Open cultures of anaerobic reactor systems convert organic wastes or biomass residues into mainly short-chain carboxylates with two to five carbon atoms. The short-chain carboxylates can be converted into the highly reduced end product methane by methanogenic consortia in anaerobic digestion. Microbial chain elongation such as via the reverse ꞵ-oxidation pathway was found as an alternative electron sink with the same anaerobic reactor microbiota. In natural ecosystems such as rumen microbial ecosystem, some anaerobic bacteria are known to produce medium-chain carboxylates (e.g., n-caproate and n-caprylate) through reverse ꞵ-oxidation. The carboxylate platform aims to recover carbon from waste streams or biomass residues by anaerobic fermentation in the form of medium-chain carboxylates. It has created great opportunities to replace chemicals derived from non-sustainable sources such as fossil feedstock. Mixed culture fermentation is commonly employed for the chain elongation processes. The diverse microbial chain elongation communities contain different functional groups involved in the processes of hydrolysis and fermentation of available organic compounds as well as the conversion of intermediates to medium-chain carboxylates. In general, the underlying metabolism and ecological interactions of the chain elongation communities are not well understood. This PhD thesis centres on the metabolism and ecological interactions in closed model ecosystems (i.e., anaerobic bioreactors) involved in microbial chain elongation with lactate. In the first chapter, a model ecosystem with reduced complexity was developed by using lactate and xylan as defined carbon sources to simulate the feedstock conditions of caproate-producing bioreactors operated with corn silage. Feeding defined carbon sources enabled balancing of electron and carbon flows. By preventing continuous inoculation, the simplified community of enrichment cultures allowed to study the metabolic and community dynamics in a clearer manner than open reactor systems. During a long-term reactor experiment, four succession stages including adaptation, stage I (high medium-chain carboxylate-producing period), transition and stage II (high butyrate-producing period) were observed. Co-occurrence networks of species based on 16S rRNA amplicon sequences and associations with process parameters were analysed to infer potential metabolic functions and microbial interactions. The results suggested that the process included diverse functions of xylan hydrolysis, xylose fermentation and chain elongation with lactate as electron donor. The inferred interactions such as cooperation between lactic acid bacteria and chain-elongating bacteria, as well as competition between medium-chain carboxylate-producing bacteria and butyrate-producing bacteria, resulted in the community development over four succession stages. In this closed model ecosystem, the chain-elongating bacteria were outcompeted by butyrate-producing bacteria under constant conditions, leading to the increase of butyrate yield at the cost of n-caproate and n-caprylate yields. The second chapter tested the effects of shortening the hydraulic retention time on the community assembly and functioning in the model ecosystems, aiming to quantitatively predict ecophysiological functions of the microbial communities. For the process performance, higher productivities and yields of n-caproate and n-caprylate were achieved by reducing the hydraulic retention time from 8 days to 2 days in two continuous reactors. A predictive model was generated by applying the random forest approach using 16S rRNA amplicon sequencing data. More than 90% accuracy in the quantitative prediction of n-caproate and n-caprylate productivities was achieved. Four inferred bioindicators belonging to the genera Olsenella, Lactobacillus, Syntrophococcus and Clostridium IV suggested their relevance to the higher carboxylate productivity at shorter hydraulic retention time. Combined with metagenomics, the recovery of metagenome-assembled genomes of these bioindicators confirmed their genetic potential to perform key steps of carboxylate production. Besides, functional redundancy in the conversion of xylan and lactate to n-butyrate, n-caproate and n-caprylate was revealed, with the relevant bioindicators increasing in relative abundance. Thus, the involved metabolic pathways were strongly coupled to the decrease in hydraulic retention time. In general, the developed machine learning framework to identify bioindicators and to quantitatively predict process performance is transferable to other ecosystem processes and microbial systems where community dynamics is linked to key functions. In the third chapter, the effects of pH increase on the chain elongation community assembly and functioning were tested based on the developed model ecosystems. The increase in pH from 5.5 to 6.0 caused fluctuations in the yields of n-butyrate, n-caproate and n-caprylate. After the pH disturbance, the carboxylate yields returned to the previous values while the communities developed to a different state, observed as decrease in diversity and evenness and increase in richness. Some taxa shifted from rare to abundant, reflecting strong selective effects of lower pH values. By applying Aitchison PCA clustering, linear mixed effect models and random forest classification, the different pH preferences of the potential chain elongators Clostridium IV and Clostridium sensu stricto