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 paper, the resistome and virulome of 33 extended drug-resistant and carbapenem-resistant Acinetobacter baumannii (XDR CRAB) strains isolated from hospitalized and ambulatory patients in Bucharest, Romania.
Abstract: Acinetobacter baumannii has emerged worldwide as a dominant pathogen in a broad range of severe infections, raising an acute need for efficient antibacterials. This is the first report on the resistome and virulome of 33 extended drug-resistant and carbapenem-resistant A. baumannii (XDR CRAB) strains isolated from hospitalized and ambulatory patients in Bucharest, Romania. A total of 33 isolates were collected and analyzed using phenotypic antibiotic susceptibility and conjugation assays, PCR, whole-genome sequencing (WGS), pulsed-field gel electrophoresis (PFGE) and MultiLocus Sequence Typing (MLST). All isolates were extensively drug-resistant (XDR), being susceptible only to colistin. The carbapenem resistance was attributed by PCR mainly to blaOXA-24 and blaOXA-23 genes. PFGE followed by MLST analysis demonstrated the presence of nine pulsotypes and six sequence types. WGS of seven XDR CRAB isolates from healthcare-associated infections demonstrated the high diversity of resistance genes repertoire, as well as of mobile genetic elements, carrying ARGs for aminoglycosides, sulphonamides and macrolides. Our data will facilitate the understanding of resistance, virulence and transmission features of XDR AB isolates from Romanian patients and might be able to contribute to the implementation of appropriate infection control measures and to develop new molecules with innovative mechanisms of action, able to fight effectively against these bugs, for limiting the spread and decreasing the infection rate and mortality.
15 citations
TL;DR: It is shown that the plasmid is able to transfer into a range of genetically diverse clinical Escherichia coli strains and that the fitness cost imposed on the host is often low, and is stably maintained under non-selective conditions across different genetic backgrounds.
Abstract: Dissemination of bacterial clones carrying plasmid-mediated resistance genes is a major factor contributing to the increasing prevalence of antibiotic resistance. Understanding the evolution of successful clones and the association to mobile resistance elements are therefore crucial. In this study, we determined the sequence of a 145 kb IncC multi-drug resistance plasmid (pK71-77-1-NDM), harbouring resistance genes to last-resort antibiotics including carbapenems. We show that the plasmid is able to transfer into a range of genetically diverse clinical Escherichia coli strains and that the fitness cost imposed on the host is often low. Moreover, the plasmid is stably maintained under non-selective conditions across different genetic backgrounds. However, we also observed a lower conjugation frequency and higher fitness cost in the E. coli sequence type (ST) 73 background, which could partially explain why this clone is associated with a lower level of antibiotic resistance than other E. coli clones. This is supported by a bioinformatical analysis showing that the ST73 background harbours plasmids less frequently than the other studied E. coli STs. Studying the evolution of antibiotic resistance in a clinical context and in diverse genetic backgrounds improves our understanding of the variability in plasmid-host associations.
15 citations
24 Sep 2020
TL;DR: This study reports the draft genome sequence of a promising fish probiotic, Bacillus subtilis strain WS1A, that possesses antimicrobial activity against Aeromonas veronii and suppressed motile aeromonas septicemia in Labeo rohita.
Abstract: This study reports the draft genome sequence of a promising fish probiotic, Bacillus subtilis strain WS1A, that possesses antimicrobial activity against Aeromonas veronii and suppressed motile Aeromonas septicemia in Labeo rohita. The de novo assembly resulted in an estimated chromosome size of 4,148,460 bp, with 4,288 open reading frames.
15 citations
01 Mar 2021
TL;DR: In this article, a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing is presented.
Abstract: Whole genome sequencing (WGS) enables complete characterization of bacterial pathogenic isolates at single nucleotide resolution, making it the ultimate tool for routine surveillance and outbreak investigation. The lack of standardization, and the variation regarding bioinformatics workflows and parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing. The workflow supports three commonly used bioinformatics approaches for the detection of genes and alleles: alignment with blast+, kmer-based read mapping with KMA, and direct read mapping with SRST2. A collection of 131 STEC isolates collected from food and human sources, extensively characterized with conventional molecular methods, was used as a validation dataset. Using a validation strategy specifically adopted to WGS, we demonstrated high performance with repeatability, reproducibility, accuracy, precision, sensitivity and specificity above 95 % for the majority of all assays. The WGS workflow is publicly available as a ‘push-button’ pipeline at https://galaxy.sciensano.be. Our validation strategy and accompanying reference dataset consisting of both conventional and WGS data can be used for characterizing the performance of various bioinformatics workflows and assays, facilitating interoperability between laboratories with different WGS and bioinformatics set-ups.
15 citations
TL;DR: The genome sequence of E. coli 657SK2 provides information on resistance mechanisms and virulence characteristics of pathogenic E. Escherichia coli harbouring blaNDM-5 and blaCTX-M-15 that are spreading into the environment via urban wastewater.
Abstract: Objectives Carbapenem-resistant Escherichia coli have emerged worldwide and represent a major challenge to effective healthcare management. Here we report the genome sequence of an NDM-5- and CTX-M-15-producing E. coli belonging to sequence type 617 isolated from wastewater treatment plant effluent in Switzerland. Methods Whole-genome sequencing of E. coli 657SK2 was performed using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology RS2 reads (C4/P6 chemistry). De novo assembly was carried out using Canu 1.6, and sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Results The genome of E. coli 657SK2 consists of a 4.9-Mbp chromosome containing blaCTX-M-15, genes associated with virulence [fyuA, hlyE, the pyelonephritis-associated pili (pap) gene cluster and the yad gene cluster], the copper resistance gene pco, and genes associated with resistance to quaternary ammonium compound (QAC) disinfectants (emrA, mdfA and sugE). A 173.9-kb multidrug resistance IncFII-FIA-FIB plasmid was detected harbouring aadA2, aadA5, blaNDM-5, blaOXA-1, cat, drfA, drfA17, the mph(A)–mrx–mphR cluster, the tetA–tetC–tetR cluster, and the virulence genes iutA and ylpA. Conclusions The genome sequence of E. coli 657SK2 provides information on resistance mechanisms and virulence characteristics of pathogenic E. coli harbouring blaNDM-5 and blaCTX-M-15 that are spreading into the environment via urban wastewater.
15 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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