GeXplore : development of e genome-wide approach to studying host-pathogen

Les technologies d'études génomiques ont été utilisées avec succès pour l’identification de facteurs de virulence bactériens ou de vaccins potentiels. Malheureusement, la complexité et les limitations intrinsèques relatives à ces approches ont entravé leur large diffusion. Nous présentons le développement d'une technologie d'étude à l'échelle du génome entier, appelée GeXplore. Elle est basée sur le ribosome display et a été conçue pour fonctionner en conditions in vitro, afin d'éviter des inconvénients d'approches telles que le phage display. Nous avons utilisé la plate-forme Illumina pour obtenir la carte-génomique de 3 espèces : S. aureus, S. gallolyticus et M. ulcerans. Les génomes obtenus ont été caractérisés et utilisés comme références pour l'analyse des banques et produits de sélection. Nous avons développé et optimisé ensuite une stratégie de clonage alternative qui permet la préparation in vitro des banques et leurs amplifications. Nous avons analysé la représentativité des banques à l'aide du NGS et observé un biais pour les séquences pauvres en Gc. Puis, nous avons amélioré la spécificité de notre méthode et l'avons validée en utilisant une interaction protéine-ligand bien caractérisée. GeXplore est ainsi capable d'identifier de multiples domaines de ligands au sein de banques génomiques provenant d'organismes pathogènes. Enfin, nous avons tenté d'identifier les immunoprotèomes de S. gallolyticus et M. ulcerans en utilisant des anticorps provenant de patients. Nous démontrons que notre méthode permet d'identifier de multiples domaines de protéines exposées à la surface présentant des propriétés potentiellement antigéniques / immunogènes.Genome-wide display technologies have been successfully used for identification of multiple bacterial virulence factors and potential vaccine candidates. Unfortunately, the relative complexity and intrinsic limitations of such approaches have hampered their wide spread in the scieniific community. Here, we present the development of a streamlined genome-wide display technology, which we term GeXplore. It is based on ribosome display and was designed 10 work under completely in vitro conditions, therefore aiming at avoiding several drawbacks of earner approaches which involve in vivo step{s) such as cell surface or phage display. First, we used Illumina platform to obtain draft genomes of S. aureus, S. gallolyticus and M.ulcerans isolates. Obtained drafts were partially characterized and used as reference sequences for selection input and output analysis. Secondly, we developed an alternative GC-assisted cloning strategy which allows for completely in vitro preparation and amplification of random genomic libraries. We analysed the coverage 01 the obtained libraries using next-qeneration sequencing and report an important source of coverage bias. Thirdly, we improved the specificity our method which was then validated using a well-characterized protein-ligand interaction. We demonstrate that GeXplore is able to identify multiple ligand-biding domains out of pathogen-derived genomic libraries. Finally, we applied GeXplore to identify immune-relevant proteomes of S. gallolyticus and M. ulcerans using patient-derived antibodies. We demonstrate that our method allows the identification of multiple surface-exposed protein domains with potentially antigenic/immunogenic properties

Draft Genome Sequences of Two Highly Erythromycin-Resistant Streptococcus gallolyticus subsp. gallolyticus Isolates Containing a Novel Tn916-Like Element, Tn6331

International audienceRecently, we reported the draft genome sequence of Streptococcus gal-lolyticus NTS31106099. It was found to contain a previously unknown putative Tn916-like conjugative transposon, Tn6263. Here, we report the draft genome sequences of two other clinical isolates, NTS31301958 and NTS31307655. Both of them contain another novel element, Tn6331, which is highly similar to Tn6263

Novel Tn916-like elements confer aminoglycoside/macrolide co-resistance in clinical isolates of Streptococcus gallolyticus ssp. gallolyticus

International audienceBackground:Streptococcus gallolyticus ssp. gallolyticus (Sgg) is a commensal bacterium and an opportunistic pathogen. In humans it has been clinically associated with the incidence of colorectal cancer (CRC) and epidemiologically recognized as an emerging cause of infective endocarditis (IE). The standard therapy of Sgg includes the administration of a penicillin in combination with an aminoglycoside. Even though penicillin-resistant isolates have still not been reported, epidemiological studies have shown that this microbe is a reservoir of multiple acquired genes, conferring resistance to tetracyclines, aminoglycosides, macrolides and glycopeptides. However, the underlying antibiotic resistance mobilome of Sgg remains poorly understood.Objectives:To investigate the mobile genetic basis of antibiotic resistance in multiresistant clinical Sgg.Methods:Isolate NTS31106099 was recovered from a patient with IE and CRC at Nantes University Hospital, France and studied by Illumina WGS and comparative genomics. Molecular epidemiology of the identified mobile element(s) was performed using antibiotic susceptibility testing (AST), PCR, PFGE and WGS. Mobility was investigated by PCR and filter mating.Results:Two novel conjugative transposons, Tn6263 and Tn6331, confer aminoglycoside/macrolide co-resistance in clinical Sgg. They display classical family Tn916/Tn1545 modular architecture and harbour an aph(3')-III→sat4→ant(6)-Ia→erm(B) multiresistance gene cluster, related to pRE25 of Enterococcus faecium. These and/or closely related elements are highly prevalent among genetically heterogeneous clinical isolates of Sgg.Conclusions:Previously unknown Tn916-like mobile genetic elements conferring aminoglycoside/macrolide co-resistance make Sgg, collectively with other gut Firmicutes such as enterococci and eubacteria, a potential laterally active reservoir of these antibiotic resistance determinants among the mammalian gastrointestinal microbiota