were identified. By constructing networks for different pH levels, the cooperation of the chain elongator Clostridium IV with lactic acid bacteria switches from Olsenella to Lactobacillus along the pH increase, revealing the plasticity of the food web of chain elongation communities. Compared with the previously observed results of decreasing the hydraulic retention time, pH increase induced dramatic shifts in the community assembly but exhibited no strong effects on community functioning in terms of medium-chain carboxylate production. High functional redundancy was indicated despite the reactors being long-term closed systems. In parallel to the reactor experiments, pure cultures of chain-elongating clostridial strains were isolated, representing three novel species. Their genomes were assembled using a hybrid short and long read sequencing approach. The three novel strains produced n-caproate, n-butyrate, iso-butyrate and acetate from lactate in batch cultivation at pH 5.5, with the confirmation of their genetic background of lactate-based chain elongation and using CoA transferase as the terminal enzyme. Their genomes show substantial genetic heterogeneity but contain highly conserved genes involved in lactate oxidation, reverse ꞵ-oxidation, hydrogen formation and either of two types of energy conservation systems (Rnf and Ech). The genetic background of lactate-based chain elongation in these isolates and other experimentally validated chain-elongating strains was analysed by comparative genomics. The chain elongation-specific core-genome was indicated to encode the pathways for reverse ꞵ-oxidation, hydrogen formation and energy conservation while chain-elongating species displayed substantial genome heterogeneity. Further research is needed to elucidate the pathways for iso-butyrate formation in these strains. In summary, model communities of chain elongation processes were enriched and further shaped by alternations of pH and hydraulic retention time in long-term bioreactor experiments. The metabolism and ecological interactions of reactor microbiota involved in microbial chain elongation with lactate were elucidated by using 16S rRNA amplicon sequencing and metagenomics coupled to network analysis, statistical modelling and machine learning, which also sparkled new insights into the relationship between microbial chain elongation community diversity and functioning. The isolation of novel chain-elongating species further expands our knowledge on the metabolism of chain elongation bacteria. Finally, a better understanding of the rules governing community assembly is key to accelerate the development of microbiota-based biotechnologies.:Abbreviations ...................................................................................................1 List of figures ...................................................................................................4 List of tables.....................................................................................................9 Zusammenfassung ........................................................................................12 Summary .......................................................................................................17 1 Introduction .................................................................................................21 1.1 Reactor microbiota................................................................................21 1.2 Carboxylate platform.............................................................................21 1.3 Microbial chain elongation ....................................................................22 1.4 Methods for investigating reactor microbiota ........................................24 1.4.1 PCR-based methods ......................................................................24 1.4.2 Metagenomics ................................................................................25 1.4.3 Culture-dependent methods...........................................................27 1.5 Aims of this study..................................................................................28 2 Research chapters......................................................................................29 2.1 Competition between butyrate fermenters and chain-elongating bacteria limits the efficiency of medium-chain carboxylate production .....................29 2.1.1 Main text.........................................................................................30 2.1.2 Supplementary information.............................................................43 2.2 Machine learning-assisted identification of bioindicators predicts medium-chain carboxylate production performance of an anaerobic mixed culture .47 2.2.1 Main text.........................................................................................48 2.2.2 Supplementary information.............................................................83 2.3 Effects of pH increase on microbial chain elongation and community dynamics in closed bioreactor ecosystems...............................................104 2.3.1 Main text.......................................................................................105 2.3.2 Supplementary information...........................................................134 2.4 Draft genome sequences of three Clostridia isolates involved in lactate-based chain elongation.............................................................................148 