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 paper, the authors performed an in-depth genetic assessment of Streptococcus uberis strains isolated from bovine clinical mastitis (CM) and performed a phylogenetic analysis to represent the evolutionary relationship among S. uberis sequences.
Abstract: Background Bovine mastitis is an important cause of economic loss in dairy farms. Streptococcus uberis is among the most frequently isolated bacterial species isolated from cows with mastitis. The aim of this study was to perform an in-depth genetic assessment of S. uberis strains isolated from bovine clinical mastitis (CM) and to perform a phylogenetic analysis to represent the evolutionary relationship among S. uberis sequences. Results A total of 159 isolates was genetically characterized using whole genome sequencing. According to the virulence determinants, all strains harbored the hasC, leuS, perR, purH, and purN virulence genes. Thirty-four resistance genes were identified in at least one strain. In terms of acquired genes, we observed that 152 (95.6 %) strains had a resistance gene to lincosamine (lnuD), 48 (30.2 %) to tetracycline (tetM), 4 (2.51 %) to tobramicine (ant6), and 1 to lincosamide (lsa(E)). MLST detected the Sequence Type (ST)797 (n = 23), while 85.5 % of the strains did not match to known STs. Conclusions Then, eleven distinct ST were identified after we submitted the new alleles to assign new STs. The other prevalent STs observed were ST1215 (n = 58), ST1219 (n = 35), and ST1213 (n = 15). And it was not possible to identify the MLST of four strains. Phylogenetic lineages indicated a high genomic diversity of S. uberis in our collection, confirming that most strains isolated from bovine mastitis have different reservoirs, typical of environmental pathogens.
5 citations
13 May 2021
TL;DR: In this article, the authors investigated potential reservoirs of ESBL-encoded genes in E. coli and found that swine and dairy cattle may be their main ESBL producers.
Abstract: Antimicrobial resistance associated with the spread of plasmid-encoded extended-spectrum β-lactamase (ESBL) genes conferring resistance to third generation cephalosporins is increasing worldwide. However, data on the population of ESBL producing E. coli in different animal sources and their antimicrobial characteristics are limited. The purpose of this study was to investigate potential reservoirs of ESBL-encoded genes in E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the United States using whole-genome sequencing (WGS). Three hundred isolates were typed into different phylogroups, characterized by BOX AIR-1 PCR and tested for resistance to antimicrobials. Of the 300 isolates, 59.7% were resistant to sulfisoxazole, 49.3% to tetracycline, 32.3% to cephalothin, 22.3% to ampicillin, 20% to streptomycin, 16% to ticarcillin; resistance to the remaining 12 antimicrobials was less than 10%. Phylogroups A and B1 were most prevalent with A (n = 92, 30%) and B1 (87 = 29%). A total of nine E. coli isolates were confirmed as ESBL producers by double-disk synergy testing and multidrug resistant (MDR) to at least three antimicrobial drug classes. Using WGS, significantly higher numbers of ESBL-E. coli were detected in swine and dairy manure than from any other animal sources, suggesting that these may be the primary animal sources for ESBL producing E. coli. These isolates carry plasmids, such as IncFIA(B), IncFII, IncX1, IncX4, IncQ1, CollRNAI, Col440I, and acquired ARGs aph(6)-Id, aph(3″)-Ib, aadA5, aph(3′)-Ia, blaCTX-M-15, blaTEM-1B, mphA, ermB, catA1, sul1, sul2, tetB, dfrA17. One of the E. coli isolates from swine with ST 410 was resistant to nine antibiotics and carried more than 28 virulence factors, and this ST has been shown to belong to an international high-risk clone. Our data suggests that ESBL producing E. coli are widely distributed in different animal sources, but swine and dairy cattle may be their main reservoir.
5 citations
TL;DR: In this paper, the authors used pulsed-field gel electrophoresis (PFGE) analysis and whole-genome sequencing (WGS) to characterize eight isolates of Y. pseudotuberculosis from zoo animals.
