistant mechanism. Polymerase chain reaction was employed to amplify genes associated with drug resistance, including β‑lactamase genes, efflux pump genes and outer membrane protein gene CarO. A few amplicons were randomly selected and sequenced. Multilocus sequence analysis (MLST) was employed in typing A. baumanni. A. baumannii was resistant to imipenem, simultaneously showing resistance to several other antimicrobials. In addtition, 13 A. baumannii were found to mediate drug resistance through operation of the efflux pump. Of the various drug resistance genes tested, blaOXA‑51 was present in 46 isolates, blaOXA‑23 gene was present in 44 isolates and blaNDM gene was found in only one strain. Other drug resistant‑associated genes, including blaKPC, blaIMP, blaOXA-24, blaOXA‑58, blaSHV, blaGIM and blaVIM were not detected. Mutation of adeS and outer membrane protein gene CarO were found in a few of the imipenem‑resistant isolates. The MLST analysis revealed that all 46 clinical isolates were clustered into 11 genotypes and the most frequent genotype was ST208. In conclusion, β‑lactamase genes, genes involved in efflux pump and mutation of outer membrane protein encoding gene may be important in mediating imipenem resistance in A. baumannii. Of the 11 different genotypes, ST11 was shared by the majority of A. baumannii, which may be due to horizontal transfer of patients from hospitals.

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.

Microbiological and molecular characterization of Corynebacterium diphtheriae isolated in Algeria between 1992 and 2015.

PubMed

Benamrouche, N; Hasnaoui, S; Badell, E; Guettou, B; Lazri, M; Guiso, N; Rahal, K

2016-12-01

The objectives of this study were to undertake the microbiological and molecular characterization of Corynebacterium diphtheriae isolates collected in Algeria during epidemic and post-epidemic periods between 1992 and 2015. Microbiological characterization includes the determination of biotype and toxigenicity status using phenotypic and genotypic methods. Antimicrobial susceptibility was determined by the E-test method. Molecular characterization was performed by multi-locus sequence typing. In total, there were 157 cases of C. diphtheriae isolates, 127 in patients with respiratory diphtheria and 30 with ozena. Isolates with a mitis biotype were predominant (122 out of 157; 77.7%) followed by belfanti (28 out of 157; 17.8%) and gravis biotype (seven out of 157; 4.5%). Toxigenic isolates were predominant in the period 1992-2006 (74 out of 134) whereas in the period 2007-2015, only non-toxigenic isolates circulated (23 out of 23). All 157 isolates were susceptible to erythromycin, gentamicin, vancomycin and cotrimoxazole. Reduced susceptibility to penicillin G, cefotaxime, tetracycline and chloramphenicol was detected in 90 (57.3%), 88 (56.1%), 112 (71.3%) and 90 (57.3%) isolates, respectively. Multi-locus sequence typing analysis indicates that sequence type 116 (ST-116) was the most frequent, with 65 out of 100 isolates analysed, in particular during the epidemic period 1992-1999 (57 out of 65 isolates). In the post-epidemic period, 2000-2015, 13 different sequence types were isolated. All belfanti isolates (ten out of 100 isolates) belonged to closely related sequence types grouped in a phylogenetically distinct eBurst group and were collected exclusively in ozena cases. In conclusion, the epidemic period was associated with ST-116 while the post-epidemic period was characterized by more diversity. Belfanti isolates are grouped in a phylogenetically distinct clonal complex. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Genotypic relationships among strains classified under the (Pasteurella) haemolytica-complex as indicated by ribotyping and multilocus enzyme electrophoresis.

PubMed

Angen, O; Caugant, D A; Olsen, J E; Bisgaard, M

1997-10-01

Two-hundred and one strains classified under the (Pasteurella) haemolytica-complex isolated from cattle, sheep, deer, pigs, hares and rabbits were investigated by ribotyping. Fifty-nine of these strains were selected for further studies using multilocus enzyme electrophoresis (MEE). A correlation between the clusters identified by ribotyping and MEE was demonstrated and the results furthermore indicated that a genetic basis exists for most clusters previously outlined by the use of quantitative evaluation of phenotypic data. The taxonomic relevance of ornithine decarboxylase and fermentation of L-arabinose, D-sorbitol and glucosides for taxonomic delineation within the (P.) haemolytica-complex was supported. A taxonomic importance was further indicated for ONPG, ONPX, ONPF, meso-inositol, D-xylose, maltose, dextrine and NPG in relation to some of the taxa. Within the porcine taxon 15, however, differences in ornithine decarboxylase did not correspond to genetic clusters. Six lineages were revealed by MEE. Lineage A contained electrophoretic types (ETs) representing biogroups 1, 3A-3H, 8A and 9, indicating a genetic relationship between these groups--an observation which was supported by ribotyping. Lineage B included biogroup 8D, 3 strains from biogroup 10 and a single strain from biogroup 1 and taxon 18/biovar 1. Lineage C contained strains allocated to biogroup 6 from ruminants and the porcine taxon 15. The similarity between these two groups was accentuated by ribotyping. Lineage D and the single isolate in lineage E contained strains allocated to biogroups 7, 10, 8B and 8C, in addition to single strains from biogroups 6 and 9. The same strains were found in the heterogenous ribotype cluster 17. Lineage F contained strains representing the leprine taxon 20 and the ruminant (P.) granulomatis. Ribotyping indicated that the ruminant biogroup 3J was affiliated with both taxon 20 and (P.) granulomatis.

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

PubMed

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

2017-08-01

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

Intra-Species Genetic Diversity and Clonal Structure of Cryptosporidium parvum in Sheep Farms in a Confined Geographical Area in Northeastern Spain.

