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Par Nadim CASSIR Culturomique: Un Nouvel Outil D'analyse De Microbiotes Impliqués Dans La Pathogénèse Ou La Transmission De

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Par Nadim CASSIR Culturomique: Un Nouvel Outil D'analyse De Microbiotes Impliqués Dans La Pathogénèse Ou La Transmission De UNIVERSITÉ D’AIX-MARSEILLE FACULTÉ DE MÉDECINE DE MARSEILLE T H È S E En vue de l’obtention du titre de Docteur de l’Université d’Aix-Marseille Discipline : Sciences de la Vie et de la Santé Spécialité : Maladies Infectieuses Par Nadim CASSIR Culturomique: un nouvel outil d’analyse de microbiotes impliqués dans la pathogénèse ou la transmission de maladies infectieuses Composition du jury : Mr le Professeur L. Papazian Université d’Aix-Marseille Président du jury Mr le Professeur J.P. Lavigne Université de Montpellier-Nîmes Rapporteur Mr le Professeur T.A. Tran Université de Montpellier-Nîmes Rapporteur Mr le Professeur B. La Scola Université d’Aix-Marseille Directeur de thèse Unité de Recherche sur les Maladies Infectieuses et Tropicales émergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, Faculté de Médecine Directeur : Pr. Didier Raoult 1 Résumé Le microbiote digestif humain joue un rôle essentiel et bénéfique pour son hôte mais il est également impliqué dans un nombre croissant de pathologies. Les connaissances sur la composition de cet écosystème ont récemment été révolutionnées grâce à l’utilisation de techniques moléculaires (pyroséquençage à haut débit ou métagénomique). Cependant, ces techniques comportent des limites importantes. C’est ainsi que le concept de « culturomique » a été introduit ; il consiste en la multiplication de milieux et conditions de culture et l’identification rapide de colonies bactériennes par spectrométrie de masse (MALDI- TOF) ou par amplification et séquençage du gène de l’ARN ribosomal 16S. Cette nouvelle approche, complémentaire à la métagénomique, permet de plus la caractérisation de souches bactériénnes vivantes par leur expression phénotypique (sensibilité aux antibiotiques, facteurs de virulence). Dans la première partie de ce travail, nous avons utilisé ces techniques complémentaires pour comparer la composition bactérienne de selles de nouveau-nés prématurés présentant une entérocolite ulcéro-nécrosante (ECUN) à celle de contrôles sains. Nous avons mis en évidence une association entre la présence de Clostridium butyricum dans les selles et la survenue d’ECUN que ce soit par méthodes de pyroséquençage et culture (15/15 vs. 2/15; P <0,0001) ou par PCR quantitative en temps réel spécifique de C. butyricum (OR = 45,4, IC à 95% [26,2-78,6]; P <0,0001). Nous avons également montré que le microbiote digestif des nouveau-nés présentant une ECUN était moins diversifié, plus acide et oxydé que celui des contrôles sains. Par ailleurs, nous avons identifié après séquençage du génome complet de toutes nos souches de C. butyricum, la présence d’un homologue du gène de la β-hémolysine. Cette toxine a précédemment été décrite chez Brachyspira hyodysenteriae, l'agent étiologique de la dysenterie porcine. Une activité cytotoxique des surnageants de culture des souches de C. butyricum a 2 été mise en évidence. Enfin une analyse des génomes complets de 34 souches de C. butyricum, nous a permis de montrer l’association entre souches clonales de C. butyricum et la survenue épidémique d’ECUN. Ces résultats soulignent l’importance de la culture bactérienne dans l’étude du microbiote digestif permettant de distinguer de nouvelles souches de bactéries pathogènes opportunistes. En effet, C. butyricum est considérée comme une bactérie commensale et certaines souches ont été utilisées comme probiotique. Ainsi, au sein d’une même espèce, différentes souches peuvent avoir des effets antagonistes. Dans la deuxième partie de ce travail, nous avons analysé par culturomique la composition du microbiote cutané de patients hospitalisés en réanimation. L’objectif était d’étudier l’impact sur la composition du microbiote cutané ainsi que le taux d’infections nosocomiales, de la toilette des patients avec des lingettes imprégnées de gluconate de Chlorhexidine (CHG) comparée à la pratique courante (eau et savon). Nous avons ainsi montré que les bactéries à Gram-négatif (BGN) étaient fréquemment disséminées au sein du microbiote cutané transitoire des patients hospitalisés en réanimation ; le réservoir serait essentiellement digestif. La toilette des patients avec des lingettes imprégnées de CHG a permis de réduire de manière significative la colonisation cutanée par des BGN (P = 0,006) ainsi que les infections nosocomiales causées par ces bactéries (P = 0,01). Cette pratique représente une opportunité importante dans le contrôle des infections nosocomiales en réanimation en particulier dans un contexte d’émergence de résistance aux antibiotiques. En conclusion, le microbiote digestif constitue un réservoir sous- estimé de bactéries pathogènes. La