Thèses sur le sujet « Microbiota intestinale (Gut microbiota) »

Le microbiote intestinal humain a été fortement corrélé avec la santé humaine et les maladies et a montré un potentiel dans les développements thérapeutiques. La métagénomique a déjà montré qu'elle était capable de générer beaucoup de données, dont certaines sont dénuées de sens et constituaient la "matière noire". Alors culturomics a été développée pour compléter la métagénomique en ciblant des espèces bactériennes précédemment non cultivées. En utilisant la culturomics, nous avons décrit le microbiote intestinal humain des Pygmées et réussi à isoler un nombre significatif d'espèces bactériennes parmi lesquelles 38 étaient de nouvelles espèces. En comparant les résultats métagénomiques aux données culturomics, on constate que seulement 26% des espèces isolées ont été récupérées par métagénomique et que jusqu'à 59% des Operational taxonomic units détectées correspondaient à de nouvelles espèces bactériennes isolées par culturomique dans cette étude ou dans les précédentes
The human gut microbiota has been correlated in general health and diseases. Thus its description became mandatory to better understand its role and therapeutic potential. However, metagenomics has previously showed to be able to generate a lot of data, of which some are meaningless and constituted the “Dark matter”. Thus, culturomics was developed to complement metagenomics by targeting previously uncultured bacterial species. Using culturomics, we described the human gut microbiota of Pygmy people and succeeded in isolating a significant number of bacterial species out of which 38 were new species. Comparing metagenomics results to culturomics data, we see that only 26% of the isolated species were recovered by metagenomics and that up to 59% of the Operational taxonomic units detected corresponded to new bacterial species isolated by culturomics either in this study or in previous ones

Food intake is controlled by a highly complex and distributed neural system integrating many sensory inputs, most notably those arising from the gastrointestinal tract in response to a meal. Reduced sensitivity to intestinal nutrients has been proposed to be partly responsible for increased energy intake and weight gain in both animals and humans during HF feeding. However, the mechanism by which a HF diet impinges on post-ingestive intestinal feedback to promote overconsumption is unclear, as is the role of diminished satiation signaling in the development of obesity. Therefore, the work of this thesis attempts to discern the role of HF feeding in nutrient-induced satiation, by addressing the impact of GI peptides and the gut microbiota in diet-induced obesity in animals prone or resistant to obesity. In the first set of experiments, we found that diet-induced obese rats exhibited a reduced responsiveness to the suppressive effects of intragastric lipid loads, compared to diet-resistant rats. This was associated with altered intestinal gut peptides and GPRs, plausibly contributing to reduced lipid-induced satiation. In the next set of experiments, we showed that OP rats develop impaired GLP-1 signaling during maintenance on a HF diet. During chow feeding, OP and OR rats had similar sensitivity to the anorectic effects of GLP-1R agonism; however, HF feeding abolished the suppressive response to exendin-4 in OP rats. This was associated with downregulation of GLP-1R in the nodose ganglia, in addition to decreased circulating GLP-1 and L-cell counts. The last set of experiments demonstrates the influence of the gut microbiota in regulating intestinal chemosensory machinery and potentially promoting adiposity in OP rats. First, GF mice exhibited increased consumption for lipid solutions with associative decreases in intestinal satiation signals and fatty acid receptors, indicating that lack of microbiota reduces post-ingestive signaling to promote overeating. Next, we found that OP rats harbor a distinct microbiota profile compared to OR rats during HF feeding. By conventionalizing GF mice with OP microbiota, we replicated the obese phenotype, and associated reductions in peripheral and central appetite-related pathways. In summary, this thesis provides evidence that the interaction of a polygenetic susceptibility to obesity coupled with HF feeding reduces sensitivity to intestinal nutrients, by altering secretion and sensitivity to satiation signals that ultimately contributing to weight gain and adiposity. Additionally, further elucidation on the ability of an aberrant gut microbiota to influence regulatory systems involved in energy regulation could provide useful information for the development of therapeutic treatments for obesity
La prise alimentaire est contrôlée par un système complexe associant des modifications du système nerveux. Ces dysfonctionnents impliquent les outils de perception, notamment ceux en provenance de l'appareil gastro-intestinal en réponse à un repas. La diminution de la sensibilité intestinale aux nutriments a été décrite en partie comme responsable de l'apport énergétique accrue et du gain de poids chez les animaux et les humains au cours d'un régime hypercalorique. Cependant, le mécanisme par lequel un régime obesogène affecte les signaux intestinaux postprandiaux favorisant la surconsommation et leurs rôles dans la diminution du signal de satiété contribuant au développent de l'obésité reste très peu étayer. Par conséquent, le travail de cette thèse a pour but de caractériser le rôle d'un régime hypercalorique dans la diminution de la satiété induite par les nutriments, d'étudier le rôle des peptides gastro-intestinaux et de microbiote au cours d'un régime hypercalorique favorisant l'obésité chez les rats OP. Dans la première série d'expériences, nous avons constaté que les rats soumises un régime hypercalorique présentaient une réponse réduite aux effets suppresseurs de charges lipidiques gastriques comparé à des rats résistants à l'obésité (OR). Cette réponse a été associée à une altération des peptides intestinaux et des GPRS contribuant à une réduction du signal du satiété. Dans une deuxième série d'expériences, nous avons démontré que les rats obeses prônes (OP) développent une déficience de la voie de