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Journal ArticleDOI

In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

01 Jul 2014-Antimicrobial Agents and Chemotherapy (American Society for Microbiology)-Vol. 58, Iss: 7, pp 3895-3903

TL;DR: Two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae are designed and developed.

AbstractIn the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match with at least at 80% nucleotide identity all replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection of Salmonella enterica serovar Typhimurium isolates. PlasmidFinder identified a total of 103 replicons and between zero and five different plasmid replicons within each of 49 S . Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens.

Topics: Plasmidome (58%), Replicon (57%), Multilocus sequence typing (56%), Sequence analysis (55%), Plasmid (53%)

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Citations
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Journal ArticleDOI
TL;DR: This work presents SRST2, a read mapping-based tool for fast and accurate detection of genes, alleles and multi-locus sequence types (MLST) from WGS data, which is highly accurate and outperforms assembly-based methods in terms of both gene detection and allele assignment.
Abstract: Rapid molecular typing of bacterial pathogens is critical for public health epidemiology, surveillance and infection control, yet routine use of whole genome sequencing (WGS) for these purposes poses significant challenges. Here we present SRST2, a read mapping-based tool for fast and accurate detection of genes, alleles and multi-locus sequence types (MLST) from WGS data. Using >900 genomes from common pathogens, we show SRST2 is highly accurate and outperforms assembly-based methods in terms of both gene detection and allele assignment. We include validation of SRST2 within a public health laboratory, and demonstrate its use for microbial genome surveillance in the hospital setting. In the face of rising threats of antimicrobial resistance and emerging virulence among bacterial pathogens, SRST2 represents a powerful tool for rapidly extracting clinically useful information from raw WGS data. Source code is available from http://katholt.github.io/srst2/.

688 citations


Journal ArticleDOI
TL;DR: The DNA sequence of K. pneumoniae isolates from around the world is determined and it is shown that there is a wide spectrum of diversity, including variation within shared sequences and gain and loss of whole genes, and there is an unrecognized association between the possession of specific gene profiles associated with virulence and antibiotic resistance.
Abstract: Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.

654 citations


Cites background from "In Silico Detection and Typing of P..."

  • ...Gene databases analyzed were AMR alleles [ARG-Annot (70)], plasmid replicons [PlasmidFinder (59)], and virulence andwzi alleles [K....

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  • ...However, screening against the PlasmidFinder database (59) identified 28 known plasmid replicons in 69/150 K....

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Journal ArticleDOI
TL;DR: The characteristics of the major types of mobile genetic elements involved in acquisition and spread of antibiotic resistance in both Gram-negative and Gram-positive bacteria are outlined, focusing on the so-called ESKAPEE group of organisms, which have become the most problematic hospital pathogens.
Abstract: SUMMARY Strains of bacteria resistant to antibiotics, particularly those that are multiresistant, are an increasing major health care problem around the world. It is now abundantly clear that both Gram-negative and Gram-positive bacteria are able to meet the evolutionary challenge of combating antimicrobial chemotherapy, often by acquiring preexisting resistance determinants from the bacterial gene pool. This is achieved through the concerted activities of mobile genetic elements able to move within or between DNA molecules, which include insertion sequences, transposons, and gene cassettes/integrons, and those that are able to transfer between bacterial cells, such as plasmids and integrative conjugative elements. Together these elements play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes. This review aims to outline the characteristics of the major types of mobile genetic elements involved in acquisition and spread of antibiotic resistance in both Gram-negative and Gram-positive bacteria, focusing on the so-called ESKAPEE group of organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli), which have become the most problematic hospital pathogens.

