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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: A holistic model for the action of the nph gene cluster is proposed which incorporates genetic architecture, uptake and metabolism of aromatic compounds, enzymatic activities and transcriptional regulation, and provides testable hypotheses for further biochemical investigations into the genes ofThe nph cluster, for potential exploitation in bioremediation.
Abstract: Rhodococcus sp.strain BUPNP1 can utilize the priority environmental pollutant 4-nitrophenol (4-NP) as its sole source of carbon and energy. In this study, genome and transcriptome sequencing were used to gain mechanistic insights into 4-NP degradation. The draft BUPNP1 genome is 5.56 Mbp and encodes 4,963 proteins, which are significantly enriched in hypothetical proteins compared to other Rhodococcus sp. A novel 4-NP catabolic 43 gene cluster "nph" was identified that encodes all the genes required for the conversion of 4-NP into acetyl-CoA and succinate, via 4-nitrocatechol. The cluster also encodes pathways for the catabolism of other diverse aromatic compounds. Comparisons between BUPN1 growing on either 4-NP or glucose resulted in significant changes in the expression of many nph cluster genes, and, during 4-NP growth, a loss of lipid inclusions. Moreover, fatty acid degradation/synthesis genes were found within the nph cluster, suggesting fatty acids may be concurrently catabolised with 4-NP. A holistic model for the action of the nph gene cluster is proposed which incorporates genetic architecture, uptake and metabolism of aromatic compounds, enzymatic activities and transcriptional regulation. The model provides testable hypotheses for further biochemical investigations into the genes of the nph cluster, for potential exploitation in bioremediation.

18 citations


Cites methods from "In Silico Detection and Typing of P..."

  • ...The presence of plasmids was tested using PlasmidFinder (Carattoli et al., 2014), family membership of regulatory proteins was obtained using P2RP (Barakat et al., 2013), while clusters of orthologous groups (COG) analysis was performed by WebMGA (Wu et al., 2011)....

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Journal ArticleDOI
TL;DR: The whole genome sequence analysis provides insight into the resistome and the discovery of new details, such as the PKS cluster, in C. indologenes MARS15, an emerging multidrug-resistant clinical strain, using the whole genome sequencing strategy.
Abstract: We decipher the resistome of Chryseobacterium indologenes MARS15, an emerging multidrug-resistant clinical strain, using the whole genome sequencing strategy. The bacterium was isolated from the sputum of a hospitalized patient with cystic fibrosis in the Timone Hospital in Marseille, France. Genome sequencing was done with Illumina MiSeq using a paired-end strategy. The in silico analysis was done by RAST, the resistome by the ARG-ANNOT database and detection of polyketide synthase (PKS) by ANTISMAH. The genome size of C. indologenes MARS15 is 4 972 580 bp with 36.4% GC content. This multidrug-resistant bacterium was resistant to all β-lactams, including imipenem, and also to colistin. The resistome of C. indologenes MARS15 includes Ambler class A and B β-lactams encoding bla CIA and bla IND-2 genes and MBL (metallo-β-lactamase) genes, the CAT (chloramphenicol acetyltransferase) gene and the multidrug efflux pump AcrB. Specific features include the presence of an urease operon, an intact prophage and a carotenoid biosynthesis pathway. Interestingly, we report for the first time in C. indologenes a PKS cluster that might be responsible for secondary metabolite biosynthesis, similar to erythromycin. The whole genome sequence analysis provides insight into the resistome and the discovery of new details, such as the PKS cluster.

18 citations


Cites methods from "In Silico Detection and Typing of P..."

  • ...Detection of plasmid was performed by PlasmidFinder [23]....