signalisation de GLP-1 au cours d'un régime hypercalorique. Sous un régime normal, les rats OP et OR avaient la même sensibilité aux effets anorexigène d'un agoniste du récepteur du GLP-1. Toutefois, le régime obesogène abolit la réponse suppressive de l'exendin-4 chez des rats OP. Ceci a été associée à une régulation négative de l'expression du GLP-1R dans les ganglions nodaux, une diminution des taux du GLP-1 circulants et du nombre des cellules L sécrétrices du GLP-1. La dernière série d'expériences démontre l'influence du microbiote intestinal dans la régulation de la chemosensibilité intestinale favorisant l'adiposité chez les rats OP. Les souris axéniques présentent une consommation accrue de solutions lipidiques associé à une diminution du signal satiétogène intestinal et des récepteurs d'acides gras. Nous avons conclu que l'absence du microbiote réduit le signal de satiété postprandial contribuant à la surconsommation des nutriments. Par la suite, nous avons identifié que les rats OP possèdent un profil du microbiote intestinal distinct de rats OR sous un régime hypercalorique. La conventionnalisation des souris axéniques avec le microbiote issu des rats OP, reproduit parfaitement le phénotype obèse avec à une réduction de la signalisation centrale et périphérique des voies contrôlant la prise alimentaire. En résumé, cette thèse apporte la preuve que l'interaction entre une prédisposition à l'obésité généralement polygénique couplée à une alimentation obesogène réduit la sensibilité intestinale aux nutriments, altérant la sécrétion et la sensibilité aux signaux de satiété. Ces effets ont pour conséquence un gain de poids et une expansion de la masse grasse. De plus, des preuves scientifiques supplémentaires sur la capacité d'un microbiote intestinal aberrant d'influencer les systèmes de régulation impliqués dans le maintien de l'homéostasie énergétique pourraient fournir des informations scientifiquement fondées afin de prévenir le développement et l'installation de l'obésité et contribuer aux progrès thérapeutiques de l'obésité

ROLE OF CART ON GUT MICROBIAL DYSBIOSIS, STUDYING THE GUT/BLOOD MICROBIOTA DURING THE FIRST TWO YEARS OF SUPPRESSIVE CART BACKGROUND Microbial dysbiosis features HIV+ individuals, both naïve and cART-treated, and is linked to anatomical/structural changes in the gastrointestinal (GI) tract, leading to microbial translocation (MT) and immune activation. Given that data on microbiota modifications during long-term therapy are lacking, we investigated gut/blood microbiota during the first 2 years of suppressive cART. METHODS We enrolled 138 HIV+ subjects. Plasma was collected at baseline (T0) and following 12 (T12) and 24 months (T24) of cART. CD8+ T-cell activation (CD38+; CD38+CD45R0+), MT (sCD14 and EndocAb) and GI damage (IFAB-P) were studied. In a sub-group of 41 patients (pts) we also evaluated GI permeability (urinary LAC/MAN test), inflammation (faecal calprotectin), 16SDNA (MT marker) and gut persistence score, metagenomic function analysis (Picrust) as well as peripheral and faecal microbiota (DNA extraction and 16S Metagenomic Sequencing; MiSeq Illumina®). For the microbiota analyses we enrolled 15 HIV- subjects as controls. All groups were analysed by Wilcoxon test, Kruskal-Wallis test and Permanova analysis. RESULTS 88% were male, 65% MSM, 6% HCV+; median age, CD4+ count, HIV RNA and duration of infection were respectively 38 years, 312/mmc, 5.03 log10cp/mL and 11.5 months. Following cART we registered a reduction of activated and activated/memory CD+8 T-cells (both with p<0.0001), an increase of EndoCab levels (p<0.0001) yet no significant changes in plasma sCD14. In contrast, an increase of I-FABP (p<0.0001) vis- à -vis a reduction of LAC/MAN test (p=0.03) and faecal calprotectin (p=0.01) were found. In faeces, cART resulted in a limited modification of the relative abundance of the microbiota, however differences between pts and controls were detected in the Firmicutes, Bacteroidetes and Actynobacteria phyla. Alpha-diversity showed higher richness in HIV+ vs controls (observed: p=0.006; Chao1: p=0.002) and these differences were maintained at T12 and T24. PCoA plot analyses showed a trend to the separation of pts and controls at all time-points yet the latter overlapped regardless of treatment status and length of cART. Lefse analyses (LDS >2.0) in HIV+ showed a significant increase of Veillonellaceae at T12 (p=0.007) and T24 (p=0.001) Desulfovibrionaceae at T24 (p=0.022) and Prevotellaceae at T24 (p=0.018). Further, many differences between pts and controls was detected in HIV+ . This persistent dysbiosis was associated with the continuous mucosal damage, despite cART introduction: I-FABP were positively correlated with Veillonellaceae both at T12 (r2=0.197; p=0.030;) and T24 (r2=0.156; p=0.017). Interestingly, when we stratified patients according to cART regimens, we found that only NNRTI-based therapy significantly reduced richness (observed: p=0.038; Chao1: p=0.006), but not evenness indexes over time. Furthermore, the relative abundance analyses showed a different profile at both family and genus levels, with NNRTI-based regimens significantly reducing the families of Coriobacteriaceae, Peptococcaceae and increasing the Veillonellaceae family. On the opposite, INSTI-based regimens resulted in decreased Peptococcaceae and increased Veillonellaceae families, as well as in higher Allisonella genus. No major effects following PI-based regimens were detected; no modifications about gut persistence score analysis as well as predicted functional metagenomic pathway analysis were found. Plasma microbiota analyses revealed no major changes of relative abundance parameters during cART and in comparison with uninfected controls. Decreased alpha-diversity was nonetheless found in HIV+ compared to controls (Shannon: p=0.02, Simpson: p=0.009) and persisted both at T12 and T24. CONCLUSIONS HIV-related modifications of the microbiota occur within the GI tract and not in the blood and are minimally affected by long-term effective cART, despite evidence of the containment of gut inflammation. These data suggest the ability of the virus to irreversibly impact the microbiological core of chronically-infected individuals.

