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: In this article , a comparative genome analysis of S. hominis ShoR14, a multidrug-resistant, methicillin-resistant blood isolate from Terengganu, Malaysia, is presented.
Abstract: Staphylococcus hominis is a coagulase-negative Staphylococcus (CoNS) commensal capable of causing serious systemic infections in humans. The emergence of multidrug-resistant S. hominis strains is of concern but little is known about the characteristics of this organism, particularly from Malaysia. Here, we present the comparative genome analysis of S. hominis ShoR14, a multidrug-resistant, methicillin-resistant blood isolate from Terengganu, Malaysia. Genomic DNA of S. hominis ShoR14 was sequenced on the Illumina platform and assembled using Unicycler v0.4.8. ShoR14 belonged to sequence type (ST) 1 which is the most prevalent ST of the S. hominis subsp. hominis. Comparative genomic analysis with closely related strains in the database with complete genome sequences, led to the discovery of a novel variant of the staphylococcal chromosome cassette mec (SCCmec) type VIII element harboring the mecA methicillin-resistance gene in ShoR14 and its possible carriage of a SCCfus element that encodes the fusidic acid resistance gene (fusC). Up to seven possible ShoR14 plasmid contigs were identified, three of which harbored resistance genes for tetracycline (tetK), chloramphenicol (catA7), macrolides, lincosamides, and streptogramin B (ermC). Additionally, we report the discovery of a novel mercury-resistant transposon, Tn7456, other genomic islands, and prophages which make up the S. hominis mobilome.
2 citations
TL;DR: Wang et al. as discussed by the authors proposed a repB sequence-based scheme for grouping IncFIB replicon into seven primary types and further into 70 subtypes, and compared with the first sequenced InFIB-4.1/4.2 single-replicon plasmids from GenBank.
Abstract: To establish a typing scheme for IncFIB replicon and to dissect genomic features of IncFIB-4.1/4.2 single-replicon plasmids.A total of 146 representative fully sequenced IncFIB-replicon-containing plasmids were selected to construct a phylogenetic tree of repBIncFIB sequences. A collection of nine IncFIB-4.1/4.2 single-replicon plasmids from China were fully sequenced here and compared with the first sequenced IncFIB-4.1/4.2 single-replicon plasmids from GenBank to dissect their genomic diversity.In this study, a repB sequence-based scheme was proposed for grouping IncFIB replicon into seven primary types and further into 70 subtypes. A collection of nine IncFIB-4.1/4.2 single-replicon plasmids were fully sequenced here and compared with the first sequenced IncFIB-4.1/4.2 single-replicon plasmids from GenBank. These 11 plasmids had small backbones and shared only three key backbone markers repB together with its iterons, parABC, and stbD. Each plasmid contained one large accessory region (LAR) inserted into the backbone, and these 11 LARs had significantly distinct profiles of mobile genetic elements (MGEs) and resistance/metabolism gene loci. Antibiotic resistance regions (ARRs; the antibiotic resistance gene-containing genetic elements) were found in seven of these 11 LARs. Besides resistance genes, ARRs carried unit or composite transposons, integrons, and putative resistance units. IncFIB-4.1/4.2 single-replicon plasmids were important vectors of drug resistance genes. This was the first report of three novel MGEs: In1776, Tn6755, and Tn6857.Data presented here provided a deeper insight into diversity and evolution of IncFIB replicon and IncFIB-4.1/4.2 single-replicon plasmids.
2 citations
TL;DR: The complete genome sequence of a Hafnia paralvei strain isolated from a lake in Switzerland in 2020 is reported in this paper , which consists of a 4.7-Mbp chromosome, a large plasmid (213 kb) harboring mcr-9, and a small plasmoid (6 kb).
Abstract: Here, we report the complete genome sequence of a Hafnia paralvei strain isolated from a lake in Switzerland in 2020. The genome consists of a 4.7-Mbp chromosome, a large plasmid (213 kb) harboring mcr-9, and a small plasmid (6 kb).
2 citations
TL;DR: PlasmidTron utilises the phenotypic data normally available in bacterial population studies, such as antibiograms, virulence factors, or geographic information, to identify sequences that are likely to represent MGEs linked to the phenotype.
Abstract: When defining bacterial populations through whole genome sequencing (WGS) the samples often have detailed associated metadata that relate to disease severity, antimicrobial resistance, or even rare biochemical traits. When comparing these bacterial populations, it is apparent that some of these phenotypes do not follow the phylogeny of the host i.e. they are genetically unlinked to the evolutionary history of the host bacterium. One possible explanation for this phenomenon is that the genes are moving independently between hosts and are likely associated with mobile genetic elements (MGE). However, identifying the element that is associated with these traits can be complex if the starting point is short read WGS data. With the increased use of next generation WGS in routine diagnostics, surveillance and epidemiology a vast amount of short read data is available and these types of associations are relatively unexplored. One way to address this would be to perform assembly de novo of the whole genome read data, including its MGEs. However, MGEs are often full of repeats and can lead to fragmented consensus sequences. Deciding which sequence is part of the chromosome, and which is part of a MGE can be ambiguous. We present PlasmidTron, which utilises the phenotypic data normally available in bacterial population studies, such as antibiograms, virulence factors, or geographic information, to identify sequences that are likely to represent MGEs linked to the phenotype. Given a set of reads, categorised into cases (showing the phenotype) and controls (phylogenetically related but phenotypically negative), PlasmidTron can be used to assemble de novo reads from each sample linked by a phenotype. A k-mer based analysis is performed to identify reads associated with a phylogenetically unlinked phenotype. These reads are then assembled de novo to produce contigs. By utilising k-mers and only assembling a fraction of the raw reads, the method is fast and scalable to large datasets. This approach has been tested on plasmids, because of their contribution to important pathogen associated traits, such as AMR, hence the name, but there is no reason why this approach cannot be utilized for any MGE that can move independently through a bacterial population. PlasmidTron is written in Python 3 and available under the open source licence GNU GPL3 from https://github.com/sanger-pathogens/plasmidtron .
2 citations
TL;DR: The data suggests that dominant lineages of E. coli are emerging as CPE clinical threats, in much the same way as occurred for ESBL lineages for E.coli carrying ESBL genes, according to recent inter-continental transmission of the ST167 lineage.
Abstract: Background: The increase in infections as a result of multi-drug resistant strains of Escherichia coli is a global health crisis. The emergence of globally disseminated lineages of E. coli carrying ESBL genes has been well characterised. An increase in strains producing carbapenemase enzymes and mobile colistin resistance is now being reported, but there is little data on emerging global lineages of such strains.
Methods: Routine screening of patients within an ICU of West China Hospital identified a potential outbreak of E. coli carrying the blaNDM-5 carbapenemase gene. Genome sequencing was performed on the strains isolated. The MLST lineage of the strains was determined and a global collection of genomes of relevant lineages utilised to characterise the emergence of two globally disseminated carbapenemase-producing Enterobacteriaceae (CPE) lineages of E. coli.
Findings: Our data describes the presence of E. coli ST167 and ST617 in a potential West China Hospital outbreak, which are globally disseminated CPE lineages of E. coli. Both lineages show extreme levels of MDR gene acquisition ranging across ESBL, CPE, and mobile colistin resistance genes. Our analysis also shows real-time switching of CPE genes in a plasmid and real-time transfer of the plasmid across lineages, as well as recent inter-continental transmission of the ST167 lineage.
Interpretation: Our data suggests that dominant lineages of E. coli are emerging as CPE clinical threats, in much the same way as occurred for ESBL lineages of E. coli.
2 citations
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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