PubMed

Ramo, Ana; Monteagudo, Luis V; Del Cacho, Emilio; Sánchez-Acedo, Caridad; Quílez, Joaquín

2016-01-01

A multilocus fragment typing approach including eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to investigate the intra-farm and intra-host genetic diversity of Cryptosporidium parvum in sheep farms in a confined area in northeastern Spain. Genomic DNA samples of 113 C. parvum isolates from diarrheic pre-weaned lambs collected in 49 meat-type sheep farms were analyzed. Loci exhibited various degrees of polymorphism, the finding of 7-9 alleles in the four most variable and discriminatory markers (ML2, Cgd6_5400, Cgd6_3940, and GP60) being remarkable. The combination of alleles at the twelve loci identified a total of 74 multilocus subtypes (MLTs) and provided a Hunter-Gaston discriminatory index of 0.988 (95% CI, 0.979-0.996). The finding that most MLTs (n = 64) were unique to individual farms evidenced that cryptosporidial infection is mainly transmitted within sheep flocks, with herd-to-herd transmission playing a secondary role. Limited intra- host variability was found, since only five isolates were genotypically mixed. In contrast, a significant intra-farm genetic diversity was seen, with the presence of multiple MLTs on more than a half of the farms (28/46), suggesting frequent mutations or genetic exchange through recombination. Comparison with a previous study in calves in northern Spain using the same 12-loci typing approach showed differences in the identity of major alleles at most loci, with a single MLT being shared between lambs and calves. Analysis of evolutionary descent by the algorithm eBURST indicated a high degree of genetic divergence, with over 41% MLTs appearing as singletons along with a high number of clonal complexes, most of them linking only two MLTs. Bayesian Structure analysis and F statistics also revealed the genetic remoteness of most C. parvum isolates and no ancestral population size was chosen. Linkage analysis evidenced a prevalent pattern of clonality within the parasite population.

Major clades of Agaricales: a multilocus phylogenetic overview.

Treesearch

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

2006-01-01

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

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

Treesearch

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

2014-01-01

Background: As it becomes increasingly possible to obtain DNA sequences of orthologous genes from diverse sets of taxa, species trees are frequently being inferred from multilocus data. However, the behavior of many methods for performing this inference has remained largely unexplored. Some methods have been proven to be consistent given certain evolutionary models,...

Analysis of multilocus zygotic associations.

PubMed

Yang, Rong-Cai

2002-05-01

While nonrandom associations between zygotes at different loci (zygotic associations) frequently occur in Hardy-Weinberg disequilibrium populations, statistical analysis of such associations has received little attention. In this article, we describe the joint distributions of zygotes at multiple loci, which are completely characterized by heterozygosities at individual loci and various multilocus zygotic associations. These zygotic associations are defined in the same fashion as the usual multilocus linkage (gametic) disequilibria on the basis of gametic and allelic frequencies. The estimation and test procedures are described with details being given for three loci. The sampling properties of the estimates are examined through Monte Carlo simulation. The estimates of three-locus associations are not free of bias due to the presence of two-locus associations and vice versa. The power of detecting the zygotic associations is small unless different loci are strongly associated and/or sample sizes are large (>100). The analysis of zygotic associations not only offers an effective means of packaging numerous genic disequilibria required for a complete characterization of multilocus structure, but also provides opportunities for making inference about evolutionary and demographic processes through a comparative assessment of zygotic association vs. gametic disequilibrium for the same set of loci in nonequilibrium populations.

Conservation genetics of the endangered Isle Royale gray wolf

USGS Publications Warehouse

Wayne, R.K.; Lehman, N.; Girman, D.; Gogan, P.J.P.; Gilbert, D.A.; Hansen, K.; Peterson, R.O.; Seal, U.S.; Eisenhawer, Andrew; Mech, L.D.; Krumenaker, R.J.

1991-01-01

The small group of wolves on Isle Royale has been studied for over three decades as a model of the relationship between large carnivores and their prey. During the last ten years the population declined from 50 individuals to as few as 12 individuals. The causes of this decline may be food shortages, disease, or reduced genetic variability. We address the issues of genetic variability and relationships of Isle Royale wolves using allozyme electrophoresis, mtDNA restriction-site analysis, and multilocus hypervariable minisatellite DNA analysis (genetic fingerprinting). Our results indicate that approximately 50% of the allozyme heterozygosity has been lost in the island population, a decline similar to that expected if no immigration had occurred from the mainland. The genetic fingerprinting data indicate that the seven sampled Isle Royale wolves are as similar as captive populations of siblings. Surprisingly, the Isle Royale wolves have an mtDNA genotype that is very rare on the mainland, being found in only one of 144 mainland wolves. This suggests that the remaining Isle Royale wolves are probably derived from a single female founder.

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.

Tools for assessing kinship, population structure, phylogeography, and interspecific hybridization in Asian carps invasive to the Mississippi River, USA: isolation and characterization of novel tetranucleotide microsatellite DNA loci in silver carp Hypophthalmichthys molitrix

USGS Publications Warehouse

King, T.L.; Eackles, M.S.; Chapman, D.C.