Maternal microbiota during pregnancy, as well as maternal disease state, may impact offspring gut bacterial colonisation. Here, we explore the impact of maternal antibiotics during gestation and/or nursing on offspring gut microbiota. Further, we investigate the effect of preconception helminth infections on maternal and infant gut microbiota. For maternal antibiotic experiments, dams were fed vancomycin, polymyxin B, or both, in drinking water during gestation, nursing or gestation plus nursing, and their offspring microbiota analysed at 14 days of life, alongside immunity in the spleens. Offspring born to vancomycin treated mothers had significantly higher relative abundance of Proteobacteria and Tenericutes while maternal oral polymyxin B led to significantly lower abundance of Proteobacteria and Deferribacteres in infants. Maternal oral vancomycin led to significant reduction in proportions of infant central memory CD4+ T cells (CD4+CD44hiCD62Lhi) regardless of antibiotic timing. Effector memory CD4+ T cells were significantly lower in pups born to dams treated with polymyxin B while nursing while proportions of central memory CD4 T cells were significantly increased in gestation only or gestation plus nursing pups. In addition, oral vancomycin in dams during nursing resulted in significantly reduced proportions of both total and follicular B cells in offspring born to antibiotic treated dams. Pups born to Vancomycin treated mothers had a significant delay in growth when infected with Respiratory Syncytial Virus (RSV). On the other hand, pups born to mothers treated with Polymyxin B during gestation or gestation plus nursing were susceptible to Nippostrongylus brasiliensis (Nb) infection. In the second study, we infected female BALB/c mice with 500Nb L3 three weeks prior to mating and examined the effect of preconception helminth infection on offspring microbiota and immunity. Preconception Nb infections led to alterations of maternal gut microbiota during pregnancy. In addition, we observed dramatic differences in offspring microbiota in pups born to previously helminth infected dams. Coriobacteriaceae were predominant in pups born to previously Nb infected dams when compared to uninfected dams. Overall, manipulation of maternal microbiota during gestation or lactation profoundly impacts offspring growth, intestinal microbiota and immunity to RSV and helminths.

La métaprotéomique s’attache à identifier et quantifier les protéines d’échantillons biologiques complexes comme le microbiote intestinal humain. L’analyse de plusieurs centaines d’échantillons revêt un intérêt évident compte tenu de la reconnaissance croissante de cet écosystème en tant que partenaire santé. Cependant, les méthodes et protocoles utilisés jusqu’à ce jour en métaprotéomique ne sont pas adaptés à des études de grande ampleur. Nous avons développé des algorithmes, évalué et comparé plusieurs approches d’identification des peptides et protéines et proposé des critères d’évaluation systématiques, avec un intérêt particulier porté sur la réplicabilité des identifications, afin de développer un pipeline de prétraitement adapté à des études d’envergure. Ce travail apporte un socle méthodologique jusqu’ici manquant dans le domaine de la métaprotéomique du microbiote intestinal humain. Nous avons également comparé des méthodes de normalisation des XIC et développé une méthodologie d’imputation des données manquantes permettant d’affiner les estimations d’abondances obtenues par la méthode de SC. Ce travail de thèse a permis de mettre en évidence des biomarqueurs microbiens potentiellement d’intérêt pour prédire la réponse à un régime amaigrissant ou pour caractériser différents phénotypes de MICI. Nous avons également analysé le métaprotéome de plus de 200 patients dans le cadre de l’ANR ProteoCardis adossée au projet MetaCardis, et s’intéressant au lien possible entre microbiote intestinal et maladies cardiovasculaires. La recherche de protéines d’intérêt parmi ces données devrait permettre de découvrir des candidats biomarqueurs de maladies cardiovasculaires
Metaproteomics focuses on identifying and quantifying proteins in complex biological samples such as the human gut microbiota. The analysis of several hundred of samples is of interest given the growing recognition of this ecosystem as a health partner. However, the methods and protocols used so far in metaproteomics are not suitable for large-scale studies. We have therefore developed algorithms, evaluated and compared several identification approaches for peptides and proteins and proposed systematic evaluation criteria, with a particular interest in the replicability of identifications, in order to develop a pre-treatment pipeline suitable for wide-ranging studies. This work bring a methodological base so far missing in the field of the metaproteomics of the human gut microbiota. Quantification of peptides and proteins by XIC has never been performed on this type of data, we have also compared normalization methods and developed a methodology for imputing missing data to refine the abundance estimations obtained by the more classical method of SC. This thesis work has highlighted microbial biomarkers of potential interest for predicting the response to a slimming diet, or to characterize various phenotypes of IBD. We have also been able to analyse the metaproteome of more than 200 patients in the framework of the ProteoCardis ANR, which is ancillary to the European project MetaCardis, and which focuses on the potential link between gut microbiota and cardiovascular diseases. The search for proteins of interest among these data should allow us to discover protective or aggravating candidate biomarkers of cardiovascular diseases

