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: Using shotgun metagenomics, soils from relatively low anthropogenic impact sites across the Namib Desert are analysed and the presence of a clinically significant extended spectrum β-lactamase (TEM-116) on a ColE1-like plasmid also carrying a metal resistance gene (arsC) is reported.
13 citations
26 Aug 2020
TL;DR: It is demonstrated that WGS-based subtyping allows better identification of sources potentially linked with human infection and emerging subpopulations, along with providing information on the risk of dissemination of plasmids and acquired antimicrobial resistance genes (AARGs).
Abstract: Serotyping has traditionally been used for subtyping of non-typhoidal Salmonella (NTS) isolates. However, its discriminatory power is limited, which impairs its use for epidemiological investigations of source attribution. Whole-genome sequencing (WGS) analysis allows more accurate subtyping of strains. However, because of the relative newness and cost of routine WGS, large-scale studies involving NTS WGS are still rare. We aimed to revisit the big picture of subtyping NTS with a public health impact by using traditional serotyping (i.e. reaction between antisera and surface antigens) and comparing the results with those obtained using WGS. For this purpose, we analysed 18 282 sequences of isolates belonging to 37 serotypes with a public health impact that were recovered in the USA between 2006 and 2017 from multiple sources, and were available at the National Center for Biotechnology Information (NCBI). Phylogenetic trees were reconstructed for each serotype using the core genome for the identification of genetic subpopulations. We demonstrated that WGS-based subtyping allows better identification of sources potentially linked with human infection and emerging subpopulations, along with providing information on the risk of dissemination of plasmids and acquired antimicrobial resistance genes (AARGs). In addition, by reconstructing a phylogenetic tree with representative isolates from all serotypes (n=370), we demonstrated genetic variability within and between serotypes, which formed monophyletic, polyphyletic and paraphyletic clades. Moreover, we found (in the entire data set) an increased detection rate for AARGs linked to key antimicrobials (such as quinolones and extended-spectrum cephalosporins) over time. The outputs of this large-scale analysis reveal new insights into the genetic diversity within and between serotypes; the polyphyly and paraphyly of certain serotypes may suggest that the subtyping of NTS to serotypes may not be sufficient. Moreover, the results and the methods presented here, leading to differentiation between genetic subpopulations based on their potential risk to public health, as well as narrowing down the possible sources of these infections, may be used as a baseline for subtyping of future NTS infections and help efforts to mitigate and prevent infections in the USA and globally.
13 citations
TL;DR: Preservation of some genetic modules of accessory genome harbored by strains from different continents in combination with great plasticity of IS and varied flow of plasmids, may be central features of the genomic structure of GC1.
Abstract: Acinetobacter baumannii is one of the most important nosocomial pathogens able to rapidly develop extensive drug resistance. Here, we study the role of accessory genome in the success of the globally disseminated clone 1 (GC1) with functional and genomic approaches. Comparative genomics was performed with available GC1 genomes (n = 106) against other A. baumannii high-risk and sporadic clones. Genetic traits related to accessory genome were found common and conserved along time as two novel regions of genome plasticity, and a CRISPR-Cas system acquired before clonal diversification located at the same loci as "sedentary" modules. Although identified within hotspot for recombination, other block of accessory genome was also "sedentary" in lineage 1 of GC1 with signs of microevolution as the AbaR0-type genomic island (GI) identified in A144 and in A155 strains which were maintained one month in independent experiments without antimicrobial pressure. The prophage YMC/09/02/B1251_ABA_BP was found to be "mobile" since, although it was shared by all GC1 genomes, it showed high intrinsic microevolution as well as mobility to different insertion sites. Interestingly, a wide variety of Insertion Sequences (IS), probably acquired by the flow of plasmids related to Rep_3 superfamily was found. These IS showed dissimilar genomic location amongst GC1 genomes presumably associated with promptly niche adaptation. On the other hand, a type VI secretion system and three efflux pumps were subjected to deep processes of genomic loss in A. baumannii but not in GC1. As a whole, these findings suggest that preservation of some genetic modules of accessory genome harbored by strains from different continents in combination with great plasticity of IS and varied flow of plasmids, may be central features of the genomic structure of GC1. Competition of A144 and A155 versus A118 (ST 404/ND) without antimicrobial pressure suggested a higher ability of GC1 to grow over a clone with sporadic behavior which explains, from an ecological perspective, the global achievement of this successful pandemic clone in the hospital habitat. Together, these data suggest an essential role of still unknown properties of "mobile" and "sedentary" accessory genome that is preserved over time under different antibiotic or stress conditions.