Abstract: Yersinia pseudotuberculosis is an important pathogen for both humans and animals. It can infect livestock, as well as pets and wild animals. During recent years, a number of reports have described the isolation of Y. pseudotuberculosis from zoo animals, mainly birds and mammals, for which the infection was mostly lethal. Between 2005 and 2019, there were at least 17 cases of deceased mammals, belonging to five different species, which suffered from a Y. pseudotuberculosis infection at the Zoo Wuppertal, Germany. Since only scarce information exists on the properties of Y. pseudotuberculosis from zoo animals, we characterized eight isolates, covering all infected species, in detail. All isolates were members of biotype 1, but belonged to five serotypes, five sequence types (STs), and seven core-genome multilocus sequence types (cgMLSTs). Using pulsed-field gel electrophoresis (PFGE) analysis and whole-genome sequencing (WGS), the seven isolates could be discriminated from each other. They differed significantly regarding their virulence genes and mobile genetic elements. While the virulence plasmid pYV existed in all serotypes (five isolates), a complete high-pathogenicity island (HPI) was detected only in the serotypes O:1a, O:1b, and O:13 (four isolates), but not in O:2a and O:2b. Similarly, the content of other plasmids and prophages varied greatly between the isolates. The data demonstrate that the deceased mammals were infected by seven individual isolates and not by a single type predominating in the zoo animals.
5 citations
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TL;DR: The Center for Genomic Epidemiology, which is located at the Technical University of Denmark, has since its beginning in 2010 developed bioinformatics methods and made them freely available as web-services and their use is the focus of this chapter.
Abstract: As whole genome sequence data of microorganisms are becoming easily accessible and cheap to produce, a transformation of the traditional methods used for typing, phenotyping and phylogenetic analysis of microorganisms is on the way. Following the anticipation that most clinical microbiological and food safety laboratories will soon have a sequencer in use on a daily basis, there is a growing need for easy-to-use bioinformatics methods that can quickly convert the sequence data into useful information on, e.g., the type of bacteria, whether it is resistant towards any types of antibiotics, and whether it is part of an outbreak. The Center for Genomic Epidemiology, which is located at the Technical University of Denmark, has since its beginning in 2010 developed such bioinformatics methods and made them freely available as web-services. These web-services and their use is the focus of this chapter.
5 citations
TL;DR: A novel MDR plasmid pSGB23 harboured by S. enterica carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. entersica MDRPlasmids identified so far, which explains its broad host range and stable inheritance.
Abstract: Salmonella enterica subspecies enterica serovar Saintpaul (S. Saintpaul) is an important gut pathogen which causes salmonellosis worldwide. Although intestinal salmonellosis is usually self-limiting, it can be life-threatening in children, the elderlies and immunocompromised patients. Appropriate antibiotic treatment is therefore required for these patients. However, the efficacy of many antibiotics on S. enterica infections has been greatly compromised due to spreading of multidrug resistance (MDR) plasmids, which poses serious threats on public health and needs to be closely monitored. In this study, we sequenced and fully characterized an S. enterica MDR plasmid pSGB23 isolated from chicken. Complete genome sequence analysis revealed that S. Saintpaul strain SGB23 harbored a 254 kb megaplasmid pSGB23, which carries 11 antibiotic resistance genes responsible for resistance to 9 classes of antibiotics and quaternary ammonium compounds that are commonly used to disinfect food processing facilities. Furthermore, we found that pSGB23 carries multiple conjugative systems, which allow it to spread into other Enterobacteriaceae spp. by self-conjugation. It also harbors multiple types of replicons and plasmid maintenance and addictive systems, which explains its broad host range and stable inheritance. We report here a novel MDR plasmid pSGB23 harboured by S. enterica. To our knowledge, it carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. enterica MDR plasmids identified so far. The isolation of pSGB23 from food sources is worrisome, while surveillance on its further spreading will be carried out based on the findings reported in this study.
5 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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