De nos jours, le changement de style de vie et la consommation excessive d'aliments riches en énergie sont associés avec l'augmentation majeure de l'incidence des maladies métaboliques comme l'obésité et le diabète de type 2. Le diabète de type 2 est caractérisé, entre autre, par une augmentation de la production hépatique de glucose responsable d'une hyperglycémie chronique. Durant ces 10 dernières années, plusieurs études ont suggéré que le microbiote intestinal pouvait être impliqué dans le développement des maladies métaboliques. Le microbiote intestinal est composé de plusieurs milliards de bactéries réparties en plus de 1000 espèces différentes qui colonisent le tractus digestif. Plusieurs études ont montré que certaines pathologies comme le diabète et l'obésité sont caractérisées par des altérations taxonomiques et fonctionnelles du microbiote intestinal. De plus, la colonisation de souris axéniques (i.e. dépourvues de microbiote) par un microbiote intestinal provenant de souris ou d'Hommes obèses/diabétiques est suffisante pour induire la pathologie. Ces résultats suggèrent que les modifications du microbiote intestinal retrouvées chez les patients obèses/diabétiques sont potentiellement impliquées dans le développement des maladies métaboliques. Cependant, l'absence de microbiote intestinal chez les souris axéniques induit des altérations structurelles et fonctionnelles de l'intestin comme une hyperperméabilité intestinale ou un système immunitaire atrophié. Dans ces conditions, il est possible de se demander si les effets délétères induits par la colonisation des souris axéniques avec un microbiote modifié peuvent être observés chez des souris conventionnelles. Pour répondre à cette question nous avons développé un nouveau protocole de transfert de microbiote intestinal dans un modèle de souris conventionnelles. Nous avons transféré le microbiote contenu dans le caecum de souris obèses (" microbiote obèse ") et celui contenu dans le caecum de souris minces (" microbiote mince ") dans des souris conventionnelles non traitées aux antibiotiques. De manière surprenante, le transfert du " microbiote obèse " a induit une diminution de la glycémie à jeun associée à une baisse de la néoglucogenèse hépatique chez les souris transplantées. A l'inverse, le transfert du " microbiote mince " n'a pas modifié la néoglucogenèse. De plus, le transfert du " microbiote obèse " a induit des modifications taxonomiques et fonctionnelles du microbiote intestinal des souris transplantées. De manière intéressante, une fois nourries avec un régime hyperlipidique les souris ayant reçu le " microbiote obèse " ont conservé une glycémie à jeun plus faible que les souris non transplantées. Encore une fois, ce phénotype résulte d'une diminution de la production hépatique de glucose caractérisée par une baisse de l'activité des enzymes néoglucogéniques phosphoenolpyruvate carboxykinase et glucose-6-phosphatase. Par ailleurs, ces souris sont également moins grasses que les souris non transplantées. En conclusion, nous avons montré que le transfert d'un " microbiote obèse " peut moduler le métabolisme hépatique et prévenir l'augmentation de la néoglucogenèse hépatique normalement induite par le régime hyperlipidique chez des souris conventionnelles. Ces travaux de thèse ont montré d'une part, que la modification du microbiote intestinal de souris conventionnelles est possible par transfert de microbiote caecal. D'autre part et contre toutes attentes, ces résultats mettent en lumière que, contrairement aux observations faites chez les souris axéniques, le transfert d'un " microbiote obèse " dans une souris conventionnelle n'induit pas les phénotypes caractéristiques des maladies métaboliques. Par ailleurs, ce modèle de transfert caecal pourrait être utile pour la compréhension du rôle des bactéries intestinales sur le développement des maladies métaboliques
Nowadays, the change of lifestyle and increase in the consumption of high-calorie foods are associated with a marked rise of the prevalence of metabolic diseases, including obesity and type 2 diabetes. Type 2 diabetes is linked, at least in part, to an increase of hepatic glucose production responsible for a fasting hyperglycemia. In the past decade, an increasing body of evidence has proposed gut microbiota as a new factor contributing to these metabolic alterations. Gut microbiota consists of trillions of bacteria identifying more than 1000 different species that inhabit our intestine. A body of work has demonstrated that multiple pathologies such as type 2 diabetes and obesity are characterized by an altered proportion and activity of the gut microbiota. In addition, the colonization of germ-free mice with the gut microbiota from either obese/diabetic humans or obese/diabetic mice transfers the phenotype. These results suggest that the modifications of the gut microbiota found in obese/diabetic patients are a potential etiologic factor for those diseases. Nevertheless, the lack of microbiota in germ-free mice determines both structural and functional alterations such as gut hyperpermeability and the atrophy of the immune system. Therefore, we could wonder whether the detrimental effects of the gut microbiota from obese/diabetic patients observed in germ-free mice may also be observed in healthy conventional mice. To address this issue, we have developed a new gut microbiota transferring process from conventional mice to other mice. We have transferred the cecal microbiota harvested from either obese ("obese microbiota") or lean ("lean microbiota") mice in antibiotic-free conventional mice. Surprisingly, the mice which