2011-01-01

We document the isolation and characterization of novel tetranucleotide microsatellite DNA markers for the invasive silver carp Hypophthalmichthys molitrix and provide the results of cross-species amplification for three additional invasive carp species: bighead (H. nobilis), grass (Ctenopharyngodon idella) and black (Mylopharyngodon piceus). In the target species these markers yielded levels of allelic diversity (average 4.4 alleles/locus) and heterozygosity (average 54.7%) sufficient to: (1) provide unique multilocus genotypes; (2) delineate kinship relationships; (3) differentiate populations/species; (4) estimate effective population sizes; and (5) provide unique demographic perspectives for control or eradication. Currently these markers are being utilized to determine the degree of introgressive hybridization between H. molitrix and H. nobilis, to quantify gene flow between different sub-basins established in the central United States, and to assess the demographic status of sub-basin groups. This information will be critically important in the management/control of these invasive species.

Characterization of extensively drug-resistant Mycobacterium tuberculosis in Nepal.

PubMed

Poudel, Ajay; Maharjan, Bhagwan; Nakajima, Chie; Fukushima, Yukari; Pandey, Basu D; Beneke, Antje; Suzuki, Yasuhiko

2013-01-01

The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global control of TB. Although molecular characterization of drug resistance-associated mutations in multidrug-resistant isolates in Nepal has been made, mutations in XDR isolates and their genotypes have not been reported previously. In this study, we identified and characterized 13 XDR Mycobacterium tuberculosis isolates from clinical isolates in Nepal. The most prevalent mutations involved in rifampicin, isoniazid, ofloxacin, and kanamycin/capreomycin resistance were Ser531Leu in rpoB gene (92.3%), Ser315Thr in katG gene (92.3%), Asp94Gly in gyrA gene (53.9%) and A1400G in rrs gene (61.5%), respectively. Spoligotyping and multilocus sequence typing revealed that 69% belonged to Beijing family, especially modern types. Further typing with 26-loci variable number of tandem repeats suggested the current spread of XDR M. tuberculosis. Our result highlights the need to reinforce the TB policy in Nepal with regard to control and detection strategies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Size asymmetry in intraspecific competition and the density-dependence of inbreeding depression in a natural plant population: a case study in cassava (Manihot esculenta Crantz, Euphorbiaceae).

PubMed

Pujol, B; McKey, D

2006-01-01

The effects of competition on the genetic composition of natural populations are not well understood. We combined demography and molecular genetics to study how intraspecific competition affects microevolution in cohorts of volunteer plants of cassava (Manihot esculenta) originating from seeds in slash-and-burn fields of Palikur Amerindians in French Guiana. In this clonally propagated crop, genotypic diversity is enhanced by the incorporation of volunteer plants into farmers' stocks of clonal propagules. Mortality of volunteer plants was density-dependent. Furthermore, the size asymmetry of intraspecific competition increased with local clustering of plants. Size of plants was correlated with their multilocus heterozygosity, and stronger size-dependence of survival in clusters of plants, compared with solitary plants, increased the magnitude of inbreeding depression when competition was severe. The density-dependence of inbreeding depression of volunteer plants helps explain the high heterozygosity of volunteers that survive to harvest time and thus become candidates for clonal propagation. This effect could help favour the maintenance of sex in this 'vegetatively' propagated crop plant.

Severe Acquired Toxoplasmosis in Immunocompetent Adult Patients in French Guiana

PubMed Central

Carme, B.; Bissuel, F.; Ajzenberg, D.; Bouyne, R.; Aznar, C.; Demar, M.; Bichat, S.; Louvel, D.; Bourbigot, A. M.; Peneau, C.; Neron, P.; Dardé, M. L.

2002-01-01

The most common presentation of symptomatic postnatally acquired toxoplasmosis in immunocompetent patients is painless cervical adenopathy. Acute visceral manifestations are associated in rare cases. We report 16 cases of severe primary toxoplasmosis diagnosed in French Guiana during a 6.5-year period. All of the subjects were immunocompetent adults hospitalized with clinical presentations consisting of a marked, nonspecific infectious syndrome accompanied by an altered general status with at least one visceral localization, mainly pulmonary involvement (14 cases). Acute toxoplasmosis was diagnosed according to the results of serological tests suggestive of recent primary infection and the absence of an alternative etiology. Recovery was rapid following specific antitoxoplasmosis treatment. Thirteen of the 16 patients had consumed game in the 2 weeks before the onset of the symptoms, and in eight cases the game was considered to have been undercooked. Toxoplasma strains, which were virulent in mice, were isolated from three patients. Microsatellite analysis showed that all of these isolates exhibited an atypical multilocus genotype, with one allele found only for isolates of this region. PMID:12409371

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.

Ustilago maydis populations tracked maize through domestication and cultivation in the Americas

PubMed Central

Munkacsi, Andrew B; Stoxen, Sam; May, Georgiana

2008-01-01

The domestication of crops and the development of agricultural societies not only brought about major changes in human interactions with the environment but also in plants' interactions with the diseases that challenge them. We evaluated the impact of the domestication of maize from teosinte and the widespread cultivation of maize on the historical demography of Ustilago maydis, a fungal pathogen of maize. To determine the evolutionary response of the pathogen's populations, we obtained multilocus genotypes for 1088 U. maydis diploid individuals from two teosinte subspecies in Mexico and from maize in Mexico and throughout the Americas. Results identified five major U. maydis populations: two in Mexico; two in South America; and one in the United States. The two populations in Mexico diverged from the other populations at times comparable to those for the domestication of maize at 6000–10 000 years before present. Maize domestication and agriculture enforced sweeping changes in U. maydis populations such that the standing variation in extant pathogen populations reflects evolution only since the time of the crop's domestication. PMID:18252671

Low occurrence of 'non-haemolytic Haemophilus haemolyticus' misidentified as Haemophilus influenzae in cystic fibrosis respiratory specimens, and frequent recurrence of persistent H. influenzae clones despite antimicrobial treatment.

