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: Genome analysis and experimental evidence suggested that AB211 has robust central carbohydrate metabolism implying that this bacterium can efficiently utilize the root exudates and other organic materials as an energy source.
Abstract: Bacillus aryabhattai AB211 is a plant growth promoting, gram positive firmicute, isolated from the rhizosphere of tea (Camellia sinensis), one of the oldest perennial crops and a major nonalcoholic beverage widely consumed all over the world. The whole genome of B. aryabhattai AB211 was sequenced, annotated and evaluated with special focus on genomic elements related to plant microbe interaction. It's genome sequence reveals the presence of a 5,403,026 bp chromosome. A total of 5226 putative protein-coding sequences, 16 rRNA, 120 tRNA, 8 ncRNAs, 58 non-protein coding genes, and 11 prophage regions were identified. Genome sequence comparisons between strain AB211 and other related environmental strains of B. aryabhattai, identified about 3558 genes conserved among all B. aryabhattai genomes analyzed. Most of the common genes involved in plant growth promotion activities were found to be present within core genes of all the genomes used for comparison, illustrating possible common plant growth promoting traits shared among all the strains of B. aryabhattai. Besides the core genes, some genes were exclusively identified in the genome of strain AB211. Functional annotation of the genes predicted in the strain AB211 revealed the presence of genes responsible for mineral phosphate solubilization, siderophores, acetoin, butanediol, exopolysaccharides, flagella biosynthesis, surface attachment/ biofilm formation, and indole acetic acid production, most of which were experimentally verified in the present study. Genome analysis and experimental evidence suggested that AB211 has robust central carbohydrate metabolism implying that this bacterium can efficiently utilize the root exudates and other organic materials as an energy source. Genes for the production of peroxidases, catalases, and superoxide dismutases, that confer resistance to oxidative stresses in plants were identified in AB211 genome. Besides these, genes for heat shock tolerance, cold shock tolerance, glycine-betaine production, and antibiotic /heavy metal resistance that enable bacteria to survive biotic/abiotic stress were also identified. Based on the genome sequence information and experimental evidence as presented in this study, strain AB211 appears to be metabolically diverse and exhibits tremendous potential as a plant growth promoting bacterium.
78 citations
TL;DR: A novel mcr-1 variant, named mCr-1.3, encoding an Ile-to-Val functional variant of MCR-1 was identified in a sequence type 155 (ST155) strain of Escherichia coli isolates of chicken origin from 13 provinces in China.
Abstract: The mcr-1 gene was detected in 5.11% (58/1136) of Escherichia coli isolates of chicken origin from 13 provinces in China. A novel mcr-1 variant, named mcr-1.3, encoding an Ile-to-Val functional variant of MCR-1 was identified in a sequence type 155 (ST155) strain. An mcr-1.3-containing IncI2 plasmid, pHeN867 (60,757 bp), was identified. The transfer of pHeN867 led to a 32-fold increase in the MIC of colistin in the recipient, exhibiting an effect on colistin resistance that was similar to that of mcr-1.
78 citations
Cites background from "In Silico Detection and Typing of P..."
...Conjugation experiments, S1-PFGE (15) and typing of plasmid, (16) showed that...
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TL;DR: The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak of a massive Salmonella enterica serovar Typhi outbreak in Zambia, and a new variant of the haplotype harboring a chromosomally translocated region containing the MDR islands of IncHI1 plasmid has emerged in Zimbabwe.
Abstract: Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar Typhi outbreak in Zambia during 2010 to 2012. Ninety-four isolates were susceptibility tested by MIC determinations. Whole-genome sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the multilocus sequence type (MLST), haplotype, plasmid replicon, antimicrobial resistance genes, and genetic relatedness by single nucleotide polymorphism (SNP) analysis and genomic deletions. The outbreak affected 2,040 patients, with a fatality rate of 0.5%. Most (83.0%) isolates were multidrug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the haplotype, H58B. Most isolates contained a chromosomally translocated region containing seven antimicrobial resistance genes, catA1, blaTEM-1, dfrA7, sul1, sul2, strA, and strB, and fragments of the incompatibility group Q1 (IncQ1) plasmid replicon, the class 1 integron, and the mer operon. The genomic analysis revealed 415 SNP differences overall and 35 deletions among 33 of the isolates subjected to whole-genome sequencing. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India by 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 of the 33 isolates sequenced belonged to a tight clonal group distinct from other H58 genomes included in the study. The small numbers of SNPs identified within this group are consistent with the short-term transmission that can be expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak, during which occasional other S. Typhi lineages, including sensitive ones, continued to cocirculate. The common view is that the emerging global S. Typhi haplotype, H58B, containing the MDR IncHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-Saharan Africa; we found that a new variant of the haplotype harboring a chromosomally translocated region containing the MDR islands of IncHI1 plasmid has emerged in Zambia. This could change the perception of the term “classical MDR typhoid” currently being solely associated with the IncHI1 plasmid. It might be more common than presently thought that S. Typhi haplotype H58B harbors the IncHI1 plasmid or a chromosomally translocated MDR region or both.