received the "obese microbiota" had a reduced fasted glycaemia compared to the mice which received the "lean microbiota". This diminution could be attributed to a decrease of the hepatic gluconeogenesis since conversion from pyruvate to glucose and phosphoenolpyruvate carboxykinase activity were lower in the liver of mice which received the "obese microbiota". Conversely, the transfer of the "lean microbiota" did not affect the hepatic gluconeogenesis. In addition, the transfer of the "obese microbiota" changed gut microbiota composition and the microbiome of recipient mice. Interestingly, mice which received the "obese microbiota" and fed a high-fat diet still exhibited reduced fed and fasted glycaemia. Once again, this phenotype was due to a decrease of hepatic gluconeogenesis characterized by a diminution of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activity. In addition, the mice which received the "obese microbiota" had less adiposity compared to the non-transferred mice. Finally, we reported that transferring the "obese microbiota" impact on hepatic metabolism and prevent HFD-increase hepatic gluconeogenesis. On the one hand, these thesis works, have demonstrated that it is possible to modify the gut microbiota by our caecal transferring process. On the other hand, our results suggest that the transfer of the "obese microbiota" in conventional mice does not induced some characteristics of metabolic diseases contrary to that it is observed in germ-free mice. Furthermore, this kind of gut microbiota transferring process may be useful for a better understanding of the etiology of metabolic diseases

La fermentation des fibres alimentaires est l’une des fonctions majeures du microbiote intestinal humain. Les bactéries fibrolytiques synthétisent un grand nombre d’enzymes, appelées Glycoside Hydrolases (GH), indispensables à la déconstruction de la grande variété structurelle des polysaccharides pariétaux que nous ingérons. Au cours de ce travail, nous avons exploré, grâce à une approche de métagénomique fonctionnelle, l’organisation et les propriétés des systèmes enzymatiques bactériens impliqués dans la dégradation des glycanes de parois végétales dans l’intestin humain.En premier lieu, nous avons cherché à déterminer si les bactéries de la muqueuse iléale étaient capables de dégrader les fibres pariétales dans un contexte sain. Cette fonction étant généralement décrite pour le microbiote colique par extrapolation de travaux menés à partir de selles humaines, nos connaissances de la dégradation des fibres dans la partie haute du tractus digestif sont donc très limitées. Un total de 20 000 clones issus du métagénome bactérien d’une partie saine de la muqueuse iléale d’un individu a été criblé pour des activités de dégradation de la carboxymethylcellulose et du xylane, deux substrats modèles des polysaccharides pariétaux. Douze clones métagénomiques positifs nous ont permis de mettre en évidence un arsenal de gènes bactériens codant pour des GH et d’autres protéines impliquées dans le métabolisme des fibres alimentaires dont certains organisés en PUL (Polysaccharide Utilization Loci), des clusters de gènes spécialisés dans la dégradation des polysaccharides complexes. Ces gènes proviennent de chromosomes bactériens assignés au genre Bacteroides ou à des espèces de Clostridiales, et codent pour des enzymes capables de dégrader également des β-glucanes à liaisons mixtes. L’étude de la prévalence de ces gènes dans les métagénomes de référence indique que plusieurs d’entre eux proviendraient de souches bactériennes plutôt spécifiques de la muqueuse iléale. De plus, certaines enzymes présentent des propriétés inédites potentiellement intéressantes dans le domaine biotechnologique. Nos recherches ont donc permis de revisiter la fonction fibrolytique du microbiote intestinal chez l’Homme et de proposer une localisation de cette fonction dès l’intestin grêle.Dans un second temps, en utilisant une approche méthodologique similaire, nous avons étudié la capacité du microbiote fécal d’un individu obèse à dégrader des polysaccharides pariétaux complexes, en général moins consommés par les individus obèses. Au total, nous avons identifié 50 clones appartenant à 14 espèces bactériennes des phyla suivants : Bacteroidetes, Firmicutes et Actinobacteria. Les inserts métagénomiques portent des gènes codant pour différentes familles de GH, impliquées dans la dégradation des polysaccharides d’intérêt. Les premières analyses de la prévalence de ces gènes chez plus d’une centaine d’individus (obèses ou non), par interrogations des catalogues de gènes microbiens de référence, suggèrent des associations avec le statut phénotypique « obèse »
Among the crucial functions of the intestinal microbiota, extracting energy from food such as dietary fibres is of major importance. Facing the huge diversity of incoming complex carbohydrates, the fibrolytic bacteria synthesize a set of diversified Carbohydrate-Active Enzymes (CAZymes) including Glycoside Hydrolases (GH) that specifically disrupt complex polysaccharides. Here, using functional metagenomic approaches, we explored the organization and properties of bacterial enzymatic systems involved in the breakdown of plant cell wall (PCW) glycans in the intestinal tract.Firstly, we investigated the capacity of the microbiota associated to the human ileum mucosa to degrade complex non-starch polysaccharides in a healthy context. This function has never been investigated in this part of the intestine, but it has been rather associated to microorganisms inhabiting the colon, due to more accessible fecal samples. Using a fosmid library derived from a healthy part of the human ileal