PubMed

Fenger, Mette G; Ridderberg, Winnie; Olesen, Hanne V; Nørskov-Lauritsen, Niels

2012-12-01

Non-influenzae commensal Haemophilus species of low pathogenicity may be difficult to discriminate from Haemophilus influenzae. We investigated the level of misidentifications in respiratory specimens from cystic fibrosis patients and evaluated the colonisation dynamics of genuine H. influenzae isolates. One hundred and ninety-two presumptive H. influenzae isolates were re-examined by assessment of marker genes sodC and fucK, and isolates with aberrant genotypes were subjected to multilocus sequence typing. Misidentifications (3%) were mainly caused by failure to identify porphyrin-synthesising strains, and only a single strain (0.5%) could be classified as 'non-haemolytic Haemophilus haemolyticus'. Sequential isolates of confirmed H. influenzae isolates from individual patients were typed by pulsed-field gel electrophoresis. Despite the routine prescription of antimicrobial therapy, the majority of H. influenzae isolates were identical with at least one of the strains cultured from the two preceding positive samples from the same patient. Copyright © 2012 Elsevier GmbH. All rights reserved.

Evidence for rare capsular switching in Streptococcus agalactiae.

PubMed

Martins, Elisabete Raquel; Melo-Cristino, José; Ramirez, Mário

2010-03-01

The polysaccharide capsule is a major antigenic factor in Streptococcus agalactiae (Lancefield group B streptococcus [GBS]). Previous observations suggest that exchange of capsular loci is likely to occur rather frequently in GBS, even though GBS is not known to be naturally transformable. We sought to identify and characterize putative capsular switching events, by means of a combination of phenotypic and genotypic methods, including pulsed-field gel electrophoretic profiling, multilocus sequence typing, and surface protein and pilus gene profiling. We show that capsular switching by horizontal gene transfer is not as frequent as previously suggested. Serotyping errors may be the main reason behind the overestimation of capsule switching, since phenotypic techniques are prone to errors of interpretation. The identified putative capsular transformants involved the acquisition of the entire capsular locus and were not restricted to the serotype-specific central genes, the previously suggested main mechanism underlying capsular switching. Our data, while questioning the frequency of capsular switching, provide clear evidence for in vivo capsular transformation in S. agalactiae, which may be of critical importance in planning future vaccination strategies against this pathogen.

Evidence for Rare Capsular Switching in Streptococcus agalactiae▿

PubMed Central

Martins, Elisabete Raquel; Melo-Cristino, José; Ramirez, Mário

2010-01-01

The polysaccharide capsule is a major antigenic factor in Streptococcus agalactiae (Lancefield group B streptococcus [GBS]). Previous observations suggest that exchange of capsular loci is likely to occur rather frequently in GBS, even though GBS is not known to be naturally transformable. We sought to identify and characterize putative capsular switching events, by means of a combination of phenotypic and genotypic methods, including pulsed-field gel electrophoretic profiling, multilocus sequence typing, and surface protein and pilus gene profiling. We show that capsular switching by horizontal gene transfer is not as frequent as previously suggested. Serotyping errors may be the main reason behind the overestimation of capsule switching, since phenotypic techniques are prone to errors of interpretation. The identified putative capsular transformants involved the acquisition of the entire capsular locus and were not restricted to the serotype-specific central genes, the previously suggested main mechanism underlying capsular switching. Our data, while questioning the frequency of capsular switching, provide clear evidence for in vivo capsular transformation in S. agalactiae, which may be of critical importance in planning future vaccination strategies against this pathogen. PMID:20023016

Isolation and characterization of microsatellite DNA loci in the threatened flat-spired three-toothed land snail Triodopsis platysayoides

USGS Publications Warehouse

King, Timothy L.; Eackles, Michael S.; Garner, B. A.; van Tuinen, M.; Arbogast, B. S.

2015-01-01

The hermaphroditic flat-spired three-tooth land snail (Triodopsis platysayoides) is endemic to a 21-km stretch of the Cheat River Gorge of northeastern West Virginia, USA. We document isolation and characterization of ten microsatellite DNA markers in this at-risk species. The markers displayed a moderate level of allelic diversity (averaging 7.1 alleles/locus) and heterozygosity (averaging 58.6 %). Allelic diversity at seven loci was sufficient to produce unique multilocus genotypes; no indication of selfing was detected in this cosexual species. Minimal deviations from Hardy–Weinberg equilibrium and no linkage disequilibrium were observed within subpopulations. All loci deviated from Hardy–Weinberg expectations when individuals from subpopulations were pooled. Microsatellite markers developed for T. platysayoides yielded sufficient genetic diversity to (1) distinguish all individuals sampled and the level of selfing; (2) be appropriate for addressing fine-scale population structuring; (3) provide novel demographic insights for the species; and (4) cross-amplify and detect allelic diversity in the congeneric T. juxtidens.

