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: It is indicated that yersiniabactin, colibactin and an expanded resistome have contributed to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America.
Abstract: The emergence and dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a worrisome public health issue compromising the treatment and outcome of infections caused by this pathogen. We performed a detailed virulome and resistome analysis of representative KPC- and/or CTX-M-producing K. pneumoniae belonging to clonal group (CG) 258 (sequence types ST11, ST258, ST340, ST437), circulating in Argentina, Brazil, Chile, Colombia and Peru; with further evaluation of the virulence behavior using the Galleria mellonella infection model. Genomic analysis of K. pneumoniae strains recovered from the human-animal-environment interface revealed a wide resistome characterized by the presence of genes and mutations conferring resistance to human and veterinary antibiotics, quaternary ammonium compounds (QACs) and heavy metals. Plasmid Inc typing revealed the presence of a wide diversity of replicon types with IncF, IncN, IncR and Col-like being frequently detected. Moreover, KPC-2-producing K. pneumoniae belonging to ST11 (KL-64 andKL-105) and ST340 (KL-15) carried multiple variants of distinct yersiniabactin siderophore (ybt) and/or genotoxic colibactin (clb) genes. In this regard, ICEKp3, ICEKp4 and ICEKp12 were identified in strains belonging to ST11 and ST340, recovered from Argentina, Brazil, Chile and Colombia; whereas ybt 17 and a novel ybt sequence type (YbST346) were identified together with clb in ICEKp10 structures from ST11 and ST258, from Brazil and Colombia, respectively. K. pneumoniae ST11 (ICEKp10/YbST346 and ICEKp4/ybt 10) strains killed 100% of wax moth larvae, in a similar way to hypervirulent K1/ST23 strain (ybt- and clb-negative) carrying the pLVPK-like plasmid, indicating enhanced virulence. In summary, our results indicate that yersiniabactin, colibactin and an expanded resistome have contributed to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. Therefore, active surveillance of hospital-associated lineages of K. pneumoniae should not only focus on clonal origin and antimicrobial resistance, but also on the virulence factors ybt and clb.
4 citations
Cites methods from "In Silico Detection and Typing of P..."
...On the other hand, since 14 of the publicly available genomes (used in this study) were only available as pre-assembled sequences, Kleborate (https://github.com/katholt/Kleborate) and PlasmidFinder were used to identify MLST, resistome, virulome and plasmid replicon genes....
[...]
...MLSTs, YbSTs, CbSTs (Lam et al., 2018a; Diancourt et al., 2005), virulome, resistome and plasmid replicon genes were screened by SRST2 (Inouye et al., 2014), using BIGSdb (Bialek-Davenet et al., 2014), ARG-Annot (Gupta et al., 2014), and PlasmidFinder (Carattoli et al., 2014) databases....
[...]
TL;DR: It is demonstrated here that routine genomic surveillance is necessary for high-resolution bacterial epidemiology of these important AMR pathogens, E. coli and K. pneumoniae, which are mainly disseminated via clonal and horizontal transfer, circulating in the East African region.