mucosa, we screened 20,000 metagenomic clones for their activities against carboxymethylcellulose and xylan chosen as models of the major PCW polysaccharides from dietary fibres. Twelve positive clones revealed a broad range of CAZyme encoding genes from Bacteroides to Clostridiales species, as well as Polysaccharide Utilization Loci (PUL). Functional GH genes were identified and break-down products examined from different polysaccharides including mixed-linkage β-glucans. Revealed CAZymes and PUL were also examined for their prevalence in human gut microbiomes. Part of them belongs to unidentified strains rather specifically established in the ileum. Others were enzymes unclassified in identified GH families or with original properties addressing novel candidates for biotechnological applications. Thus, we evidenced for the first time that the ileal mucosa associated-microbiota encompasses the enzymatic potential for PCW complex polysaccharide degradation that might start in the small intestine.In a second time, by using the same methodology, we harvested the enzymatic capacities of the fecal microbiota from an obese person to disrupt complex polysaccharides from dietary fibres usually consumed in lower quantity in obese people. This study aimed at examining the links between genes encoding enzymes specifically dedicated to PCW complex carbohydrates and the obese phenotypic status using reference microbial gene catalogs. We screened a fecal metagenomic library from an obese individual on representative PCW substrates and identified 50 clones belonging to 14 different species from the Bacteroidetes, Firmicutes and Actinobacteria phyla. The metagenomic inserts harbor genes encoding enzymes from GH families specific from complex carbohydrate degradation. First querying of the prevalence of these genes in hundreds individuals (obese and control), using catalogs of reference microbial genes, suggest associations with the "obese" phenotypic status

Dissertação de Mestrado Integrado em Medicina Veterinária
Investigations of the relationships between the gut microbiota and gastrointestinal parasitic nematodes are attracting growing interest by the scientific community. These studies have however been carried out mainly in humans and experimental animals, while knowledge of the make-up of the gut commensal microbiota in presence or absence of infection by parasitic nematodes in domestic animals is limited. In this study, we investigate the qualitative and quantitative impact that infections by a widespread parasite of cats (i.e. Toxocara cati) exert on the gut microbiota of feline hosts. The faecal microbiota of cats with patent infection by T. cati (= Tc+), as well as that of negative controls (= Tc-) was examined via high-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data. A total of 2,325,366 useable high-quality sequences were generated from the faecal samples analysed in this study and subjected to further bioinformatics analyses, which led to the identification of 128 OTUs and nine bacterial phyla, respectively. The phylum Firmicutes was predominant in all samples analysed (mean of 53.0%), followed by the phyla Proteobacteria (13.8%), Actinobacteria (13.7%) and Bacteroidetes (10.1%). Among others, bacteria of the order Lactobacillales, the family Enterococcaceae and genera Enterococcus and Dorea showed a trend towards increased abundance in Tc+ compared with Tc- samples, while no significant differences in OTU richness and diversity were recorded between Tc+ and Tcsamples (P = 0.485 and P = 0.581, respectively). However, Canonical Correlation and Redundancy Analyses were able to separate samples by infection status (P = 0.030 and P = 0.015, respectively), which suggests a correlation between the latter and the composition of the feline faecal microbiota.
RESUMO - Alterações na microbiota intestinal felina associadas a infecções por Toxocara cati - A investigação das interações entre a microbiota intestinal e os nematodes intestinais tem vindo a atrair o interesse da comunidade cientifica. No entanto, a maioria destes estudos tem sido desenvolvida em humanos e animais de laboratório, e deste modo o conhecimento da composição da microbiota comensal do intestino na presença de nematodes intestinais é reduzido. Neste estudo, foi investigado o impacto qualitativo e quantitativo da infeção pelo parasita comum dos gatos (i.e. Toxocara cati) na microbiota intestinal do hospedeiro felino. A microbiota fecal de gatos com infeção patente por T. cati (Tc+), bem como controlos negativos (Tc-) foi avaliada através de sequenciação de alto débito da região hiper-variável V3-V4 do gene 16S, seguido de análise bioinformática e bioestatística. Das amostras fecais incluídas no estudo foram obtidas um total de 2 325 366 sequências de alta qualidade e sujeitas a analise bioinformática, o que levou à identificação de 128 OTUs e nove filos bacterianos. O filo Firmicutes foi encontrado em predominância em todas as amostras (média de 53,0%), seguido do filo Proteobacteria (13,8%), Actinobacteria (13,7%) e por fim Bacteroidetes (10,1%). A abundância de determinados grupos de bactérias tendeu a aumentar nas amostras Tc+ quando comparadas com as amostras Tc-, tais como a ordem Lactobacillales, a família Enterococaceae e o género Enterococcus e Dorea. No entanto, a riqueza e diversidade das OTUs não apresentou diferenças significativas entre as amostras Tc+ e Tc- (P=0,485 e P=0,581, respetivamente). Todavia, a análise canónica de redundância demonstrou uma separação das amostras de acordo com o estado de infeção (P=0,030 e P=0,015, respetivamente), o que sugere uma correlação entre este e a composição da microbiota fecal felina.