2.5 Three novel Clostridia isolates produce n-caproate and iso-butyrate from lactate: comparative genomics of chain-elongating bacteria ....................151 2.5.1 Main text.......................................................................................152 2.5.2 Supplementary information...........................................................192 3 General discussion ...................................................................................196 3.1 Understanding microbial community assembly in model ecosystems 196 3.2 Linking microbial community structure to functioning..........................199 3.3 Moving from intriguing science to real-world practice – Microbiota-based biotechnology ...........................................................................................200 4 References ...............................................................................................202 5 Appendix...................................................................................................208 5.1 Declaration of authorship....................................................................208 5.2 Coauthor contributions........................................................................209 5.3 Curriculum Vitae .................................................................................213 5.4 List of publications and conference contributions ...............................215 5.5 Acknowledgements.............................................................................218

Les cancers du rein métastatiques résistants aux inhibiteurs de tyrosine kinase peuvent faire l’objet de traitements fondés sur le blocage des points de contrôles immunitaires (ICB). Cependant, les ICB induisent des réponses chez une minorité de patients, et des efforts sont en cours pour identifier les mécanismes à l'origine de la résistance. Les ICB compromettent l'intégrité de la barrière intestinale, affectant ainsi la composition du microbiote, favorisant ainsi soit l'accumulation intestinale, soit la translocation de commensaux immunogènes capables de moduler le tonus immunitaire systémique et de reprogrammer le microenvironnement tumoral. Pour déterminer si des profils microbiens distincts du microbiote intestinal pourraient expliquer la résistance aux ICB, ma thèse a montré que dans une cohorte de 249 patients atteints de cancer traités par ICB que la prescription d’antibiotiques (ATB) a significativement diminué la survie sans progression (PFS) et la survie globale (OS) par rapport aux patients sans ATB. Nous avons confirmé ces données en analysant 121 cencers du rein traités par ICB à Gustave Roussy et 239 cancers du poumon traités par ICB au Memorial Sloan Kettering Cancer Center. Nous avons validé que la dysbiose liée à l'ATB diminue l'activité des ICB. Par la suite, nous avons analysé de manière prospective les microbiotes fécaux de 100 patients atteints de tumeurs sensibles à l'anti-PD-1 en utilisant la métagénomique (MG). Nous avons démontré que les bactéries intestinales présentes avant le traitement anti-PD-1 étaient systématiquement différentes entre les patients qui ont répondu (R) au traitement et les patients qui n’ont pas (NR). Entre les R, nous avons observé une surreprésentation d’Akkermansia muciniphila. Au sein d'une large cohorte de patients RCC (n = 85) traités avec un anticorps anti-PD-1, nous avons analysé le microbiote fécal de 69 patients. Des empreintes de MG spécifiques étaient liées aux meilleures réponses et à la survie sans progression. A. muciniphila et Bacteroides spp étaient plus abondants chez les R. Pour valider la pertinence de ces constatations, nous avons mis en évidence deux principaux éléments de preuve. Premièrement, nous avons démontré que chez les patients atteints de NSCLC, la présence de lymphocytes T CD4 + et CD8 + à mémoire spécifiques de l’IFNγ + vis-à-vis de A. muciniphila prédit une PFS plus longue. Deuxièmement, une transplantation de microbiote fécal (FMT) a été réalisée à l'aide de selles de patient pour recoloniser des souris axéniques ou traitées par ATB dans deux modèles murins. Les matières fécales de R entrainaient une réponse immunitaire plus forte contre la tumeur par rapport aux matières fécales de NR. Ensuite, une supplémentation orale d'A. muciniphila post-FMT avec des matières fécales de NR restaurait l'efficacité de l'anti- PD-1. Dans ce contexte, les cellules dendritiques sécrétaient plus d'IL-12, augmentant le recrutement de lymphocytes T CCR9 + CXCR3 + CD4 + à partir des ganglions lymphatiques mésentériques jusque dans les lits tumoraux, ainsi qu'une augmentation du rapport CD4 + /Treg dans le lit tumoral de souris co-traitées avec mAb anti-PD-1 et A. muciniphila. Enfin, nous avons montré qu'une supplémentation orale avec Bacteroides (B. salyersiae mais pas B. xylanisolvens) ou A. muciniphila pourrait restaurer l'efficacité des ICB dans un modèle FMT- défavorable/dysbiotique". La découverte de bactéries immunogènes capables de prédire et d'accroître les avantages cliniques de l'ICB contribuera au développement de nouveaux outils de biomarqueurs et d'un futur concept thérapeutique, grâce auxquels le traitement du cancer peut être amélioré par la modulation du microbiote intestinal. / Metastatic RCC resistant to tyrosine kinase inhibitors are amenable to new therapies based on immune checkpoint blockade (ICB). However, ICB induces responses in a sizeable minority of patients and efforts are ongoing