Abstract: Background Urinary tract infection (UTI) develops after a pathogen adhered to the inner lining of the urinary tract. Cases of UTIs are predominantly caused by several Gram-negative bacteria and account for high morbidity in the clinical and community settings. Of greater concern are the strains carrying antimicrobial resistance (AMR)-conferring genes. The gravity of UTI is also determined by a spectrum of other virulence factors. This study represents a pilot project to investigate the burden of AMR among uropathogens in East Africa. Methods We examined bacterial samples isolated in 2017-2018 from in- and out-patients in Kenya (KY) and Uganda (UG) that presented with clinical symptoms of UTI. We reconstructed the evolutionary history of the strains, investigated their population structure and performed comparitive analysis their pangenome contents. Results We found 55 Escherichia coli and 19 Klebsiella pneumoniae strains confirmed uropathogenic following screening for the prevalence of UTI virulence genes including fimH, iutA, feoA/B/C, mrkD and foc. We identified 18 different sequence types in E. coli population while all K. pneumoniae strains belong to ST11. The most prevalent E. coli sequence types were ST131 (26%), ST335/1193 (10%) and ST10 (6%). Diverse plasmid types were observed in both collections such as Incompatibility (IncF/IncH/IncQ1/IncX4) and Col groups. Pangenome analysis of each set revealed a total of 2,862 and 3,464 genes comprised the core genome of E. coli and K. pneumoniae population, respectively. Among these are AMR determinants including fluoroquinolone resistance-conferring genes aac(3)-Ib-cr and other significant genes: aad, tet, sul1, sul2, and cat, which are associated with aminoglycoside, tetracycline, sulfonamide and chloramphenicol resistance. Accessory genomes of both species collection were detected several β-lactamase genes, blaCTX-M, blaTEM and blaOXAor blaNDM.Overall, 93% are multi-drug resistant in the E. coli collection while 100% of the K. pneumoniae strains contained genes that are associated with resistance to 3 or more antibiotic classes. Conclusions Our findings illustrate the abundant resistome and virulome repertoire in uropathogenic E. coli and K. pneumoniae, which are mainly disseminated via clonal and horizontal transfer, circulating in the East African region. We further demonstrate here that routine genomic surveillance is necessary for high-resolution bacterial epidemiology of these important AMR pathogens.
4 citations
TL;DR: In this paper, the authors characterized 77 representative Colombian S. Typhi isolates collected between 1997 and 2018 using pulse field gel electrophoresis (PFGE; the accepted genotyping method in Latin America) and whole genome sequencing (WGS) and found that the main S. typhi clades circulating in Colombia were clades 2.5 and 3.5.
Abstract: Little is known about the genetic diversity of Salmonella enterica serovar Typhi (S. Typhi) circulating in Latin America. It has been observed that typhoid fever is still endemic in this part of the world; however, a lack of standardized blood culture surveillance across Latin American makes estimating the true disease burden problematic. The Colombian National Health Service established a surveillance system for tracking bacterial pathogens, including S. Typhi, in 2006. Here, we characterized 77 representative Colombian S. Typhi isolates collected between 1997 and 2018 using pulse field gel electrophoresis (PFGE; the accepted genotyping method in Latin America) and whole genome sequencing (WGS). We found that the main S. Typhi clades circulating in Colombia were clades 2.5 and 3.5. Notably, the sequenced S. Typhi isolates from Colombia were closely related in a global phylogeny. Consequently, these data suggest that these are endemic clades circulating in Colombia. We found that AMR in S. Typhi in Colombia was uncommon, with a small subset of organisms exhibiting mutations associated with reduced susceptibility to fluoroquinolones. This is the first time that S. Typhi isolated from Colombia have been characterized by WGS, and after comparing these data with those generated using PFGE, we conclude that PFGE is unsuitable for tracking S. Typhi clones and mapping transmission. The genetic diversity of pathogens such as S. Typhi is limited in Latin America and should be targeted for future surveillance studies incorporating WGS.
4 citations
TL;DR: In this article, the authors characterized resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum β-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and determined their phylogenetic relatedness.
