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Journal ArticleDOI

In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

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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Citations
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Journal ArticleDOI
TL;DR: It is confirmed that plastic litter, serve as novel reservoir for human multidrug resistant pathogenic bacteria that combined with poor sanitation and waste handling, may lead to transmission of infectious diseases and antimicrobial resistance.

31 citations

Journal ArticleDOI
23 Oct 2019
TL;DR: These resistant genes/plasmids were conjugative, and they could be co-conjugated, conferring resistance to multiple types of antibiotics, including the carbapenems and colistin, to the recipient Escherichia coli strains.
Abstract: The emergence of carbapenem-resistant and colistin-resistant Enterobacteriaceae represents a great risk for public health. In this study, the phenotypical and genetic characteristics of eight carbapenem-resistant and colistin-resistant isolates from pig farms in China were determined by the broth microdilution method and whole genome sequencing. Antimicrobial susceptibility testing showed that the eight carbapenem-resistant and colistin-resistant strains were resistant to three aminoglycosides, twelve β-lactams, one of the phenicols, one of the tetracyclines, and one of the fluoroquinolones tested, simultaneously. The prediction of acquired resistant genes using the whole genome sequences revealed the co-existence of blaNDM-1 and mcr-1 as well as the other genes that were responsible for the multidrug-resistant phenotypes. Bioinformatics analysis also showed that the carbapenem-resistant gene blaNDM-1 was located on a putative IncFII-type plasmid, which also carried the other acquired resistant genes identified, including fosA3, blaTEM-1B and rmtB, while the colistin-resistant gene mcr-1 was carried by a putative IncX4-type plasmid. Finally, we found that these resistant genes/plasmids were conjugative, and they could be co-conjugated, conferring resistance to multiple types of antibiotics, including the carbapenems and colistin, to the recipient Escherichia coli strains.

31 citations

Journal ArticleDOI
TL;DR: The use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions, and threshold parameters in W GS methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics.
Abstract: Whole-genome sequencing (WGS)-based typing methods have emerged as promising and highly discriminative epidemiological tools. In this study, we combined gene-by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) approaches to investigate the genetic relatedness of a well-characterized collection of OXA-48-producing Klebsiella pneumoniae isolates. We included isolates from the predominant sequence type ST405 (n = 31) OXA-48-producing K. pneumoniae clone and isolates from ST101 (n = 3), ST14 (n = 1), ST17 (n = 1), and ST1233 (n = 1), obtained from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) schemes from Institut Pasteur's BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) were used. Allele differences or allelic mismatches and the genetic distance, as the proportion of allele differences, were used to interpret the results from a gene-by-gene approach, whereas the number of SNPs was used for the cgSNP analysis. We observed between 0-10 and 0-14 allele differences among the predominant ST405 using cgMLST and wgMLST from SeqSphere+, respectively, and <2 allelic mismatches when using Institut Pasteur's BIGSdb-Kp cgMLST scheme. For ST101, we observed 14 and 54 allele differences when using cgMLST and wgMLST SeqSphere+, respectively, and 2-5 allelic mismatches for BIGSdb-Kp cgMLST. A low genetic distance (<0.0035, a previously established threshold for epidemiological link) was generally in concordance with a low number of allele differences (<8) when using the SeqSphere+ cgMLST scheme. The cgSNP analysis showed 6-29 SNPs in isolates with identical allelic SeqSphere+ cgMLST profiles and 16-61 cgSNPs among ST405 isolates. Furthermore, comparison of WGS-based typing results with previously obtained MLST and pulsed-field gel electrophoresis (PFGE) data showed some differences, demonstrating the different molecular principles underlying these techniques. In conclusion, the use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions. Furthermore, threshold parameters in WGS-based typing methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics.

