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.
Citations
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TL;DR: W Whole genome sequencing of the Aeromonas salmonicida strain revealed 13 antibiotic resistance genes, including the ESBL CTX-M-3 and the unusual β-lactamase SCO-1.
Abstract: We report the selection in a 15-year-old boy of a multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Aeromonas salmonicida after medicinal leech therapy that required an antibiotic prophylaxis based on piperacillin/tazobactam and cotrimoxazole. Whole genome sequencing of the strain indeed revealed 13 antibiotic resistance genes, including the ESBL CTX-M-3 and the unusual β-lactamase SCO-1.
17 citations
Cites methods from "In Silico Detection and Typing of P..."
...The search for plasmid replicons using PlasmidFinder [10] returned negative for contigs obtained with both IDBA-UD and plasmid-SPAdes....
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TL;DR: How chromosome conformation capture and methylome analyses have the potential to simultaneously study various types of mobile elements and their associated hosts and how they can be used to address outstanding questions in the field of horizontal gene transfer in microbial communities are discussed.
Abstract: Horizontal gene transfer is an important mechanism of microbial evolution and is often driven by the movement of mobile genetic elements between cells. Due to the fact that microbes live within communities, various mechanisms of horizontal gene transfer and types of mobile elements can co-occur. However, the ways in which horizontal gene transfer impacts and is impacted by communities containing diverse mobile elements has been challenging to address. Thus, the field would benefit from incorporating community-level information and novel approaches alongside existing methods. Emerging technologies for tracking mobile elements and assigning them to host organisms provide promise for understanding the web of potential DNA transfers in diverse microbial communities more comprehensively. Compared to existing experimental approaches, chromosome conformation capture and methylome analyses have the potential to simultaneously study various types of mobile elements and their associated hosts. We also briefly discuss how fermented food microbiomes, given their experimental tractability and moderate species complexity, make ideal models to which to apply the techniques discussed herein and how they can be used to address outstanding questions in the field of horizontal gene transfer in microbial communities.
17 citations
07 Nov 2018
TL;DR: No single gene accounts for the C′ resistance of the isolates sequenced in this study, which means that no single OM constituent can account for resistance, which is likely to depend on biophysical properties of the target bilayer.
Abstract: The capacity to resist the bactericidal action of complement (C') is a strong but poorly understood virulence trait in Klebsiella spp. Killing requires activation of one or more C' pathways, assembly of C5b-9 membrane attack complexes (MACs) on the surface of the outer membrane (OM), and penetration of MACs into the target bilayer. We interrogated whole-genome sequences of 164 Klebsiella isolates from three tertiary hospitals in Thailand for genes encoding surface-located macromolecules considered to play a role in determination of C' resistance. Most isolates (154/164) were identified as Klebsiella pneumoniae, and the collection conformed to previously established population structures and antibiotic resistance patterns. The distribution of sequence types (STs) and capsular (K) types were also typical of global populations. The majority (64%) of isolates were resistant to C', and the remainder were either rapidly or slowly killed. All isolates carried genes encoding capsular polysaccharides (K antigens), which have been strongly linked to C' resistance. In contrast to previous reports, there were no differences in the amount of capsule produced by C'-resistant isolates compared to C'-susceptible isolates, nor was there any correlation between serum reactivity and the presence of hypermucoviscous capsules. Similarly, there were no correlations between the presence of genes specifying lipopolysaccharide O-side chains or major OM proteins. Some virulence factors were found more frequently in C'-resistant isolates but were considered to reflect clonal ST expansion. Thus, no single gene accounts for the C' resistance of the isolates sequenced in this study.IMPORTANCE Multidrug-resistant Klebsiella pneumoniae is responsible for an increasing proportion of nosocomial infections, and emerging hypervirulent K. pneumoniae clones now cause severe community-acquired infections in otherwise healthy individuals. These bacteria are adept at circumventing immune defenses, and most survive and grow in serum; their capacity to avoid C'-mediated destruction is correlated with their invasive potential. Killing of Gram-negative bacteria occurs following activation of the C' cascades and stable deposition of C5b-9 MACs onto the OM. For Klebsiella, studies with mutants and conjugants have invoked capsules, lipopolysaccharide O-side chains, and OM proteins as determinants of C' resistance, although the precise roles of the macromolecules are unclear. In this study, we sequenced 164 Klebsiella isolates with different C' susceptibilities to identify genes involved in resistance. We conclude that no single OM constituent can account for resistance, which is likely to depend on biophysical properties of the target bilayer.
17 citations
Cites methods from "In Silico Detection and Typing of P..."
...Plasmids were detected with the PLasmidFinder database (87) using Ariba software....
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TL;DR: In this article, a 40-year-old female with Pandrug-resistant K. pneumoniae infection of cardiovascular implantable electronic device (CIED) and right-sided infective endocarditis was treated with synergistic combinations of aztreonam with ǫ-ceftazidime-avibactam.
Abstract: Pandrug-resistant (PDR) K. pneumoniae refractory to conventional treatment has been reported worldwide, causing a huge burden on the healthcare system, patient safety and the economy. K. pneumoniae is a prominent opportunistic pathogen causing hospital-acquired and community-acquired infections, but is rarely associated with infective endocarditis. Currently, there are sparse data guiding the optimal regimen when commonly used antibiotics fail, notably for the treatment of endocarditis infections. Here we report our experience in treating a 40-year-old female with PDR K. pneumoniae infection of cardiovascular implantable electronic device (CIED) and right-sided infective endocarditis. Initial susceptibility testing of the incriminated pathogen showed an apparent susceptibility to colistin but the prolonged course of colistin, gentamicin and meropenem did not resolve the infection. However, the synergistic combinations of aztreonam with ceftazidime-avibactam was able to overcome resistance and clear the infection rapidly. Genome sequencing showed that the PDR K. pneumoniae isolate belongs to the international high-risk clone ST14. The isolate harbored genes encoding NDM-1, OXA-48, CTX-M-14b, SHV-28 and OXA-1, explaining resistance to all β-lactams, including carbapenems. It carried the armA gene conferring resistance to all clinically important aminoglycosides and had alterations in GyrA, ParC and MgrB, explaining resistance to ciprofloxacin and colistin.
17 citations
TL;DR: This work has identified a sequence type 131 strain harboring blaIMP-14 within a class 1 integron, itself nested within an ∼54-kb multidrug resistance region on an epidemic IncA/C2 plasmid.
Abstract: The blaIMP-14 carbapenem resistance gene has largely previously been observed in Pseudomonas aeruginosa and Acinetobacter spp. As part of global surveillance and sequencing of carbapenem-resistant Escherichia coli, we identified a sequence type 131 strain harboring blaIMP-14 within a class 1 integron, itself nested within an ∼54-kb multidrug resistance region on an epidemic IncA/C2 plasmid. The emergence of blaIMP-14 in this context in the ST131 lineage is of potential clinical concern.
17 citations
Cites background from "In Silico Detection and Typing of P..."
...In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing....
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...ence to the ResFinder (17), PlasmidFinder (18), and ISFinder...
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...Additional annotation focused on resistance genes and insertion sequences was performed with reference to the ResFinder (17), PlasmidFinder (18), and ISFinder (19) databases....
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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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