in order to identify mechanisms driving resistance. ICB compromise the integrity of the intestinal barrier, hereby affecting the composition of the intestinal microbiome, thereby promoting either the intestinal accumulation or the translocation of immunogenic commensals capable of modulating the systemic immune tone and reprogramming the tumor microenvironment. To address whether distinct microbial patterns of the intestinal microbiome could account for the resistance to ICB in RCC, during my PhD I showed in a cohort of 249 patients with cancers treated with ICB that antibiotic (ATB) prescription in a therapeutic window of -2 months up to +1 month after starting ICB significantly decreased progression-free survival (PFS) and overall survival (OS) compared to patients without ATB. We confirmed this data analyzing 121 RCC treated with ICB at Gustave Roussy and 239 NSCLC treated with ICB at Memorial Sloan Kettering Cancer Center. We validated that ATB-related dysbiosis decreases activity of ICB. More precisely, patients in RCC ATB group treated with ICB had a higher rate of primary progressive disease. Subsequently, we prospectively analyzed the fecal microbiomes of 100 patients with tumours amenable to anti-PD-1 mAb using quantitative metagenomics (MG) by shotgun sequencing. We demonstrated that gut bacteria present before anti-PD-1 therapy was consistently different between patients who responded (R) to treatment and patients who did not (NR). In R we observed an overrepresentation of un- and classified Firmicutes. The commensal most significantly associated with favorable clinical outcome and PFS was Akkermansia muciniphila. Within a large cohort of RCC patients (n = 85) treated in the NIVOREN study with anti-PD-1 Ab at Gustave Roussy, we analyzed the fecal microbiome of 69 patients. Specific MG-fingerprints were related to best responses and PFS. A. muciniphila and Bacteroides spp were more abundant in R (Derosa et al. ASCO Merit Award 2018). To validate the relevance of these findings, we brought up two major lines of evidence. First, we demonstrated that in NSCLC patients, the presence of specific IFNγ+ memory CD4+ and CD8+ T cells toward A. muciniphila predicted a longer PFS. Secondly, fecal microbiota transplantation (FMT) was performed using patient feces to recolonize germ-free or ATB-treated mice in two tumor models (MCA-205 and RENCA, the renal cell carcinoma model). Feces from R conveyed a stronger immune response against the tumor compared to feces from NR. Next, in MCA-205 model oral supplementation with A. muciniphila post-FMT with NR feces restored the efficacy of PD-1 Abs. In this setting, dendritic cells secreted more IL-12, increasing the recruitment of CCR9+CXCR3+CD4+ T lymphocytes from the mesenteric lymph nodes into tumor beds as well as an increase of CD4+/Treg ratio within the tumor bed of mice co-treated with anti-PD-1 mAb and A. muciniphila. Finally, we showed that oral supplementation with Bacteroides (B. salyersiae but not B. xylanisolvens) or A. muciniphila could restore the efficacy of ICB in "unfavorable/dysbiotic" FMT. The discovery of immunogenic bacteria capable of predicting and increasing clinical benefit of ICB will help for the development of novel biomarker tools and a future therapeutic concept, whereby treatment of cancer can be improved by the modulation of gut microbiome.

Le Virus Respiratoire Syncytial (VRS) est responsable de 70 % des cas de bronchiolite chez les enfants de moins de cinq ans. La survenue de bronchiolites sévères chez le nourrisson est un facteur de risque de développement d’asthme en grandissant. Aucun vaccin contre le VRS n’est disponible chez l’Homme. Le système immunitaire inné est la première ligne de défense de l’organisme contre les infections. De plus, en interaction avec la flore bactérienne commensale des poumons, l’immunité innée participe à la maturation de la réponse immunitaire adaptative qui confère à l’individu une protection sur le long terme vis-à-vis des pathogènes. Afin d’expliquer la susceptibilité néonatale au VRS, nous avons caractérisé un nouveau mécanisme de contrôle de la réponse innée antivirale lors de l’infection de souriceaux. Nous avons également testé une nouvelle approche de modulation de la réponse immunitaire au VRS par le microbiote pulmonaire. Ainsi, mieux comprendre les mécanismes immunologiques et virologiques responsables de bronchiolites sévères en période néonatale permettra de développer des moyens de lutte sûrs et efficaces contre l’infection par le VRS. / Respiratory Syncytial Virus (RSV) is responsible for 70 % of bronchiolitis in children under five years old. Severe bronchiolitis in infants is a risk factor for asthma development. No vaccine against RSV is available in humans. The innate immune system is the first line of defense against infections. Moreover, in interaction with lung microbiota, innate immunity shapes adaptive immune response responsible for long-term protection against pathogens. To explain the susceptibility of young children to RSV, we characterized a novel regulatory mechanism of the innate antiviral response during neonatal RSV infection in the murine model. We also tested a new approach for modulating immune responses to RSV by the pulmonary microbiota. Thus, a better understanding of immunological and virological mechanisms responsible for severe bronchiolitis during the neonatal period will allow the development of safe and effective therapeutic strategies against RSV infection.

