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: Understanding the evolution of antibiotic resistance at the molecular level as a functional tool for bioinformatic-based drug design is an important step in the fight against antibiotic resistance.
Abstract: The use of antibiotics to manage infectious diseases dates back to ancient civilization, but the lack of a clear distinction between the therapeutic and toxic dose has been a major challenge. This precipitates the notion that antibiotic resistance was from time immemorial, principally because of a lack of adequate knowledge of therapeutic doses and continuous exposure of these bacteria to suboptimal plasma concentration of antibiotics. With the discovery of penicillin by Alexander Fleming in 1924, a milestone in bacterial infections' treatment was achieved. This forms the foundation for the modern era of antibiotic drugs. Antibiotics such as penicillins, cephalosporins, quinolones, tetracycline, macrolides, sulphonamides, aminoglycosides and glycopeptides are the mainstay in managing severe bacterial infections, but resistant strains of bacteria have emerged and hampered the progress of research in this field. Recently, new approaches to research involving bacteria resistance to antibiotics have appeared; these involve combining the molecular understanding of bacteria systems with the knowledge of bioinformatics. Consequently, many molecules have been developed to curb resistance associated with different bacterial infections. However, because of increased emphasis on the clinical relevance of antibiotics, the synergy between in silico study and in vivo study is well cemented and this facilitates the discovery of potent antibiotics. In this review, we seek to give an overview of earlier reviews and molecular and structural understanding of bacteria resistance to antibiotics, while focusing on the recent bioinformatics approach to antibacterial drug discovery.
36 citations
TL;DR: Combining genetic context characterization with other molecular epidemiology methods, the molecular epidemiological links between genetically unrelated bacterial species are established by linking their acquisition of an IncN2 or IncA/C plasmid carrying blaNDM-1 for carbapenem resistance.
Abstract: The carbapenem resistance determinant blaNDM-1 has been found in various Gram-negative bacteria and upon different plasmid replicon types (Inc). Here, we present four patients within two hospitals in Pakistan harboring between two and four NDM-1-producing Gram-negative bacilli of different species coresident in their stool samples. We characterize the blaNDM-1 genetic contexts of these 11 NDM-1-producing Gram-negative bacilli in addition to other antimicrobial resistance mechanisms, plasmid replicon profiles, and sequence types (STs) in order to understand the underlying acquisition mechanisms of carbapenem resistance within these bacteria. Two common plasmid types (IncN2 and IncA/C) were identified to carry blaNDM-1 among the six different bacterial species isolated from the four patients. Two of these strains were novel Citrobacter freundii ST 20 and ST 21. The same IncN2-type blaNDM-1 genetic context was found in all four patients and within four different species. The IncA/C-type blaNDM-1 genetic context was found in two different species and in two of the four patients. Combining genetic context characterization with other molecular epidemiology methods, we were able to establish the molecular epidemiological links between genetically unrelated bacterial species by linking their acquisition of an IncN2 or IncA/C plasmid carrying blaNDM-1 for carbapenem resistance. By combining plasmid characterization and in-depth genetic context assessment, this analysis highlights the importance of plasmids in antimicrobial resistance. It also provides a novel approach for investigating the underlying mechanisms of blaNDM-1-related spread between bacterial species and genera via plasmids.
36 citations
Cites background from "In Silico Detection and Typing of P..."
...1 (26), databases (available at the Center of Genomic...
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TL;DR: Animal isolates showed a higher degree of antimicrobial resistance with greater MDR but human isolates formed more biofilm and had greater swarming motility as well as increased virulence to the nematode C. elegans.
