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 first draft genome sequence of a Salmonella enterica serovar Typhi clinical isolate from Pakistan exhibiting resistance to cefepime (a fourth-generation cephalosporin) and fluoroquinolone antibiotics, two of the last-generation therapies against this pathogen.
Abstract: Typhoid is endemic in developing countries. We report here the first draft genome sequence of a Salmonella enterica serovar Typhi clinical isolate from Pakistan exhibiting resistance to cefepime (a fourth-generation cephalosporin) and fluoroquinolone antibiotics, two of the last-generation therapies against this pathogen. The genome is ~4.8 Mb, with two putative plasmids.
11 citations
Cites background from "In Silico Detection and Typing of P..."
...dk/services/PlasmidFinder/) (9) identified Received 31 July 2017 Accepted 18 September 2017 Published 19 October 2017...
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TL;DR: The antimicrobial susceptibility phenotypes and genomic features of two hypermucoviscous K. pneumoniae Kp isolates recovered from RTx recipients with asymptomatic bacteriuria are described.
Abstract: Klebsiella pneumoniae (Kp) is one of the most important etiological factors of urinary tract infections in renal transplant (RTx) recipients. We described the antimicrobial susceptibility phenotypes and genomic features of two hypermucoviscous (HM) Kp isolates recovered from RTx recipients with asymptomatic bacteriuria (ABU). Using whole genome sequencing (WGS) data, we showed that the strains belong to the ST152 lineage with the KL149 capsular serotype, but without rmpA/magA genes, which is typical for HM+ hypervirulent Kp. These new strains carried virulence-associated genes that predispose for urinary tract infections (UTIs). Likewise, both strains carried the ecp gene encoding pilus common for extended-spectrum β-lactamase (ESBL) Escherichia coli. Although the two ST152 isolates were closely related and differed by only nine single nucleotide polymorphisms (SNPs) in their chromosomes, they had different plasmid compositions and chromosomal elements, with isolate KP28872 carrying an ESBL plasmid and an integrative conjugative element. These two isolates are an example of the high plasticity of the K. pneumoniae accessory genome. The identification of patients with ABU matched with the correct epidemiological profiling of isolates could facilitate interventions to prevent or rapidly treat K. pneumoniae infections.
11 citations
TL;DR: There was a clear correlation between CTX-R and CIP-R, largely attributable to the dominance of the high-risk pandemic clones, ST131 and ST1193 in this study.
Abstract: Objectives Third-generation cephalosporin-resistant Escherichia coli from community-acquired urinary tract infections (UTI) have been increasingly reported worldwide. In this study we sought to determine and characterise the mechanisms of cefotaxime-resistance (CTX-R) employed by urinary E. coli obtained from primary care over a 12-month period, in Bristol and surrounding counties in the South West of England. Methods Cephalexin resistant (Ceph-R) E. coli isolates were identified directly from general practice (GP) referred urine samples using disc susceptibility testing as per standard diagnostic procedures. CTX-R was determined by subsequent plating onto MIC breakpoint plates. β-Lactamase genes were detected by PCR. Whole Genome Sequencing (WGS) was performed on 225 urinary isolates and analyses were performed using the Centre for Genomic Epidemiology platform. Patient information provided by the referring GPs was reviewed. Results During the study period, Ceph-R E. coli (n=900) were obtained directly from urines from 146 GPs. Seventy-percent (626/900) of isolates were CTX-R. WGS of 225 non-duplicate isolates identified that the most common mechanism of CTX-R was blaCTX-M carriage (185/225; 82.2%), predominantly blaCTX-M-15 (114/185; 61.6%), followed by carriage of plasmid mediated AmpCs (pAmpCs) (17/225; 7.6%), ESBL blaSHV variants (6/225; 2.7%), AmpC hyperproduction (13/225; 5.8%), or a combination of both blaCTX-M and pAmpC carriage (4/225; 1.8%). Forty-four sequence types (STs) were identified with ST131 representing 101/225 (45.0%) of sequenced isolates, within which the blaCTX-M-15-positive clade C2 was dominant (54/101; 53.5%). Ciprofloxacin-resistance (CIP-R) was observed in 128/225 (56.9%) of sequenced CTX-R isolates – predominantly associated with fluoroquinolone-resistant clones ST131 and ST1193. Conclusions Most Ceph-R urinary E. colis were CTX-R, predominantly caused by blaCTX-M carriage. There was a clear correlation between CTX-R and CIP-R, largely attributable to the dominance of the high-risk pandemic clones, ST131 and ST1193 in this study. This localised epidemiological data provides greater resolution than regional data and can be valuable for informing treatment choices in the primary care setting.
