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: The phenotypic comparisons, fatty acid methyl esters compositions, genetic rep PCR fingerprint and detailed whole-cell MALDI-TOF mass spectrometry proteomic profiles permitted the differentiation of Polish strains from the type strains of all other known species of the genus Pectobacterium.
Abstract: Gram-stain-negative, rod-shaped pectinolytic bacteria strains designated as DPMP315T, DPMP316, DPMP317 and DPMP318 isolated from groundwater sampled from a vegetable field in the North of Poland, were subjected to the polyphasic analyses. Multilocus sequence analyses based on five housekeeping genes (gyrA, recA, recN, rpoA and rpoS) revealed their distinctiveness from the other species of the genus, simultaneously indicating that the newly described species,
Pectobacterium punjabense
, as well as
Pectobacterium parmentieri
and
P. wasabiae
, to be the closest relatives. In silico DNA–DNA hybridization (<43.1 %) and average nucleotide identity (<92.5 %) values of strain DPMP315T with other type strains of species of the genus
Pectobacterium
supported the delineation of the novel strain as representing a novel species. The phenotypic comparisons, fatty acid methyl esters compositions, genetic rep PCR fingerprint and detailed whole-cell MALDI-TOF mass spectrometry proteomic profiles permitted the differentiation of Polish strains from the type strains of all other known species of the genus
Pectobacterium
. The results of polyphasic analyses performed for four Polish strains are the basis for the distinction of the novel species. Here, we propose to establish DPMP315T as a type strain (=PCM3006T=LMG 31077T) with the name Pectobacterium polonicum sp. nov.
28 citations
TL;DR: This study offers a comprehensive way to decipher plasmid-bacterium associations and demonstrates that the CTX-M-encoding ST131 Group1 and Group2 plasmids are clade-restricted and presumably less transmissible, potentially contributing to ST131 clonal superiority.
Abstract: Extraintestinal multidrug resistant Escherichia coli sequence type (ST) 131 is a worldwide pandemic pathogen and a major cause of urinary tract and bloodstream infections. The role of this pandemic lineage in multidrug resistance plasmid dissemination is still scarce. We herein performed a meta-analysis on E. coli ST131 whole-genome sequence (WGS) databases to unravel ST131 plasmidome and specifically to decipher CTX-M encoding plasmids-clade associations. We mined 880 ST131 WGS data and proved that CTX-M-27-encoding IncF[F1:A2:B20] (Group1) plasmids are strictly found in clade C1, whereas CTX-M-15-encoding IncF[F2:A1:B-] (Group2) plasmids exist only in clade C2 suggesting strong plasmid-clade adaptations. Specific Col-like replicons (Col156, Col(MG828), and Col8282) were also found to be clade C1-associated. BLAST-based search revealed that Group1 and Group2 plasmids are narrow-host-range and restricted to E.coli. Among a collection of 20 newly sequenced Israeli ST131 CTX-M-encoding plasmids (2003–2016), Group1 and Group2 plasmids were dominant and associated with the expected clades. We found, for the first time in ST131, a CTX-M-15-encoding phage-like plasmid group (Group3) and followed its spread in the WGS data. This study offers a comprehensive way to decipher plasmid-bacterium associations and demonstrates that the CTX-M-encoding ST131 Group1 and Group2 plasmids are clade-restricted and presumably less transmissible, potentially contributing to ST131 clonal superiority.
28 citations
TL;DR: The benefits and limitations of using different metagenomic protocols are elaborated, the relative merits of various sequencing technologies are discussed, and relevant bioinformatics tools and pipelines are highlighted to predict the presence of MGEs and their microbial hosts.
28 citations
TL;DR: A retrospective genomic study led to the identification of two carbapenem-resistant K. pneumoniae isolates carrying bla KPC-2 genes on non-conjugative plasmids.
Abstract: A retrospective genomic study led to the identification of two carbapenem-resistant K. pneumoniae isolates (KPN535 and KPC45) carrying bla KPC-2 genes on non-conjugative plasmids.….
28 citations
Cites background from "In Silico Detection and Typing of P..."
...(MGD2) plasmid (NC_003789) (3), and carrying relaxase and mobC genes....
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TL;DR: The utility of large-scale genome-based MLST as a quick and effective approach to narrow the scope of in-depth comparative genomic analysis and consequently provide new insights into the fine scale of pathogen evolution and population structure is highlighted.
Abstract: Typhoid fever, caused by Salmonella enterica serovar Typhi, remains an important public health burden in Southeast Asia and other endemic countries. Various genotyping methods have been applied to study the genetic variations of this human-restricted pathogen. Multilocus sequence typing (MLST) is one of the widely accepted methods, and recently, there is a growing interest in the re-application of MLST in the post-genomic era. In this study, we provide the global MLST distribution of S. Typhi utilizing both publicly available 1,826 S. Typhi genome sequences in addition to performing conventional MLST on S. Typhi strains isolated from various endemic regions spanning over a century. Our global MLST analysis confirms the predominance of two sequence types (ST1 and ST2) co-existing in the endemic regions. Interestingly, S. Typhi strains with ST8 are currently confined within the African continent. Comparative genomic analyses of ST8 and other rare STs with genomes of ST1/ST2 revealed unique mutations in important virulence genes such as flhB, sipC, and tviD that may explain the variations that differentiate between seemingly successful (widespread) and unsuccessful (poor dissemination) S. Typhi populations. Large scale whole-genome phylogeny demonstrated evidence of phylogeographical structuring and showed that ST8 may have diverged from the earlier ancestral population of ST1 and ST2, which later lost some of its fitness advantages, leading to poor worldwide dissemination. In response to the unprecedented increase in genomic data, this study demonstrates and highlights the utility of large-scale genome-based MLST as a quick and effective approach to narrow the scope of in-depth comparative genomic analysis and consequently provide new insights into the fine scale of pathogen evolution and population structure.
28 citations
Cites methods from "In Silico Detection and Typing of P..."
...4 (Carattoli et al., 2014) and validated with manual inspection....
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...For plasmid analyses, the plasmid replicons were detected using PlasmidFinder 1.3 database (Carattoli et al., 2014) and plasmid MLST was determined using pMLST1....
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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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