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: Assessment of the effect of different antimicrobial treatments on faecal shedding and diversity of ESBL-producing Escherichia coli in horses found transmission of ES BL-EC in the hospital was suspected, highlighting the importance of antimicrobial stewardship and infection control practices in equine medicine.
7 citations
TL;DR: Results show that in vivo selection of gyrA mutants occurred in mallards during exposure to ciprofloxacin at concentrations previously found in aquatic environments, highlighting the potential for enrichment of resistant bacteria in wildlife and underline the importance of reducing antibiotic pollution in the environment.
Abstract: Emergence and selection of antibiotic resistance following exposure to high antibiotic concentrations have been repeatedly shown in clinical and agricultural settings, whereas the role of the weak selective pressures exerted by antibiotic levels below the MIC (sub-MIC) in aquatic environments due to anthropogenic contamination remains unclear. Here, we studied how exposure to sub-MIC levels of ciprofloxacin enriches for Escherichia coli with reduced susceptibility to ciprofloxacin using a mallard colonization model. Mallards were inoculated with two isogenic extended-spectrum-β-lactamase (ESBL)-encoding E. coli strains, differing only by a gyrA mutation that results in increased MICs of ciprofloxacin, and exposed to different levels of ciprofloxacin in their swimming water. Changes in the ratios of mutant to parental strains excreted in feces over time and ESBL plasmid spread within the gut microbiota from individual birds were investigated. Results show that in vivo selection of gyrA mutants occurred in mallards during exposure to ciprofloxacin at concentrations previously found in aquatic environments. During colonization, resistance plasmids were readily transferred between strains in the intestines of the mallards, but conjugation frequencies were not affected by ciprofloxacin exposure. Our results highlight the potential for enrichment of resistant bacteria in wildlife and underline the importance of reducing antibiotic pollution in the environment.
7 citations
Cites methods from "In Silico Detection and Typing of P..."
...Sequences were analyzed with CLC Genomics Workbench v12 (Qiagen) by de novo assembly, and contigs were analyzed for resistance genes, plasmid replicons, and virulence genes by the ResFinder (51), PlasmidFinder (52), and VirulenceFinder (53) applications....
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...Whole-genome sequencing was performed on an Illumina MiSeq using the Nextera XT library prep kit (Illumina) and sequenced with a paired-end read length of 100bp. Sequences were analyzed with CLC Genomics Workbench v12 (Qiagen) by de novo assembly, and contigs were analyzed for resistance genes, plasmid replicons, and virulence genes by the ResFinder (51), PlasmidFinder (52), and VirulenceFinder (53) applications....
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TL;DR: This paper performed a phylogenomic analysis of 299 E. coli ST127 isolates of diverse epidemiological origin to characterize their population structure, genetic determinants of virulence, antimicrobial resistance, and repertoire of mobile genetic elements with a focus on plasmids.
Abstract: Escherichia coli ST127, a recently emerged global pathogen noted for high virulence gene carriage, is a leading cause of urinary tract and blood stream infections. ST127 is frequently isolated from humans and companion animals; however, it is unclear if they are distinct or related populations of ST127. We performed a phylogenomic analysis of 299 E. coli ST127 of diverse epidemiological origin to characterize their population structure, genetic determinants of virulence, antimicrobial resistance, and repertoire of mobile genetic elements with a focus on plasmids. The core gene phylogeny was divided into 13 clusters, the largest of which (BAP4) contained the majority of human and companion animal origin isolates. This dominant cluster displayed genetic differences to the remainder of the phylogeny, most notably alternative gene alleles encoding important virulence factors including lipid A, flagella, and K capsule. Furthermore, numerous close genetic linkages (<30 SNPs) between human and companion animal isolates were observed within the cluster. Carriage of antimicrobial resistance genes in the collection was limited, but virulence gene carriage was extensive. We found evidence of pUTI89-like virulence plasmid carriage in over a third of isolates, localised to four of the major phylogenetic clusters. Our study supports global scale repetitive transfer of E. coli ST127 lineages between humans and companion animals, particularly within the dominant BAP4 cluster.