Abstract: Salmonella enterica subsp. enterica serovar Dublin (S. Dublin), a cattle adapted serovar causes enteritis, and systemic disease in bovines. The invasive index of this serovar far exceeds that of the other serovars and human infections often present as fatal or highly resistant infections. In this, observational study, phenotypic properties of human and bovine-derived isolates of S. Dublin along with antibiogram of common antimicrobials were evaluated. The multiplex PCR confirmed isolates were genotyped using 7-gene legacy MLST. MIC assay was done by broth microdilution method. Previously published protocols were used to assess the motility, biofilm formation and morphotype. Vi antigen was agglutinated using commercial antiserum. Caenorhabditis elegans infection model was used to evaluate the virulence potiential. Phenotyping experiments were done in duplicates while virulence assay was done in triplicates. Whole-genome sequencing was used to predict the genes responsible for acquired resistance and a genotype-phenotype comparison was made. We evaluated 96 bovine and 10 human isolates in this study. All the isolates belonged to ST10 in eBG53 and were negative for Vi-antigen. The swarming motility, biofilm formation and morphotype were variable in the isolates of both groups. Resistance to sulfamethoxazole, ampicillin, chloramphenicol, tetracycline was > 90% in animal isolates whereas resistance to sulfamethoxazole was > 70% in human isolates. MDR was also higher in animal isolates. Human isolates were significantly (P < 0.0001) more virulent than animal isolates on C. elegans infection model. The genomic comparison based on the core SNPs showed a high degree of homogeneity between the isolates. The carriage of IncA/C2 plasmid was seen as a typical feature of isolates from the bovine hosts. Human isolates showed more diversity in the phenotypic assays. Animal isolates showed a higher degree of antimicrobial resistance with greater MDR but human isolates formed more biofilm and had greater swarming motility as well as increased virulence to the nematode C. elegans. The carriage of IncA/C2 plasmid could contribute to the distinguishing feature of the bovine isolates. The tandem use of genotypic-phenotypic assays improves the understanding of diversity and differential behaviour of the same serovar from unrelated host sources.
36 citations
TL;DR: In this paper, the occurrence of mcr-1 -harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr1 plasmids were determined.
Abstract: Objectives To determine the occurrence of mcr-1 -harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr-1 plasmids. Methods Enrichment and selective culture of 387 archived porcine caecal contents and recovery from archive of 1109 E. coli isolates to identify colistin-resistant bacteria by testing for the presence of mcr-1 by PCR and RT-PCR. mcr-1 -harbouring E. coli were characterized by WGS and compared with other available mcr-1 WGS. Results Using selective isolation following enrichment, the occurrence of mcr-1 E. coli in caeca from healthy pigs at slaughter from unique farms in GB was 0.6% (95% CI 0%-1.5%) in 2015. mcr-1 E. coli were also detected in isolates from two porcine veterinary diagnostic submissions in 2015. All isolates prior to 2015 were negative. WGS analysis of the four mcr-1 -positive E. coli indicated no other antimicrobial resistance (AMR) genes were linked to mcr-1 -plasmid-bearing contigs, despite all harbouring multiple AMR genes. The sequence similarity between mcr-1 -plasmid-bearing contigs identified and those found in GB, Chinese and South African human isolates and Danish, French and Estonian livestock-associated isolates was 90%-99%. Conclusions mcr-1- harbouring plasmids were diverse, implying transposable elements are involved in mcr-1 transmission in GB. The low number of mcr-1 -positive E. coli isolates identified suggested mcr-1 is currently uncommon in E. coli from pigs within GB. The high sequence similarity between mcr-1 plasmid draft genomes identified in pig E. coli and plasmids found in human and livestock-associated isolates globally requires further investigation to understand the full implications.
36 citations
TL;DR: Diverse STs of E. faecalis, including strains associated with common nosocomial infections are circulating in the healthcare facility of Saudi Arabia and carried multi-drug resistance, which has important implications for infection control.
Abstract: Enterococcus faecalis is a ubiquitous member of the gut microbiota and has emerged as a life- threatening multidrug-resistant (MDR) nosocomial pathogen. The aim of this study was to survey the prevalence of multidrug-resistant and epidemiologically important strains of E. faecalis in the western region of Saudi Arabia using phenotypic and whole genome sequencing approaches. In total, 155 patients positive for E. faecalis infection were included in this study. The isolates were identified by MALDI-TOF, and screen for antimicrobial resistance using VITEK-2 system. Genome sequencing was performed with paired-end strategy using MiSeq platform. Seventeen sequence types (STs) were identified through multilocus sequence typing (MLST) analysis of the E. faecalis genomes, including two novels STs (ST862 and ST863). The most common STs in the Saudi patients were ST179 and ST16 from clonal complex 16 (CC16). Around 96% (n = 149) isolates were MDR. The antibiotics quinupristin/dalfopristin, clindamycin, and erythromycin demonstrated almost no coverage, and high-level streptomycin, gentamycin, and ciprofloxacin demonstrated suboptimal coverage. Low resistance was observed against vancomycin, linezolid, and ampicillin. Moreover, 34 antimicrobial resistance genes and variants, and three families of insertion sequences were found in the E. faecalis genomes, which likely contributed to the observed antimicrobial resistance. Twenty-two virulence factors, which were mainly associated with biofilm formation, endocarditis, cell adherence, and colonization, were detected in the isolates. Diverse STs of E. faecalis, including strains associated with common nosocomial infections are circulating in the healthcare facility of Saudi Arabia and carried multi-drug resistance, which has important implications for infection control.
35 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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