La réduction du taux de cholestérol (CH) sanguin est un point clé dans la lutte contre les maladies cardiovasculaires. L’efficacité contrastée des médicaments disponibles actuellement ainsi que l’intérêt porté autour du microbiote intestinal dans la régulation de la physiologie de l’hôte nous amènent à envisager cette voie comme alternative thérapeutique. La production de coprostanol (CO), dérivé très peu absorbé du CH, par des bactéries de ce microbiote a été corrélée positivement à une faible cholestérolémie. Les objectifs de cette thèse sont i) d’isoler et d’identifier de nouvelles souches bactériennes ayant cette activité, ii) d’identifier les gènes bactériens responsables de cette transformation et iii) de détereminer l’impact de ce métabolisme sur la physiologie de l’hôte. Nous avons isolé 22 nouvelles souches productrices de CO à partir des selles d’un individu en produisant beaucoup. Nous avons choisi les souches Bacteroides sp. D8 et Bacteroides sp. BV pour la construction de deux banques génomiques et huit autres pour des essais d’implantation in vivo dans le tractus gastro-intestinal (TGI) de souris axéniques. Nous avons identifié 55 clones potentiellement positifs par le criblage fonctionnel des banques génomiques. Leurs analyses supplémentaires devraient nous apporter des informations sur les gènes impliqués dans cette activité. Toutes les bactéries sélectionnées sont capables de coloniser le TGI de la souris axénique. La souche Parabacteroides distasonis est la meilleure souche productrice de CO in vivo. Nous avons testé son effet sur la cholestéolémie chez des souris axéniques soumises à un régime riche en CH sur 11 semaines en comparaison avec une souche non productrice in vitro, B. dorei, et avec des souris conventionnalisées comme contrôle. La souche B. dorei produit du CO in vivo, soulignant l’importance de l’environnement dans l’activité de production de CO déjà supposée d’après la littérature et nos résultats in vitro. Des gènes impliqués dans l’excrétion du CH de l’organisme vers les selles sont surexprimés chez ces souris et celles colonisées avec P. distasonis. Cependant seules ces dernières présentent une cholestérolémie plus faible que les souris conventionnalisées. Le mécanisme impliqué semble indépendant de la production de CO et de l’excrétion de CH car les mêmes quantités de ces composés sont retrouvées dans les selles indépendamment du statut bactérien. Les concentrations en acides biliaires totaux dans la bile et dans les selles sont supérieures pour les souris monocolonisées comparées au conventionnalisées. Les selles des souris colonisées avec P. distasonis présentent plus d’acides urso- et chénodésoxycholiques que les souris conventionnalisées et plus d’acide cholique que les souris colonisées avec B. dorei. En conclusion, nous avons isolé de nouvelles souches et identifier des clones potentiellement positifs. Les études in vivo tendent à montrer que l’activité de production de coprostanol n’a pas d’effet sur la cholestérolémie. En revanche, la souche P. distasonis semble diminuer la cholestérolémie par un mécanisme encore inconnu. / Cholesterol (CH) level management is a keystone to limit cardiovascular diseases. The contrasted efficiency of the drugs currently available as well as the interest around the intestinal microbiota in regulating the host physiology lead us to consider this pathway as a therapeutic alternative. The production of coprostanol (CO), a very poorly absorbed CH derivative, by bacteria of this microbiota has been positively correlated with low CH plasma level. The aims of this thesis are (i) isolate and identify new bacterial strains possessing this activity, (ii) identify the bacterial genes responsible for this transformation and (iii) determine the impact of this metabolism on host physiology. We isolated 22 new strains producing CO from the stools of a high-coprostanol producing individual. We chose Bacteroides sp. D8 and Bacteroides sp. BV for the construction of two genomic libraries and eight others for in vivo implantation tests in the gastrointestinal tract (GIT) of germ-free mice. We identified 55 potentially positive clones by functional screening of these genomic libraries. Their additional analyzes should provide us with information about the genes involved in this activity. All selected bacteria are capable of colonizing the GIT of germ-free mice. Parabacteroides distasonis is the best strain producing CO in vivo. We tested its effect on blood cholesterol level in germ-free mice subjected to an 11-week CH-rich diet compared to an in vitro non-producing strain, B. dorei, and with conventionalized mice as control. The B. dorei strain produces CO in vivo, emphasizing the importance of the environment in the CO production activity already assumed from the literature and our results in vitro. Genes involved in the excretion of CH from body to feces are overexpressed in these mice and those colonized with P. distasonis. However, only the latter have lower cholesterolemia than conventional mice. The mechanism involved appears to be independent of CO production and CH excretion because the same amounts of these compounds are found in feces independently of bacterial status. Total biliary acids concentrations in bile and feces are higher for monocolonized mice compared to conventionalized mice. The feces of mice colonized with P. distasonis exhibited more urso- and chenodeoxycholic acids than conventionalized mice and more cholic acid than mice colonized with B. dorei. In conclusion, we have isolated new strains and identified potentially positive clones. In vivo studies tend to show that coprostanol production activity has no effect on plasma cholesterol. In contrast, P. distasonis seems to decrease plasma cholesterol by a still unknown mechanism.