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Els trastorns funcionals gastrointestinals (síndrome de l'intestí irritable) i la malaltia inflamatòria intestinal presenten una inflamació crònica del tracte gastrointestinal. Encara que la seva patogènia no es coneix totalment, totes dues malalties resulten de la interacció de la microbiota amb factors ambientals que convergeixen en individus genèticament susceptibles, donant lloc a una activació anòmala del sistema immune. Els antibiòtics amb activitat immunomoduladora són una opció terapèutica interessant per la seva acció simultània davant la microbiota i la reacció immunitària. La rifaximina és un antibiòtic no absorbible aprovat pel tractament de la síndrome de l'intestí irritable amb diarrea i amb efectes beneficiosos en la malaltia inflamatòria intestinal. No obstant, es desconeix el seu mecanisme d'acció i la contribució dels seus efectes antimicrobians i immunomoduladors. Aquest treball explora el mecanisme d'acció de la rifaximina en ratolins sans i durant la inflamació intestinal aguda. Es valoraren els efectes de la rifaximina en animals sans i en animals amb colitis aguda, utilitzant el model de colitis induïda per dextrà sulfat sòdic (DSS, 3% en aigua, 5 dies). Els ratolins es tractaren preventivament amb rifaximina o doxiciclina (control positiu). Es valoraren els signes clínics d'inflamació del còlon (macroscòpicament i histopatologia) en la necròpsia. La microbiota ceco-còlica, luminal i adherida a l'epiteli, es caracteritzarà mitjançant: hibridació in situ fluorescent (FISH), polimorfismes de longitud de fragments de restricció (T-RFLP) i seqüenciació del 16S ARNr. La resposta immune local i els mecanismes de interacció hoste-microbiota es valoraren determinant canvis en l'expressió gènica (RT-qPCR) de citocines pro- i antiinflamatòries, pèptids antimicrobians i receptors de tipus Toll (TLRs). En animals sans, la rifaximina no va modificar els recomptes totals, la biodiversitat o la adherència a la paret del còlon dels bacteris ceco-còlics. Només es van observar canvis menors en l'expressió de TLRs o d'altres marcadors de tipus immune. Els animals que van rebre DSS van mostrar signes clínics indicatius del desenvolupament de colitis, amb regulació a l'alça en l'expressió de marcadors immunes, incloent el pèptid antimicrobià RegIIIɣ. La rifaximina no va afectar el curs clínic de la colitis ni l'expressió d'aquests marcadors. Per contra, la doxiciclina va atenuar les dues respostes. Independentment del tractament aplicat, només es van observar canvis menors en l'expressió de TLRs. L'expressió del receptor X de pregnà (PXR) va disminuir durant la inducció de la colitis, un canvi no modificat per la rifaximina, però que es va prevenir amb la doxiciclina. La colitis va induir una disbiosi caracteritzada per un augment de Verrucomicrobia i Deferribacteres amb un descens de Bacteroidetes, mantenint-se la diversitat de tipus alfa. Durant el tractament amb rifaximina, amb o sense colitis, es va mantenir la biodiversitat, amb una composició bacteriana molt similar a l'observada en animals sans. Els animals tractats amb doxiciclina van mostrar canvis extensos en la microbiota, amb similituds entre els grups inflamat i no inflamat. Concretament, la doxiciclina va reduir els grups Clostridiales patogènics, mentre que va augmentar els Clostridia grup XIVa, Lactobacillus i Ruminococcus, considerats grups beneficiosos. Aquests resultats mostren que en animals sans la rifaximina presenta una activitat antimicrobiana i immunomoduladora limitada. En estat de colitis, la rifaximina no manifesta efectes immunomoduladors ni antimicrobians consistents amb una activitat antiinflamatòria. No obstant això, aquestes accions no es poden excloure en humans, explicant els efectes beneficiosos observats clínicament. Tenint en compte que en humans la rifaximina actuaria a través dels PXR, és possible que el model de colitis induïda per DSS en ratolí no sigui l'adequat per estudiar el seu mecanisme d'acció. (...)
Los trastornos funcionales gastrointestinales (síndrome del intestino irritable) y la enfermedad inflamatoria intestinal presentan una inflamación crónica del tracto gastrointestinal. Aunque su patogenia no se conoce totalmente, ambas enfermedades resultan de la interacción de la microbiota con factores ambientales que convergen en individuos genéticamente susceptibles, dando lugar a una activación anómala del sistema inmune. Los antibióticos con actividad inmunomoduladora son una opción terapéutica interesante por su acción simultánea frente la microbiota y la reacción inmunitaria. La rifaximina es un antibiótico no absorbible aprobado para el tratamiento del síndrome del intestino irritable con diarrea y con efectos beneficiosos en la enfermedad inflamatoria intestinal. Sin embargo, se desconoce su mecanismo de acción y la contribución de sus efectos antimicrobianos e inmunomoduladores. Este trabajo explora el mecanismo de acción de la rifaximina en ratones sanos y durante la inflamación intestinal aguda. Se valoraron los efectos de la rifaximina en animales sanos y en animales con colitis aguda, utilizando el modelo de colitis inducida por dextrano sulfato sódico (DSS, 3% en agua, 5 días). Los ratones se trataron preventivamente con rifaximina o doxiciclina (control positivo). Se valoraron los signos clínicos de inflamación y el estado del colon (macroscópicamente e histopatología) en el momento de la necropsia. La microbiota ceco-cólica, luminal y adherida al epitelio, se caracterizó mediante: hibridación in situ fluorescente (FISH), polimorfismos de longitud de fragmentos de restricción (T-RFLP) y secuenciación del 16S ARNr. La respuesta inmune local y los mecanismos de interacción hospedador-microbiota se valoraron determinando cambios en la expresión génica (RT-qPCR) de citoquinas pro- y anti-inflamatorias, péptidos antimicrobianos y receptores de tipo Toll (TLRs). En animales sanos, la rifaximina no modificó los recuentos totales de bacterias ceco-cólicas, su biodiversidad o la adherencia bacteriana a la pared del colon. Sólo se observaron cambios menores en la expresión de TLRs o de otros marcadores de tipo inmune. Los animales que recibieron DSS mostraron signos clínicos indicativos del desarrollo de colitis, con regulación al alza en la expresión de marcadores inmunes, incluyendo al péptido antimicrobiano RegIIIɣ. La rifaximina no afectó el curso clínico de la colitis ni la expresión de dichos marcadores. Por el contrario, la doxiciclina atenuó ambas respuestas. Con independencia del tratamiento aplicado, sólo se observaron cambios menores en la expresión de TLRs. La expresión del receptor X de pregnano (PXR) disminuyó durante la inducción de la colitis, un cambio no modificado por la rifaximina, pero que se previno con la doxiciclina. La colitis indujo una disbiosis caracterizada por un aumento de Verrucomicrobia y Deferribacteres con un descenso de Bacteroidetes, manteniéndose la diversidad de tipo alfa. Durante el tratamiento con rifaximina, con o sin colitis, se mantuvo la biodiversidad, con una composición bacteriana muy similar a la observada en animales sanos. Los animales tratados con doxiciclina mostraron cambios extensos en la microbiota, con similitudes entre los grupos inflamado y no inflamado. Concretamente, la doxiciclina redujo los grupos Clostridiales patogénicos, mientras que aumentó los Clostridia grupo XIVa, Lactobacillus and Ruminococcus, considerados grupos beneficiosos. Estos resultados muestran que en animales sanos la rifaximina presenta una actividad antimicrobiana e inmunomoduladora limitada. En estado de colitis, la rifaximina no manifiesta efectos inmunomoduladores ni antimicrobianos consistentes con una actividad anti-inflamatoria. Sin embargo, estas acciones no pueden excluirse en humanos, explicando los efectos beneficiosos observados clínicamente. (...)