GENETIC STRUCTURE OF TRIATOMA INFESTANS POPULATIONS IN RURAL COMMUNITIES OF SANTIAGO DEL ESTERO, NORTHERN ARGENTINA

PubMed Central

Marcet, PL; Mora, MS; Cutrera, AP; Jones, L; Gürtler, RE; Kitron, U; Dotson, EM

2008-01-01

To gain an understanding of the genetic structure and dispersal dynamics of T. infestans populations, we analyzed the multilocus genotype of 10 microsatellite loci for 352 T. infestans collected in 21 houses of 11 rural communities in October 2002. Genetic structure was analyzed at the community and house compound levels. Analysis revealed that vector control actions affected the genetic structure of T. infestans populations. Bug populations from communities under sustained vector control (core area) were highly structured and genetic differentiation between neighboring house compounds was significant. In contrast, bug populations from communities with sporadic vector control actions were more homogeneous and lacked defined genetic clusters. Genetic differentiation between population pairs did not fit a model of isolation by distance at the microgeographical level. Evidence consistent with flight or walking bug dispersal was detected within and among communities, dispersal was more female-biased in the core area and results suggested that houses received immigrants from more than one source. Putative sources and mechanisms of re-infestation are described. These data may be use to design improved vector control strategies PMID:18773972

Population Structure of Pseudocercospora fijiensis in Costa Rica Reveals Shared Haplotype Diversity with Southeast Asian Populations.

PubMed

Saville, Amanda; Charles, Melodi; Chavan, Suchitra; Muñoz, Miguel; Gómez-Alpizar, Luis; Ristaino, Jean Beagle

2017-12-01

Pseudocercospora fijiensis is the causal pathogen of black Sigatoka, a devastating disease of banana that can cause 20 to 80% yield loss in the absence of fungicides in banana crops. The genetic structure of populations of P. fijiensis in Costa Rica was examined and compared with Honduran and global populations to better understand migration patterns and inform management strategies. In total, 118 isolates of P. fijiensis collected from Costa Rica and Honduras from 2010 to 2014 were analyzed using multilocus genotyping of six loci and compared with a previously published global dataset of populations of P. fijiensis. The Costa Rican and Honduran populations shared haplotype diversity with haplotypes from Southeast Asia, Oceania, and the Americas but not Africa for all but one of the six loci studied. Gene flow and shared haplotype diversity was found in Honduran and Costa Rican populations of the pathogen. The data indicate that the haplotypic diversity observed in Costa Rican populations of P. fijiensis is derived from dispersal from initial outbreak sources in Honduras and admixtures between genetically differentiated sources from Southeast Asia, Oceania, and the Americas.

Direct detection of Trichomonas vaginalis virus in Trichomonas vaginalis positive clinical samples from the Netherlands.

PubMed

Jehee, Ivo; van der Veer, Charlotte; Himschoot, Michelle; Hermans, Mirjam; Bruisten, Sylvia

2017-12-01

Trichomonas vaginalis is the most common sexually transmitted parasitical infection worldwide. T. vaginalis can carry a virus: Trichomonas vaginalis virus (TVV). To date, four TVV species have been described. Few studies have investigated TVV prevalence and its clinical importance. We have developed a nested reverse-transcriptase PCR, with novel, type specific primers to directly detect TVV RNA in T. vaginalis positive clinical samples. A total of 119T. vaginalis positive clinical samples were collected in Amsterdam and "s-Hertogenbosch, the Netherlands, from 2012 to 2016. For all samples T. vaginalis was genotyped using multi-locus sequence typing. The T. vaginalis positive samples segregated into a two-genotype population: type I (n=64) and type II (n=55). All were tested for TVV with the new TVV PCR. We detected 3 of the 4 TVV species. Sequencing of the amplified products showed high homology with published TVV genomes (82-100%). Half of the T. vaginalis clinical samples (n=60, 50.4%) were infected with one or more TVV species, with a preponderance for TVV infections in T. vaginalis type I (n=44, 73.3%). Clinical data was available for a subset of samples (n=34) and we observed an association between testing positive for (any) TVV and reporting urogenital symptoms (p=0.023). The nested RT-PCR allowed for direct detection of TVV in T. vaginalis positive clinical samples. This may be helpful in studies and clinical settings, since T. vaginalis disease and/or treatment outcome may be influenced by the protozoa"s virus. Copyright © 2017 Elsevier B.V. All rights reserved.

Increase in Genetic Diversity of Haemophilus influenzae Serotype b (Hib) Strains after Introduction of Hib Vaccination in The Netherlands

PubMed Central

Schouls, Leo M.; van der Ende, Arie; van de Pol, Ingrid; Schot, Corrie; Spanjaard, Lodewijk; Vauterin, Paul; Wilderbeek, Dorus; Witteveen, Sandra

2005-01-01

Recently, there has been an increase in The Netherlands in the number of cases of invasive disease caused by Haemophilus influenzae serotype b (Hib). To study a possible change in the Hib population that could explain the rise in incidence, a multiple-locus variable number tandem repeats analysis (MLVA) was developed to genotype H. influenzae isolates. The MLVA enabled the differentiation of H. influenzae serotype b strains with higher discriminatory power than multilocus sequence typing (MLST). MLVA profiles of noncapsulated H. influenzae and H. influenzae serotype f strains were more heterogeneous than serotype b strains and were distinct from Hib, although some overlap occurred. The MLVA was used to genotype a collection of 520 H. influenzae serotype b strains isolated from patients in The Netherlands with invasive disease. The strains were collected from 1983 from 2002, covering a time period of 10 years before and 9 years after the introduction of the Hib vaccine in the Dutch national vaccination program. MLVA revealed a sharp increase in genetic diversity of Hib strains isolated from neonates to 4-year-old patients after 1993, when the Hib vaccine was introduced. Hib strains isolated from patients older than 4 years in age were genetically diverse, and no significant change in diversity was seen after the introduction of the vaccine. These observations suggest that after the introduction of the Hib vaccine young children no longer constitute the reservoir for Hib and that they are infected by adults carrying genetically diverse Hib strains. PMID:15956392

Expression of Curli by Escherichia coli O157:H7 Strains Isolated from Patients during Outbreaks Is Different from Similar Strains Isolated from Leafy Green Production Environments.