Au cours des dernières décennies, le régime alimentaire a subi une transition sans précédent, avec une augmentation de la consommation de protéines, de lipides et de glucides simples, et la diminution des apports en fibres. Par ailleurs, au-delà de la composition, la structure des aliments joue un rôle essentiel sur les cinétiques de digestibilité et la biodisponibilité des nutriments, modulant ainsi leur accessibilité pour microbiote dans le côlon. L’impact de la structure d’une matrice alimentaire complexe, formée de protéines et de lipides, sur le microbiote a été analysé de façon intégrée et a montré in vivo que la structure seule, dans le contexte d’un régime équilibré, pouvait altérer la composition du microbiote dans les zones distales et proximales que sont l’iléon et le cæcum. L’émulsion de protéines natives en phase liquide continue avec de fines gouttelettes protéolipidiques a arboré des protéines moins digestibles que l’émulsion de protéines dénaturées, en phase gélifiée, solide, avec de grandes gouttelettes. D’autre part, les lipides de l’émulsion solide étaient, à l’inverse, moins digestibles. Les protéines non digérées de l’émulsion liquide ont favorisé in vivo, les communautés de Lactobacillus et de Copprococcus tout en activant plus fortement les métabolismes de protéolyse. Inversement, les communautés de Bifidobacterium et d’Akkermansia muciniphila ont vu leurs abondances augmenter chez les rats consommant l’émulsion solide. Le deuxième objectif de ce travail de thèse a alors été d'analyser la capacité d'espèces prévalentes du microbiote intestinal humain à métaboliser des protéines non digérées. Nous avons montré, par le suivi des cinétiques de croissance et des productions de métabolites spécifiques, que les protéines du lait étaient une source d'énergie pour B. caccae, P. distasonis, B. longum et B. cocccoides en milieu pauvre ainsi qu'en milieu riche. Dans ces mêmes conditions, le transcriptome de B. caccae a montré la sur-expression de gènes codant pour des peptidases de specifités différentes, pour la production d'indoles, de GABA et de fimbriae. Ces travaux apportent des informations nouvelles sur l'impact de la structure sur l'écosystème digestif, et ouvre des portes pour le développement de nouveaux aliments. / Over the past decades, diet in developed countries has undergone an unprecedented transition, with increased intakes of protein, fat and high glycemic index carbohydrates. The first goal of this PhD work was to investigate how, beyond its composition, the food structure itself could play a part in nutrient digestibility and bioavailability, and consequently modulate the microbiota. We showed in vivo that the structure of proteino-lipidic emulsions modulated peptides transporters, and protein fermentation. The native proteins emulsion in a continuous liquid phase, with fine proteolipid droplets, was less digestible and led to more protein fermentation. It modified the gut microbiota composition in the distal and proximal intestinal sections and increased Lactobacillus and Coprococcus communities. A second in vivo study, using 15N labelled emulsions allowed us to disentangle the digestibility from the transit time effect. The second objective of the PhD was to characterize the capacity of prevalent human gut bacterial species to use undigested proteins as energy source. By monitoring growth kinetics and the production of specific metabolites, we showed that B. caccae, P. distasonis, B. longum et B. cocccoides could use whey protein as energy source. In addition we measured in B. caccae transcriptome, the over-expression of genes encoding for distinct peptidases, but also of GABA and indole pathways, and fimbriae biosynthesis. These data provide new insights on the relationships between food structure and the digestive ecosystem and could lead to the design of new functional food.

Résumé : Les régimes hyperprotéiques sont couramment consommés mais les conséquences au niveau du gros intestin sont peu connues. L’objectif de la thèse était d’étudier les effets des régimes hyperprotéiques et des métabolites bactériens dérivés des acides aminés sur la muqueuse du gros intestinUne série d’expérimentations animales et in vitro a permis de montrer que deux métabolites bactériens dérivés des acides aminés (le sulfure d’hydrogène et le p-cresol) sont toxiques pour l’épithélium lorsqu’ils sont présents en concentration élevée. Les résultats obtenus lors d’une étude clinique montrent que la quantité et la qualité des protéines alimentaires n’ont pas d’effets marqués sur la composition du microbiote fécal mais modifient les concentrations fécales et urinaires en métabolites bactériens.Ces modifications de l’environnement luminal du gros intestin n’étaient pas associées à une augmentation de la cytotoxicité des eaux fécales in vitro. Néanmoins, dans la muqueuse rectale, l’augmentation de l’apport en protéines a régulé l’expression de gènes impliqués dans le maintien de l’homéostasie et ces effets étaient distincts en fonction de la source de protéines utilisée. Toutefois, le niveau d’apport en protéines n’avait pas d’effet sur les paramètres inflammatoires et histologiques dans la muqueuse. Ces résultats ont été complétés par une étude chez le rat montrant qu’un régime hyperprotéique modifie le transcriptome dans les colonocytes mais n’a pas d’effets délétères en termes d’intégrité de l’ADN, de renouvellement de l’épithélium et de fonction barrière. / Abstract: High-protein diets are frequently consumed but the consequences for the large intestine are not well described. The objective of this thesis was to evaluate the effects of high-protein diets and of amino-acid derived bacterial metabolites on the large intestine mucosa. Animal and in vitro studies showed that two amino acid derived bacterial metabolites (hydrogen sulfide and p-cresol) are toxic for the epithelium when present at high concentration. The results obtained in a clinical trial indicate that quantity and quality of dietary protein do not have major effects on the fecal microbiota composition but modify the fecal and urinary concentration of bacterial metabolites.These changes in luminal environment were not associated with an increase in fecal water cytotoxicity in vitro. Nevertheless, in the rectal mucosa, the increase in protein intake regulated the expression of genes implicated in homeostatic processes and these effects were modulated by the source of protein. However, the level of protein intake had no effect on immune and histological parameters in the mucosa. These results were completed with a study in rats showing a clear transcriptome profile in colonocytes induced by a high-protein diet but that was not associated with detrimental effects in terms of DNA integrity, epithelial renewal and barrier function.

