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 diversity, prevalence and transmission dynamics of carbapenemase-encoding plasmids are investigated using long-read sequencing of representative isolates from this collection in combination with short-read data from all isolates.
Abstract: The incidence of Klebsiella pneumoniae infections that are resistant to carbapenems, a last-line class of antibiotics, has been rapidly increasing. The primary mechanism of carbapenem resistance is production of carbapenemase enzymes, which are most frequently encoded on plasmids by blaOXA-48-like, blaVIM, blaNDM and blaKPC genes. Using short-read sequence data, we previously analysed genomes of 1717 isolates from the K. pneumoniae species complex submitted during the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE). Here, we investigated the diversity, prevalence and transmission dynamics of carbapenemase-encoding plasmids using long-read sequencing of representative isolates (n=79) from this collection in combination with short-read data from all isolates. We highlight three major patterns by which carbapenemase genes have disseminated via plasmids. First, blaOXA-48-like genes have spread across diverse lineages primarily via a highly conserved, epidemic pOXA-48-like plasmid. Second, blaVIM and blaNDM genes have spread via transient associations of diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one clonal lineage (ST258/512) despite frequent mobilisation between pre-existing yet diverse plasmids within the lineage. Despite contrasts in these three modes of carbapenemase gene spread, which can be summarised as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage, all are underpinned by significant propagation along high-risk clonal lineages.
4 citations
Cites background from "In Silico Detection and Typing of P..."
...The assemblies contained 1-8 plasmid replicons (median, 4), which are sequences used for defining plasmid incompatibility (Inc) groups (Carattoli et al. 2014)....
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...6.1 (Hunt et al. 2017) with the PlasmidFinder database (Carattoli et al. 2014)....
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TL;DR: The WGS and annotation of a Campylobacter coli strain, FNW20G12, which was isolated from milk in the United States in 1997 and carries multidrug resistance is reported and presented to aid in the epidemiology study of C. coli antimicrobial resistance and role in foodborne outbreak.
Abstract: As the most prevalent bacterial cause of human gastroenteritis, food-borne Campylobacter infections pose a serious threat to public health. Whole Genome Sequencing (WGS) is a tool providing quick and inexpensive approaches for analysis of food-borne pathogen epidemics. Here we report the WGS and annotation of a Campylobacter coli strain, FNW20G12, which was isolated from milk in the United States in 1997 and carries multidrug resistance. The draft genome of FNW20G12 (DDBJ/ENA/GenBank accession number LWIH00000000) contains 1, 855,435 bp (GC content 31.4%) with 1902 annotated coding regions, 48 RNAs and resistance to aminoglycoside, beta-lactams, tetracycline, as well as fluoroquinolones. There are very few genome reports of C. coli from dairy products with multidrug resistance. Here the draft genome of FNW20G12, a C. coli strain isolated from raw milk, is presented to aid in the epidemiology study of C. coli antimicrobial resistance and role in foodborne outbreak.
4 citations
Cites methods from "In Silico Detection and Typing of P..."
...[10] A. Carattoli, E. Zankari, A. Garcia-Fernandez, M. Voldby Larsen, O. Lund, L. Villa, F. Moller Aarestrup, H. Hasman, In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing....
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...There was no plasmid replicon region identified in FNW20G12 genome using PlasmidFinder [10]....
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TL;DR: This study data demonstrates that two prevalent blaCTX-M genes have been circulating in S. sonnei in Korea over the last 10 years, and suggests a large phylogenetic distance between the S.Sonnei isolates related to overseas travel and those acquired domestically in Korea.
Abstract: Background Multidrug-resistant Shigella isolates have recently emerged as a serious public health threat worldwide. In particular, overseas travel is a risk factor for acquisition of antimicrobial-resistant Shigella strains. To explore the role of travel in the spread of cefotaxime-resistant Shigella sonnei in Korea, we screened 751 Shigella spp. isolates from 2007 to 2016 through the National Surveillance system, and 28 cephalosporin-resistant S. sonnei isolates were identified. Methods For cephalosporin-resistant S. sonnei isolates, epidemiological and molecular analyses (plasmid structure analysis, pulsed-field gel electrophoresis (PFGE) and high-quality single-nucleotide polymorphisms (hqSNPs) based on whole-genome sequencing (WGS)) were conducted to investigate the source of infection and transmission route. Results Among the 28 cefotaxime-resistant S. sonnei strains, 18 were isolated from travellers returning from Asia, including Vietnam (n = 11). Molecular analysis of 18 blaCTX-M-type isolates revealed that 15 contain CTX-M-15; 50% of isolates from domestic patients contain CTX-M-14. Analysis of the genetic environments of the blaCTX-M-14 and blaCTX-M-15 genes revealed different genetic organization surrounding the blaCTX-M genes. Additionally, PFGE and hqSNP results suggested a large phylogenetic distance between the S. sonnei isolates related to overseas travel and those acquired domestically in Korea. Conclusion Our study data demonstrates that two prevalent blaCTX-M genes, blaCTX-M-14 and blaCTX-M-15, have been circulating in S. sonnei in Korea over the last 10 years. Recently, international travellers are at a high risk for acquisition of CTX-M-15-producing S. sonnei in Korea.
4 citations
TL;DR: The mechanism of gene amplification, and the subsequent hyperproduction, of blaTEM-1B is an important consideration when using genomic data to predict resistance/susceptibility to TZP.
Abstract: A novel phenotype of Escherichia coli and Klebsiella pneumoniae resistant to piperacillin/tazobactam (TZP), but susceptible to carbapenems and 3rd generation cephalosporins has recently emerged. The resistance mechanism of this phenotype has been identified as hyperproduction of the β-lactamase blaTEM, however the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. We sought to understand this mechanism by focussing on a pair of isolates obtained from an individual patient across two infection episodes and displaying within-patient evolution to TZP resistance. Following confirmation that the two isolates were clonal, we found that the TZP-resistant isolate hyperproduced a β-lactamase but lacked mutations within β-lactamase promoter regions. Hybrid assembly of long and short sequencing reads of the two isolates revealed both harboured a novel IS26-flanked composite transposon containing several antibiotic resistance genes, including blaTEM-1B, which was designated Tn6762. These resistance genes are also found to be present on a translocatable unit which had excised from Tn6762 in the TZP-resistant isolate. By replicating the evolutionary event leading to TZP resistance we were able to observe excision of the translocatable unit from Tn6762 following exposure to TZP and capture the TU in a plasmid containing a copy of IS26. Subsequent amplification of the TU, and by extension blaTEM-1B, leads to β-lactamase hyperproduction and TZP resistance. Despite a significant increase in gene copy number (P value =
4 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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