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: An analysis of 39 whole-genome sequences comprising three Comamonas species from aquatic environments in South Australia that were recovered on media supplemented with carbapenems makes several key observations, including the identification of a subset of C. denitrificans isolates that harbored genomic islands encodingcarbapenemase blaGES-5 or extended-spectrum β-lactamases blaOXA alleles.
Abstract: Antimicrobial resistance (AMR) poses a global public health threat, and the increase in resistance to “last-resort drugs,” such as carbapenems, is alarming. Wastewater has been flagged as a hot spot for AMR evolution. Comamonas spp. are among the most common bacteria in wastewater and play a role in its bioaugmentation. ABSTRACT Comamonas spp. are Gram-negative bacteria that catabolize a wide range of organic and inorganic substrates. Comamonas spp. are abundant in aquatic and soil environments, including wastewater, and can cause opportunistic infections in humans. Because of their potential in wastewater bioaugmentation and bioremediation strategies, the identification of Comamonas species harboring genes encoding carbapenemases and other clinically important antibiotic resistance genes warrant further investigation. Here, we present an analysis of 39 whole-genome sequences comprising three Comamonas species from aquatic environments in South Australia that were recovered on media supplemented with carbapenems. The analysis includes a detailed description of 33 Comamonas denitrificans isolates, some of which carried chromosomally acquired blaGES-5, blaOXA, and aminoglycoside resistance (aadA) genes located on putative genomic islands (GIs). All blaGES-5- and blaOXA-containing GIs appear to be unique to this Australian collection of C. denitrificans. Notably, most open reading frames (ORFs) within the GIs, including all antimicrobial resistance (AMR) genes, had adjacent attC sites, indicating that these ORFs are mobile gene cassettes. One C. denitrificans isolate carried an IncP-1 plasmid with genes involved in xenobiotic degradation and response to oxidative stress. Our assessment of the sequences highlights the very distant nature of C. denitrificans to the other Comamonas species and its apparent disposition to acquire antimicrobial resistance genes on putative genomic islands. IMPORTANCE Antimicrobial resistance (AMR) poses a global public health threat, and the increase in resistance to “last-resort drugs,” such as carbapenems, is alarming. Wastewater has been flagged as a hot spot for AMR evolution. Comamonas spp. are among the most common bacteria in wastewater and play a role in its bioaugmentation. While the ability of Comamonas species to catabolize a wide range of organic and inorganic substrates is well documented, some species are also opportunistic pathogens. However, data regarding AMR in Comamonas spp. are limited. Here, through the genomic analyses of 39 carbapenem-resistant Comamonas isolates, we make several key observations, including the identification of a subset of C. denitrificans isolates that harbored genomic islands encoding carbapenemase blaGES-5 or extended-spectrum β-lactamase blaOXA alleles. Given the importance of Comamonas species in potential wastewater bioaugmentation and bioremediation strategies, as well as their status as emerging pathogens, the acquisition of critically important antibiotic resistance genes on genomic islands warrants future monitoring.
6 citations
TL;DR: Although some acquired genes were lost over time, the retention of 12 resistance genes and inactivation of mgrB provided resistance to 13 classes of antibiotics, the first description of K. pneumoniae ST14 resistant to both carbapenem and colistin in South Korea is described.
6 citations
TL;DR: The findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China, and suggests that multiple mobilization events might have occurred in South America.
Abstract: OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.
6 citations
Cites result from "In Silico Detection and Typing of P..."
...In-silico analysis showed that both pKP64477a and pKP64216a belonged to IncFIIk group of incompatibility group, while pPM64421 was a non-typable plasmid according to PlasmidFinder analysis.(19) pKP64477a and pKP26216a showed a distinct backbone, when compared with pPM464421a, except for the region in which rmtB-1 was inserted....
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TL;DR: The dynamic nature of Type I-F CRISPR in E. coli of phylogenetic group B2 is presented and new insights into the application ofCRISPR-based typing in the species are provided.
6 citations
TL;DR: W Whole genome sequencing of Slovenian carbapenem-resistant K. pneumoniae isolates revealed multiple clusters of STs, two of which were involved in the first hospital outbreak of carbAPenem producing K. tuberculosis in Slovenia, highlighting the importance of whole genome sequencing in detection and/or characterization of hospital outbreaks and surveillance of drug-resistant bacterial clones.
Abstract: Objectives The first hospital outbreak of carbapenemase-producing Enterobacteriaceae in Slovenia occurred in 2014–2016. Whole genome sequencing was used to analyse the population of carbapenem-resistant Klebsiella pneumoniae collected in Slovenia in 2014–2017, including OXA-48 and/or NDM-1 producing strains from the outbreak. Methods A total of 32 K. pneumoniae isolates were analysed using short-read sequencing. Multi-locus sequence typing and core genome multi-locus sequence typing were used to infer genetic relatedness. Antimicrobial resistance markers, virulence factors, plasmid content and wzi types were determined. Long-read sequencing was used for six isolates for detailed analysis of plasmids and their possible transmission. Results Overall, we detected 10 different sequence types (STs), the most common being ST437 (40.6%). Isolates from the initial outbreak belonged to ST437 (12/16) and ST147 (4/16). A second outbreak of four ST15 isolates was discovered. A new ST (ST3390) and two new wzi types (wzi-556, wzi-559) were identified. blaOXA-48 was found in 17 (53.1%) isolates, blaNDM-1 in five (15.6%), and a combination of blaOXA-48/NDM-1 in seven (21.9%) isolates. Identical plasmids carrying blaOXA-48 were found in outbreak isolates sequenced with long-read sequencing technology. Conclusions Whole genome sequencing of Slovenian carbapenem-resistant K. pneumoniae isolates revealed multiple clusters of STs, two of which were involved in the first hospital outbreak of carbapenem producing K. pneumoniae in Slovenia. Transmission of the plasmid carrying blaOXA-48 between two STs was likely to have occurred. A previously unidentified second outbreak was also discovered, highlighting the importance of whole genome sequencing in detection and/or characterization of hospital outbreaks and surveillance of drug-resistant bacterial clones.
6 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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