De plus en plus d’évidences suggèrent que la composition du microbiote intestinal pourrait jouer un rôle dans certaines pathologies comme l’hypertension, l’obésité, le diabète et plusieurs autres. Le but de cette étude est de démontrer que le microbiote intestinal modulé par des diètes enrichies en acide gras oméga-3 (w-3) ou en acide gras oméga-6 (w-6) avec un supplément ou non en probiotiques, pourrait influencer différemment la taille de l’infarctus du myocarde. Pour ce faire, des transplantations de microbiote provenant de rats nourris avec une diète w-3 ou w-6 combiné avec la prise ou non probiotiques ont été effectuées pendant 10 jours sur des rats dont le microbiote a été supprimé antérieurement par antibiothérapie. Ensuite, l’artère coronaire antérieure a été occluse pendant 30 minutes sur les rats transplantés et la taille de leur infarctus a été mesurée après 24 heures de reperfusion. La résistance intestinale, la concentration plasmatique de LPS (lipopolysaccharides), l’accumulation myocardique des neutrophiles, l’activation de la voie NF-kB (nuclear factor-kappa B), l’activation de la voie de cardioprotection RISK (Reperfusion Injury Salvage Kinase) et la composition du microbiote sont les autres paramètres qui ont été mesurés. Nos résultats démontrent une taille d’infarctus plus importante chez les animaux transplantés avec le microbiote w-6 sans probiotiques comparativement aux autres groupes incluant le groupe ayant reçu le microbiote w-6 avec probiotiques. Ces résultats indiquent qu’un microbiote provenant d’une diète enrichie en w-6 produit des effets délétères qui augmentent la taille de l’infarctus du myocarde comparativement à un microbiote provenant d’une diète enrichie en w-3 et que la prise de probiotiques permet de diminuer la taille de l’infarctus. / A lot of evidences suggest that the composition of the gut microbiota may play a role in certain conditions such as hypertension, obesity, diabetes and many others. The aim of our experiment is to demonstrate that the intestinal microbiota modulated by diets enriched in omega-3 fatty acid (w-3) or in omega-6 fatty acid (w-6) with or without a supplement in probiotics, could influence differently the size of a myocardial infarction. To do this, microbiota transplants from rats fed a w-3 or w-6 diet combined or not with intake of probiotics were performed for 10 days on rats whose microbiota had previously been suppressed by antibiotic therapy. Then, the anterior coronary artery was occluded for 30 minutes in the transplanted rats and the size of their infarction was measured after 24 hours of reperfusion. Intestinal resistance, plasma LPS (lipopolysaccharides) concentration, myocardial accumulation of neutrophils, activation of the NF-kB (nuclear factor-kappa B) pathway, activation of the RISK (Reperfusion Injury Salvage Kinase) cardioprotection pathway and microbiota composition are the other parameters that were measured. Our results demonstrate a larger infarct size in animals transplanted with the w-6 microbiota without probiotics compared to other groups (including the w-6 microbiota with probiotics group. These results indicate that a microbiota from a diet enriched in w-6 produces deleterious effects that increase the size of the myocardial infarction compared to a microbiota from a diet enriched in w-3 and taking probiotics can reduce the size of the myocardial infarction.