11 citations
TL;DR: Both transposons and vertical transmission to contribute to the transformation of blaKPC-2 are suggested, which strongly suggest strict implementation of infection control of CRKA, in healthcare facilities.
Abstract: Purpose This study aimed to evaluate the molecular characteristics and prevalence of clinical carbapenem-resistant Klebsiella aerogenes (CRKA), collected during an outbreak in a Chinese tertiary hospital. Methods Antimicrobial susceptibility test, using 17 antibiotics, was performed on 14 CRKA isolates. The strains were examined for the presence of β-lactamase genes by PCR, and efflux pump phenotype was determined by efflux pump inhibition test. Presence of outer-membrane porins was examined. Clonal relatedness among the isolates was investigated by pulsed-field gel electrophoresis (PFGE). S1 nuclease-PFGE and plasmid incompatibility group analysis were performed to determine plasmids, and the genetic environment of bla KPC-2 was analyzed. Epidemiological data were collected via chart review. Results The 14 CRKA isolates were all resistant to carbapenems; five distinct groups (PFGE types A-E) were observed. All 14 isolates carried the bla KPC-2 gene. S1 nuclease-PFGE indicated the size of bla KPC-2-carrying plasmids to range from 20 kb to 200 kb, and the 14 plasmids belonged to various incompatibility groups. The most frequent genetic environment of bla KPC-2 was Tn1721- bla KPC-2-ΔTn3-IS26. PFGE type A group, including 11 KPC-2-producing clinical isolates, was primarily responsible for dissemination. Conclusion Our findings suggest both transposons and vertical transmission to contribute to the transformation of bla KPC-2. The results strongly suggest strict implementation of infection control of CRKA, in healthcare facilities.
11 citations
Cites methods from "In Silico Detection and Typing of P..."
...by PlasmidFinder, and plasmid typing was performed using pMSI software.(24)...
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TL;DR: Enterotoxin C and enterotoxin-like L carrying S. aureus are associated with bovine mastitis and these may also be important virulence factors for human mastitis, according to whole-genome sequencing findings.
Abstract: Denmark is a low prevalence country with regard to methicillin resistant Staphylococcus aureus (MRSA). In 2008 and 2014, two neonatal wards in the Copenhagen area experienced outbreaks with a typical community acquired MRSA belonging to the same spa type and sequence type (t015:ST45) and both were PVL and ACME negative. In outbreak 1, the isolates harbored SCCmec IVa and in outbreak 2 SCCmec V. The clinical presentation differed between the two outbreaks, as none of five MRSA positive mothers in outbreak 1 had mastitis vs. five of six MRSA positive mothers in outbreak 2 (p < 0.02). To investigate if whole-genome sequencing could identify virulence genes associated with mastitis, t015:ST45 isolates from Denmark (N = 101) were whole-genome sequenced. Sequence analysis confirmed two separate outbreaks with no sign of sustained spread into the community. Analysis of the accessory genome between isolates from the two outbreaks revealed a S. aureus pathogenicity island containing enterotoxin C and enterotoxin-like L only in isolates from outbreak 2. Enterotoxin C and enterotoxin-like L carrying S. aureus are associated with bovine mastitis and our findings indicate that these may also be important virulence factors for human mastitis.
11 citations
Cites methods from "In Silico Detection and Typing of P..."
...Virulence and resistance genes and plasmid replicons were identified with the online tools VirulenceFinder (Joensen et al., 2014), ResFinder (Zankari et al., 2012), and PlasmidFinder (Carattoli et al., 2014), respectively, available from the homepage http://genomicepidemiology.org/....
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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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