Genome-wide high-throughput screening to investigate essential genes involved in methicillin-resistant Staphylococcus aureus Sequence Type 398 survival.

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.This work was funded in part by the Danish Ministry of Food, Agriculture and Fisheries (Grant no. 3304-FVFP-09-F-002-1) and The Technical University of DenmarkThis is the final published version distributed under a Creative Commons Attribution License, which can also be found on the publisher's website at: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.008901

Characterization and genetic variation of vibrio cholerae isolated from clinical and environmental sources in Thailand

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

The Lake Chad Basin, an Isolated and Persistent Reservoir of Vibrio cholerae O1: A Genomic Insight into the Outbreak in Cameroon, 2010

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

Is the Evolution of Salmonella enterica subsp. enterica Linked to Restriction-Modification Systems?

Salmonella enterica subsp. enterica bacteria are highly diverse foodborne pathogens that are subdivided into more than 1,500 serovars. The diversity is believed to result from mutational evolution, as well as intra- and interspecies recombination that potentially could be influenced by restriction-modification (RM) systems. The aim of this study was to investigate whether RM systems were linked to the evolution of Salmonella enterica subsp. enterica. The study included 221 Salmonella enterica genomes, of which 68 were de novo sequenced and 153 were public available genomes from ENA. The data set covered 97 different serovars of Salmonella enterica subsp. enterica and an additional five genomes from four other Salmonella subspecies as an outgroup for constructing the phylogenetic trees. The phylogenetic trees were constructed based on multiple alignment of core genes, as well as the presence or absence of pangenes. The topology of the trees was compared to the presence of RM systems, antimicrobial resistance (AMR) genes, Salmonella pathogenicity islands (SPIs), and plasmid replicons. We did not observe any correlation between evolution and the RM systems in S. enterica subsp. enterica. However, sublineage correlations and serovar-specific patterns were observed. Additionally, we conclude that plasmid replicons, SPIs, and AMR were all better correlated to serovars than to RM systems. This study suggests a limited influence of RM systems on the evolution of Salmonella enterica subsp. enterica, which could be due to the conjugational mode of horizontal gene transfer in Salmonella. Thus, we conclude that other factors must be involved in shaping the evolution of bacteria. IMPORTANCE The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica

An Assessment of Different Genomic Approaches for Inferring Phylogeny of Listeria monocytogenes

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

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage

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.info:eu-repo/semantics/publishedVersio

Evaluation of whole genome sequencing for outbreak detection of Salmonella enterica

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

The COMPARE Data Hubs

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