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, a multilocus sequence typing (MLST) analysis revealed 24 distinct sequence types identified within the isolates, among which the most frequently represented were ST76 (16%) and ST70 (11%).
Abstract: Klebsiella pneumoniae is recognized as a common cause of nosocomial infections and outbreaks causing pneumonia, septicemia, and urinary tract infections. This opportunistic bacterium shows an increasing acquisition of antibiotic-resistance genes, which complicates treatment of infections. Hence, fast reliable strain typing methods are paramount for the study of this opportunistic pathogen's multi-drug resistance genetic profiles. In this study, thirty-eight strains of K. pneumoniae isolated from the blood of pediatric patients were characterized by whole-genome sequencing and genomic clustering methods. Genes encoding β-lactamase were found in all the bacterial isolates, among which the blaSHV variant was the most prevalent (53%). Moreover, genes encoding virulence factors such as fimbriae, capsule, outer membrane proteins, T4SS and siderophores were investigated. Additionally, a multi-locus sequence typing (MLST) analysis revealed 24 distinct sequence types identified within the isolates, among which the most frequently represented were ST76 (16%) and ST70 (11%). Based on LPS structure, serotypes O1 and O3 were the most prevalent, accounting for approximately 63% of all infections. The virulence capsular types K10, K136, and K2 were present in 16, 13, and 8% of the isolates, respectively. Phylogenomic analysis based on virtual genome fingerprints correlated with the MLST data. The phylogenomic reconstruction also denoted association between strains with a higher abundance of virulence genes and virulent serotypes compared to strains that do not possess these traits. This study highlights the value of whole-genomic sequencing in the surveillance of virulence attributes among clinical K. pneumoniae strains.
7 citations
TL;DR: In this paper, S. Dublin serotype serotype was characterized by analyzing phenotypic and genotypic antimicrobial resistance profiles, the presence of plasmids, and phylogenetic relationships.
Abstract: Salmonella enterica subspecies enterica serotype Dublin is a host-adapted serotype in cattle, associated with enteritis and systemic disease. The primary clinical manifestation of Salmonella Dublin infection in cattle, especially calves, is respiratory disease. While rare in humans, it can cause severe illness, including bacteremia, with hospitalization and death. In the United States, S. Dublin has become one of the most multidrug-resistant serotypes. The objective of this study was to characterize S. Dublin isolates from sick cattle by analyzing phenotypic and genotypic antimicrobial resistance (AMR) profiles, the presence of plasmids, and phylogenetic relationships. S. Dublin isolates (n = 140) were selected from submissions to the NVSL for Salmonella serotyping (2014-2017) from 21 states. Isolates were tested for susceptibility against 14 class-representative antimicrobial drugs. Resistance profiles were determined using the ABRicate with Resfinder and NCBI databases, AMRFinder and PointFinder. Plasmids were detected using ABRicate with PlasmidFinder. Phylogeny was determined using vSNP. We found 98% of the isolates were resistant to more than 4 antimicrobials. Only 1 isolate was pan-susceptible and had no predicted AMR genes. All S. Dublin isolates were susceptible to azithromycin and meropenem. They showed 96% resistance to sulfonamides, 97% to tetracyclines, 95% to aminoglycosides and 85% to beta-lactams. The most common AMR genes were: sulf2 and tetA (98.6%), aph(6)-Id (97.9%), aph(3'')-Ib, (97.1%), floR (94.3%), and blaCMY-2 (85.7%). All quinolone resistant isolates presented mutations in gyrA. Ten plasmid types were identified among all isolates with IncA/C2, IncX1, and IncFII(S) being the most frequent. The S. Dublin isolates show low genomic genetic diversity. This study provided antimicrobial susceptibility and genomic insight into S. Dublin clinical isolates from cattle in the U.S. Further sequence analysis integrating food and human origin S. Dublin isolates may provide valuable insight on increased virulence observed in humans.
7 citations
TL;DR: This study highlights the link between humans, domestic animals, and wildlife, showing that the current knowledge on this ecological context is lacking and that the potential health risks are underestimated.