PubMed

Ravva, Subbarao V; Sarreal, Chester Z; Cooley, Michael B

2016-01-01

We previously reported that the strains of Escherichia coli O157:H7 (EcO157) that survived longer in austere soil environment lacked expression of curli, a fitness trait linked with intestinal colonization. In addition, the proportion of curli-positive variants of EcO157 decreased with repeated soil exposure. Here we evaluated 84 and 176 clinical strains from outbreaks and sporadic infections in the US, plus 211 animal fecal and environmental strains for curli expression. These shiga-toxigenic strains were from 328 different genotypes, as characterized by multi-locus variable-number tandem-repeat analysis (MLVA). More than half of the fecal strains (human and animal) and a significant proportion of environmental isolates (82%) were found to lack curli expression. EcO157 strains from several outbreaks linked with the consumption of contaminated apple juice, produce, hamburgers, steak, and beef were also found to lack curli expression. Phylogenetic analysis of fecal strains indicates curli expression is distributed throughout the population. However, a significant proportion of animal fecal isolates (84%) gave no curli expression compared to human fecal isolates (58%). In addition, analysis of environmental isolates indicated nearly exclusive clustering of curli expression to a single branch of the minimal spanning tree. This indicates that curli expression depends primarily upon the type of environmental exposure and the isolation source, although genotypic differences also contribute to clonal variation in curli. Furthermore, curli-deficient phenotype appears to be a selective trait for survival of EcO157 in agricultural environments.

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

Echinocandin Resistance in Candida Species Isolates from Liver Transplant Recipients.

PubMed

Prigent, Gwénolé; Aït-Ammar, Nawel; Levesque, Eric; Fekkar, Arnaud; Costa, Jean-Marc; El Anbassi, Sarra; Foulet, Françoise; Duvoux, Christophe; Merle, Jean-Claude; Dannaoui, Eric; Botterel, Françoise

2017-02-01

Liver transplant recipients are at risk of invasive fungal infections, especially candidiasis. Echinocandin is recommended as prophylactic treatment but is increasingly associated with resistance. Our aim was to assess echinocandin drug resistance in Candida spp. isolated from liver transplant recipients treated with this antifungal class. For this, all liver-transplanted patients in a University Hospital (Créteil, France) between January and June of 2013 and 2015 were included. Susceptibilities of Candida isolates to echinocandins were tested by Etest and the EUCAST reference method. Isolates were analyzed by FKS sequencing and genotyped based on microsatellites or multilocus sequence typing (MLST) profiles. Ninety-four patients were included, and 39 patients were colonized or infected and treated with echinocandin. Echinocandin resistance appeared in 3 (8%) of the treated patients within 1 month of treatment. One patient was colonized by resistant Candida glabrata, one by resistant Candida dubliniensis, and one by resistant Candida albicans Molecular analysis found three mutations in FKS2 HS1 (F659S, S663A, and D666E) for C. glabrata and one mutation in FKS1 HS1 (S645P) for C. dubliniensis and C. albicans Susceptible and resistant isolates belonged to the same genotype. To our knowledge, this is the first study on echinocandin resistance in Candida spp. in a liver transplant population. Most resistant isolates were found around/in digestive sites, perhaps due to lower diffusion of echinocandin in these sites. This work documents the risk of emergence of resistance to echinocandin, even after short-term treatment. Copyright © 2017 Prigent et al.

Genomic scan as a tool for assessing the genetic component of phenotypic variance in wild populations.

PubMed

Herrera, Carlos M

2012-01-01

Methods for estimating quantitative trait heritability in wild populations have been developed in recent years which take advantage of the increased availability of genetic markers to reconstruct pedigrees or estimate relatedness between individuals, but their application to real-world data is not exempt from difficulties. This chapter describes a recent marker-based technique which, by adopting a genomic scan approach and focusing on the relationship between phenotypes and genotypes at the individual level, avoids the problems inherent to marker-based estimators of relatedness. This method allows the quantification of the genetic component of phenotypic variance ("degree of genetic determination" or "heritability in the broad sense") in wild populations and is applicable whenever phenotypic trait values and multilocus data for a large number of genetic markers (e.g., amplified fragment length polymorphisms, AFLPs) are simultaneously available for a sample of individuals from the same population. The method proceeds by first identifying those markers whose variation across individuals is significantly correlated with individual phenotypic differences ("adaptive loci"). The proportion of phenotypic variance in the sample that is statistically accounted for by individual differences in adaptive loci is then estimated by fitting a linear model to the data, with trait value as the dependent variable and scores of adaptive loci as independent ones. The method can be easily extended to accommodate quantitative or qualitative information on biologically relevant features of the environment experienced by each sampled individual, in which case estimates of the environmental and genotype × environment components of phenotypic variance can also be obtained.

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.

Genetic identification of Theobroma cacao L. trees with high Criollo ancestry in Soconusco, Chiapas, Mexico.

