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X2018;Nissabacter Archeti’ AIX-MARSEILLE UNIVERSITE FACULTE DE MEDECINE-LA TIMONE ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE Présentée et soutenue le 24 Novembre 2017 Par En vue de l’obtention du grade de Docteur de l’Université Aix-Marseille Spécialité : Génomique et Bio-informatique REAL-TIME GENOMICS TO DECIPHER ATYPICAL BACTERIA IN CLINICAL MICROBIOLOGY COMPOSITION DU JURY Président du Jury Professeur Anthony Levasseur Examinateur Professeur Ruimy Raymond Rapporteur1 Professeur Marie Kempf Rapporteur2 Professeur Estelle Jumas-Bilak Directeur de Thèse Professeur Jean-Marc Rolain Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes URMITE CNSR-IRD UMR7278, IHU MEDITERRANEE INFECTION AIX-MARSEILLE UNIVERSITE FACULTE DE MEDECINE-LA TIMONE ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE Présentée et soutenue le 24 Novembre 2017 Par En vue de l’obtention du grade de Docteur de l’Université Aix-Marseille Spécialité : Génomique et Bio-informatique REAL-TIME GENOMICS TO DECIPHER ATYPICAL BACTERIA IN CLINICAL MICROBIOLOGY COMPOSITION DU JURY Président du Jury Professeur Anthony Levasseur Examinateur Professeur Ruimy Raymond Rapporteur1 Professeur Marie Kempf Rapporteur2 Professeur Estelle Jumas-Bilak Directeur de Thèse Professeur Jean-Marc Rolain Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes URMITE CNSR-IRD UMR7278, IHU MEDITERRANEE INFECTION 1 CONTENT Avant-propos Résumé /Abstract Introduction Chapter I: Review Articles I: Real-time genomics and the impact of bacterial genome recombination in clinical microbiology Kodjovi D. Mlaga, Seydina M. Diene, R. Ruimy, J-M Rolain. (Submitted in Genome Biology and Evolution) Chapter II: Comparative genomic applied in clinical microbiology Articles II: Using MALDI-TOF MS typing method to decipher outbreak: the case of Staphylococcus saprophyticus causing urinary tract infections (UTIs) in Marseille, France. Kodjovi D. Mlaga, Grégory Dubourg, Cedric Abat, Hervé Chaudet, Laurène Lotte, Seydina M. Diene, Didier Raoult, Raymond Ruimy and Jean-Marc Rolain. European Journal of Clinical Microbiology & Infectious Diseases. pp. 1–7, Aug. 2017. Articles III: Comparative genomic analysis of Staphylococcus saprophyticus reveals a drift from “saprophytic” to “pathogenic” bacteria due to extensive genomic recombination. Kodjovi D. Mlaga, Seydina M. Diene, Ruimy Raymond, Jean-Marc Rolain (Submitted in BMC genomics) Articles IV: Extensive comparative genomic analysis of Enterococcus faecalis and Enterococcus faecium reveals a direct association between absence of CRISPR systems, the presence of anti-endonuclease (ardA) and acquisition of vancomycin resistance genes in E. faecium. Kodjovi D. Mlaga, Seydina M. Diene, Vincent Garcia, Philippe Colson, Ruimy Raymond, Didier Raoult, Jean-Marc Rolain (draft manuscript) Chapter III: Taxonogenomics applied in new species/genus description Article V: ‘Nissabacter archeti’, gen. nov, sp. nov., a new member of Enterobacteriaceae family, isolated from pustule scalp at Archet 2 Hospital, Nice. Kodjovi D. Mlaga, Jean-Marc Rolain, Ruimy. Raymond New Microbes New Infect. 2017 May; 17: 81–83. Article IV: Phenotypic and genomic description of ‘Nissabacter archeti’, gen. nov., sp. nov., a new member of Enterobacteriaceae family, isolated from human pustule scalp at Archet 2 Hospital, Nice, France. Kodjovi D. Mlaga, Romain Lotte, Henri Montaudié, Jean- 2 Marc Rolain, Ruimy. Raymond (Submitted in International Journal of Systematic and Evolutionary Microbiology) Chapter IV: Conclusion and Perspectives Posters and Presentation Acknowledgments 3 Avant-propos Le format de presentation de cette thèse correcpond à la recommendation de la specialité Genomique et bio-informatique a l’interieur du Master des Sciences de la vie et de la Santé qui dépend de l’Ecole Doctorale des Sciences de la vie et de la santé de Marseille. Le candidat est amené à respecter les règles qui lui sont imposées et qui comportent un format de thèses utilisé dans le Nord de l’Europe et qui permet un meilleur rangement que les thèses traditionnelles. Par ailleurs, la partie introduction et bibliographie est remplacée par une revue envoyée dans un journal afin de permettre une evaluation exterieure de la qualité de la revue et de permettre a l’étudiant de commencer le plus tôt possible une bibliographie exhaustive sur le demaine de cette thèse. Par ailleurs, la thèses est présentée sur article publié, accepté, ou soumis associé d’un bref commentaire donnant le sens général du travail. Cette forme de présentation a paru plus en adéquation avec les exigences de la competition internatinales et permet de se concentrer sur des travaux qui bénéficieront d’une diffuion internationale. Professeur Didier RAOULT 4 Résumé Le développement du « Next-Generation Sequencing » (NGS) et l'amélioration de « Whole Genome Sequencing » (WGS) ont contribué a l’amélioration de l'analyse des données du séquençage des génomes microbiens à haut-débit. Le concept de «Génomique en Temps Réel» (GTR) initialement inventé par des sociétés bio-informatiques pour une analyse génomique profonde a progressivement intégré les processus diagnostiques de routine en microbiologie clinique pour déchiffrer l’evolution genomique bacterienne, la detection et l’analyse des déterminants de resistance aux agents antimicrobiens, la taxono-genomique et la surveillance systématique des épidémies. L'augmentation de la quantité de données de séquençage mise à disposition dans GenBank nécessitera des outils d’analyse bien établis et intégrés pour l'analyse des données de séquençage du génome, afin de fournir des résultats précis pour une bonne gestion des patients. L'émergence de souches multiresistantes et de pathogenes de plus en plus virulents sont devenus une menace sérieuse pour la santé humaine et animale. La recombinaison génomique au travers des mutations ponctuelles, le transfert horizontal de gène et la perte de gènes ont enormement contribué à l'adaptation et à l'évolution des bactéries dans les divers environnements et hôtes; résultant de l'acquisition des déterminants de la résistance antimicrobienne, de la virulence et de nouveau profile métabolique. Un diagnostic rapide est l'approche la plus efficace pour prévenir et contrôler les infections ou les maladies microbiennes, par conséquent, pour parvenir à une thérapeutique efficace. De nos jours, les outils diagnostiques de routine en microbiologie ont montré leur limite dans la gestion des infections ou d’épidemie due à des pathogènes atypiques, les facteurs de virulences et la classification taxonomique. Les progrès récents dans les technologies de séquençage ont donné aux laboratoires de microbiologie l'accès au WGS. L'objectif de notre thèse est d'appliquer les approches génomiques en temps réel (GTR) pour déchiffrer les caractéristiques génomiques et la recombinaison génomique des bactéries atypiques ainsi que leur impact sur les maladies infectieuses. Le premier projet de notre thèses consiste à déchiffrer une epidemie communautaire due à Staphylococcus saprophyticus impliqué dans les infections des voies 5 urinaires (UTI) en utilisant la technologie de MALDI-TOF MS et une analyse comparative des génomes entiers de Staphylococcus saprophyticus d’origine clinique et non-clinique pour comprendre leur évolution génomique. Le deuxième projet est une analyse comparative de l'évolution du génome d'Enterococcus fecalis et d'Enterococcus faecium isolés de l'homme, des animaux et de l'environnement pour décrypter la différence de propagation et l'acquisition de déterminants antimicrobiens, specialement les genes de resistance à la vancomycine; et un dernier projet axé sur la description d'un nouveau genre Nissabacter et sa première espèce Nissabacter archeti une nouvelle branche phylogéniques de la famille des Enterobacteriaceae à l'aide de la taxono-genomique. 6 Abstract The development of Next-Generation Sequencing (NGS) and the improvement in whole genome sequencing (WGS) have contributed to improving the analysis of high-throughput microbial genome sequence data. The concept of “Real-Time Genomics” (RTG) initially coined by computational biology companies for in-depth genomic analysis has recently toward becoming an integral part of routine diagnostic processes in clinical microbiology to decipher antimicrobial resistance determinants, taxono-genomics, and routine outbreak surveillance. The increase in the amount of sequencing data made available in Genbank will require well- established and integrated computational tools for genome sequencing data analysis, to provide accurate results for correct patient management. The emergence of multidrug resistance and extensive virulent pathogens have become a severe threat to human health. Genome recombination through point mutations, horizontal gene transfer and gene loss have contributed the adaptation and the evolution of bacteria in the various environment and host niches significantly; resulting in the acquisition of antimicrobial resistance determinants, virulence and metabolism pattern. A timely diagnosis is the most effective approach to prevent and control microbial infections or diseases, consequently, to achieve an effective therapy. Nowadays, microbiology routine diagnostic tools have shown their limitation in the management of atypical bacteria infection or outbreak as far as multi-drug resistance bacteria, novel virulence or toxin genes and taxonomic classification are concerned. The recent advances in sequencing technologies have given microbiology laboratories access to whole genome sequencing.
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