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: This is the first report of coexistence of two blaNDM–5-carrying plasmids in a single bacterial isolate, highlighting the genetic complexity of NDM-5 carbapenemase circulation, and the urgent need for continued active surveillance.
Abstract: The emergence of carbapenem-resistant Enterobacteriaceae poses a significant threat to public health worldwide. Here, we reported a multidrug-resistant Escherichia coli strain with two different blaNDM-5-carrying plasmids from China. Illumina short-read and MinION long-read whole genome sequencing were performed. Genomic analysis found that one blaNDM-5 gene together with mphA was located on a 55-kb IncX3 plasmid, while the other blaNDM-5 gene was on a novel 68-kb IncFII plasmid. Susceptibility testing and quantitative reverse transcription PCR results further indicated that the transconjugants with the IncX3 plasmid exhibited higher-level carbapenem resistance and expression of blaNDM-5 than those with both plasmids or the IncFII plasmid. Two other β-lactamase genes (blaCTX-M-15 and blaOXA-1) were also detected on another 160-kb IncF plasmid. This is the first report of coexistence of two blaNDM-5-carrying plasmids in a single bacterial isolate, highlighting the genetic complexity of NDM-5 carbapenemase circulation, and the urgent need for continued active surveillance.
11 citations
TL;DR: This work studied a group of plasmids from enterobacteria that encode phylogenetically related mobilization functions that populate the previously non-described MOBQ4 relaxase family and found that MPFI conjugative plasmsids were the most efficient helpers for MOBZ4 conjugatives dissemination among clinically relevant enterob bacteria.
Abstract: Plasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. In this work, we studied a group of plasmids from enterobacteria that encode phylogenetically related mobilization functions that populate the previously non-described MOBQ4 relaxase family. These plasmids encode two transfer genes: mobA coding for the MOBQ4 relaxase; and mobC, which is non-essential but enhances the plasmid mobilization frequency. The origin of transfer is located between these two divergently transcribed mob genes. We found that MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative dissemination among clinically relevant enterobacteria. While highly similar in their mobilization module, two sub-groups with unrelated replicons (Rep_3 and ColE2) can be distinguished in this plasmid family. These subgroups can stably coexist (are compatible) and transfer independently, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such a strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.
11 citations
TL;DR: Clinical attention should be paid to the importance of E. coli ST131 in respiratory infections and the spread of blaKPC–2, the genetic context of which suggests that the carbapenem resistance was due to the acquisition by E.coli ST131 of transmissible plasmids pE0272_KPC and pE0171-KPC carrying bla kPC– 2.
Abstract: Escherichia coli sequence type 131 (ST131) is well known for its multidrug resistance profile. Carbapenems have been considered the treatment of choice for E. coli ST131 infections and resistance to carbapenems is now emerging due to the acquisition of carbapenemase. 45 carbapenem-resistant E. coli isolates were collected in a hospital. The resistance mechanisms, plasmid profiles and genetic relatedness of these strains were determined. Phylogenetic relationships between these strains were assessed by molecular profiling and aligned with patient clinical details. The genetic context of blaKPC-2 was analyzed to trace the potential dissemination of blaKPC-2.45 carbapenem-resistant E. coli ST131strains are closely related. Initially prevalent only in the ward E02, ST131 subsequently spread to other wards and multiple lineages evolved independently, resulting in a respiratory infection outbreak of carbapenem-resistant E. coli ST131. Eight of the 30 patients died within 28 days of the first E. coli ST131 isolation. The blaKPC-2 positive plasmid profiles suggest that the carbapenem resistance was due to the acquisition of transmissible plasmids pE0272_KPC and pE0171_KPC carrying blaKPC-2 by E. coli ST131. Additionally, diverse multidrug resistance elements were transferred and rearranged between these plasmids mediated by IS26. E. coli ST131 has become a multidrug resistant epidemic clone that can cause hospital-acquired respiratory infections resulting in mortality. Our research indicates that clinical attention should be paid to the importance of E. coli ST131 in respiratory infections. The emergence of blaKPC in E. coli ST131 may herald the beginning of the high prevalence of carbapenem resistance in Chinese communities and hospitals.
11 citations
TL;DR: The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes and warrants isolation and antibiotic testing of enteric pathogens before treating macaques with quinolones prophylactically or therapeutically.
Abstract: Non-human primates (NHPs) for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants isolation and antibiotic testing of enteric pathogens before treating macaques with quinolones prophylactically or therapeutically.
11 citations
Cites methods from "In Silico Detection and Typing of P..."
...VirulenceFinder 1.5 (Joensen et al., 2014), ResFinder 2.1 (Zankari et al., 2012), and PlasmidFinder 1.3 (Carattoli et al., 2014) were used to identify virulence factors, antibiotic resistance genes, and plasmids, respectively....
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...3 (Carattoli et al., 2014) were used to identify virulence factors, antibiotic resistance genes, and plasmids, respectively....
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TL;DR: In this article , the authors tracked the evolution of the clinically relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalized patients.
Abstract: Antimicrobial resistance (AMR) in bacteria is a major threat to public health; one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been studied extensively in vitro but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here, we tracked the evolution of the clinically relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalized patients. Combining genomic and experimental approaches, we first characterized plasmid diversity and the genotypic and phenotypic effects of multiple plasmid mutations on a common genetic background. Second, using cutting-edge genomic editing in wild-type multidrug-resistant enterobacteria, we dissected three cases of within-patient plasmid-mediated AMR evolution. Our results revealed compensatory evolution of plasmid-associated fitness cost and the evolution of enhanced plasmid-mediated AMR in bacteria evolving in the gut of hospitalized patients. Crucially, we observed that the evolution of pOXA-48-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness. This study highlights the need to develop new evolution-informed approaches to tackle plasmid-mediated AMR dissemination.
11 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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