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.
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TL;DR: This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDR K. pneumoniae strains.
Abstract: Background: Klebsiella pneumoniae is a recognised agent of multidrug-resistant (MDR) healthcare-associated infections, however individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin (iuc) and salmochelin (iro) are associated with invasive disease and are common amongst hypervirulent K. pneumoniae clones that cause severe community-associated infections such as liver abscess and pneumonia. Concerningly iuc has also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in the K. pneumoniae population. Methods: Here we examined the genetic diversity, distribution and mobilisation of iuc and iro loci among 2503 K. pneumoniae genomes using comparative genomics approaches, and developed tools for tracking them via genomic surveillance. Results: Iro and iuc were detected at low prevalence (<10%). Considerable genetic diversity was observed, resolving into five iro and six iuc lineages that show distinct patterns of mobilisation and dissemination in the K. pneumoniae population. The major burden of iuc and iro amongst the genomes analysed was due to two linked lineages (iuc1/iro1, 74% and iuc2/iro2, 14%), each carried by a distinct non-self-transmissible IncFIBK virulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy (rmpA) determinants and include all previously described virulence plasmids of K. pneumoniae. The other iuc and iro lineages were associated with diverse plasmids, including some carrying FII conjugative transfer regions and some imported from E. coli; the exceptions were iro3 (mobilised by ICEKp1), and iuc4 (fixed in the chromosome of K. pneumoniae subspecies rhinoscleromatis). Iro/iuc MGEs appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc), but were also detected at low prevalence in others such as MDR strain ST258. Conclusions: Iuc and iro are mobilised in K. pneumoniae via a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDR K. pneumoniae strains.
9 citations
Cites methods from "In Silico Detection and Typing of P..."
...were identified using BLASTn search of the PlasmidFinder database (40)....
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TL;DR: This article used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major ETEC lineages with manually curated chromosomes and plasmids.
Abstract: Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 deaths annually, with the highest rates of burden, ca 75 million cases per year, amongst children under 5 years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. We used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. We confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades, suggesting that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.
9 citations
17 Jan 2019
TL;DR: The draft genome sequence of P. rettgeri NVIT03 is presented, the most common bacterial symbiont of the insect hymenopteran model Nasonia vitripennis, which is also part of the Sarcophaga bullata pupal microbiome that N Masonia spp.
Abstract: Providencia rettgeri is a common insect-associated Gram-negative bacterium. Here, we present the draft genome sequence of P. rettgeri NVIT03, the most common bacterial symbiont of the insect hymenopteran model Nasonia vitripennis. This symbiont is also part of the Sarcophaga bullata pupal microbiome that Nasonia spp. parasitize and that critically influences the development of the wasp.
9 citations
31 Oct 2019
TL;DR: Pantoea agglomerans strain C1 has plant growth-promoting (PGP) traits and exhibits antimicrobial activity and the genome comprises 4.8 Mb, 4,696 protein-coding sequences, and a G+C content of 55.2%.
Abstract: Pantoea agglomerans strain C1 has plant growth-promoting (PGP) traits and exhibits antimicrobial activity. The genome comprises 4.8 Mb, 4,696 protein-coding sequences, and a G+C content of 55.2%.
9 citations
Cites methods from "In Silico Detection and Typing of P..."
...No plasmid was detected in the genome by using Plasmid Finder (15)....
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TL;DR: Cattle are a reservoir of diverse salmonellae with shared serovars with humans, but WGST does not support zoonotic transmission.
Abstract: The sources and modes of transmission of non-typhoidal Salmonella particularly zoonotic transmission are poorly understood in Africa. This study compared phenotypic and genotypic characteristics of Salmonellae isolated from cattle and humans. Faecal samples of diarrhoeic patients (n = 234), and a healthy population (n = 160), beef cattle at slaughter (n = 250), farms (n = 72) and market (n = 100) were cultured for salmonellae and serotyping and antimicrobial susceptibility were determined. Whole-genome sequence typing (WGST) of selected isolates and bioinformatic analysis were used to identify the multilocus sequence type (MLST), plasmid replicons, antimicrobial resistance genes and genetic relatedness by single nucleotide polymorphism (SNP) analysis. The Salmonella isolates, diarrhoeic patients (n = 17), healthy population (n = 13), cattle (abattoir, n = 67; farms, n = 10; market n = 5), revealed 49 serovars; some serovars were common to humans and cattle. Rare serovars were prevalent: Colindale (cattle and humans); Rubislaw and Bredeney (humans); and Dublin, Give, Eastbourne, Hadar, Marseille, Sundsvall, Bergen, Ekotedo, Carno and Ealing (cattle). The sequence types (ST) include ST 584, ST 198, ST 562 and ST 512 for S. Colindale, S. Kentucky S. Rubislaw and S. Urbana, respectively. Clonal cluster shared by cattle and human WGST isolates was not found. Antimicrobial resistance rates were generally low and towards only chloramphenicol, ampicillin, gentamicin, ciprofloxacin, tetracycline and streptomycin, range 2.7% (chloramphenicol) to 8.9% (streptomycin). Multiply resistant isolates included serovars Kentucky, 4,5,12:i:- and Typhimurium. The study presents a baseline description of the prevalence, serotypes, antimicrobial resistance phenotypes and genetic relatedness of Salmonella isolated from healthy and diarrhoeic humans, and cattle at harvest, on farm and at market. Cattle are a reservoir of diverse salmonellae with shared serovars with humans, but WGST does not support zoonotic transmission. Further study with larger samples is recommended to determine whether epidemiological link exists between cattle and humans.
9 citations
Cites methods from "In Silico Detection and Typing of P..."
...…and acquired antimicrobial resistance genes using the pipelines mlst (version 1.7), plasmidfinder (version 1.2, 80% threshold for %ID) and resfinder (version 2.1, 80% threshold for %ID/60% minimum length) available from CGE (Carattoli et al., 2014; Larsen et al., 2012; Zankari et al., 2012)....
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...Single nucleotide polymorphisms (SNPs) were determined using the pipeline csi phylogeny (version 1.4) available from the CGE (http://cge.cbs.dtu.dk/services/all.php)....
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...The assembled sequences were analysed to identify the MLST sequence type (ST) for Salmonella enterica, plasmid replicons and acquired antimicrobial resistance genes using the pipelines mlst (version 1.7), plasmidfinder (version 1.2, 80% threshold for %ID) and resfinder (version 2.1, 80% threshold for %ID/60% minimum length) available from CGE (Carattoli et al., 2014; Larsen et al., 2012; Zankari et al., 2012)....
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...The quality of the reads was assessed using the FastQC quality control tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), reads with a quality score of below 20 were filtered out, and all the remaining reads were assembled using the assembler pipeline (version 1.4) available from the Center for Genomic Epidemiology (CGE) (http://cge.cbs.dtu.dk/services/all.php)....
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References
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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)....
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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)....
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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....
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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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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)....
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