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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31 May 2022
TL;DR: In this paper , the plasmid pOXA-48 was tracked in a large collection of enterobacterial clones isolated from the gut of hospitalised patients, and the authors observed that the evolution of plasmID-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness.
Abstract: Abstract Antibiotic resistance (AMR) in bacteria is a major threat to public health, and one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been extensively studied in vitro , but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here, we tracked the evolution of the clinically-relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalised patients. Combining genomic and experimental approaches, we first characterized plasmid diversity and the genotypic and phenotypic effects of multiple plasmid mutations on a common genetic background. Second, using cutting-edge genomic editing in wild-type multidrug resistant enterobacteria, we dissected three cases of within-patient plasmid-mediated AMR evolution. Our results revealed, for the first time, compensatory evolution of plasmid-associated fitness cost, as well as the evolution of enhanced plasmid-mediated AMR, in bacteria evolving within the gut of hospitalised patients. Crucially, we observed that the evolution of plasmid-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness. This study highlights the need to develop new evolution-informed approaches to tackle plasmid-mediated AMR dissemination.
3 citations
TL;DR: Osdaghi et al. as mentioned in this paper presented the whole genome resources of 17 Curtobacterium flaccumfaciens Strains including Pathotypes of C. betae, C. oortii, and C. poinsettiae.
Abstract: HomeMolecular Plant-Microbe Interactions®Vol. 35, No. 4Whole Genome Resources of 17 Curtobacterium flaccumfaciens Strains Including Pathotypes of C. flaccumfaciens pv. betae, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseWhole Genome Resources of 17 Curtobacterium flaccumfaciens Strains Including Pathotypes of C. flaccumfaciens pv. betae, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiaeEbrahim Osdaghi, Geraldine Taghouti, Cecile Dutrieux, S. Mohsen Taghavi, Amal Fazliarab, Martial Briand, Marion Fischer-Le Saux, Perrine Portier, and Marie-Agnes JacquesEbrahim Osdaghi†Corresponding authors: E. Osdaghi; E-mail Address: eosdaghi@ut.ac.ir, P. Portier; E-mail Address: perrine.portier@inrae.fr, and M.-A. Jacques; E-mail Address: marie-agnes.jacques@inrae.frhttps://orcid.org/0000-0002-0359-0398Department of Plant Protection, College of Agriculture, University of Tehran, Karaj 31587-77871, IranSearch for more papers by this author, Geraldine TaghoutiUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, FranceSearch for more papers by this author, Cecile DutrieuxUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, FranceSearch for more papers by this author, S. Mohsen TaghaviDepartment of Plant Protection, School of Agriculture, Shiraz University, Shiraz 71441-65186, IranSearch for more papers by this author, Amal FazliarabIranian Sugarcane Research and Training Institute (ISCRTI), Ahvaz, Khuzestan, IranSearch for more papers by this author, Martial BriandUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, FranceSearch for more papers by this author, Marion Fischer-Le SauxUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, FranceSearch for more papers by this author, Perrine Portier†Corresponding authors: E. Osdaghi; E-mail Address: eosdaghi@ut.ac.ir, P. Portier; E-mail Address: perrine.portier@inrae.fr, and M.-A. Jacques; E-mail Address: marie-agnes.jacques@inrae.frUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, FranceSearch for more papers by this author, and Marie-Agnes Jacques†Corresponding authors: E. Osdaghi; E-mail Address: eosdaghi@ut.ac.ir, P. Portier; E-mail Address: perrine.portier@inrae.fr, and M.