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 nationwide study on the occurrence of extended-spectrum β-lactamase (ESBL)/AmpC in nonhospitalized horses in the Netherlands was performed and investigated the molecular characteristics, geographical distribution throughout the Netherlands, and potential risk factors for fecal ESBL carriage in horses.
Abstract: A nationwide study on the occurrence of extended-spectrum β-lactamase (ESBL)/AmpC in nonhospitalized horses in the Netherlands was performed. Molecular characterization was done, and questionnaires were analyzed to identify factors associated with carriage. In total, 796 horse owners were approached; 281 of these submitted a fecal sample from their horse(s), resulting in 362 samples. All samples were cultured qualitatively in Luria-Bertani (LB) broth and subsequently on MacConkey agar, both supplemented with 1 mg/liter cefotaxime (LB+ and MC+). Positive samples were subsequently cultured quantitatively on MC+. Initial extended-spectrum-β-lactamase (ESBL)/AmpC screening was performed by PCR, followed by whole-genome sequencing on selected strains. Associations between ESBL/AmpC carriage and questionnaire items were analyzed using a univariate generalized estimating equation (GEE) regression analysis, followed by a multiple GEE model for relevant factors. In total, 39 of 362 samples (11%) were determined to be positive for ESBL/AmpC. blaCTX-M-1-carrying isolates were obtained from 77% of positive samples (n = 30). Other ESBL/AmpC genes observed included blaCTX-M-2, blaCTX-M-14, blaCTX-M-15, blaCTX-M-32, blaSHV-12, blaCMY-2, and blaACT-10 A high association between the presence of blaCTX-M-1 and IncHI1 plasmids was observed (46% of samples; n = 18). Based on core genome analysis (n = 48 isolates), six Escherichia coli clusters were identified, three of which represented 80% of the isolates. A negative association between ESBL/AmpC carriage and horses being in contact with other horses at a different site was observed. The presence of a dog on the premises and housing in a more densely human-populated region were positively associated.IMPORTANCE Extended-spectrum β-lactamases (ESBLs) are widespread in human and animal populations and in the environment. Many different ESBL variants exist. The dissemination of ESBLs within and between populations and the environment is also largely influenced by genetic mobile elements (e.g., plasmids) that facilitate spread of these ESBLs. In order to identify potential attributable ESBL sources for, e.g., the human population, it is important to identify the different ESBL variants, the bacteria carrying them, and the potential risk factors for ESBL carriage from other potential sources. This nationwide study focuses on ESBL carriage in the open horse population and investigated the molecular characteristics, geographical distribution throughout the Netherlands, and potential risk factors for fecal ESBL carriage in horses. These data can be used for future attribution studies in order to reduce potential transmission of ESBL-producing bacteria between sources.
9 citations
Cites methods from "In Silico Detection and Typing of P..."
...Resistance genes and plasmid incompatibility groups were determined with ResFinder (33) and PlasmidFinder (34) by using the ABRicate tool (35)....
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TL;DR: A new transposon, Tn6696, has been detected on a blaNDM–1-carrying plasmid recovered from multidrug-resistant E. cloacae ssp.
Abstract: Background: Enterobacter cloacae (E. cloacae) is a pathogen which is responsible for serious nosocomial infections. A gene which plays an important role in resistance to carbapenems in E. cloacae is new Delhi metallo-β-lactamase 1 (NDM-1). Currently, the spread of E. cloacae which carry a plasmid containing blaNDM-1 is a serious public threat. Methods: A multidrug-resistant clone of E. cloacae, CBG15936, was recovered in 2017 in Guangzhou, China. PCR, S1-pulsed-field gel electrophoresis, and Southern blotting were performed to identify and locate the blaNDM-1 gene. Susceptibility testing and conjugation experiments were also performed. Illumina HiSeq and Nanopore sequencers were used to perform whole genome sequencing. Results: Strain CBG15936 belongs to ST932 and is resistant to carbapenems. Correspondingly, a blaNDM-1 gene was found in the IncN1 plasmid of this strain. This plasmid is approximately ~62 kb in length and has a conjugation frequency of 1.68 × 10−3 events per donor cell. A new transposon, Tn6696, was also identified on this plasmid. Tn6696 consists of an intact qnrS1-carrying Tn6292 element, an inverted 8.3-kb Tn3000 remnant, ISkpn19, ΔtnpA, and IS26. Conclusion: A new transposon, Tn6696, has been detected on a blaNDM-1-carrying plasmid recovered from multidrug-resistant E. cloacae CBG15936 from China. This finding provides a new perspective regarding the potential for blaNDM-1 to undergo horizontal transfer among drug-resistant bacteria.