Abstract: Simple Summary Klebsiella pneumoniae is an opportunistic bacterium known as a nosocomial pathogen, but it is able to colonize a multitude of ecological niches, both free-living and host-associated. For this reason, the presence of antimicrobial resistance genes in wildlife is an indicator that resistant bacteria of human- or livestock-origin are widespread in the environment. Wild animals can act as efficient reservoirs and epidemiological links between humans, livestock, and natural environments. Despite the renewal of interest in epidemiology, there is a critical lack of an unbiased ecological approaches to defining reservoirs and sources of infections. The aims of the present study were to investigate the occurrence of K. pneumoniae in wildlife, to characterize antimicrobial features through phenotypic and whole genome sequencing approaches, and to identify similar available sequences that could suggest a transmission event. Abstract Despite Klebsiella pneumoniae being widely recognized as a nosocomial pathogen, there is a critical lack in defining its reservoirs and sources of infections. Most studies on risk factors have focused on multidrug-resistant (MDR) isolates and clinically-oriented questions. Over a two-year period, we sampled 131 wild animals including mammal and bird species from three regions of Central Italy. All typical colonies isolated from the analytical portions were confirmed by real-time PCR and identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). All confirmed K. pneumoniae isolates were tested for antimicrobial susceptibility to 29 antimicrobials and subjected to whole genome sequencing. Typical colonies were detected in 17 samples (13%), which were identified as K. pneumoniae (n = 16) and as K. quasipneumoniae (n = 1) by MALDI-TOF MS. The antimicrobial susceptibility profile showed that all the isolates were resistant to β-lactams (ceftobiprole, cloxacillin, cefazolin) and tetracycline; resistance to ertapenem and trimethoprim was observed and nine out of 16 K. pneumoniae isolates (56.2%) were classified as MDR. Genomic characterization allowed the detection of fluoroquinolone resistance-associated efflux pumps, fosfomycin and β-lactamase resistance genes, and virulence genes in the overall dataset. The cluster analysis of two isolates detected from wild boar with available clinical genomes showed the closest similarity. This study highlights the link between humans, domestic animals, and wildlife, showing that the current knowledge on this ecological context is lacking and that the potential health risks are underestimated.
7 citations
TL;DR: In this article, the authors investigated fitness effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into Escherichia coli.
Abstract: (1) Background: Resistance plasmids are under selective conditions beneficial for the bacterial host, but in the absence of selective pressure, this carriage may cause fitness costs. Compensation of this fitness burden is important to obtain competitive ability under antibiotic-free conditions. In this study, we investigated fitness effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into Escherichia coli. (2) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to E. coli J53. Growth rates were compared for all transconjugants and the recipient. Selected transconjugants were challenged in long-term growth experiments. Growth rates were assessed at different time points during growth for 500 generations. Whole-genome sequencing (WGS) of initial and evolved transconjugants was determined. Results: Most plasmid acquisitions resulted in growth differences, ranging from −4.5% to 7.2%. Transfer of a single blaCMY-16-carrying plasmid resulted in a growth burden and a growth benefit in independent mating. Long-term growth led to a compensation of fitness burdens and benefits. Analyzing WGS revealed genomic changes caused by Single Nucleotide Polymorphisms (SNPs) and insertion sequences over time. Conclusions: Fitness effects associated with plasmid acquisitions were variable. Potential compensatory mutations identified in transconjugants’ genomes after 500 generations give interesting insights into aspects of plasmid–host adaptations.
7 citations
TL;DR: In this paper, the authors characterized the genome of a C. freundii strain with an IncX3 plasmid encoding both the bla NDM-1 and bla SHV-12 genes.
Abstract: Background The emergence of multidrug-resistant Citrobacter freundii poses daunting challenges to the treatment of clinical infections. The purpose of this study was to characterize the genome of a C. freundii strain with an IncX3 plasmid encoding both the bla NDM-1 and bla SHV-12 genes. Methods Strain ZT01-0079 was isolated from a clinical urine sample. The Vitek2 system was used for identification and antimicrobial susceptibility testing. The presence of bla NDM-1 was detected by PCR and sequencing. Conjugation experiments and Southern blotting were performed to determine the transferability of the bla NDM-1- carrying plasmid. Nanopore and Illumina sequencing were performed to better understand the genomic characteristics of the strain. Results Strain ZT01-0079 was identified as C. freundii, and the coexistence of bla NDM-1 and multiple drug resistance genes was confirmed. Electrophoresis and Southern blotting showed that bla NDM-1 was located on a ~53kb IncX3 plasmid. The NDM-1-encoding plasmid was successfully transferred at a frequency of 1.68×10-3. Both the bla NDM-1 and bla SHV-12 genes were located on the self-transferable IncX3 plasmid. Conclusion The rapid spread of the IncX3 plasmid highlights the importance of continuous monitoring of the prevalence of NDM-1-encoding Enterobacteriaceae. Mutations of existing carbapenem resistance genes will bring formidable challenges to clinical treatment.
7 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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