75 citations
TL;DR: Evaluating the safety of raw vegetable products present on the German market regarding toxin-producing Bacillus cereus sensu lato (s.l.) group bacteria showed that B. cereus s.l. strains encoding toxin genes occur in products sold on theGerman market and that these may pose a health risk to the consumer if present at elevated levels.
Abstract: This study aimed to evaluate the safety of raw vegetable products present on the German market regarding toxin-producing Bacillus cereus sensu lato (s.l.) group bacteria. A total of 147 B. cereus s.l. group strains isolated from cucumbers, carrots, herbs, salad leaves and ready-to-eat mixed salad leaves were analyzed. Their toxinogenic potential was assessed by multiplex PCR targeting the hemolysin BL (hbl) component D (hblD), non-hemolytic enterotoxin (nhe) component A (nheA), cytotoxin K-2 (cytK-2) and the cereulide (ces) toxin genes. In addition, a serological test was used to detect Hbl and Nhe toxins. On the basis of PCR and serological results, none of the strains were positive for the cereulide protein/genes, while 91.2, 83.0 and 37.4% were positive for the Hbl, Nhe and CytK toxins or their genes, respectively. Numerous strains produced multiple toxins. Generally, strains showed resistance against the β-lactam antibiotics such as penicillin G and cefotaxim (100%), as well as amoxicillin/clavulanic acid combination and ampicillin (99.3%). Most strains were susceptible to ciprofloxacin (99.3%), chloramphenicol (98.6%), amikacin (98.0%), imipenem (93.9%), erythromycin (91.8%), gentamicin (88.4%), tetracycline (76.2%) and trimethoprim/sulfamethoxazole combination (52.4%). The genomes of eight selected strains were sequenced. The toxin gene profiles detected by PCR and serological test mostly agreed with those from whole-genome sequence data. Our study showed that B. cereus s.l. strains encoding toxin genes occur in products sold on the German market and that these may pose a health risk to the consumer if present at elevated levels. Furthermore, a small percentage of these strains harbor antibiotic resistance genes. The presence of these bacteria in fresh produce should, therefore, be monitored to guarantee their safety.
74 citations
Cites methods from "In Silico Detection and Typing of P..."
...Zankari E. Comparison of the web tools ARG-ANNOT and ResFinder for detection of resistance genes in bacteria....
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...cereus-group strains using the PATRIC [43] database (bold highlights acquired resistance genes identified by ResFinder [45])....
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...The acquired antibiotic resistance genes were identified using the ResFinder server (v. 3.0) [44]....
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...Antibiotic resistance genes identified on the genomes of B. cereus-group strains using the PATRIC [43] database (bold highlights acquired resistance genes identified by ResFinder [45])....
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18 Feb 2020
TL;DR: The relevant approaches for phylogenomic studies for outbreak studies are described and an overview of selected tools for the characterization of foodborne pathogens based on WGS data are given.
Abstract: Whole genome sequencing (WGS) of foodborne pathogens has become an effective method for investigating the information contained in the genome sequence of bacterial pathogens. In addition, its highly discriminative power enables the comparison of genetic relatedness between bacteria even on a sub-species level. For this reason, WGS is being implemented worldwide and across sectors (human, veterinary, food, and environment) for the investigation of disease outbreaks, source attribution, and improved risk characterization models. In order to extract relevant information from the large quantity and complex data produced by WGS, a host of bioinformatics tools has been developed, allowing users to analyze and interpret sequencing data, starting from simple gene-searches to complex phylogenetic studies. Depending on the research question, the complexity of the dataset and their bioinformatics skill set, users can choose between a great variety of tools for the analysis of WGS data. In this review, we describe the relevant approaches for phylogenomic studies for outbreak studies and give an overview of selected tools for the characterization of foodborne pathogens based on WGS data. Despite the efforts of the last years, harmonization and standardization of typing tools are still urgently needed to allow for an easy comparison of data between laboratories, moving towards a one health worldwide surveillance system for foodborne pathogens.
72 citations
Cites background from "In Silico Detection and Typing of P..."
...Carattoli A, Zankari E, Garcia-Fernandez A, Voldby Larsen M, Lund O, Villa L, et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing....
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...bacteriaceae and some Gram-positive bacteria, but lacks information on plasmids from a broad range of other bacteria [96]....
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...PlasmidFinder further provides information on the similarity values of the requested sequence to a closely related reference....
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...elements can also provoke homologous or nonhomologous recombination with the chromosome leading to an exchange of small or large DNA sequences [96]....
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...PlasmidFinder is well established for in silico analysis of plasmids from Enterobacteriaceae and some Gram-positive bacteria, but lacks information on plasmids from a broad range of other bacteria [96]....
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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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