9 citations
Cites methods from "In Silico Detection and Typing of P..."
...Plasmid replicon type was analyzed by using PlasmidFinder (Carattoli et al., 2014)....
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TL;DR: In this paper, the authors used next-generation sequencing (NGS) technology to map genes associated with antimicrobial resistance (AMR) and to identify multilocus sequence types (MLST).
Abstract: Carbapenem-resistant Acinetobacter baumannii (A. baumannii, CRAb) is an emerging global threat for healthcare systems, particularly in Southeast Asia. Next-generation sequencing (NGS) technology was employed to map genes associated with antimicrobial resistance (AMR) and to identify multilocus sequence types (MLST). Eleven strains isolated from humans in Vietnam were sequenced, and their AMR genes and MLST were compared to published genomes of strains originating from Southeast Asia, i.e., Thailand (n = 49), Myanmar (n = 38), Malaysia (n = 11), Singapore (n = 4) and Taiwan (n = 1). Ten out of eleven Vietnamese strains were CRAb and were susceptible only to colistin. All strains harbored ant(3")-IIa, armA, aph(6)-Id and aph(3") genes conferring resistance to aminoglycosides, and blaOXA-51 variants and blaADC-25 conferring resistance to s-lactams. More than half of the strains harbored genes that confer resistance to tetracyclines, sulfonamides and macrolides. The strains showed high diversity, where six were assigned to sequence type (ST)/2, and two were allocated to two new STs (ST/1411-1412). MLST analyses of 108 strains from Southeast Asia identified 19 sequence types (ST), and ST/2 was the most prevalent found in 62 strains. A broad range of AMR genes was identified mediating resistance to s-lactams, including cephalosporins and carbapenems (e.g., blaOXA-51-like, blaOXA-23, blaADC-25, blaADC-73, blaTEM-1, blaNDM-1), aminoglycosides (e.g., ant(3")-IIa, aph(3")-Ib, aph(6)-Id, armA and aph(3')-Ia), phenicoles (e.g., catB8), tetracyclines (e.g., tet.B and tet.39), sulfonamides (e.g., sul.1 and sul.2), macrolides and lincosamide (e.g., mph.E, msr.E and abaF). MLST and core genome MLST (cgMLST) showed an extreme diversity among the strains. Several strains isolated from different countries clustered together by cgMLST; however, different clusters shared the same ST. Developing an action plan on AMR, increasing awareness and prohibiting the selling of antibiotics without prescription must be mandatory for this region. Such efforts are critical for enforcing targeted policies on the rational use of carbapenem compounds and controlling AMR dissemination and emergence in general.
9 citations
TL;DR: It is suggested that attaining significant improvements in AMR meat safety requires the identification and removal (or treatment) of product harboring MDR NTS, instead of screening for Salmonella spp.
Abstract: Multi-drug resistant (MDR) non-typhoidal Salmonella (NTS) is a public health concern globally. This study reports the phenotypic and genotypic antimicrobial resistance (AMR) profiles of NTS isolates from bovine lymph nodes (n = 48) and ground beef (n = 29). Furthermore, we compared genotypic AMR data of our isolates with those of publicly available NTS genomes from Mexico (n = 2400). The probability of finding MDR isolates was higher in ground beef than in lymph nodes:χ2 = 12.0, P = 0.0005. The most common resistant phenotypes involved tetracycline (40.3%), carbenicillin (26.0%), amoxicillin-clavulanic acid (20.8%), chloramphenicol (19.5%) and trimethoprim-sulfamethoxazole (16.9%), while more than 55% of the isolates showed decreased susceptibility to ciprofloxacin and 26% were MDR. Conversely, resistance to cephalosporins and carbapenems was infrequent (0–9%). MDR phenotypes were strongly associated with NTS serovar (χ2 = 24.5, P<0.0001), with Typhimurium accounting for 40% of MDR strains. Most of these (9/10), carried Salmonella genomic island 1, which harbors a class-1 integron with multiple AMR genes (aadA2, blaCARB-2, floR, sul1, tetG) that confer a penta-resistant phenotype. MDR phenotypes were also associated with mutations in the ramR gene (χ2 = 17.7, P<0.0001). Among public NTS isolates from Mexico, those from cattle and poultry had the highest proportion of MDR genotypes. Our results suggest that attaining significant improvements in AMR meat safety requires the identification and removal (or treatment) of product harboring MDR NTS, instead of screening for Salmonella spp. or for isolates showing resistance to individual antibiotics. In that sense, massive integration of whole genome sequencing (WGS) technologies in AMR surveillance provides the shortest path to accomplish these goals.
9 citations
01 Jan 2019
TL;DR: Assessing the Clinical Significance of LUT Microbiota Variability Following Vaginal Intercourse and assessing the Ability of Urinary Isolates to Adhere to Urothelial Cells.
