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: It is confirmed that ESBL-EC, including those that are capable of causing human infection, are present in recreational waters where there is a potential for human exposure and subsequent gut colonisation and infection in bathers.
Abstract: Extended spectrum β-lactamase producing Escherichia coli (ESBL-EC) are excreted via effluents and sewage into the environment where they can re-contaminate humans and animals. The aim of this observational study was to detect and quantify ESBL-EC in recreational water and wastewater, and perform a genetic and phenotypic comparative analysis of the environmental strains with geographically associated human urinary ESBL-EC. Recreational fresh- and saltwater samples from four different beaches and wastewater samples from a nearby sewage plant were filtered and cultured on differential and ESBL-selective media. After antimicrobial susceptibility testing and multi-locus variable number of tandem repeats assay (MLVA), selected ESBL-EC strains from recreational water were characterized by whole genome sequencing (WGS) and compared to wastewater and human urine isolates from people living in the same area. We detected ESBL-EC in recreational water samples on 8/20 occasions (40%), representing all sites. The ratio of ESBL-EC to total number of E. coli colony forming units varied from 0 to 3.8%. ESBL-EC were present in all wastewater samples in ratios of 0.56-0.75%. ST131 was most prevalent in urine and wastewater samples, while ST10 dominated in water samples. Eight STs and identical ESBL-EC MLVA-types were detected in all compartments. Clinical ESBL-EC isolates were more likely to be multidrug-resistant (p<0.001). This study confirms that ESBL-EC, including those that are capable of causing human infection, are present in recreational waters where there is a potential for human exposure and subsequent gut colonisation and infection in bathers. Multidrug-resistant E. coli strains are present in urban aquatic environments even in countries where antibiotic consumption in both humans and animals is highly restricted.
62 citations
TL;DR: This longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.
Abstract: Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.
61 citations
TL;DR: It is demonstrated that the in vivo proximity ligation method Hi-C can determine the in situ host range of ARGs, plasmids, and integrons in a wastewater sample by physically linking them to their host chromosomes.
Abstract: The rapid spread of antibiotic resistance is a serious human health threat. A range of environments have been identified as reservoirs of the antibiotic resistance genes (ARGs) found in pathogens. However, we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrated that the in vivo proximity ligation method Hi-C can determine the in situ host range of ARGs, plasmids, and integrons in a wastewater sample by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile elements and host genomes, mostly validating this method. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
61 citations
01 Feb 2018
TL;DR: It is indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings and provides insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.
Abstract: The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.
61 citations
TL;DR: It is shown that the occurrence at a high rate of colistin resistance in human faecal E. coli is the result of two distinct evolutionary pathways, i.e. the occurrence of chromosomal mutations in an endogenous E. Escherichia coli population and the rare acquisition of exogenous mcr-1-bearing strains probably of animal origin.
Abstract: Background Beyond plasmid-encoded resistance (mcr genes) prevalence in strain collections, large epidemiological studies to estimate the human burden of colistin-resistant Escherichia coli gut carriage are lacking. Objectives To evaluate the prevalence of colistin-resistant E. coli carriage in inpatients and decipher the molecular support of resistance and the genetic background of the strains. Methods During a 3 month period in 2017, we prospectively screened patients in six Parisian hospitals for rectal carriage of colistin-resistant E. coli using a selective medium, a biochemical confirmatory test and MIC determination. WGS of the resistant strains and their corresponding plasmids was performed. Results Among the 1217 screened patients, 153 colistin-resistant E. coli strains were isolated from 152 patients (12.5%). The mcr-1 gene was identified in only seven isolates (4.6%) on different plasmid scaffolds. The genetic background of these MCR-1 producers argued for an animal origin. Conversely, the remaining 146 colistin-resistant E. coli exhibited a phylogenetic distribution corresponding to human gut commensal/clinical population structure (B2 and D phylogroup predominance); 72.6% of those isolates harboured convergent mutations in the PmrA and PmrB proteins, constituting a two-component system shown to be associated with colistin resistance. Conclusions We showed that the occurrence at a high rate of colistin resistance in human faecal E. coli is the result of two distinct evolutionary pathways, i.e. the occurrence of chromosomal mutations in an endogenous E. coli population and the rare acquisition of exogenous mcr-1-bearing strains probably of animal origin. The involved selective pressures need to be identified in order to develop preventative strategies.
61 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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