The gut microbiota plays a major part in maintaining the health of a human host. Countless of crucial functions in the body, including immune responses, cell signaling and energy metabolism to name a few, are conducted by the gut microbiota and its metabolites. Accordingly, it is of interest to gain knowledge on what can alter the gut microbiota, as these alterations by extension can give rise to adverse health effects. In this study, the impact of polychlorinated biphenyl (PCB)- and organochlorine pesticide (OCP) exposure on tricarboxylic acid (TCA) cycle metabolites, short-chain fatty acids (SCFAs) and bile acids, as well as other polar and semi-polar metabolites, which are all related to the gut microbiota, were investigated. An in vitro fermentation of faecal samples exposed to a PCB/OCP mixture was performed, and liquid chromatography-time of flight mass spectrometry (LC-qToF-MS) targeted and non-targeted approaches were applied to the extracts. The results obtained suggested that PCBs and OCPs most likely have an effect on the levels of several features of the gut metabolome with either increased or decreased levels upon exposure. Bile acids and TCA metabolites appear to follow a trend of decreasing levels, while no apparent effects could be seen for the SCFAs. Furthermore, distinct concentrations of the PCB/OCP mixture appear to induce different changes in gut microbiota functioning, which highlights the importance of performing dose-response studies when exploring biological effects of these compounds. The identification of different metabolite profiles during fermentation also allows for the possibility of further investigation of potential biomarkers to assess PCB/OCP exposure.

Phenobarbital (PB) is one of the most important antiseizure drugs (ASDs) to treat canine idiopathic epilepsy (IE). The effect of PB on the taxonomic changes in gastrointestinal microbiota (GIM) and their functions is less known, which may explain parts of its pharmacokinetic and pharmacodynamic properties, especially its antiseizure effect and drug responsiveness or drug resistance as well as its effect on behavioral comorbidities. Fecal samples of 12 dogs with IE were collected prior to the initiation of PB treatment and 90 days after oral PB treatment. The fecal samples were analyzed using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based dysbiosis index (DI), and quantification of short-chain fatty acids (SCFAs). Behavioral comorbidities were evaluated using standardized online questionnaires, namely, a canine behavioral assessment and research questionnaire (cBARQ), canine cognitive dysfunction rating scale (CCDR), and an attention deficit hyperactivity disorder (ADHD) questionnaire. The results revealed no significant changes in alpha and beta diversity or in the DI, whereas only the abundance of Clostridiales was significantly decreased after PB treatment. Fecal SCFA measurement showed a significant increase in total fecal SCFA concentration and the concentrations of propionate and butyrate, while acetate concentrations revealed an upward trend after 90 days of treatment. In addition, the PB-Responder (PB-R) group had significantly higher butyrate levels compared to the PB-Non-Responder (PB-NR) group. Metagenomics of functional pathway genes demonstrated a significant increase in genes in trehalose biosynthesis, ribosomal synthesis, and gluconeogenesis, but a decrease in V-ATPase-related oxidative phosphorylation. For behavioral assessment, cBARQ analysis showed improvement in stranger-directed fear, non-social fear, and trainability, while there were no differences in ADHD-like behavior and canine cognitive dysfunction (CCD) scores after 90 days of PB treatment. While only very minor shifts in bacterial taxonomy were detected, the higher SCFA concentrations after PB treatment could be one of the key differences between PB-R and PB-NR. These results suggest functional changes in GIM in canine IE treatment.

Background: Urinary tract infection (UTI) is a common bacterial infection that is usually diagnosed by symptoms such as dysuria and frequency, and the golden standard is to take a urine culture to identify bacteria that may cause UTI. This method was founded with the idea that normal urine is sterile, but this is now being questioned because of growing evidence of a urinary microbiota thus giving a new approach to methods for UTI diagnosis. Aim: To identify and re-evaluate findings of bacteria from urine cultures in the ongoing paradigm shift of a potential urinary microbiome, and dysbiosis as a cause for UTI. Materials and Methods: This study used MALDI-TOF MS to identify approximately 250 bacteria isolates that had been cultured by Expanded Quantitative Urine Culture (EQUC) from 162 women with symptoms of cystitis. EQUC had allowed the bacteria to grow in both CO2 and anaerobic conditions, which differs from standard techniques.   Results and Conclusion: Escherichia coli and Enterococcus faecalis dominated the results of most frequently identified bacteria. However, other bacteria were commonly present within the same culture which is traditionally considered as contamination but may now indicate a urinary flora. Anaerobic bacteria – such as Porphyromonas sp. – were also identified, but their connection to UTI is unclear. Lactobacillus sp. – which are associated with a healthy flora in women – were found in urine cultures and often in smaller quantities which could suggest dysbiosis. More research on Lactobacillus sp. and their correlation with UTI is suggested for a more accurate indication of urinary dysbiosis in women.