7 citations
TL;DR: Examining the FII-33 group, which has been associated with multidrug resistance and particularly carbapenem resistance in clinically significant members of the Enterobacterales in China, provides insight into the evolution of this important plasmid group, and develops resources for rapidly typing them to the sublineage level in complete or draft genome sequences.
Abstract: Effective genomic surveillance of antibiotic-resistant bacterial pathogens must consider plasmids, which are frequently implicated in the accumulation and transfer of resistance genes between bacterial strains or species. However, the evolution of plasmids is complex, and simple typing or comparison tools cannot accurately determine whether plasmids belong to the same sublineages. ABSTRACT We examined 185 complete, publicly available FII-33 plasmid sequences, characterizing their backbone and various insertions. The variable characteristic insertions facilitated evolutionary reconstruction for this plasmid group, beginning with the acquisition of a primary resistance region (PRR) over 10 years ago. FII-33 plasmids have evolved by acquiring additional resistance genes in the PRR via translocatable elements and by forming cointegrates with plasmids of other types. In all cases, IS26 is suspected to have mediated cointegration. Plasmid cointegration has contributed to the accumulation of resistance genes and may have increased the transmissibility, stability, and host range of the original FII-33 lineage. A particularly important sublineage was formed by a replicative IS26 cointegration event that fused an FII-33 plasmid with a blaKPC-2-containing R-type plasmid, interrupting the FII-33 traI gene encoding the conjugative relaxase. The FII-33:R cointegrate arose in the Klebsiella pneumoniae ST11 clone and remains largely confined there due to the abolition of transfer ability by the FII-33:R cointegration event. However, in some cases FII-33:R cointegrates have fused with additional plasmids and acquired complete transfer regions or oriT sequences that might restore their ability to transfer horizontally. Cointegration events across FII-33 plasmid sublineages have involved plasmids of at least 15 different types. This suggests that plasmid cointegration occurs readily and is more common than previously appreciated, raising questions about the effects of cointegrate formation on plasmid host range, stability, and capacity for horizontal transfer. Resources are provided for detecting and characterizing FII-33 plasmid sublineages from complete or draft genome sequences. IMPORTANCE Effective genomic surveillance of antibiotic-resistant bacterial pathogens must consider plasmids, which are frequently implicated in the accumulation and transfer of resistance genes between bacterial strains or species. However, the evolution of plasmids is complex, and simple typing or comparison tools cannot accurately determine whether plasmids belong to the same sublineages. This precludes precise tracking of plasmid movement in bacterial populations. We have examined the FII-33 group, which has been associated with multidrug resistance and particularly carbapenem resistance in clinically significant members of the Enterobacterales in China. Our analysis has provided insight into the evolution of this important plasmid group, allowing us to develop resources for rapidly typing them to the sublineage level in complete or draft genome sequences. Our approach will improve detection and characterization of FII-33 plasmids and facilitate surveillance within and outside China. The approach can serve as a model for similar studies of other plasmid types.
7 citations
TL;DR: The genome sequence of a multidrug-resistant Klebsiella pneumoniae strain, which caused an outbreak in a neonatal ward in 2011, is reported.
Abstract: We report here the genome sequence of a multidrug-resistant Klebsiella pneumoniae strain, which caused an outbreak in a neonatal ward in 2011. The genome consists of a single chromosome (5,278 kb) and three plasmids (362 kb, 5 kb, and 4 kb).
7 citations
Cites background from "In Silico Detection and Typing of P..."
...Furthermore, the 362-kb plasmid exhibits four replication-related rep genes, including repC (Kpn23412_5167), which is identical to the repC in the reference sequence for replicon type IncQ1 (6), and repA (Kpn23412_5166)....
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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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