13 citations
Cites background from "In Silico Detection and Typing of P..."
...The search of replication initiation proteins previously described (Carattoli et al., 2014; Hamidian and Hall, 2018a; Salto et al., 2018), revealed the predominance of the Rep_3 superfamily (17/27) in the GC1 Group 1 genomes (Supplementary Table 4)....
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TL;DR: In this article, the authors examined 106 E. coli strains carrying the ter operon from various animals and found that ter operons were clearly classified into four subtypes, ter-type 1-4, at the nucleotide sequence level.
Abstract: Tellurite is highly toxic to most bacteria owing to its strong oxidative ability. However, some bacteria demonstrate tellurite resistance. In particular, some Escherichia coli strains, including Shiga toxin-producing E. coli O157:H7, are known to be resistant to tellurite. This resistance is involved in ter operon, which is usually located on a prophage-like element of the chromosome. The characteristics of the ter operon have been investigated mainly by genome analysis of pathogenic E. coli; however, the distribution and structural characteristics of the ter operon in other E. coli are almost unknown. To clarify these points, we examined 106 E. coli strains carrying the ter operon from various animals. The draft genomes of 34 representative strains revealed that ter operons were clearly classified into four subtypes, ter-type 1-4, at the nucleotide sequence level. Complete genomic sequences revealed that operons belonging to three ter-types (1, 3, and 4) were located on the prophage-like elements on the chromosome, whereas the ter-type 2 operon was located on the IncHI2 plasmid. The positions of the tRNASer, tRNAMet, and tRNAPhe indicated the insertion sites of elements carrying the ter operons. Using the PCR method developed in this study, 106 strains were classified as type 1 (n = 66), 2 (n = 13), 3 (n = 8), and 4 (n = 17), and two strains carried both types 1 and 2. Furthermore, significant differences in the minimum inhibitory concentration (MIC) of tellurite were observed between strains carrying ter-type 4 and the others (p < 0.05). The ter-type was also closely related to the isolation source, with types 2 and 4 associated with chickens and deer, respectively. This study provided new insights related not only to genetic characteristics of the ter operons, but also to phenotypic and ecological characteristics that may be related to the diversity of the operon.
13 citations
TL;DR: In this paper, the authors evaluated the profile, virulence, and gene resistance to antibiotics of presumptive C. sakazakii strains previously isolated from powdered and dairy formulas (P-DF).
Abstract: Cronobacter sakazakii is an enteropathogen that causes neonatal meningitis, septicemia, and necrotizing enteritis in preterm infants and newborns with a mortality rate of 40% to 80%. Powdered and dairy formulas (P-DF) have been implicated as transmission vehicles and the presence of this pathogen in P-DF led to product recalls in Chile in 2017. The objective of this study was to evaluate the profile, virulence, and gene resistance to antibiotics of presumptive C. sakazakii strains previously isolated from P-DF. The strains were re-identified by MALDI-TOF-MS and by whole genome sequencing (WGS). The in vitro detection of virulence genes was evaluated using PCR probes and adherence and invasion ability assays using the Mouse Neuroblastoma N1E-115 cell line. Antibiotic resistance profiles were determined using the disk diffusion method. In silico, plasmids, the virulence and resistant genes were detected using RidomSeqSphere+, PlasmidFinder, Comprehensive Antibiotic Resistance Database platform, and AMRFinderPlus. The CRISPR-cas system was searched with CRISPRCasFinder. Average Nucleotide Identity (ANI), ribosomal MLST and core genome MLST identified seven strains as C. sakazakii and one strain as Franconibacter helveticus. All C. sakazakii strains exhibited adherence, however, only 50% were able to invade. The C. sakazakii strains were positive for six virulence factors. The inv gene was identified in the genome of all C. sakazakii strains, despite being negative using PCR probes. Thirty-one virulence genes were detected by WGS; these were grouped as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, invasion, plasminogen activator, colonization, transcriptional regulator, survival in macrophages, and the use of sialic acid and toxin genes. Five strains were resistant to cephalothin, four to ampicillin, and two strains to ceftazidime and amoxicillin/clavulanic acid. We detected the mcr-9.1 and blaCSA genes conferring resistance to colistin and cephalothin, respectively. In addition, isolates carried the IncFII(pECLA), Col440I, and Col(pHHAD28) plasmids. All C. sakazakii strains showed CRISPR matrices and three strains had two type I-E and I-F matrices. The use of WGS led to the identification of C. sakazakii and determination of multiple antibiotic resistance and virulence genes. These findings support the decision to recall all the powdered and dairy formulas involved in Chile in 2017.
13 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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