Abstract: The purpose of the study was to characterize the resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum β-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and to determine their phylogenetic relatedness. The isolates were from Algeria, isolated at the University Hospital Establishment of Oran, between 2011 and 2012. ESBL-KP isolates (n = 193) were screened for several antibiotic resistance genes (ARGs) using qPCR followed by Pulsed-Field Gel Electrophoresis (PFGE). Representative isolates were selected from PFGE clusters and subjected to whole-genome sequencing (WGS). Genomic characterization of the WGS data by studying prophages, CRISPR-Cas systems, Multi-Locus Sequence Typing (MLST), serotype, ARGs, virulence genes, plasmid replicons, and their pMLST. Phylogenetic and comparative genomic were done using core genome MLST and SNP-Based analysis. Generally, the ESBL-KP isolates were polyclonal. The whole genome sequences of nineteen isolates were taken of main PFGE clusters. Sixteen sequence types (ST) were found including high-risk clones ST14, ST23, ST37, and ST147. Serotypes K1 (n = 1), K2 (n = 2), K3 (n = 1), K31 (n = 1), K62 (n = 1), and K151 (n = 1) are associated with hyper-virulence. CRISPR-Cas system was found in 47.4%, typed I-E and I-E*. About ARGs, from 193 ESBL-KP, the majority of strains were multidrug-resistant, the CTX-M-1 enzyme was predominant (99%) and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes was high with aac(6')-lb-cr (72.5%) and qnr's (65.8%). From 19 sequenced isolates we identified ESBL, AmpC, and carbapenemase genes: blaCTX-M-15 (n = 19), blaOXA-48 (n = 1), blaCMY-2 (n = 2), and blaCMY-16 (n = 2), as well as non-ESBL genes: qnrB1 (n = 12), qnrS1 (n = 1) and armA (n = 2). We found IncF, IncN, IncL/M, IncA/C2, and Col replicon types, at least once per isolate. This study is the first to report qnrS in ESBL-KP in Algeria. Our analysis shows the concerning co-existence of virulence and resistance genes and would support that genomic surveillance should be a high priority in the hospital environment.
4 citations
TL;DR:
Abstract: While Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, including pneumonia and pyogenic liver abscess, little is known about the population structure of this bacterium. We collected 232 isolates from carriers, pyogenic liver abscess patients, and pneumonia patients, and the isolates from different sources had their own sequence types. ABSTRACT While Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, including pneumonia and pyogenic liver abscess, little is known about the population structure of this bacterium. In this study, we investigated the prevalence and molecular characteristics of K. pneumoniae isolates from carriers, pyogenic liver abscess patients, and pneumonia patients, and genomic and phenotypic assays were used to determine the differences among the isolates. A total of 232 K. pneumoniae isolates were subtyped into 74 sequence types (STs). The isolates from different sources had their own STs, and the predominant subtypes in liver abscess and pneumonia patients were ST23 and ST11, respectively. Pangenome analysis also distinguished three phylogroups that were consistent with the isolate sources. The isolates collected from liver abscess patients carried significantly more virulence factors, and those from pneumonia patients harbored significantly more resistance genes and replicons. Almost all isolate STs (93/97 [95.88%]) from liver abscesses strongly correlated with the virulence factor salmochelin, while most pneumonia isolate STs (52/53 [98.11%]) from pneumonia did not correlate with salmochelin. The isolates collected from liver abscesses showed higher virulence in the cytotoxicity and mouse models. These data provide genomic support for the proposal that isolates collected from carriers, liver abscess patients, and pneumonia patients have distinct genomic features. Isolates from the different sources are largely nonoverlapping, suggesting that different patients may be infected via different sources. Further studies on the pathogenic mechanisms of salmochelin and other virulence factors will be required. IMPORTANCE While Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, including pneumonia and pyogenic liver abscess, little is known about the population structure of this bacterium. We collected 232 isolates from carriers, pyogenic liver abscess patients, and pneumonia patients, and the isolates from different sources had their own sequence types. Pangenome analysis also distinguished three phylogroups that were consistent with the isolate sources. The isolates collected from liver abscess patients carried significantly more virulence factors, and those from pneumonia patients harbored significantly more resistance genes and replicons. Besides, there was a strong link between salmochelin and liver abscess. The isolates collected from liver abscesses also showed higher virulence in the cytotoxicity and mouse models. Isolates collected from different sources have distinct genomic features, suggesting that different patients may be infected via different sources.
4 citations
References
More filters
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.
3,956 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)....
[...]
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.
2,934 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)....
[...]
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.
2,163 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....
[...]
...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)....
[...]
...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....
[...]
...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....
[...]
...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: 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
1,943 citations
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.
1,620 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)....
[...]