PubMed

Vázquez-Ovando, J A; Molina-Freaner, F; Nuñez-Farfán, J; Ovando-Medina, I; Salvador-Figueroa, M

2014-12-12

Criollo-type cacao trees are an important pool of genes with potential to be used in cacao breeding and selection programs. For that reason, we assessed the diversity and population structure of Criollo-type trees (108 cultivars with Criollo phenotypic characteristics and 10 Criollo references) using 12 simple sequence repeat (SSR) markers. Cultivars were selected from 7 demes in the Soconusco region of southern Mexico. SSRs amplified 74 alleles with an average of 3.6 alleles per population. The overall populations showed an average observed heterozygosity of 0.28, indicating heterozygote deficiency (average fixation index F = 0.50). However, moderate allelic diversity was found within populations (Shannon index for all populations I = 0.97). Bayesian method analysis determined 2 genetic clusters (K = 2) within individuals. In concordance, an assignment test grouped 37 multilocus genotypes (including 10 references) into a first cluster (Criollo), 54 into a second (presumably Amelonado), and 27 admixed individuals unassigned at the 90% threshold likely corresponding to the Trinitario genotype. This classification was supported by the principal coordinate analysis and analysis of molecular variance, which showed 12% of variation among populations (FST = 0.123, P < 0.0001). Sampled demes sites (1- 7) in the Soconusco region did not show any evidence of clustering by geographic location, and this was supported by the Mantel test (Rxy = 0.54, P = 0.120). Individuals with high Criollo lineage planted in Soconusco farms could be an important reservoir of genes for future breeding programs searching for fine, taste, flavor, and aroma cocoa.

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds

PubMed Central

Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa

2015-01-01

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates. PMID:25809972

Spatial variation in anthropogenic mortality induces a source-sink system in a hunted mesopredator.

PubMed

Minnie, Liaan; Zalewski, Andrzej; Zalewska, Hanna; Kerley, Graham I H

2018-04-01

Lethal carnivore management is a prevailing strategy to reduce livestock predation. Intensity of lethal management varies according to land-use, where carnivores are more intensively hunted on farms relative to reserves. Variations in hunting intensity may result in the formation of a source-sink system where carnivores disperse from high-density to low-density areas. Few studies quantify dispersal between supposed sources and sinks-a fundamental requirement for source-sink systems. We used the black-backed jackal (Canis mesomelas) as a model to determine if heterogeneous anthropogenic mortality induces a source-sink system. We analysed 12 microsatellite loci from 554 individuals from lightly hunted and previously unhunted reserves, as well as heavily hunted livestock- and game farms. Bayesian genotype assignment showed that jackal populations displayed a hierarchical population structure. We identified two genetically distinct populations at the regional level and nine distinct subpopulations at the local level, with each cluster corresponding to distinct land-use types separated by various dispersal barriers. Migration, estimated using Bayesian multilocus genotyping, between reserves and farms was asymmetric and heterogeneous anthropogenic mortality induced source-sink dynamics via compensatory immigration. Additionally some heavily hunted populations also acted as source populations, exporting individuals to other heavily hunted populations. This indicates that heterogeneous anthropogenic mortality results in the formation of a complex series of interconnected sources and sinks. Thus, lethal management of mesopredators may not be an effective long-term strategy in reducing livestock predation, as dispersal and, more importantly, compensatory immigration may continue to affect population reduction efforts as long as dispersal from other areas persists.

Predominance of ST320 among Streptococcus pneumoniae serotype 19A isolates from 10 Asian countries.

PubMed

Shin, Juyoun; Baek, Jin Yang; Kim, So Hyun; Song, Jae-Hoon; Ko, Kwan Soo

2011-05-01

After 7-valent pneumococcal conjugate vaccine (PCV7) introduction, non-vaccine serotypes such as 19A are increasing among Streptococcus pneumoniae. However, only limited data on 19A S. pneumoniae are available in Asian countries. Out of 1637 S. pneumoniae clinical pneumonia isolates collected during 2008 and 2009 from 10 Asian countries (Korea, Malaysia, Taiwan, Thailand, Saudi Arabia, Hong Kong, India, Japan, the Philippines and Vietnam), 91 serotype 19A S. pneumoniae isolates were identified. Capsular swelling reaction identified serotype 19A isolates. Antimicrobial susceptibility testing was performed on the serotype 19A isolates using the broth microdilution method, and the genotypes of the isolates were assessed using multilocus sequence typing. Thirty different sequence types (STs) were identified. The most prevalent clone was ST320 (46 isolates, 51.1%). ST320 was found in Hong Kong, India, Korea, Malaysia, Saudi Arabia and Taiwan. ST320 isolates were mostly multidrug resistant (MDR) and showed significantly higher resistance rates than other STs for cefuroxime, clindamycin, and trimethoprim/sulfamethoxazole. Although diverse clones were identified among 19A S. pneumoniae isolates, MDR ST320 was the predominant clone in Asian countries. Its predominance, even in countries with no or low coverage of PCV7, may indicate that its emergence and dissemination was due to more than just vaccine selection pressure in Asian countries. A longitudinal investigation of the change of serotypes and genotypes since the introduction of PCV7 is required to understand the emergence and dissemination mechanisms of a certain clone of 19A S. pneumoniae isolates.

Co-Infection and Genetic Diversity of Tick-Borne Pathogens in Roe Deer from Poland

PubMed Central

Werszko, Joanna; Cydzik, Krystian; Bajer, Anna; Michalik, Jerzy; Behnke, Jerzy M.