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Journal ArticleDOI
TL;DR: In this article, the authors determined the prevalence and genetic characteristics of ESBL-producing Escherichia coli in retail raw meats from Singapore markets and confirmed ESBL isolates using the double-disc synergy test.
Abstract: Objectives To determine the prevalence and genetic characteristics of ESBL-producing Escherichia coli in retail raw meats from Singapore markets. Methods A total of 634 raw meat (chicken, pork and beef) samples were collected from markets in Singapore during June 2017-October 2018. The samples were enriched overnight and then incubated on Brilliance™ ESBL Agar. Presumptive ESBL isolates were confirmed using the double-disc synergy test. Confirmed ESBL-producing E. coli were sent for WGS and bioinformatic analysis was performed. Results The prevalence of ESBL-producing E. coli in chicken, pork and beef meats was 51.2% (109/213), 26.9% (58/216) and 7.3% (15/205), respectively. A total of 225 ESBL-producing E. coli were isolated from 184 samples. β-Lactam resistance genes were detected in all isolates. After β-lactam resistance genes, the most common antimicrobial resistance genes detected were aminoglycoside resistance genes (92.4%). One hundred and seventy-two (76.4%), 102 (45.3%) and 52 (23.1%) isolates carried blaCTX-M genes, blaTEM genes and blaSHV genes, respectively. blaCTX-M-55 (57/225, 25.3%) and blaCTX-M-65 (40/225, 17.8%) were the most frequent ESBL genes. Colistin resistance genes (including mcr-1, mcr-3 and mcr-5) were found in 15.6% of all isolates. Conclusions This study indicates that ESBL-producing E. coli are widely found in retail raw meats, especially chicken, in Singapore. Occurrence of MDR (resistance to at least three classes of antimicrobial) and colistin resistance genes in retail raw meat suggests potential food safety and public health risks.

18 citations

Journal ArticleDOI
09 Oct 2020-PLOS ONE
TL;DR: A comprehensive molecular characterization of putative CR-EC strains from Oman shows polyclonal population structure with OXA-48 and NDM as the only carbapenemases in CR- EC from Oman.
Abstract: The prevalence of carbapenem-resistant Enterobacterales (CRE) in the Arabian Peninsula is predicted to be high, as suggested from published case reports. Of particular concern, is carbapenem-resistant E. coli (CR-EC), due to the importance of this species as a community pathogen. Herein, we conducted a comprehensive molecular characterization of putative CR-EC strains from Oman. We aim to establish a baseline for future molecular monitoring. We performed whole-genome sequencing (WGS) for 35 putative CR-EC. Isolates were obtained from patients at multiple centers in 2015. Genetic relatedness was investigated using several typing approaches such as MLST, SNP calling, phylogroup and CRISPR typing. Maxiuium likelihood SNP-tree was performed by RAxML after variant calling and removal of recombination regions with Snippy and Gubbins, respectively. Resistance genes, plasmid replicon types, virulence genes, and prophage were also characterised. The online databases CGE, CRISPRcasFinder, Phaster and EnteroBase were used for the in silico analyses. Screening for mutations in genes regulating the expression of porins and efflux pump as well as mutations lead to fluoroquinolones resistance were performed with CLC Genomics Workbench. The genetic diversity suggests a polyclonal population structure with 21 sequence types (ST), of which ST38 being the most prevalent (11%). SNPs analysis revealed possible transmission episodes. Whereas, CRISPR typing helped to spot outlier strains belonged to phylogroups other than B2 which was CRISPR-free. The virulent phylogroups B2 and D were detected in 4 and 9 isolates, respectively. In some strains bacteriophages acted as vectors for virulence genes. Regarding resistance to β-lactam, 22 were carbapenemase producers, 3 carbapenem non-susceptible but carbapenemase-negative, 9 resistant to expanded-spectrum cephalosporins, and one isolate with susceptibility to cephalosporins and carbapenems. Thirteen out of the 22 (59%) carbapenemase-producing isolates were NDM and 7 (23%) were OXA-48-like which mirrors the situation in Indian subcontinent. Two isolates co-produced NDM and OXA-48-like enzymes. In total, 80% (28/35) were CTX-M-15 producers and 23% (8/35) featured AmpC. The high-risk subclones ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC were detected, the latter associated with NDM. To our knowledge, this is the first report of ST1193-H64Rx subclone with NDM. In conclusion, strains showed polyclonal population structure with OXA-48 and NDM as the only carbapenemases in CR-EC from Oman. We detected the high-risk subclone ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC. The latter was reported with carbapenemase gene for the first time here.

18 citations

Journal ArticleDOI
TL;DR: These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE, a cohort of ESBL-E bacteraemia cases in Houston, TX, USA.
Abstract: Background Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. Objectives To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. Methods This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively. Results Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. Conclusions These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

18 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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