-A. Jacques; E-mail Address: marie-agnes.jacques@inrae.frUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, FranceSearch for more papers by this author AffiliationsAuthors and Affiliations Ebrahim Osdaghi1 † Geraldine Taghouti2 Cecile Dutrieux2 S. Mohsen Taghavi3 Amal Fazliarab4 Martial Briand2 Marion Fischer-Le Saux2 Perrine Portier2 † Marie-Agnes Jacques2 † 1Department of Plant Protection, College of Agriculture, University of Tehran, Karaj 31587-77871, Iran 2University of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, France 3Department of Plant Protection, School of Agriculture, Shiraz University, Shiraz 71441-65186, Iran 4Iranian Sugarcane Research and Training Institute (ISCRTI), Ahvaz, Khuzestan, Iran Published Online:14 Mar 2022https://doi.org/10.1094/MPMI-11-21-0282-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Curtobacterium flaccumfaciens complex species in the family Microbacteriaceae encompasses a group of plant-pathogenic actinobacterial strains affecting annual crops and ornamental plants. The species includes five pathovars, namely C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. ilicis, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae. Despite the economic importance of C. flaccumfaciens, its members have rarely been investigated for their phylogenetic relationships, molecular characteristics, and virulence repertories, due, in part, to the lack of whole-genome resources. Here, we present the whole-genome sequence of 17 C. flaccumfaciens strains representing members of four pathovars isolated from different plant species in a diverse geographical and temporal span. The genomic data presented in this study will pave the way for research on the comparative genomics, phylogenomics, and taxonomy of C. flaccumfaciens and extend our understanding of the virulence features of the species.Gram-positive actinobacterial plant pathogens in the family Microbacteriaceae comprise a series of economically important agents infecting several annual crops, ornamental plants, and vegetables (Dye and Kemp 1977; Jacques et al. 2012). Plant-pathogenic members in this group include Clavibacter spp., Rathayibacter spp., Leifsonia xyli, and Curtobacterium flaccumfaciens (Vidaver and Davis 1988). The latter species is a complex taxon comprising several pathovars as well as nonpathogenic, environmental, and, in some cases, clinical species (Francis et al. 2011). Among the gram-positive bacterial plant pathogens, C. flaccumfaciens is the least-studied member in terms of genomic features and pathogenicity determinants (Chen et al. 2021; Thapa et al. 2019). Plant-pathogenic members of C. flaccumfaciens are subdivided into five pathovars based on their host of isolation, pathogenicity, and host range (Collins and Jones 1983; Davis and Vidaver 2001; Davis 1986, 2001). Pathovars of the species include C. flaccumfaciens pv. flaccumfaciens, causing bacterial wilt of dry beans (Hedges 1922), C. flaccumfaciens pv. poinsettiae, causing bacterial canker of poinsettia (Pirone and Bender 1941), C. flaccumfaciens pv. betae, the agent of silvering disease of red beet (Keyworth et al. 1956), C. flaccumfaciens pv. ilicis, causing bacterial blight of American holly (Mandel et al. 1961; Young et al. 2004), and C. flaccumfaciens pv. oortii, the agent of bacterial wilt and spot of tulip (Saaltink and Maas Geesteranus 1969). Two additional pathovars including ‘C. flaccumfaciens pv. basellae’, the causal agent of bacterial leaf spot of malabar spinach (Basella alba or B. ruba) (Chen et al. 2000), and ‘C. flaccumfaciens pv. beticola’, the causal agent of bacterial leaf spot of sugar beet (Chen et al. 2007) were also proposed. However, the Committee on the Taxonomy of Plant Pathogenic bacteria (Bull et al. 2010) has not accepted them to date as valid taxa.Among these pathovars, bacterial wilt of dry beans caused by C. flaccumfaciens pv. flaccumfaciens and bacterial