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 applied whole-genome sequence analysis to 982 animal-derived Escherichia coli samples collected in China from the 1970s to 2019, finding that the number of AMR genes per isolate doubled, including those conferring resistance to critically important agents for both veterinary (florfenicol and norfloxacin) and human medicine (colistin, cephalosporins and meropenem).
Abstract: Antimicrobial use in livestock production is linked to the emergence and spread of antimicrobial resistance (AMR), but large-scale studies on AMR changes in livestock isolates remain scarce. Here we applied whole-genome sequence analysis to 982 animal-derived Escherichia coli samples collected in China from the 1970s to 2019, finding that the number of AMR genes (ARGs) per isolate doubled—including those conferring resistance to critically important agents for both veterinary (florfenicol and norfloxacin) and human medicine (colistin, cephalosporins and meropenem). Plasmids of incompatibility groups IncC, IncHI2, IncK, IncI and IncX increased distinctly in the past 50 years, acting as highly effective vehicles for ARG spread. Using antimicrobials of the same class, or even unrelated classes, may co-select for mobile genetic elements carrying multiple co-existing ARGs. Prohibiting or strictly curtailing antimicrobial use in livestock is therefore urgently needed to reduce the growing threat from AMR. Genomic analyses reveal Escherichia coli samples from livestock in China have a third more plasmids than 50 years ago, contributing to the spread of antimicrobial resistance.
24 citations
TL;DR: In this paper, the authors applied methods used in AMR monitoring, supplemented by colistin resistance screening, to investigate the AMR status of Escherichia coli from free-living birds coming from natural habitats and rescue centers.
Abstract: Antimicrobial resistance (AMR) is one of the most important global health concerns; therefore, the identification of AMR reservoirs and vectors is essential. Attention should be paid to the recognition of potential hazards associated with wildlife as this field still seems to be incompletely explored. In this context, the role of free-living birds as AMR carriers is noteworthy. Therefore, we applied methods used in AMR monitoring, supplemented by colistin resistance screening, to investigate the AMR status of Escherichia coli from free-living birds coming from natural habitats and rescue centers. Whole-genome sequencing (WGS) of strains enabled to determine resistance mechanisms and investigate their epidemiological relationships and virulence potential. As far as we know, this study is one of the few that applied WGS of that number (n = 71) of strains coming from a wild avian reservoir. The primary concerns arising from our study relate to resistance and its determinants toward antimicrobial classes of the highest priority for the treatment of critical infections in people, e.g., cephalosporins, quinolones, polymyxins, and aminoglycosides, as well as fosfomycin. Among the numerous determinants, bla CTX-M-15, bla CMY-2, bla SHV-12, bla TEM-1B, qnrS1, qnrB19, mcr-1, fosA7, aac(3)-IIa, ant(3")-Ia, and aph(6)-Id and chromosomal gyrA, parC, and parE mutations were identified. Fifty-two sequence types (STs) noted among 71 E. coli included the global lineages ST131, ST10, and ST224 as well as the three novel STs 11104, 11105, and 11194. Numerous virulence factors were noted with the prevailing terC, gad, ompT, iss, traT, lpfA, and sitA. Single E. coli was Shiga toxin-producing. Our study shows that the clonal spread of E. coli lineages of public and animal health relevance is a serious avian-associated hazard.
24 citations
13 Dec 2018
TL;DR: It is confirmed that genetically diverse antimicrobial resistant enterococci harboring virulence factors can be promoted by the use of certain antimicrobials in feed and suchEnterococci could persist in broiler chickens and their litter, potentially contaminating the soil upon land application.