31 citations

Journal ArticleDOI
TL;DR: The role of entry exclusion in the apparent incompatibility between IncA and IncC plasmids is unraveled while shedding light on the importance of the TraG subunit substitution used by SGI1 to evade entry exclusion.
Abstract: Conjugative plasmids of incompatibility group C (IncC), formerly known as A/C 2 , disseminate antibiotic resistance genes globally in diverse pathogenic species of Gammaproteobacteria. Salmonella genomic island 1 (SGI1) can be mobilized by IncC plasmids and was recently shown to reshape the conjugative type IV secretion system (T4SS) encoded by these plasmids to evade entry exclusion. Entry exclusion blocks DNA translocation between cells containing identical or highly similar plasmids. Here, we report that the protein encoded by the entry exclusion gene of IncC plasmids ( eexC ) mediates entry exclusion in recipient cells through recognition of the IncC-encoded TraG C protein in donor cells. Phylogenetic analyses based on EexC and TraG C homologs predicted the existence of at least three different exclusion groups among IncC-related conjugative plasmids. Mating assays using Eex proteins encoded by representative IncC and IncA (former A/C 1 ) and related untyped plasmids confirmed these predictions and showed that the IncC and IncA plasmids belong to the C exclusion group, thereby explaining their apparent incompatibility despite their compatible replicons. Representatives of the two other exclusion groups (D and E) are untyped conjugative plasmids found in Aeromonas sp. Finally, we determined through domain swapping that the carboxyl terminus of the EexC and EexE proteins controls the specificity of these exclusion groups. Together, these results unravel the role of entry exclusion in the apparent incompatibility between IncA and IncC plasmids while shedding light on the importance of the TraG subunit substitution used by SGI1 to evade entry exclusion. IMPORTANCE IncA and IncC conjugative plasmids drive antibiotic resistance dissemination among several pathogenic species of Gammaproteobacteria due to the diversity of drug resistance genes that they carry and their ability to mobilize antibiotic resistance-conferring genomic islands such as SGI1 of Salmonella enterica. While historically grouped as “IncA/C,” IncA and IncC replicons were recently confirmed to be compatible and to abolish each other’s entry into the cell in which they reside during conjugative transfer. The significance of our study is in identifying an entry exclusion system that is shared by IncA and IncC plasmids. It impedes DNA transfer to recipient cells bearing a plasmid of either incompatibility group. The entry exclusion protein of this system is unrelated to any other known entry exclusion proteins.

31 citations

Journal ArticleDOI
01 Sep 2019
TL;DR: This work characterized two highly related ST131-H22 strains, one from a healthy pig and the other from a human infection, which were closely related to a faecal strain isolated in 2010 from a geographically distinct, healthy human in New South Wales, Australia.
Abstract: The interplay between food production animals, humans and the environment with respect to the transmission of drug-resistant pathogens is widely debated and poorly understood. Pandemic uropathogenic Escherichia coli ST131-H30Rx, with conserved fluoroquinolone and cephalosporin resistance, are not frequently identified in animals. However, the phylogenetic precursor lineage ST131-H22 in animals and associated meat products is being reported with increasing frequency. Here we characterized two highly related ST131-H22 strains, one from a healthy pig and the other from a human infection (in 2007 and 2009, respectively). We used both long and short genome sequencing and compared them to ST131-H22 genome sequences available in public repositories. Even within the context of H22 strains, the two strains in question were highly related, separated by only 20 core SNPs. Furthermore, they were closely related to a faecal strain isolated in 2010 from a geographically distinct, healthy human in New South Wales, Australia. The porcine and hospital strains carried highly similar HI2-ST3 multidrug resistant plasmids with differences in the hospital strain arising due to IS-mediated insertions and rearrangements. Near identical ColV plasmids were also present in both strains, further supporting their shared evolutionary history. This work highlights the importance of adopting a One Health approach to genomic surveillance to gain insights into pathogen evolution and spread.

31 citations

References
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Journal ArticleDOI
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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Journal ArticleDOI
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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Journal ArticleDOI
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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Journal ArticleDOI
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

Journal ArticleDOI
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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