canker of poinsettia caused by C. flaccumfaciens pv. poinsettiae are economically important, the former one being included as a quarantine pathogen in the Annex II part A of European Regulation 2019/2072 and in the A2 list of the European and Mediterranean Plant Protection Organization and the latter one being included in the alert lists of EPPO (EPPO 2011; Osdaghi et al. 2020a). While molecular diagnostic tests are available to detect and identify C. flaccumfaciens pv. flaccumfaciens (Tegli et al. 2020), quarantined members of the species require considerable cost and effort to prevent the risk of global spread and introduction into areas yet free of the disease (Harveson et al. 2015; Osdaghi et al. 2020a). Members of C. flaccumfaciens are well-known for variability in colony pigmentation and morphology possessing a wide range of colony variants including orange, pink, purple, red, and yellow phenotypes in either fluidal, mucoid, or dry form on culture media (Harveson and Vidaver 2008; Osdaghi and Lak 2015; Osdaghi et al. 2016). As a complex species, population structure and taxonomy of C. flaccumfaciens has rarely been examined using high throughput genomics techniques, leaving the phylogenetic relationships of different pathovars to each other uninvestigated. Further, due to the lack of whole-genome resources from different pathovars of C. flaccumfaciens, pathogenicity-associated genomic features and virulence strategies of its members are still almost entirely unknown. Unlike the other plant-pathogenic actinobacteria, for which the nucleotide sequence data from a wide range of strains are available (Ansari et al. 2019; Jacques et al. 2012; Osdaghi et al. 2020b), whole-genome sequence data of only a few C. flaccumfaciens strains are published in the literature (Chen et al. 2021). Among the pathotype strains of the species, C. flaccumfaciens pv. flaccumfaciens is the only one for which a whole-genome sequence is available (Gonçalves et al. 2019).DNA fingerprinting-based investigations (Agarkova et al. 2012; Osdaghi et al. 2018a) and multilocus sequence analysis (MLSA) (Gonçalves et al. 2019: Osdaghi et al. 2018b) suggested high genetic diversity among plant pathogenic members of C. flaccumfaciens either in an intrapathovar or interpathovar level. For instance, it has been noted that yellow-pigmented strains of the dry bean pathogen C. flaccumfaciens pv. flaccumfaciens are genetically and phylogenetically distinct from those of red- and orange-pigmented strains of the same pathovar (Osdaghi et al. 2018a, b). Interestingly, MLSA-based results showed that the red- and orange-pigmented strains of the latter pathogen are phylogenetically more related to the poinsettia pathogen C. flaccumfaciens pv. poinsettiae than to the pathotype strain of C. flaccumfaciens pv. flaccumfaciens. All these findings led us to conclude that an inclusive genome sequencing effort is warranted to shed light on the taxonomic structure, phylogenetic relationships, and genomic repertoires of C. flaccumfaciens members. Thus, the main objective of this study was to provide whole-genome resources for 17 C. flaccumfaciens strains, covering nearly the entire genetic diversity of the species, including the pathotype strains of C. flaccumfaciens pv. betae, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae.C. flaccumfaciens strains representing members of different pathovars with various colony morphology (pigmentation) and diverse geographic origins were obtained from the French Collection for Plant-Associated Bacteria CIRM-CFBP (Table 1). The strains were streaked onto yeast-extract peptone glucose agar medium, were resuspended in sterile distilled water and stored at 4°C for further use, and were stored in 15% glycerol at −70°C for long-term purposes. Bacterial DNAs were extracted using the Wizard genomic DNA purification kit (Promega, Madison WI, U.S.A.). The 17 strains were sequenced using NovaSeq 