microbiologie moderne incluant les nouvelles méthodes de culture permet d’étendre de manière considérable les connaissances sur la composition de cet écosystème et son implication en pathologie humaine. Mots clés : microbiote digestif, entérocolite nécrosante, Clostridium butyricum, microbiote cutané, infections nosocomiales, réanimation 3 Abstract The gut microbiota plays an important and beneficial role towards its host but is also involved in a growing number of diseases. Knowledge of the composition of this ecosystem have recently been revolutionized by the use of metagenomics. However, this technique induces several bias. Thus, the concept of "culturomics" has been introduced; it consists of the multiplication of culture conditions and the rapid identification of bacterial colonies by mass spectrometry (MALDI-TOF) or by PCR 16S RNA gene sequencing. This new approach, complementary to the metagenomic allows further characterization of bacterial live strains by their biochemical properties and virulence factors. In the first part of this work, we used these complementary techniques to compare the bacterial composition of stools from preterm neonates with necrotizing enterocolitis (NEC) with healthy controls. We demonstrated an association between the presence of Clostridium butyricum in stools and the occurrence of NEC by pyrosequencing and culture (15/15 vs. 2/15, P <0.0001) and quantitative real-time PCR specific for C. butyricum (OR = 45.4, 95% CI [26.2 to 78.6]; P <0.0001). We also showed that the gut microbiota of preterm neonates with NEC was less diversified, more acidic and oxidized than that of healthy controls. Furthermore, we have found after sequencing the complete genome of all our strains of C. butyricum, the presence of a homologue of the β-hemolysin (toxin) gene also shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. C. butyricum culture supernatants were cytotoxic. Finally an analysis of complete genomes of 34 C. butyricum strains, led us to conclude on an association between clonal strains of C. butyricum and outbreaks of ECUN. These results highlighted the importance of the bacterial culture in the study of the gut microbiota to distinguish new strains of opportunistic pathogenic bacteria. Indeed, 4 C. butyricum is considered a commensal bacterium and some strains have been used as a probiotic. Thus, within the same species, different strains can have antagonistic effects. In the second part of this work, we analyzed by culturomic the skin microbiota composition of patients hospitalized in the intensive care unit (ICU). The objective was to study the impact on the skin microbiota and the nosocomial infections rate, of Chlorhexidine gluconate (CHG) daily bathing compared to the current practice (water and soap). We have shown that Gram-negative bacteria (GNB) were frequently isolated and spread in the skin of ICU patients ; the reservoir beeing primarily digestive. CHG daily bathing was associated with a significant reduction of skin colonization by GNB (P = 0.006) and nosocomial infections caused by these bacteria (P = 0.01). This practice represents therefore an important opportunity in the field of infection control knowing the emergence of antibiotic resistance among GNB. However, potential collateral damages (change of skin microbiota, emergence of resistant strains CHG) merit caution before spreading the use of CHG daily bathing. In conclusion, the gut microbiota is an underestimated reservoir of pathogenic bacteria. Modern microbiology including new culture- based methods is currently extending exponentially our knowledge on gut microbiota giving rise to new insights into the pathogenesis or the transmission of infectious diseases. Keywords: gut microbiota, necrotizing enterocolitis, Clostridium butyricum, skin microbiota, healthcare associated infections, intensive care unit, Gram- negative bacteria 5 Table des matières Résumé .................................................................................................. 2 Abstract ................................................................................................. 4 Avant propos ......................................................................................... 9 Introduction ........................................................................................ 13 Partie I: Rôle du microbiote digestif et de Clostridium butyricum dans la pathogénèse de l’entérocolite nécrosante chez les nouveau- nés prématurés .................................................................................. 19 Chapitre I: Microbiote digestif et entérocolite nécrosante chez les nouveau- nés prématurés ............................................................................................... 21 Publication n°1: Gut Microbiota and the Pathogenesis of Necrotizing Enterocolitis in Preterm Neonates ..............................................................
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