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: A multidrug-resistant Escherichia coli strain (B2C) was isolated from a river water sample in Brazil after the Mariana mining dam disaster as mentioned in this paper , and the genome was sequenced using the Illumina MiSeq platform, and de novo assembled using Unicycler.
3 citations
TL;DR: The presence of antibiotic resistance genes is concerning, as prior studies have found transmission between animals, and these findings are important to understand the circulation of K. variicola in healthy livestock animals.
Abstract: Klebsiella variicola is an opportunistic pathogen in humans. It also has been associated with bovine mastitis, which can have significant economic effects. ABSTRACT Klebsiella variicola, a member of Klebsiella pneumoniae complex, is found to infect plants, insects, and animals and is considered an emerging pathogen in humans. While antibiotic resistance is often prevalent among K. variicola isolates from humans, this has not been thoroughly investigated in isolates from nonhuman sources. Prior evidence suggests that K. variicola can be transmitted between agricultural products as well as between animals, and the use of antibiotics in agriculture has increased antibiotic resistance in other emerging pathogens. Furthermore, in animals that contain K. variicola as a normal member of the rumen microbiota, the same bacteria can also cause infections, such as clinical mastitis in dairy cows. Here, we describe K. variicola UFMG-H9 and UFMG-H10, both isolated from the urine of healthy Gyr heifers. These two genomes represent the first isolates from the urine of cattle and exhibit greater similarity with strains from the human urinary tract than isolates from bovine fecal or milk samples. Unique to the UFMG-H9 genome is the presence of flagellar genes, the first such observation for K. variicola. Neither of the sampled animals had symptoms associated with K. variicola infection, even though genes associated with virulence and antibiotic resistance were identified in both strains. Both strains were resistant to amoxicillin, erythromycin, and vancomycin, and UFMG-H10 is resistant to fosfomycin. The observed resistances emphasize the concern regarding the emergence of this species as a human pathogen given its circulation in healthy livestock animals. IMPORTANCE Klebsiella variicola is an opportunistic pathogen in humans. It also has been associated with bovine mastitis, which can have significant economic effects. While numerous isolates have been sequenced from human infections, only 12 have been sequenced from cattle (fecal and milk samples) to date. Recently, we discovered the presence of K. variicola in the urine of two healthy heifers, the first identification of K. variicola in the bovine urinary tract and the first confirmed K. variicola isolate encoding for flagella-mediated motility. Here, we present the genome sequences and analysis of these isolates. The bovine urinary genomes are more similar to isolates from the human urinary tract than they are to other isolates from cattle, suggesting niche specialization. The presence of antibiotic resistance genes is concerning, as prior studies have found transmission between animals. These findings are important to understand the circulation of K. variicola in healthy livestock animals.
3 citations
TL;DR: The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions, and the close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare.
Abstract: OBJECTIVES
To describe the population genetics and antibiotic resistance gene distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates causing infections in three Mediterranean countries.
METHODS
Isolates were collected during the 2013-17 AIDA clinical trial in six hospitals in Israel, Greece and Italy. WGS, bioinformatic characterization and antibiotic resistance profiling were performed.
RESULTS
In the 247 CRAB isolates characterized in this study, ST distribution varied by country: 29/31 (93.5%) Greek isolates, 34/41 (82.9%) Italian isolates and 70/175 (40.0%) Israeli isolates belonged to ST2. The identified ST2 isolates included eight distinct clades: 2C, 2D and 2H were significantly more common in Italy, while 2F was unique to Greece. The uncommon ST3 was not present among Greek isolates and constituted only 5/41 (12%) Italian isolates. On the other hand, it was much more common among Israeli isolates: 78/175 (44.6%) belonged to ST3. The vast majority of isolates, 240/247 (97.2%), were found to harbour acquired carbapenemases, primarily blaOXA-23. The chromosomal oxaAb (blaOXA-51-like) and ampC genes characteristic of this organism were also ubiquitous. Most (96.4%) ST3 isolates carried a broad-host-range plasmid IncP1α.