Abstract: The objective of this study was to compare the resistance phenotypes to genotypes of enterococci from broiler and to evaluate the persistence and distribution of resistant genotypes in broiler fed bambermycin (BAM), penicillin (PEN), salinomycin (SAL), bacitracin (BAC) or a salinomycin/bacitracin combination (SALBAC) for 35 days. A total of 95 enterococci from cloacal (n=40), cecal (n=38) and litter collected on day 36 (n=17) samples were isolated weekly from day 7 to 36. All isolates were identified by API-20 Strep and their antimicrobial susceptibilities were evaluated using the Sensititre system with the commercially available NARMS’s plates of Gram positive bacteria. Whole genome sequencing (WGS) was used to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. All isolates were further characterized for hemolysin production (HEM), bile salt hydrolysis (BSH) and gelatinase (GEL) activities. Of the 95 isolates, E. faecium (n = 58) and E. faecalis (n = 24) were the most common Enterococcus species identified. Significant differences in the level of resistance for the E. faecium isolates to ciprofloxacin, macrolide, penicillin and tetracycline were observed among treatments. The bcrR, mefA and aac(6) genes were higher in BAM treatment than the other groups whereas bcrR, ermA, ermB, aphA(3) and tetL were more prevalent in PEN and BAC treatments. Overall, E. faecium isolates showed higher prevalence of antimicrobial resistance, but E. faecalis from litter also exhibited a significant level of resistance. A range of 4 to 15 different virulence genes was detected in E. faecalis. All isolates from litter but one (94.1%) showed BSH activities while 52.9% of them produced GEL. HEM activity was observed only in isolates collected on Day 7 (n= 9) and Day 14 (n= 1). This study confirmed that genetically diverse antimicrobial resistant enterococci harboring virulence factors can be promoted by the use of certain antimicrobials in feed and such enterococci could persist in broiler chickens and their litter, potentially contaminating the soil upon land application. This study underscores the need for ongoing monitoring the AMR enterococci.
24 citations
TL;DR: WGS provided a wealth of data on prevalence of AMR genotypes and plasmid persistence absent from phenotypic data and, also, demonstrated the importance of culture media for detecting ESBL E. coli.
Abstract: To tackle the problem of antimicrobial resistance (AMR) surveillance programmes are in place within Europe applying phenotypic methods, but there are plans for implementing whole genome sequencing (WGS). We tested the benefits of WGS using Escherichia coli collected from pig surveillance performed between 2013 to 2017. WGS was performed on 498 E. coli producing ESBL and AmpC enzymes, recovered from pig caeca on MacConkey + cefotaxime (McC + CTX) agar, as recommended by the European Commission, or ESBL agar, used additionally by United Kingdom. Our results indicated WGS was extremely useful for monitoring trends for specific ESBL genes, as well as a plethora of AMR genotypes, helping to establish their prevalence and co-linkage to certain plasmids. Recovery of isolates with multi-drug resistance (MDR) genotypes was lower from McC + CTX than ESBL agar. The most widespread ESBL genes belonged to the blaCTX-M family. blaCTX-M-1 dominated all years, and was common in two highly stable IncI1 MDR plasmids harbouring (blaCTX-M-1,sul2, tetA) or (blaCTX-M-1, aadA5, sul2, dfrA17), in isolates which were phylogenetically dissimilar, suggesting plasmid transmission. Therefore, WGS provided a wealth of data on prevalence of AMR genotypes and plasmid persistence absent from phenotypic data and, also, demonstrated the importance of culture media for detecting ESBL E. coli.
24 citations
TL;DR: This study performed a study based on phenotypic and WGS data of isolates from poultry farms, chicken carcasses and humans to understand the importance of S. enterica in the broiler industry in Ecuador and identified a pESI-related megaplasmid identified in Ecuadorian samples.
Abstract: Salmonella enterica is one of the most important foodborne pathogens around the world. In the last years, S. enterica serovar Infantis has become an important emerging pathogen in many countries, often as multidrug resistant clones. To understand the importance of S. enterica in the broiler industry in Ecuador, we performed a study based on phenotypic and WGS data of isolates from poultry farms, chicken carcasses and humans. We showed a high prevalence of S. enterica in poultry farms (41.4%) and chicken carcasses (55.5%), but a low prevalence (1.98%) in human samples. S. Infantis was shown to be the most prevalent serovar with a 98.2, 97.8, and 50% in farms, foods, and humans, respectively, presenting multidrug resistant patterns. All sequenced S. Infantis isolates belonged to ST32. For the first time, a pESI-related megaplasmid was identified in Ecuadorian samples. This plasmid contains genes of antimicrobial resistance, virulence factors, and environmental stress tolerance. Genomic analysis showed a low divergence of S. Infantis strains in the three analyzed components. The results from this study provide important information about genetic elements that may help understand the molecular epidemiology of S. Infantis in Ecuador.
24 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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