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.
Abstract: In 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.
Citations
More filters
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.
1,162 citations
University of Melbourne1, University of Oxford2, Cornell University3, United States Army Medical Research Institute of Infectious Diseases4, Cardiff University5, University Health System6, Erasmus University Medical Center7, Pasteur Institute8, University of California, San Francisco9, University of Queensland10, University of Amsterdam11, Airlangga University12, Mahosot Hospital13, Wellcome Trust14, University of London15
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.
879 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....
[...]
...However, screening against the PlasmidFinder database (59) identified 28 known plasmid replicons in 69/150 K....
[...]
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/.
820 citations
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
654 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)....
[...]
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.
478 citations
Cites methods from "In Silico Detection and Typing of P..."
...Plasmid replicons were identified using ARIBA and the PlasmidFinder database (30)....
[...]
References
More filters
TL;DR: Multiple events of mobilization, transposition and replicon fusion generate the complexity observed in qnr-positive isolates that are emerging worldwide.
Abstract: Objectives: The aim of this study was to identify and characterize plasmids carrying qnrS1, qnrB2 and qnrB19 genes identified in Salmonella strains from The Netherlands. The identification of plasmids may help to follow the dissemination of these resistance genes in different countries and environments. Methods: Plasmids from 33 qnr-positive Salmonella strains were transferred to Escherichia coli and analysed by restriction, Southern blot hybridization, PCR and sequencing of resistance determinants. They were also assigned to incompatibility groups by PCR-based replicon typing, including three additional PCR assays for the IncU, IncR and ColE groups. The collection included isolates from humans and one from chicken meat. Results: Five IncN plasmids carrying qnrS1, qnrB2 and qnrB19 genes were identified in Salmonella enterica Bredeney, Typhimurium PT507, Kentucky and Saintpaul. qnrS1 genes were also located on three further plasmid types, belonging to the ColE (in Salmonella Corvallis and Anatum), IncR (in Salmonella Montevideo) and IncHI2 (in Salmonella Stanley) groups. Conclusions: Multiple events of mobilization, transposition and replicon fusion generate the complexity observed in qnr-positive isolates that are emerging worldwide. Despite the fact that the occurrence of qnr genes in bacteria from animals is scarcely reported, these genes are associated with genetic elements and located on plasmids that are recurrent in animal isolates.
252 citations
"In Silico Detection and Typing of P..." refers methods in this paper
...Two replicon sequences (probes Q1 and Q2) were devised to recognize the repA gene of 7 small IncQ-like plasmids, and one replicon sequence [probe P(6)] was devised to detect the small IncP plasmid pRIO-5 (see Table S1 for the list of plasmids recognized by the probes in Table 3)....
[...]
...P(6)_1__JF785550 1477–2282 repA This study...
[...]
TL;DR: Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content and their outer membrane protein profiles; the virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.
Abstract: Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains. Images
239 citations
TL;DR: It is demonstrated that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among beta-lactamase producers isolated from animals and humans.
Abstract: Received 17 January 2008; returned 11 February 2008; revised 28 February 2008; accepted 29 February 2008 Objectives: Plasmids belonging to incompatibility group I1 (IncI1) are widespread in Enterobacteriaceae and are characterized by the presence of a cluster of genes encoding the type IV pili, contributing to the virulence of Shiga-toxigenic Escherichia coli. Recently, IncI1 plasmids were identified in E. coli and Salmonella strains of animal origin as responsible for the dissemination of b-lactamase genes. Plasmid multilocus sequence typing (pMLST) was developed to discern naturally occurring IncI1 plasmids in homogeneous groups according to their allele assortment. Methods: pMLST was developed by selecting multiple target genes on the available complete IncI1 plasmid DNA sequences. Sixteen plasmids, all assigned to the IncI1 group by the PCR-based replicon typing method, were included in this study. They were analysed for b-lactamase genes and typed by restriction fragment length polymorphism (RFLP) and pMLST. Results: Sixteen plasmids identified in E. coli and Salmonella isolated from animals and humans in different countries carried blaCMY-2, blaCTX-M-15, blaCTX-M-1, blaCTX-M-14, blaTEM-52, blaSHV-12 or blaTEM-1 b-lactamase genes. These plasmids were classified by RFLP in nine different groups corresponding to the nine sequence types determined by pMLST. Conclusions: The pMLST method was suitable for rapid and easy subtyping of IncI1 plasmids. This study demonstrates that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among b-lactamase producers isolated from animals and humans.
230 citations
TL;DR: Heterogeneity among fi− R factors has already been indicated, and it is sometimes assumed that all fi+ R factors have I pili9,10, but this work indicates that some fi−R factors determine pili similar to those specified by col I.
Abstract: TRANSMISSIBLE R factors in bacteria are divisible into two main classes, fi+ (fertility inhibition +) and fi−, according to their effect on F mediated conjugation1. fi+ R factors determine F-like pili2. Some fi− R factors determine pili similar to those specified by col I (I-type pili)3 which can be recognized as receptors for I-specific phages like If1 (ref. 4). Although heterogeneity among fi− R factors has already been indicated5–8, it is sometimes assumed that all fi− R factors have I pili9,10.
222 citations
"In Silico Detection and Typing of P..." refers methods in this paper
...This method was initially developed to detect the replicons of plasmids belonging to the 18 major incompatibility (Inc) groups of Enterobacteriaceae species (3)....
[...]
TL;DR: It is revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged and this revised typing procedure can be used to better discern the occurrence of IncX type plasmids among enterobacterial populations.
222 citations