Les microorganismes anaérobies représentent la population majoritaire de notre tube digestif et ont un impact remarquable sur notre santé. Leur culture demeure à ce jour longue, fastidieuse et coûteuse et nombreux sont ceux qui restent incultivables. Or la culture est un outil indispensable pour l'étude du microbiote digestif. Ainsi, le laboratoire dans lequel ma thèse s’est déroulée a créé un nouveau concept de culture « Microbial Culturomics » qui a permis d’isoler 193 nouvelles espèces bactériennes anaérobies. Un travail sur l’utilisation des antioxydants pour permettre la culture aérobie des bactéries anaérobies a également été amorcé : une optimisation des techniques de culture prometteuse autour de laquelle mes travaux ont vu le jour. Notre premier projet a consisté à développer un dispositif de culture innovant permettant la culture des archaea méthanogènes en aérobiose et en absence de source externe de dihydrogène. Notre deuxième projet a consisté à élaborer un flacon d’hémoculture unique dans lequel la croissance de toutes les bactéries, aérobies et anaérobies, pouvaient être détectées. Notre troisième projet quant à lui repose sur la comparaison du mode de culture anaérobie et de celui en aérobie avec les antioxydants à travers l’exemple de trois souches bactériennes strictement anaérobies. L’utilisation des antioxydants pour faciliter la culture des microorganismes anaérobies a donc apporter des résultats très prometteurs qui pourrait être utilisés, après validation par des études multicentriques dans les laboratoires de microbiologie clinique et environnementaux. / Anaerobic microorganisms are characterized by their ability to grow and survive in the absence of oxygen. Indeed free oxygen molecules are not used for their metabolism and can be toxic to varying degrees, sometimes leading to cell death. Although it is known that these microorganisms are the predominant in our digestive microbiota and that they have a great impact on our health, their culture remain long, fastidious, costly, and in most cases impossible. Becteria culture is an indispensable tool for isolating strains, performing studies from living models, and identifying new ones. Thus, the laboratory in which my thesis tooks place created a new concept of culture "Microbial Culturomics" which made it possible to isolate 193 new anaerobic bacterial species. A work based on the use of antioxidants to enable the aerobic culture of anaerobic bacteria was also initiated: a promising optimization of the culture techniques from which my work was born. Our first project consisted in developing an innovative culture device allowing the cultivation of methanogenic archaea in aerobic and without an external source of dihydrogen. In our second project, we performed a single culture bottle in which the growth of all bacteria, aerobic and anaerobic, could be detected. Our third project was based on the comparison of anaerobic and aerobic culture with antioxidants through the example of three strictly anaerobic bacterial strains.Therefore the use of antioxidants enable to facilitate anaerobic bacteria cultivation. These results are very encouraging for clinical and environmental microbiology laboratories.

[Truncated abstract] In Australia, there is pressure to reduce the amount of methane produced by ruminant livestock because they are the single largest source of methane emitted from anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In addition, methane production represents a loss of gross energy intake to the animal. The organisms that are responsible for methane production in the animal gut are a distinct group of Archaea called methanogens. Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic). The types and number of methanogens, as well as rumen ciliates and their symbiotic interactions, influence the amount of methane produced from the rumen. These factors in turn are affected by many factors, including diet and ruminal retention time. In this thesis, I tested the general hypothesis that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, including their association with ruminal ciliates. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into four groups. ... The change in DGGE banding patterns and Shannon indices when sheep were fed grain indicated that the types of methanogens changed when sheep were fed low and high grain diets, but their diversity did not. In contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All except one, which was 98% similar to Cycloposthium sp. not found previously in the rumen, matched the sequences for previously identified rumen ciliates. Some of the rumen ciliates identified were not present in sheep fed the high grain diet. On a high grain diet, methanogens associate endosymbiotically with rumen ciliates to get better access to hydrogen. It appears that the association between methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen and not the generic composition of the ciliate population. Furthermore, endosymbiotic methanogens appear to produce less methane than methanogens in other niches. The pot scrubbers did not change ruminal retention time but they did reduce the acetate/propionate measurements observed in sheep on the high grain treatment. The reason why pot scrubbers had this effect remains unknown, but it is interesting to consider that some physical interaction has occurred between the pot scrubbers, the grain and the sheep that has improved the fermentation parameters in sheep fed a high grain diet. The results from this study have advanced our understanding of the interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen in response to dietary changes and mechanical challenges. Extending this work to look more specifically at the species of methanogens that are most closely linked to high methane production and how they interact with the ruminal ciliates will be critical for manipulating enteric greenhouse gas emissions.

Les méthanogènes sont des Archaea anaérobies stricts, connus pour être les seuls êtres vivants capables de produire du méthane comme sous-produit de leur métabolisme. Notre revue de littérature a montré qu’outre les espèces environnementales, l’ordre des Methanomassiliicoccales ne comptait à ce jour qu’une seule espèce Methanomassiliicoccus luminyensis isolée et cultivée dans notre laboratoire. Aucours de notre thèse, nous avons développé une nouvelle méthode permettant la culture et l’isolement des méthanogènes en absence de source externe de dihydrogène (H2) d’une part et d’autre part une méthode de génotypage Multispacer Sequence Typing (MST) basée sur le séquençage d’espaces intergéniques pour typer Methanobrevibacter smithii et M. oralis. Par la suite, nous avons mis en évidence la présence de méthanogènes en situation pathologique chez l’homme. Nous avons détecté et isolé pour la première fois au sein de flores anaérobies, M. oralis à partir d’échantillons d’abcès cérébraux, et de sinusite d’une part, et d’autre part M. smithii à partir d’un échantillon de patient souffrant d’abcès para-vertébral. Enfin, dans la cinquième partie de notre thèse, nous avons testé in vitro la sensibilité de cinq méthanogènes associés aux flores humaines à la lovastatine qui est une pro-drogue utilisée pour abaisser la concentration de cholestérol chez l’homme dans le cadre de certaines pathologies. Les cinq méthanogènes se sont avérées sensibles à une concentration minimale inhibitrice de 1µg/mL, après activation par hydrolyse de la lovastatine par des bactéries anaérobies du microbiote digestif, via une l’inhibition de la croissance et de la production du méthane. / Methanogens are strict anaerobic Archaea, known to the only being able to producing methane gas as a byproduct. Methanogens which are not detected in clinical microbiology laboratories were present in oral, digestive, vaginal and cutaneous microbiota of human. Only five species on the thirteen known in human have been cultivated before the beginning of our thesis. In our thesis, we initially reviewed the state of knowledge about methanogens in human microbiota, particularly the new order Methanomassiliicoccales of methylotrophic methanogens, the as member of human microbiota. In the second part of this work, we performing new method for methanogens culture and isolation without any dihydrogen atmosphere, by co-cultured in tubes methanogens with Bacteroides thetaiotaomicron, which produces hydrogen. We also developed Multispacer Sequence Typing (MST), a genotyping method based on intergenic spacers sequencing, to genotype M. oralis and Methanobrevibacter. Smithii. We demonstrated that methanogens could be part of polymicrobial infection in the case of brain and sinusal abscesses, and also in skeletal muscle abscess, by isolating for the first time M. oralis and M. smithii in these pathologies, using culture-based and molecular-based approaches, and suggested that methanogens could be considered as human opportunistic or emerging pathogens. Finally, we tested the in vitro susceptibility of lovastatin which is a prodrug used as a powerful serum cholesterol-lowering drug in some human diseases and showed that it’s inhibits growth and methane production in human-associated methanogens without affecting intestinal bacteria.