2013-01-01

Abstract Wild species are essential hosts for maintaining Ixodes ticks and the tick-borne diseases. The aim of our study was to estimate the prevalence, the rate of co-infection with Babesia, Bartonella, and Anaplasma phagocytophilum, and the molecular diversity of tick-borne pathogens in roe deer in Poland. Almost half of the tested samples provided evidence of infection with at least 1 species. A. phagocytophilum (37.3%) was the most common and Bartonella (13.4%) the rarest infection. A total of 18.3% of all positive samples from roe deer were infected with at least 2 pathogens, and one-third of those were co-infected with A. phagocytophilum, Bartonella, and Babesia species. On the basis of multilocus molecular studies we conclude that: (1) Two different genetic variants of A. phagocytophilum, zoonotic and nonzoonotic, are widely distributed in Polish roe deer population; (2) the roe deer is the host for zoonotic Babesia (Bab. venatorum, Bab. divergens), closely related or identical with strains/species found in humans; (3) our Bab. capreoli and Bab. divergens isolates differed from reported genotypes at 2 conserved base positions, i.e., positions 631 and 663; and (4) this is the first description of Bart. schoenbuchensis infections in roe deer in Poland. We present 1 of the first complex epidemiological studies on the prevalence of Babesia, Bartonella, and A. phagocytophilum in naturally infected populations of roe deer. These game animals clearly have an important role as reservoir hosts of tick-borne pathogens, but the pathogenicity and zoonotic potential of the parasite genotypes hosted by roe deer requires further detailed investigation. PMID:23473225

The use (or misuse) of microsatellite allelic distances in the context of inbreeding and conservation genetics.

PubMed

Hansson, Bengt

2010-03-01

In line with inbreeding theory, genetic diversity at a set of molecular markers may explain variation in fitness-associated traits in partially inbred populations, and such associations will appear as 'genotype-fitness correlations'. An individual genetic diversity index specifically used for microsatellites is 'mean d(2)', i.e. the mean squared distance between alleles. The original hypothesis for mean d(2)-fitness correlations assumes that mean d(2) captures fitness effects at both ends of the inbreeding-outbreeding spectrum. This hypothesis received strong criticism from work showing that even a plain diversity estimate such as multi-locus heterozygosity (MLH) outperforms mean d(2) as a predictor of the inbreeding coefficient and fitness in most realistic situations. Despite this critique, the mean d(2)-approach is still used frequently in ecological and evolutionary research, producing results suggesting that mean d(2) sometimes provides a stronger prediction of fitness than does MLH. In light of the critique, such results are unexpected, but potential explanations for them may exist (at least hypothetically), including scenarios based on close linkage and recent admixture. Nevertheless, a major caveat is that it is very difficult to predict a priori if mean d(2) will improve the genotype-fitness correlation, which in turn makes objective interpretations difficult. Mean d(2)-fitness associations are potentially interesting, but the fact that we cannot easily understand them is problematic and should be thoroughly addressed in each study. Therefore, instead of hastily reached interpretations of mean d(2)-fitness correlations, conclusions need support from complementary analyses, e.g. verifying admixture of genetically structured populations.

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

PubMed

Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa; Hirai, Itaru

2015-06-01

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Occurrence of Diverse AbGRI1-Type Genomic Islands in Acinetobacter baumannii Global Clone 2 Isolates from South Korea.

PubMed

Kim, Dae Hun; Jung, Sook-In; Kwon, Ki Tae; Ko, Kwan Soo

2017-02-01

In this study, we analyzed the frequency of the AbGRI1-type genomic island (GI) and its association with genotypes. We obtained 130 Acinetobacter baumannii isolates causing bloodstream infections from patients in South Korea. Antimicrobial susceptibility testing and multilocus sequence typing were performed. The presence of AbGRI1-type GIs and their structures were determined by sequential PCR and sequencing. Ninety-eight isolates (75.3%) representing 14 sequence types (STs) belonged to clonal complex 208 (CC208), corresponding to global clone 2 (GC2). AbGRI1-type GIs interrupted the comM gene in 107 isolates (82.4%). Four types of GIs were identified: Tn6022 (50 isolates; 46.7%), AbaR4 (23 isolates; 21.5%), Tn6166 (10 isolates; 9.3%), and Tn6166/Tn2006 (24 isolates; 22.4%). In the 50 isolates with Tn6022, Tn2006 or Tn2008B, both containing ISAba1-bla OXA-23 , was present in sites other than GIs in 3 or 28 isolates, respectively. In the 10 isolates with Tn6166, Tn2008B was identified in one isolate. AbGRI1-type GIs were identified nearly exclusively in CC208 isolates, with the exception of nine non-CC208 isolates (AbaR4 in eight ST229 isolates and Tn6022 in one ST1244 isolate). Within CC208 isolates, there was evidence of frequent recombination events, in both housekeeping genes and AbGRI1-type GIs, contributing to genotype diversification and the emergence of carbapenem resistance. Copyright © 2017 American Society for Microbiology.

Genotyping of Mycobacterium leprae for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil

PubMed Central

Fontes, Amanda N. B.; Lima, Luana N. G. C.; Mota, Rosa M. S.; Almeida, Rosa L. F.; Pontes, Maria A.; Gonçalves, Heitor de S.; Frota, Cristiane C.; Vissa, Varalakshmi D.; Brennan, Patrick J.; Guimaraes, Ricardo J. P. S.; Kendall, Carl; Kerr, Ligia R. F. S.; Suffys, Philip N.

2017-01-01

Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1–4 typing of Mycobacterium leprae in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from M. leprae in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique M. leprae genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation. PMID:29244821

Genotyping of Mycobacterium leprae for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil.

PubMed

Fontes, Amanda N B; Lima, Luana N G C; Mota, Rosa M S; Almeida, Rosa L F; Pontes, Maria A; Gonçalves, Heitor de S; Frota, Cristiane C; Vissa, Varalakshmi D; Brennan, Patrick J; Guimaraes, Ricardo J P S; Kendall, Carl; Kerr, Ligia R F S; Suffys, Philip N

2017-12-01

Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1-4 typing of Mycobacterium leprae in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from M. leprae in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique M. leprae genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation.