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1.
J Antimicrob Chemother ; 75(12): 3491-3500, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32780112

RESUMO

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Animais , Antibacterianos/farmacologia , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Fenótipo
2.
J Clin Microbiol ; 53(1): 262-72, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25392358

RESUMO

Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar Typhi outbreak in Zambia during 2010 to 2012. Ninety-four isolates were susceptibility tested by MIC determinations. Whole-genome sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the multilocus sequence type (MLST), haplotype, plasmid replicon, antimicrobial resistance genes, and genetic relatedness by single nucleotide polymorphism (SNP) analysis and genomic deletions. The outbreak affected 2,040 patients, with a fatality rate of 0.5%. Most (83.0%) isolates were multidrug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the haplotype, H58B. Most isolates contained a chromosomally translocated region containing seven antimicrobial resistance genes, catA1, blaTEM-1, dfrA7, sul1, sul2, strA, and strB, and fragments of the incompatibility group Q1 (IncQ1) plasmid replicon, the class 1 integron, and the mer operon. The genomic analysis revealed 415 SNP differences overall and 35 deletions among 33 of the isolates subjected to whole-genome sequencing. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India by 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 of the 33 isolates sequenced belonged to a tight clonal group distinct from other H58 genomes included in the study. The small numbers of SNPs identified within this group are consistent with the short-term transmission that can be expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak, during which occasional other S. Typhi lineages, including sensitive ones, continued to cocirculate. The common view is that the emerging global S. Typhi haplotype, H58B, containing the MDR IncHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-Saharan Africa; we found that a new variant of the haplotype harboring a chromosomally translocated region containing the MDR islands of IncHI1 plasmid has emerged in Zambia. This could change the perception of the term "classical MDR typhoid" currently being solely associated with the IncHI1 plasmid. It might be more common than presently thought that S. Typhi haplotype H58B harbors the IncHI1 plasmid or a chromosomally translocated MDR region or both.


Assuntos
Surtos de Doenças , Farmacorresistência Bacteriana Múltipla , Genoma Bacteriano , Genômica , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/genética , Febre Tifoide/epidemiologia , Febre Tifoide/microbiologia , Antibacterianos/farmacologia , Criança , Pré-Escolar , Cromossomos Bacterianos , Conjugação Genética , Evolução Molecular , Feminino , Ordem dos Genes , Genes Bacterianos , Haplótipos , História do Século XXI , Humanos , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Mutação , Filogenia , Plasmídeos , Polimorfismo de Nucleotídeo Único , Salmonella typhi/classificação , Deleção de Sequência , Translocação Genética , Febre Tifoide/história , Zâmbia/epidemiologia
3.
Euro Surveill ; 20(49)2015.
Artigo em Inglês | MEDLINE | ID: mdl-26676364

RESUMO

The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2, an incX4 replicon was found to be linked to mcr-1. This report follows a recent detection of mcr-1 in E. coli from animals, food and humans in China.


Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Infecções por Escherichia coli/sangue , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Animais , Galinhas , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Genótipo , Humanos , Carne/microbiologia , Plasmídeos
4.
J Clin Microbiol ; 52(5): 1501-10, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24574290

RESUMO

Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.


Assuntos
Infecções por Escherichia coli/diagnóstico , Escherichia coli Shiga Toxigênica/genética , Adesinas Bacterianas/genética , Técnicas de Tipagem Bacteriana/métodos , Dinamarca , Surtos de Doenças , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Genoma Bacteriano/genética , Estudo de Associação Genômica Ampla/métodos , Filogenia , Análise de Sequência de DNA/métodos , Toxina Shiga I/genética , Toxina Shiga II/genética , Virulência/genética
5.
BMC Genomics ; 13: 577, 2012 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-23114024

RESUMO

BACKGROUND: Escherichia coli exists in commensal and pathogenic forms. By measuring the variation of individual genes across more than a hundred sequenced genomes, gene variation can be studied in detail, including the number of mutations found for any given gene. This knowledge will be useful for creating better phylogenies, for determination of molecular clocks and for improved typing techniques. RESULTS: We find 3,051 gene clusters/families present in at least 95% of the genomes and 1,702 gene clusters present in 100% of the genomes. The former 'soft core' of about 3,000 gene families is perhaps more biologically relevant, especially considering that many of these genome sequences are draft quality. The E. coli pan-genome for this set of isolates contains 16,373 gene clusters.A core-gene tree, based on alignment and a pan-genome tree based on gene presence/absence, maps the relatedness of the 186 sequenced E. coli genomes. The core-gene tree displays high confidence and divides the E. coli strains into the observed MLST type clades and also separates defined phylotypes. CONCLUSION: The results of comparing a large and diverse E. coli dataset support the theory that reliable and good resolution phylogenies can be inferred from the core-genome. The results further suggest that the resolution at the isolate level may, subsequently be improved by targeting more variable genes. The use of whole genome sequencing will make it possible to eliminate, or at least reduce, the need for several typing steps used in traditional epidemiology.


Assuntos
Técnicas de Tipagem Bacteriana , Escherichia coli/genética , Genoma Bacteriano/genética , Tipagem de Sequências Multilocus , Sequência de Bases , Mapeamento Cromossômico , Variação Genética , Genômica , Família Multigênica , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Shigella/genética
6.
BMC Genomics ; 13 Suppl 7: S6, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23281601

RESUMO

BACKGROUND: The advances and decreasing economical cost of whole genome sequencing (WGS), will soon make this technology available for routine infectious disease epidemiology. In epidemiological studies, outbreak isolates have very little diversity and require extensive genomic analysis to differentiate and classify isolates. One of the successfully and broadly used methods is analysis of single nucletide polymorphisms (SNPs). Currently, there are different tools and methods to identify SNPs including various options and cut-off values. Furthermore, all current methods require bioinformatic skills. Thus, we lack a standard and simple automatic tool to determine SNPs and construct phylogenetic tree from WGS data. RESULTS: Here we introduce snpTree, a server for online-automatic SNPs analysis. This tool is composed of different SNPs analysis suites, perl and python scripts. snpTree can identify SNPs and construct phylogenetic trees from WGS as well as from assembled genomes or contigs. WGS data in fastq format are aligned to reference genomes by BWA while contigs in fasta format are processed by Nucmer. SNPs are concatenated based on position on reference genome and a tree is constructed from concatenated SNPs using FastTree and a perl script. The online server was implemented by HTML, Java and python script.The server was evaluated using four published bacterial WGS data sets (V. cholerae, S. aureus CC398, S. Typhimurium and M. tuberculosis). The evaluation results for the first three cases was consistent and concordant for both raw reads and assembled genomes. In the latter case the original publication involved extensive filtering of SNPs, which could not be repeated using snpTree. CONCLUSIONS: The snpTree server is an easy to use option for rapid standardised and automatic SNP analysis in epidemiological studies also for users with limited bioinformatic experience. The web server is freely accessible at http://www.cbs.dtu.dk/services/snpTree-1.0/.


Assuntos
Bactérias/genética , Genoma Bacteriano , Polimorfismo de Nucleotídeo Único , Bactérias/classificação , Bases de Dados Genéticas , Internet , Mycobacterium tuberculosis/genética , Salmonella typhimurium/genética , Software , Staphylococcus aureus/genética , Interface Usuário-Computador , Vibrio cholerae/genética
7.
Sci Rep ; 9(1): 11624, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406241

RESUMO

Community level genetic information can be essential to direct health measures and study demographic tendencies but is subject to considerable ethical and legal challenges. These concerns become less pronounced when analyzing urban sewage samples, which are ab ovo anonymous by their pooled nature. We were able to detect traces of the human mitochondrial DNA (mtDNA) in urban sewage samples and to estimate the distribution of human mtDNA haplogroups. An expectation maximization approach was used to determine mtDNA haplogroup mixture proportions for samples collected at each different geographic location. Our results show reasonable agreement with both previous studies of ancient evolution or migration and current US census data; and are also readily reproducible and highly robust. Our approach presents a promising alternative for sample collection in studies focusing on the ethnic and genetic composition of populations or diseases associated with different mtDNA haplogroups and genotypes.


Assuntos
DNA Mitocondrial/genética , Haplótipos , Esgotos , População Urbana , Evolução Molecular , Humanos , Filogenia , Análise de Componente Principal , Reprodutibilidade dos Testes , Processos Estocásticos
8.
Nat Commun ; 10(1): 1124, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850636

RESUMO

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.


Assuntos
Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos , Metagenoma , Esgotos/microbiologia , África , Ásia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Monitoramento Epidemiológico , Europa (Continente) , Humanos , Metagenômica/métodos , Consórcios Microbianos/efeitos dos fármacos , Consórcios Microbianos/genética , América do Norte , Oceania , Saúde da População , Fatores Socioeconômicos , América do Sul
9.
Database (Oxford) ; 20192019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31868882

RESUMO

Data sharing enables research communities to exchange findings and build upon the knowledge that arises from their discoveries. Areas of public and animal health as well as food safety would benefit from rapid data sharing when it comes to emergencies. However, ethical, regulatory and institutional challenges, as well as lack of suitable platforms which provide an infrastructure for data sharing in structured formats, often lead to data not being shared or at most shared in form of supplementary materials in journal publications. Here, we describe an informatics platform that includes workflows for structured data storage, managing and pre-publication sharing of pathogen sequencing data and its analysis interpretations with relevant stakeholders.


Assuntos
Bases de Dados Factuais , Disseminação de Informação , Bactérias/classificação , Metagenômica , Filogenia , Interface Usuário-Computador
10.
PLoS One ; 12(1): e0169324, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28103259

RESUMO

Cholera is still an important public health problem in several countries, including Thailand. In this study, a collection of clinical and environmental V. cholerae serogroup O1, O139, and non-O1/non-O139 strains originating from Thailand (1983 to 2013) was characterized to determine phenotypic and genotypic traits and to investigate the genetic relatedness. Using a combination of conventional methods and whole genome sequencing (WGS), 78 V. cholerae strains were identified. WGS was used to determine the serogroup, biotype, virulence, mobile genetic elements, and antimicrobial resistance genes using online bioinformatics tools. In addition, phenotypic antimicrobial resistance was determined by the minimal inhibitory concentration (MIC) test. The 78 V. cholerae strains belonged to the following serogroups O1: (n = 44), O139 (n = 16) and non-O1/non-O139 (n = 18). Interestingly, we found that the typical El Tor O1 strains were the major cause of clinical cholera during 1983-2000 with two Classical O1 strains detected in 2000. In 2004-2010, the El Tor variant strains revealed genotypes of the Classical biotype possessing either only ctxB or both ctxB and rstR while they harbored tcpA of the El Tor biotype. Thirty O1 and eleven O139 clinical strains carried CTXϕ (Cholera toxin) and tcpA as well four different pathogenic islands (PAIs). Beside non-O1/non-O139, the O1 environmental strains also presented chxA and Type Three Secretion System (TTSS). The in silico MultiLocus Sequence Typing (MLST) discriminated the O1 and O139 clinical strains from other serogroups and environmental strains. ST69 was dominant in the clinical strains belonging to the 7th pandemic clone. Non-O1/non-O139 and environmental strains showed various novel STs indicating genetic variation. Multidrug-resistant (MDR) strains were observed and conferred resistance to ampicillin, azithromycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim and harboured variants of the SXT elements. For the first time since 1986, the presence of V. cholerae O1 Classical was reported causing cholera outbreaks in Thailand. In addition, we found that V. cholerae O1 El Tor variant and O139 were pre-dominating the pathogenic strains in Thailand. Using WGS and bioinformatic tools to analyze both historical and contemporary V. cholerae circulating in Thailand provided a more detailed understanding of the V. cholerae epidemiology, which ultimately could be applied for control measures and management of cholera in Thailand.


Assuntos
Cólera/microbiologia , Variação Genética , Vibrio cholerae/genética , Vibrio cholerae/isolamento & purificação , Cólera/epidemiologia , Surtos de Doenças , Farmacorresistência Bacteriana/genética , Microbiologia Ambiental , Genes Bacterianos , Ilhas Genômicas , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Filogenia , Sorotipagem , Tailândia/epidemiologia , Vibrio cholerae/patogenicidade , Vibrio cholerae O1/genética , Vibrio cholerae O1/isolamento & purificação , Vibrio cholerae O1/patogenicidade , Vibrio cholerae O139/genética , Vibrio cholerae O139/isolamento & purificação , Vibrio cholerae O139/patogenicidade , Vibrio cholerae não O1/genética , Vibrio cholerae não O1/isolamento & purificação , Vibrio cholerae não O1/patogenicidade , Virulência/genética
11.
Front Microbiol ; 8: 2351, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29238330

RESUMO

Background/objectives: Whole genome sequencing (WGS) has proven to be a powerful subtyping tool for foodborne pathogenic bacteria like L. monocytogenes. The interests of genome-scale analysis for national surveillance, outbreak detection or source tracking has been largely documented. The genomic data however can be exploited with many different bioinformatics methods like single nucleotide polymorphism (SNP), core-genome multi locus sequence typing (cgMLST), whole-genome multi locus sequence typing (wgMLST) or multi locus predicted protein sequence typing (MLPPST) on either core-genome (cgMLPPST) or pan-genome (wgMLPPST). Currently, there are little comparisons studies of these different analytical approaches. Our objective was to assess and compare different genomic methods that can be implemented in order to cluster isolates of L. monocytogenes. Methods: The clustering methods were evaluated on a collection of 207 L. monocytogenes genomes of food origin representative of the genetic diversity of the Anses collection. The trees were then compared using robust statistical analyses. Results: The backward comparability between conventional typing methods and genomic methods revealed a near-perfect concordance. The importance of selecting a proper reference when calling SNPs was highlighted, although distances between strains remained identical. The analysis also revealed that the topology of the phylogenetic trees between wgMLST and cgMLST were remarkably similar. The comparison between SNP and cgMLST or SNP and wgMLST approaches showed that the topologies of phylogenic trees were statistically similar with an almost equivalent clustering. Conclusion: Our study revealed high concordance between wgMLST, cgMLST, and SNP approaches which are all suitable for typing of L. monocytogenes. The comparable clustering is an important observation considering that the two approaches have been variously implemented among reference laboratories.

12.
PLoS One ; 11(5): e0155691, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27191718

RESUMO

The prevalence of reported cholera was relatively low around the Lake Chad basin until 1991. Since then, cholera outbreaks have been reported every couple of years. The objective of this study was to investigate the 2010/2011 Vibrio cholerae outbreak in Cameroon to gain insight into the genomic make-up of the V. cholerae strains responsible for the outbreak. Twenty-four strains were isolated and whole genome sequenced. Known virulence genes, resistance genes and integrating conjugative element (ICE) elements were identified and annotated. A global phylogeny (378 genomes) was inferred using a single nucleotide polymorphism (SNP) analysis. The Cameroon outbreak was found to be clonal and clustered distant from the other African strains. In addition, a subset of the strains contained a deletion that was found in the ICE element causing less resistance. These results suggest that V. cholerae is endemic in the Lake Chad basin and different from other African strains.


Assuntos
Cólera/epidemiologia , Cólera/microbiologia , Surtos de Doenças , Reservatórios de Doenças , Lagos/microbiologia , Vibrio cholerae O1/genética , Antibacterianos/farmacologia , Camarões/epidemiologia , Cólera/história , Genoma Bacteriano , Genótipo , História do Século XXI , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Sorogrupo , Vibrio cholerae O1/classificação , Vibrio cholerae O1/efeitos dos fármacos , Vibrio cholerae O1/isolamento & purificação
13.
PLoS One ; 9(8): e104984, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25110940

RESUMO

Whole genome sequencing (WGS) shows great potential for real-time monitoring and identification of infectious disease outbreaks. However, rapid and reliable comparison of data generated in multiple laboratories and using multiple technologies is essential. So far studies have focused on using one technology because each technology has a systematic bias making integration of data generated from different platforms difficult. We developed two different procedures for identifying variable sites and inferring phylogenies in WGS data across multiple platforms. The methods were evaluated on three bacterial data sets and sequenced on three different platforms (Illumina, 454, Ion Torrent). We show that the methods are able to overcome the systematic biases caused by the sequencers and infer the expected phylogenies. It is concluded that the cause of the success of these new procedures is due to a validation of all informative sites that are included in the analysis. The procedures are available as web tools.


Assuntos
Genoma Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Salmonella typhimurium/genética , Análise de Sequência de DNA/métodos , Staphylococcus aureus/genética , Sequência de Bases , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Salmonella typhimurium/isolamento & purificação , Staphylococcus aureus/isolamento & purificação
14.
PLoS One ; 9(2): e87991, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24505344

RESUMO

Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly 'real-time' monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE revealing that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.


Assuntos
Genoma Bacteriano/genética , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/genética , Salmonella enterica/genética , DNA Bacteriano/genética , Surtos de Doenças , Estudo de Associação Genômica Ampla/métodos , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA/métodos
15.
PLoS One ; 9(2): e89018, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24563689

RESUMO

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) Sequence Type 398 (ST398) is an opportunistic pathogen that is able to colonize and cause disease in several animal species including humans. To better understand the adaptation, evolution, transmission and pathogenic capacity, further investigations into the importance of the different genes harboured by LA-MRSA ST398 are required. In this study we generated a genome-wide transposon mutant library in an LA-MRSA ST398 isolate to evaluate genes important for bacterial survival in laboratory and host-specific environments. The transposon mutant library consisted of approximately 1 million mutants with around 140,000 unique insertion sites and an average number of unique inserts per gene of 44.8. We identified LA-MRSA ST398 essential genes comparable to other high-throughput S. aureus essential gene studies. As ST398 is the most common MRSA isolated from pigs, the transposon mutant library was screened in whole porcine blood. Twenty-four genes were specifically identified as important for bacterial survival in porcine blood. Mutations in 23 of these genes resulted in attenuated bacterial fitness. Seven of the 23 genes were of unknown function, whereas 16 genes were annotated with functions predominantly related to carbon metabolism, pH shock and a variety of regulations and only indirectly to virulence factors. Mutations in one gene of unknown function resulted in a hypercompetitive mutant. Further evaluation of these genes is required to determine their specific relevance in blood survival.


Assuntos
Ensaios de Triagem em Larga Escala , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/microbiologia , Animais , Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Biblioteca Gênica , Genes Essenciais , Genoma , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Staphylococcus aureus Resistente à Meticilina/classificação , Mutação , Plasmídeos/metabolismo , Análise de Sequência de DNA , Suínos , Fatores de Virulência
16.
Nat Biotechnol ; 32(8): 822-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24997787

RESUMO

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.


Assuntos
Metagenômica , Análise por Conglomerados , Bases de Dados Genéticas
17.
mBio ; 2(4): e00157-11, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21862630

RESUMO

Cholera continues to be an important cause of human infections, and outbreaks are often observed after natural disasters, such as the one following the 2010 earthquake in Haiti. Once the cholera outbreak was confirmed, rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. We used whole-genome sequence typing (WGST), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing to characterize 24 recent Vibrio cholerae isolates from Nepal and evaluate the suggested epidemiological link with the Haitian outbreak. The isolates were obtained from 30 July to 1 November 2010 from five different districts in Nepal. We compared the 24 genomes to 10 previously sequenced V. cholerae isolates, including 3 from the Haitian outbreak (began July 2010). Antimicrobial susceptibility and PFGE patterns were consistent with an epidemiological link between the isolates from Nepal and Haiti. WGST showed that all 24 V. cholerae isolates from Nepal belonged to a single monophyletic group that also contained isolates from Bangladesh and Haiti. The Nepalese isolates were divided into four closely related clusters. One cluster contained three Nepalese isolates and three Haitian isolates that were almost identical, with only 1- or 2-bp differences. Results in this study are consistent with Nepal as the origin of the Haitian outbreak. This highlights how rapidly infectious diseases might be transmitted globally through international travel and how public health officials need advanced molecular tools along with standard epidemiological analyses to quickly determine the sources of outbreaks.


Assuntos
Cólera/epidemiologia , Cólera/microbiologia , Variação Genética , Vibrio cholerae/genética , Vibrio cholerae/isolamento & purificação , Adolescente , Adulto , Antibacterianos/farmacologia , Criança , Surtos de Doenças , Feminino , Haiti/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Nepal/epidemiologia , Filogenia , Vibrio cholerae/classificação , Vibrio cholerae/efeitos dos fármacos , Adulto Jovem
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