591 citations


Journal ArticleDOI
TL;DR: The data highlight the complex evolution of MDR and XDR K. pneumoniae, involving transfer and spread of ARGs, and epidemic plasmids in highly disseminating successful clones, and a need for future genomic and translational studies to decipher specific targets in HiR clones to design targeted prevention and treatment.
Abstract: Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen affecting humans and a major source for hospital infections associated with high morbidity and mortality due to limited treatment options We summarize the wide resistome of this pathogen, which encompasses plentiful chromosomal and plasmid-encoded antibiotic resistance genes (ARGs) Under antibiotic selective pressure, K pneumoniae continuously accumulates ARGs, by de novo mutations, and via acquisition of plasmids and transferable genetic elements, leading to extremely drug resistant (XDR) strains harboring a 'super resistome' In the last two decades, numerous high-risk (HiR) MDR and XDR K pneumoniae sequence types have emerged showing superior ability to cause multicontinent outbreaks, and continuous global dissemination The data highlight the complex evolution of MDR and XDR K pneumoniae, involving transfer and spread of ARGs, and epidemic plasmids in highly disseminating successful clones With the worldwide catastrophe of antibiotic resistance and the urgent need to identify the main pathogens that pose a threat on the future of infectious diseases, further studies are warranted to determine the epidemic traits and plasmid acquisition in K pneumoniae There is a need for future genomic and translational studies to decipher specific targets in HiR clones to design targeted prevention and treatment

377 citations


Cites methods from "In Silico Detection and Typing of P..."

  • ...We identified the replicons on these plasmids using the PlasmidFinder database (https://cge.cbs.dtu.dk/services/PlasmidFinder/) (Carattoli et al. 2014)....

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Journal ArticleDOI
07 Mar 2018-Mbio
TL;DR: The first large-scale emergence and spread of a novel extensively drug-resistant S. Typhi clone in Sindh, Pakistan is reported, highlighting the evolving threat of antibiotic resistance in S. typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases.
Abstract: Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum β-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S Typhi clone in Sindh, Pakistan. The XDR S Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S Typhi observed here adds urgency to the need for typhoid prevention measures.

361 citations


Cites methods from "In Silico Detection and Typing of P..."

  • ...Plasmid replicons were identified using ARIBA and the PlasmidFinder database (30)....

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References
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Journal ArticleDOI
TL;DR: A web server providing a convenient way of identifying acquired antimicrobial resistance genes in completely sequenced isolates was created, and the method was evaluated on WGS chromosomes and plasmids of 30 isolates.
Abstract: Objectives Identification of antimicrobial resistance genes is important for understanding the underlying mechanisms and the epidemiology of antimicrobial resistance. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available in routine diagnostic laboratories and is anticipated to substitute traditional methods for resistance gene identification. Thus, the current challenge is to extract the relevant information from the large amount of generated data.

3,131 citations


"In Silico Detection and Typing of P..." refers methods in this paper

  • ...To extract the relevant information from the large amount of data generated, a Web-based tool, ResFinder, for the identification of acquired or intrinsically present antimicrobial resistance genes in whole-genome data was recently developed (15)....

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Journal ArticleDOI
TL;DR: NCBI’s Conserved Domain Database (CDD) is a resource for the annotation of protein sequences with the location of conserved domain footprints, and functional sites inferred from these footprints.
Abstract: NCBI's Conserved Domain Database (CDD) is a resource for the annotation of protein sequences with the location of conserved domain footprints, and functional sites inferred from these footprints. CDD includes manually curated domain models that make use of protein 3D structure to refine domain models and provide insights into sequence/structure/function relationships. Manually curated models are organized hierarchically if they describe domain families that are clearly related by common descent. As CDD also imports domain family models from a variety of external sources, it is a partially redundant collection. To simplify protein annotation, redundant models and models describing homologous families are clustered into superfamilies. By default, domain footprints are annotated with the corresponding superfamily designation, on top of which specific annotation may indicate high-confidence assignment of family membership. Pre-computed domain annotation is available for proteins in the Entrez/Protein dataset, and a novel interface, Batch CD-Search, allows the computation and download of annotation for large sets of protein queries. CDD can be accessed via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

2,698 citations


"In Silico Detection and Typing of P..." refers background in this paper

  • ...In particular, the replicase proteins showing the pfam02387 or pfam01051 conserved domains were assigned to the FII and FIB groups, respectively (31)....