CONCLUSIONS
The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions. The close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare. The clustering of antibiotic resistance genes and plasmids that is unique to each clade/ST, and nearly uniform within clades/STs, suggests that horizontal transmission is rare but crucial to the clade's/ST's success.
3 citations
18 Mar 2022
TL;DR: In this paper , the authors characterized the genomic diversity of 100 clinical non-typhoidal Salmonella collected from 93 patients in 2001 from the eastern, and in 2006-2018 from the western regions of The Gambia respectively.
Abstract: Non-typhoidal Salmonella associated with multidrug resistance cause invasive disease in sub-Saharan Africa. Specific lineages of serovars Typhimurium and Enteritidis have been implicated. Here we characterized the genomic diversity of 100 clinical non-typhoidal Salmonella collected from 93 patients in 2001 from the eastern, and in 2006-2018 from the western regions of The Gambia respectively. A total of 93 isolates (64 invasive, 23 gastroenteritis and six other sites) representing a single infection episode were phenotypically tested for antimicrobial susceptibility using the Kirby-Bauer disc diffusion technique. Whole genome sequencing of 100 isolates was performed using Illumina, and the reads were assembled and analysed using SPAdes. The Salmonella in Silico Typing Resource (SISTR) was used for serotyping. SNP differences among the 93 isolates were determined using Roary, and phylogenetic analysis was performed in the context of 495 African strains from the European Nucleotide Archive. Salmonella serovars Typhimurium (26/64; 30.6 %) and Enteritidis (13/64; 20.3 %) were associated with invasive disease, whilst other serovars were mainly responsible for gastroenteritis (17/23; 73.9 %). The presence of three major serovar Enteritidis clades was confirmed, including the invasive West African clade, which made up more than half (11/16; 68.8 %) of the genomes. Multidrug resistance was confined among the serovar Enteritidis West African clade. The presence of this epidemic virulent clade has potential for spread of resistance and thus important implications for systematic patient management. Surveillance and epidemiological investigations to inform control are warranted.
3 citations
TL;DR: This work surveyed a large number of bacterial genomes sampled from infections at a single hospital in a systematic and unbiased manner, and was able to track the independent transfer of MGEs over time, expanding the understanding of HGT in healthcare settings and can inform efforts to limit the spread of drug-resistant pathogens in hospitals.
Abstract: Multidrug-resistant bacterial pathogens pose a serious public health threat, especially in hospital settings. Horizontal gene transfer (HGT) of mobile genetic elements (MGEs) contributes to this threat by facilitating the rapid spread of genes conferring antibiotic resistance, enhanced virulence, and environmental persistence between nosocomial pathogens. Despite recent advances in microbial genomics, studies of HGT in hospital settings remain limited in scope. The objective of this study was to identify and track the movement of MGEs within a single hospital system using unbiased methods. We screened the genomes of 2,173 bacterial isolates from healthcare-associated infections collected over an 18-month time period to identify nucleotide regions that were identical in the genomes of bacteria belonging to distinct genera. These putative MGEs were found in 196 isolates belonging to 11 different genera; they grouped into 51 clusters of related elements, and they were most often shared between related genera. To resolve the genomic locations of the most prevalent MGEs, we performed long-read sequencing on a subset of representative isolates and generated highly contiguous, hybrid-assembled genomes. Many of these genomes contained plasmids and chromosomal elements encoding one or more of the MGEs we identified, which were often arranged in a mosaic fashion. We then tracked the appearance of ten MGE-bearing plasmids in all 2,173 genomes, and found evidence supporting the transfer of plasmids between patients independent from bacterial transmission. Finally, we identified two instances of likely plasmid transfer across genera within individual patients. In one instance, the plasmid appeared to have subsequently transferred to a second patient. By surveying a large number of bacterial genomes sampled from infections at a single hospital in a systematic and unbiased manner, we were able to track the independent transfer of MGEs over time. This work expands our understanding of HGT in healthcare settings, and can inform efforts to limit the spread of drug-resistant pathogens in hospitals.
3 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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