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: In this article, the authors characterized ESBL/pAmpC-producing E. coli collected from food samples and found that the blaCTX-M-1 gene and its flanking sequences were located within the shufflon of the type IV pilus region in diverse orientations.
44 citations
TL;DR: Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids.
Abstract: Objectives: This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective blaSHV-12-carrying plasmids and sequencing one of these plasmids completely. Methods: Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed blaSHV-12-carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the blaSHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing. Results: Among the 23 SHV-12-positive E. coli, some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All blaSHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 (n=17), IncK (n=3), IncF (n=1), IncX3 (n=1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1-blaSHV-12-carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to β-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX-psp-aadA2-cmlA1-aadA1-qacI gene cassette array), and to tetracycline. A novel blaSHV-12 environment was detected and whole plasmid sequencing revealed a Tn21-derived-blaSHV12-ΔTn1721 resistance complex. Conclusions: Results from this study suggest that the dissemination of blaSHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
44 citations
TL;DR: In this paper, genomic and transcriptomic data of 4 Pseudomonas fluorescens strains (2 blue-pigmenting and 2 non-P. fluorescence) were analyzed to evaluate the presence and the expression of blue strain-specific genes.
44 citations
Cites methods from "In Silico Detection and Typing of P..."
...G-C contentwas obtained using Artemis release 16.0.0 (Carver et al., 2012); the presence of plasmids was investigated using Plasmid finder 1.2 (Carattoli et al., 2014)....
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TL;DR: CompASS, a novel and comprehensive database compiling 12,084 complete plasmids with associated metadata from 1571 distinct species isolated worldwide over more than 100 years, provides a comprehensive picture of the different IncF subtypes by identifying 20 different profiles in 849 IncF multireplicons, which were mostly associated with Enterobacteriaceae.
Abstract: Plasmids are genetic elements that enable rapid adaptation and evolution by transferring genes conferring selective advantages to their hosts. Conjugative plasmids are predominantly responsible for the global dissemination of antimicrobial resistance, representing an important threat to global health. As the number of plasmid sequences grows exponentially, it becomes critical to depict the global diversity and decipher the distribution of circulating plasmids in the bacterial community. To this end, we created COMPASS, a novel and comprehensive database compiling 12,084 complete plasmids with associated metadata from 1571 distinct species isolated worldwide over more than 100 years. The curation of the database allowed us to identify identical plasmids across different bacteria revealing mainly intraspecies dissemination and rare cases of horizontal transmission. We outlined and analyzed all relevant features, plasmid properties, host range and characterized their replication and mobilization systems. After an exhaustive comparison of PlasmidFinder and MOB-typer, the MOB-typer-based analysis revealed that the current knowledge embedded in the current typing schemes fails to classify all the plasmid sequences collected in COMPASS. We were able to categorize 6828 and 5229 plasmids by replicon and MOB typing, respectively, mostly associated with Proteobacteria and Firmicutes. We then searched for the presence of multiple core genes involved in replication and propagation. Our results showed that 2403 plasmids carried multiple replicons that were distributed in 206 bacterial species. The co-integration of replicon types from different incompatibility (Inc) groups is an adaptive mechanism, which plays an important role in plasmid survival and dissemination by extending their host range. Our results highlight the crucial role of IncF alleles (present in 56% of all multireplicons) and revealed that IncH, IncR, and IncU replicons were also frequently carried in multireplicons. Here, we provided a comprehensive picture of the different IncF subtypes by identifying 20 different profiles in 849 IncF multireplicons, which were mostly associated with Enterobacteriaceae. These results could provide the basis for a novel IncF plasmid nomenclature based on different allelic profiles.
44 citations
Cites background or methods from "In Silico Detection and Typing of P..."
...Replicon typing was also performed using the conventional in silico tool PlasmidFinder (version October/2019)3 with recommended parameters (Carattoli et al., 2014) and the results were compared to those obtained with MOB-typer....
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...The term “replicon” can refer to any DNA sequence that is capable of replicating as a unit such as a plasmid or can refer more precisely, like in this study to specific regions or genes encoding functions that enable the replication (Carattoli et al., 2014)....
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TL;DR: A high diversity of lineages associated with AMR, including ESBL and fluoroquinolone resistance, in Malawi is revealed, highlighting the value of longitudinal bacteraemia surveillance coupled with detailed molecular epidemiology in all settings, including low-income settings, in describing the global epidemiology of ESBL resistance.
Abstract: Objectives: Efforts to treat Escherichia coli infections are increasingly being compromised by the rapid, global spread of antimicrobial resistance (AMR). Whilst AMR in E. coli has been extensively investigated in resource-rich settings, in sub-Saharan Africa molecular patterns of AMR are not well described. In this study, we have begun to explore the population structure and molecular determinants of AMR amongst E. coli isolates from Malawi. Methods: Ninety-four E. coli isolates from patients admitted to Queen's Hospital, Malawi, were whole-genome sequenced. The isolates were selected on the basis of diversity of phenotypic resistance profiles and clinical source of isolation (blood, CSF and rectal swab). Sequence data were analysed using comparative genomics and phylogenetics. Results: Our results revealed the presence of five clades, which were strongly associated with E. coli phylogroups A, B1, B2, D and F. We identified 43 multilocus STs, of which ST131 (14.9%) and ST12 (9.6%) were the most common. We identified 25 AMR genes. The most common ESBL gene was bla CTX-M-15 and it was present in all five phylogroups and 11 STs, and most commonly detected in ST391 (4/4 isolates), ST648 (3/3 isolates) and ST131 [3/14 (21.4%) isolates]. Conclusions: This study has revealed a high diversity of lineages associated with AMR, including ESBL and fluoroquinolone resistance, in Malawi. The data highlight the value of longitudinal bacteraemia surveillance coupled with detailed molecular epidemiology in all settings, including low-income settings, in describing the global epidemiology of ESBL resistance.
44 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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