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Journal ArticleDOI
TL;DR: Results indicated that the inc/rep PCR method demonstrates high specificity and sensitivity in detecting replicons on reference plasmids and also revealed the presence of recurrent and common plasmid in epidemiologically unrelated Salmonella isolates of different serotypes.
Abstract: The epidemiological importance of tracing plasmids conferring drug resistance prompted us to develop a PCR method based on replicons (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae. Eighteen pairs of primers were designed to perform 5 multiplex- and 3 simplex-PCRs, recognizing FIA, FIB, FIC, HI1, HI2, I1-Igamma, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. The specificity of the method was tested on a collection of 61 reference plasmids and on 20 Salmonella enterica strains of different serotypes isolated in Italy. Results indicated that the inc/rep PCR method demonstrates high specificity and sensitivity in detecting replicons on reference plasmids and also revealed the presence of recurrent and common plasmids in epidemiologically unrelated Salmonella isolates of different serotypes. These results suggest that the method is potentially applicable to a large number of strains to trace the diffusion of specific multi-drug resistance plasmids in different environments.

1,989 citations


"In Silico Detection and Typing of P..." refers methods in this paper

  • ...A collection of 24 previously characterized and fully FIG 1 Numbers of fully sequenced plasmids (y axis) classified into incompatibility groups occurring in the different bacterial species of the Enterobacteriaceae family....

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  • ...Since 2005, a PCR-based replicon typing (PBRT) scheme has been available that targets in multiplex PCRs the replicons of the major plasmid families occurring in members of the family Enterobacteriaceae (2)....

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  • ...Here, we present two free, easy-to-use Web tools, PlasmidFinder and pMLST, to analyze and classify plasmids from bacterial species of the family Enterobacteriaceae....

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  • ...Here, we describe the design of two new easy-to-use Web tools useful for the rapid identification of plasmids in Enterobacteriaceae species that are of interest for epidemiological and clinical microbiology investigations of the plasmid-associated spread of antimicrobial resistance....

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  • ...This method was initially developed to detect the replicons of plasmids belonging to the 18 major incompatibility (Inc) groups of Enterobacteriaceae species (3)....

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Journal ArticleDOI
TL;DR: The Bacterial Isolate Genome Sequence Database (BIGSDB) represents a freely available resource that will assist the broader community in the elucidation of the structure and function of bacteria by means of a population genomics approach.
Abstract: The opportunities for bacterial population genomics that are being realised by the application of parallel nucleotide sequencing require novel bioinformatics platforms These must be capable of the storage, retrieval, and analysis of linked phenotypic and genotypic information in an accessible, scalable and computationally efficient manner The Bacterial Isolate Genome Sequence Database (BIGSDB) is a scalable, open source, web-accessible database system that meets these needs, enabling phenotype and sequence data, which can range from a single sequence read to whole genome data, to be efficiently linked for a limitless number of bacterial specimens The system builds on the widely used mlstdbNet software, developed for the storage and distribution of multilocus sequence typing (MLST) data, and incorporates the capacity to define and identify any number of loci and genetic variants at those loci within the stored nucleotide sequences These loci can be further organised into 'schemes' for isolate characterisation or for evolutionary or functional analyses Isolates and loci can be indexed by multiple names and any number of alternative schemes can be accommodated, enabling cross-referencing of different studies and approaches LIMS functionality of the software enables linkage to and organisation of laboratory samples The data are easily linked to external databases and fine-grained authentication of access permits multiple users to participate in community annotation by setting up or contributing to different schemes within the database Some of the applications of BIGSDB are illustrated with the genera Neisseria and Streptococcus The BIGSDB source code and documentation are available at http://pubmlstorg/software/database/bigsdb/ Genomic data can be used to characterise bacterial isolates in many different ways but it can also be efficiently exploited for evolutionary or functional studies BIGSDB represents a freely available resource that will assist the broader community in the elucidation of the structure and function of bacteria by means of a population genomics approach

1,661 citations


Journal ArticleDOI
TL;DR: A Web-based method for MLST of 66 bacterial species based on whole-genome sequencing data that enables investigators to determine the sequence types of their isolates on the basis of WGS data.
Abstract: Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the “gold standard” of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

1,253 citations


"In Silico Detection and Typing of P..." refers methods in this paper

  • ...If raw sequence reads are uploaded, they are first assembled (after the sequencing platform is given by the user) as described previously (16)....

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