Draft Genome Sequences of Two Highly Erythromycin-Resistant Streptococcus gallolyticus subsp. gallolyticus Isolates Containing a Novel Tn916-Like Element, Tn6331

International audienceRecently, we reported the draft genome sequence of Streptococcus gal-lolyticus NTS31106099. It was found to contain a previously unknown putative Tn916-like conjugative transposon, Tn6263. Here, we report the draft genome sequences of two other clinical isolates, NTS31301958 and NTS31307655. Both of them contain another novel element, Tn6331, which is highly similar to Tn6263

Draft Genome Sequence of Erythromycin-Resistant Streptococcus gallolyticus subsp. gallolyticus NTS 31106099 Isolated from a Patient with Infective Endocarditis and Colorectal Cancer

International audienceStreptococcus gallolyticus subsp. gallolyticus is known for its close association with infective endocarditis and colorectal cancer in humans. Here, we report the draft genome sequence of highly erythromycin-resistant strain NTS 31106099 isolated from a patient with infective endocarditis and colorectal cancer. Citation Kambarev S, Caté C, Corvec S, Pecorari F. 2015. Draft genome sequence of erythromycin-resistant Streptococcus gallolyticus subsp. gallolyticus NTS 31106099 isolated from a patient with infective endocarditis and colorectal cancer

Draft Genome Sequence of Highly Rifampin-Resistant Propionibacterium namnetense NTS 31307302T Isolated from a Patient with a Bone Infection

International audiencePropionibacterium namnetense was recently described as a potential bone pathogen, which is closely related to Propionibacte-rium acnes, a skin commensal microorganism. Here, we report the draft genome sequence of the highly rifampin-resistant strain NTS 31307302 T isolated from a patient with a tibia infection

In vitro emergence of fluoroquinolone resistance in Cutibacterium (formerly Propionibacterium ) acnes and molecular characterization of mutations in the gyrA gene

International audienceIn vitro occurrence of levofloxacin (LVX) resistance in C. acnes and characterization of its molecular background were investigated. The mutation frequency was determined by inoculation of 108 cfu of C. acnes ATCC 11827 (LVX MIC = 0.25 mg/L) on LVX-containing agar plates. The progressive emergence of resistance was studied by a second exposure to increasing LVX concentrations. For mutants, the QRDR regions including the gyrA and parC genes were sequenced and compared to both C. acnes ATCC 11827 and C. acnes KPA171202 reference sequences (NC006085). The importance of the efflux pump system in resistance was investigated by using inhibitors on selected resistant mutants with no mutation in the QRDR. C. acnes growth was observed on LVX-containing plates with mutation frequencies of 3. 8 cfu × 10-8 (8 × MIC) and 1.6 cfu × 10-7 (4 × MIC). LVX resistance emerged progressively after one-step or two-step assays. In LVX-resistant isolates, the MIC ranged from 0.75 to >32 mg/L. Mutations were detected exclusively in the gyrA gene. Ten genotypes were identified: G99 C, G99 D, D100N, D100 H, D100 G, S101L, S101W, A102 P, D105 H and A105 G. Mutants S101L and S101W were always associated with a high level of resistance. Mutants with no mutation in the QRDR were more susceptible when incubated with an efflux pump inhibitor (phenyl-arginine β-naphthylamide) only, suggesting, for the first time, the expression of such a system in C. acnes LVX-resistant mutants

Draft Genome Sequence of an Erythromycin-Resistant Propionibacterium acnes Isolate Recovered from Folliculitis of the Scalp

International audiencePropionibacterium acnes is now well-known and recognized for its implication in the pathogenesis of acne vulgaris. Here, we report the draft genome sequence of an erythromycin-resistant P. acnes strain isolated from a case of folliculitis of the scalp belonging to phylotype IA1 and sequence type 18 (ST18)

Draft Genome Sequence of Mycobacterium ulcerans S4018 Isolated from a Patient with an Active Buruli Ulcer in Benin, Africa

International audienceCurrently, there are only two publicly available genomes of Mycobacte-rium ulcerans—the causative agent of the neglected, but devastating, tropical disease Buruli ulcer. Here, we report the draft genome sequence of isolate S4018, recovered from an active cutaneous lesion of a patient with Buruli ulcer in Benin, Africa

Draft Genome Sequences of Four Propionibacterium acnes Strains Isolated from Implant-Related Infections

International audiencePropionibacterium acnes was previously described as a potential implant-related pathogen. Here, we report the draft genome sequence of four P. acnes strains, isolated from spine material, hip arthroplasty, and knee arthroplasty infections in France belonging to different sequence types (ST18, ST27, and ST36)