6000 Illumina with Nextera XT Preparation kit and paired-end 150 bp (Institut du Cerveau, CHU Pitié-Salpêtrière, Paris). Genome assembly was performed using SPAdes 1 (Bankevich et al. 2012) after a quality filtration with CheckM (Parks et al. 2015). For each strain, sequencing coverage, genome size, number of contigs, and G+C percentage are summarized in Table 1. Genome annotation was performed using the GeneMarkS+ suite implemented in the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline with default settings (Borodovsky and Lomsadze 2014). Total number of genes and coding sequences were determined for all the genomes (Table 1).Table 1. Source, place, and date of isolation, and genomic information of the Curtobacterium flaccumfaciens strains used in this studyStrainOther namesTaxonHostYearCountryColony color (pigmentation)Genome size (bp)No. ofG+C% contentCoverage ×Plasmid/phagebGenBank accession numberCDSsaGenesContigsPlasmid FinderSourceFinderPhagecPlasmidCFBP 2402PTICMP 2594PT, LMG 3596PT, NCPPB 374PTC. flaccumfaciens pv. betaeBeta vulgaris1955U.K.Yellow-Dry3,768,0773,5343,5895070.9219ND45, 49, 50NDJAHEXD000000000CFBP 1384PTNCPPB 2113PT, ATCC 25283PT, ICMP 2632PT, LMG 3702PTC. flaccumfaciens pv. oortiiTulipa gesneriana1967NetherlandsYellow-Fluidal4,024,4223,8213,8786470.7249ND7, 54, 59, 61, 63, 64NDJAHEXC000000000CFBP 2403PTICMP 2566PT, ATCC 9682PT, NCPPB 854PTC. flaccumfaciens pv. poinsettiaeEuphorbia pulcherrimaMissingU.S.A.Orange-Dry3,616,0773,3963,4505471.0219ND39, 53NDJAHEXB000000000CFBP 3400PD 1751C. flaccumfaciens pv. oortiiZantedeschia aethiopica1990NetherlandsYellow-Dry3,773,5923,5593,6144871.0249NDNDNDJAHEXA000000000CFBP 3423NCPPB 2344, ATCC 23827C. flaccumfaciens pv. flaccumfaciensPhaseolus vulgaris1957U.S.A.Yellow-Fluidal3,906,8943,6823,7395270.5204ND25, 41, 43, 46, 51, 5240, 44, 45JAHEWZ000000000CFBP 3422NCPPB 2343, ATCC 12813C. flaccumfaciens pv. flaccumfaciensPhaseolus vulgaris1956U.S.A.Orange-Dry3,587,1233,3493,4044571.0234ND2, 44NDJAHEWY000000000CFBP 3417NCPPB 558C. flaccumfaciens pv. flaccumfaciensPhaseolus vulgaris1958U.S.A.Orange-Fluidal3,731,9363,5493,6044870.8264ND15, 22, 40, 46, 47, 4836JAHEWX000000000CFBP 3401LMG 7238, PDDCC 4735C. flaccumfaciens pv. betaeBeta vulgarisMissingU.K.Yellow-Dry3,771,8933,5403,5955270.9310ND9, 17, 19, 40, 42, 43, 46, 47, 48, 49, 50NDJAHEWW000000000CFBP 3415LMG 7321C. flaccumfaciens pv. poinsettiaeEuphorbia pulcherrimaMissingU.S.A.Orange-Fluidal3,693,8183,4643,5184370.9332ND40, 42, 43NDJAHEWU000000000CFBP 8818Tom50, ICMP 22062C. flaccumfaciens pv. flaccumfaciensSolanum lycopersicum2015IranRed-Dry3,718,2763,4973,5537370.9189ND19, 56, 59, 63, 67NDJAHEWT000000000CFBP 881950R, ICMP 22071C. flaccumfaciens pv. flaccumfaciensPhaseolus vulgaris2014IranRed-Fluidal3,722,5623,5043,5566370.8219ND24, 44, 48, 53, 57, 60NDJAHEWS000000000CFBP 8820P990, ICMP 22053C. flaccumfaciens pv. flaccumfaciensCapsicum annuum2015IranYellow-Fluidal3,841,5553,6183,6735670.9234ND24, 52, 55, 56NDJAHEWR000000000CFBP 8821Cmmeg20, ICMP 22056C. flaccumfaciensSolanum melongena2014IranYellow-Fluidal3,667,5573,4173,4704771.0189ND46, 47NDJAHEWQ000000000CFBP 8822Xeu15, ICMP 21400C. flaccumfaciensCapsicum annuum2013IranYellow-Fluidal3,715,9763,4953,5494771.0219ND46, 47NDJAHEWP000000000CFBP 8823Cff156C. flaccumfaciens pv. flaccumfaciensPhaseolus vulgaris2015IranOrange-Fluidal3,749,4033,5193,5766570.8227ND12, 23, 53, 56, 59, 64NDJAHEWO000000000CFBP 8824G105, ICMP 22064C. flaccumfaciensSolanum lycopersicum2015IranRed-Fluidal3,603,5413,3693,4223571.0287ND8, 33, 35NDJAHEWN000000000CFBP 8825Tom827, ICMP 22084C. flaccumfaciensSolanum lycopersicum2015IranYellow-Fluidal3,707,673 3,4873,5424771.0189ND25, 45, 46NDJAHEWM000000000aCDS = coding sequences.bPresence of plasmids was evaluated using both PlasmidFinder and SourceFinder services, while the presence of phages was evaluated using SourceFinder. ND = Not detected.cThe numbers indicate the contig number in the