Abstract: xix CHAPTER I: INTRODUCTION AND LITERATURE REVIEW 1 The Human Microbiota 1 The Role of the Human Microbiota 2 Measuring the Microbiome 3 Analyzing Microbiome Data 5 “Urine Denial”: The Concept of Bladder Sterility 7 The Discovery of the Female Urinary Microbiota and Microbiome 9 Urine is Not Sterile: Now What? 13 Correlations with Health and Disease 16 Description of Urinary Bacterial Isolates 21 Other Urinary Microorganisms 25 Description of the Male Urinary Microbiota 25 Correlations with Health and Disease 28 Correlations with Lifestyle and Preliminary Measures of Stability 30 Differences between the Male and Female Urinary Microbiota 31 The Microbiome is Shaped by Environment 31 The GI Microbiota and Lifestyle 33 Description of GI Tract Physiology 33 Correlations between the GI Microbiota and Lifestyle 34 Temporal Dynamics of the GI Microbiota 36 The Vaginal Microbiota and Lifestyle 39 Description of Vaginal Tract Physiology 39 Correlations between the Vaginal Microbiota, BV Risk, and Lifestyle 40 Temporal Dynamics of the Vaginal Microbiota 41 UTI Risk and Lifestyle 44 Current Understanding of UTI and an Overview of Risk Factors 44 vii Probiotics and UTI Risk 45 Correlations between UTI Risk and Lifestyle 47 Correlations between UTI Risk and Urine Properties 53 Longitudinal Assessment of the Lower Urinary Tract Microbiota 54 Rational and Problems 55 What Specimen to Collect? 56 Preliminary Findings 63 Summary 64 CHAPTER II: MATERIALS AND METHODS 67 Overview of Clinical Studies 67 MAGnUM Study 68 ProFUM Study 70 PARTNER Study 76 Bacterial Identification Methods 80 Specimen Collection 80 Expanded Culture Protocol 81 16S Sequencing Protocol 82 Dipstick Urinalysis 85 Storage of Bacterial Isolates 85 Statistical Analyses 85 Whole Genome Sequencing 86 DNA Extraction and Library Prep 86 Assembly and Annotation 87 Bacterial Assays: Experimental Procedures 88 Bacterial Strains and Growth Conditions 88 Urothelial Cell Adherence Assays 93 Biofilm Assays 94 Growth Competition Assays 95 CHAPTER III: FEASIBILITY PROJECT: MAGnUM STUDY 96 Overview of MAGnUM Study 96 Longitudinal Assessment of the Microbiota of the Male LUT 96 Longitudinal Assessment of the Microbiota of the Female LUT 100 Summary 106 CHAPTER IV: EFFECT OF INTRINSIC LIFESTYLE FACTORS ON LUT MICROBIOTA 109 Overview of ProFUM Study 109 Screening Eligible Participants 109 viii ProFUM Specimen Integrity 112 Qualitative Description of the Longitudinal LUT Microbiota and Microbiome 115 Measuring the Overall Stability of the LUT Microbiota 122 Menstruation and LUT Microbiota Variability 125 Urine Properties and Constituents and LUT Microbiota Variability 138 Summary 143 CHAPTER V: EFFECT OF EXTRINSIC LIFESTYLE FACTORS ON LUT MICROBIOTA 147 Sexual Activity and LUT Microbiota Variability 147 Hygiene and LUT Microbiota Variability 159 Oral Probiotics and LUT Microbiota Variability 163 Summary 165 CHAPTER VI: MECHANISTIC DETERMINATION AND SIGNIFICANCE OF LUT MICROBIOTA VARIABILITY AND VAGINAL INTERCOURSE 168 Introduction and Rationale 168 Assessing the Clinical Significance of LUT Microbiota Variability Following Vaginal Intercourse 170 Effect of Urinary Isolates on UPEC Adherence to Urothelial Cells 170 Effect of Urinary Isolates on UPEC Biofilm Formation 172 Effect of Urinary Isolates on UPEC Growth 175 Assessing the Ability of Urinary Isolates to Adhere to Urothelial Cells 181 Assessing the Biological Significance of LUT Microbiota Variability Following Vaginal Intercourse 183 Overview of PARTNER Study 183 Description of Microbiota Findings 184 Measuring Clonal Relatedness of S. mitis Isolates 190 Summary 191 CHAPTER VII: DISCUSSION 194 Brief Summary 194 Discussion of Chapter III 194 Discussion of Chapter IV 199 Discussion of Chapter V 208 Discussion of Chapter VI 215 Significance and Implications 220 Future Directions 224
9 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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