FASTA files (in the GenBank database) in which the corresponding sequence was detected.Table 1. Source, place, and date of isolation, and genomic information of the Curtobacterium flaccumfaciens strains used in this studyView as image HTML Whole-genome sequences of the 17 C. flaccumfaciens strains obtained in this study were assembled in contigs varying in number from 35 to 73, while sequencing coverage was between 189× and 332× (Table 1). Genome size of the strains ranged from 3,587,123 bp in C. flaccumfaciens pv. flaccumfaciens CFBP 3422 to 4,024,422 bp in the pathotype strain of C. flaccumfaciens pv. oortii CFBP 1384PT, while G+C% content was between 70.5 and 71.0% (Table 1). It has previously been shown that a number of C. flaccumfaciens strains possess plasmids harboring various biological functions (Chen et al. 2021; Hendrick et al. 1984; Vaghefi et al. 2021). Hence, the genome sequences obtained in this study were investigated for the presence of plasmid and phage sequences via online services PlasmidFinder 2.0 and SourceFinder 1.0 (Carattoli et al. 2014). While PlasmidFinder did not find any plasmid in the genome sequences, SourceFinder suggested the presence of plasmid in the C. flaccumfaciens pv. flaccumfaciens strains CFBP 3423 and CFBP 3417. Phage fragments were also frequently detected in all the genomes except C. flaccumfaciens pv. oortii CFBP 3400, as detailed in Table 1. Agarose gel–based plasmid profiling is recommended to decipher the precise plasmid profile of the strains sequenced in this study. Furthermore, a preliminary average nucleotide identity (ANI) and digital DNA-DNA hybridization calculation among the strains sequenced in this study suggest a need for an in-depth taxonomic investigation on the phylogenetic relationships of the C. flaccumfaciens members. For instance, ANI between the type strain of C. flaccumfaciens CFBP 3418T and the pathotype strain of C. flaccumfaciens pv. poinsettiae CFBP 2403PT was 93.5%, which was below the accepted threshold (95 to 96%) for definition of prokaryotic species (Kim et al. 2014). A comprehensive multiphasic taxonomic study is ongoing to re-evaluate the taxonomy of C. flaccumfaciens complex species.Data AvailabilityThe whole-genome shotgun sequences obtained in this study are deposited at the NCBI GenBank database under the accession numbers shown in Table 1. Furthermore, a pure culture of the 17 strains is available in CIRM-CFBP culture collection.AcknowledgmentsWe thank C. Dutrieux and A. Lathus at CIRM-CFBP for preservation of the bacterial strains.The author(s) declare no conflict of interest.Literature CitedAgarkova, I. V., Lambrecht, P. A., Vidaver, A. K., and Harveson, R. M. 2012. Genetic diversity among Curtobacterium flaccumfaciens pv. flaccumfaciens populations in the American high plains. 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This is an open access article distributed under the CC BY-NC-ND 4.0 International license.DetailsFiguresLiterature CitedRelated Vol. 35, No. 4 April 2022ISSN:0894-0282e-ISSN:1943-7706 Download Metrics Downloaded 867 times Article History Issue Date: 12 Apr 2022Published: 14 Mar 2022First Look: 12 Jan 2022Accepted: 10 Jan 2022 Pages: 352-356 InformationCopyright © 2022 The Author(s).This is an open access article distributed under the CC BY-NC-ND 4.0 International license.FundingInstitut National de la Recherche AgronomiqueGrant/Award Number: CIRM 2020College of Agriculture Natural Resources, University of TehranGrant/Award Number: CA16107Keywordsactinobacteriabacterial wilt of beanscoryneform bacteriaMicrobacteriaceaequarantine pathogenbacterial pathogenesisThe author(s) declare no conflict of interest.PDF download
3 citations
Dissertation•
01 Apr 2016
TL;DR: This chapter discusses molecular typing techniques and whole-genome sequencing analysis in Staphylococcus, a worldwide concern in the clinical setting, and its role in the evolution of Methicillin resistance.
Abstract: ............... ....................................................................................................................... ix RESUMO................. ...................................................................................................................... xv THESIS OUTLINE..... ...................................................................................................................... xxi LIST OF ABBREVIATIONS ............................................................................................................ xxiii Chapter I: General Introduction .................................................................................................... 1 1. Staphylococcus in the clinical setting: a worldwide concern .................................................... 3 1.1. The genus Staphylococcus and clinically relevant species ............................................. 3 1.2. Molecular typing techniques and whole-genome sequencing analysis in Staphylococcus....... ....................................................................................................................... 5 1.2.1. Pulsed-field gel electrophoresis (PFGE) ............................................................... 5 1.2.2. Multilocus sequence typing (MLST) ..................................................................... 7 1.2.3. Whole-genome sequence analysis ...................................................................... 7 1.2.3.1. Historical prespective ................................................................................ 7 1.2.3.2. Whole-genome sequencing analysis ....................................................... 12 1.2.3.2.1. Closed reference genome-based analysis .................................... 12 1.2.3.2.2. De novo assembly ......................................................................... 13 1.2.3.2.3. Downstream analysis ................................................................... 14 1.3 The opportunistic pathogen Staphylococcus epidermidis ............................................. 15 1.3.1. Staphylococcus epidermidis as a commensal .................................................... 15 1.3.2. Staphylococcus epidermidis as a pathogen ....................................................... 17 1.3.3. Molecular epidemiology and population structure ........................................... 19 1.3.4. Virulence genes .................................................................................................. 23 2. Methicillin resistance: a crucial event in the evolution of Staphylococcus ............................. 27 2.1. Mechanism of resistance .............................................................................................. 28 2.2. The staphylococcal cassette chromosome mec (SCCmec) ........................................... 29 2.2.1. Historical perspective ........................................................................................ 29 2.2.2. Basic structure and diversity .............................................................................. 29 2.2.3. Transfer and mobility ......................................................................................... 31 2.2.4. The mec complex ............................................................................................... 32 2.2.5. The ccr complex ................................................................................................. 34
3 citations
Cites background from "In Silico Detection and Typing of P..."
...fr/), rep genes with Plasmid finder (233) (https://cge....
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Dissertation•
29 Apr 2020
TL;DR: Evaluated bivalve mollusks as tools for monitoring Escherichia coli and associated AR, in the marine environment in Norway to generate knowledge regarding the prevalence of antibiotic and heavy metal resistance, and associated resistance genes, among other things.
Abstract: Antibiotic resistance (AR) is a major global health concern, especially in clinical and veterinary settings. Environmental niches, including the aquatic environment, serve as a source of and/or a dissemination route for antibiotic resistance genes (ARGs) and resistant bacteria. Bivalves are suspension feeders that actively filter, retain and concentrates particles from their surrounding water, including free living or particlebound bacteria. The main aim of this thesis was to evaluate bivalve mollusks as tools for monitoring Escherichia coli and associated AR, in the marine environment in Norway. Sampling of bivalves were conducted from several sites along the Norwegian coast and the samples were examined for the presence of E. coli, according to the most probable number (MPN) EU reference method. More than half (61%) of the samples were positive for E. coli, and a selection of 200 E. coli isolates were further identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDITOF MS). The majority (90%) were confirmed as E. coli, while the remaining isolates (10%) were identified as other species mostly belonging to the Enterobacteriaceae family. The isolates were antibiotic susceptibility tested (AST) using the disk diffusion method recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Seventy-five bacterial isolates (38%) showed phenotypic resistance to at least one antibiotic, while multidrug-resistance was observed in eight isolates (4%). Based on resistance phenotypes, selected E. coli isolates were subjected to whole-genome sequencing (WGS). Two isolates revealed to carry CTX-M-type extended-spectrum β-lactamases (ESBLs). Accordingly, the two E. coli isolates were subjected to long-read sequencing, and a hybrid de novo assembly using long-reads and short-reads to obtain complete and closed genome sequences. One isolate harbored four identical chromosomal copies of the blaCTX-M-14 gene, while the other isolate carried the blaCTX-M-15 gene on a conjugative plasmid. Another aim of this thesis was to generate knowledge regarding the prevalence of antibiotic and heavy metal resistance, and associated resistance genes, among
3 citations
Cites methods from "In Silico Detection and Typing of P..."
...Plasmid replicons 26 were typed using PlasmidFinder v.2.0 (Carattoli et al., 2014) as well as BLASTP analysis of the replication initiation (Rep) sequence against the NCBI database....
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TL;DR: The prevalence of carbapenemase-producing Enterobacterales has become a significant threat to public health and the number of cases is expected to increase in the coming years.
Abstract: The prevalence of carbapenemase-producing Enterobacterales has become a significant threat to public health.…
3 citations
Cites background from "In Silico Detection and Typing of P..."
...The blaKPC-2-carrying plasmid pM92-KPC2 could not be assigned to any known incompatibility group by PlasmidFinder (4)....
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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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