Inflammation of the gastrointestinal tract is a common component of functional gastrointestinal disorders (irritable bowel syndrome, IBS and inflammatory bowel disease, IBD). Evidences indicate that both arise because of a convergence of altered microbiota and external environmental factors in genetically susceptible individuals, leading to abnormal immune responses and the development of persistent inflammation, through a mechanism not fully understood. Given the important role of the microbiota and the immune system in their pathogenesis, immunomodulatory antibiotics are an interesting therapeutic approach, targeting simultaneously the microbiota and the exacerbated inflammatory response. Rifaximin is a non-absorbable antibiotic approved for the treatment of IBS with diarrhea and with beneficial effects in IBD. However, the mechanisms mediating these effects and the exact contribution of its antimicrobial and immunomodulatory activities are not fully understood. This work explores the mechanisms of action of rifaximin modulating gut microbiota and the local immune system in a healthy state and during acute intestinal inflammation using a dextran sulfate sodium (DSS)-induced colitis mouse model. First, healthy female mice were treated with either vehicle or rifaximin during 7 or 14 days. In a second study, colitis was induced with DSS (3% in water, 5 days). Mice were treated, in a preventive manner, with either rifaximin or doxycycline, serving as positive control. Daily clinical signs were recorded. At necropsy, colonic inflammation was assessed (macroscopic signs and histopathology). Luminal and wall-adhered ceco-colonic microbiota were characterized by fluorescent in situ hybridization (FISH), terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequencing. Local immune responses and host-bacterial interactions were determined assessing the expression (RT-qPCR) of pro- and anti-inflammatory cytokines, antimicrobial peptides and Toll-like receptors (TLRs). In healthy mice, rifaximin did not modify neither total ceco-colonic bacterial counts not microbial biodiversity. Moreover, rifaximin was associated to a minor upregulation of TLRs expression, without changes in the expression of immune-related markers. Animals receiving DSS showed clinical signs indicative of the development of colitis. Rifaximin did not affect the clinical course of colitis, while doxycycline attenuated clinical signs. Similarly, colitis-associated up-regulation of immune-related markers was not affected by rifaximin, while doxycycline completely prevented this response. As it relates to host-bacterial interaction markers, colitis selectively up-regulated the antimicrobial peptide RegIIIɣ, while it had minor effects on TLRs expression. Rifaximin did not affect colitis-associated RegIIIɣ up-regulation, while doxycycline completely normalized its expression. The pregnane X receptor (PXR) was down-regulated during colitis, a change not affected by rifaximin but prevented by doxycycline. DSS-induced colitis was associated to a dysbiotic state characterized by an increase in Verrucomicrobia and Deferribacteres and a simultaneous decrease in Bacteroidetes; with a maintenance of alpha diversity. During rifaximin treatment, with or without colitis, bacterial richness was maintained, with a bacterial composition closely related to that observed in healthy animals, vs. that observed during colitis. Doxycycline-treated animals showed extensive changes in their microbiota, with similarities between the inflamed and non-inflamed conditions. Particularly, doxycycline reduced pathogenic Clostridiales, while increased Clostridia cluster XIVa-related, Lactobacillus and Ruminococcus, considered beneficial groups. (...)

L’intestin humain est colonisé par un grand nombre de microorganismes qui ont des associations intimes avec notre organisme. L’application de technologies de séquençage à haut débit a permis d’identifier un grand nombre de membres de la communauté tant au niveau phylogénétique qu’au niveau du métagénome. Ce dernier a permis de définir un ensemble de référence de plusieurs millions de gènes principalement bactériens et a servi de base à l'élaboration d'approches omiques permettant de cibler la taxonomie, l'activité et la fonction du microbiome humain. La disponibilité d'informations exhaustives sur le génome de nombreux microbes, y compris des espèces non cultivables dans des écosystèmes microbiens complexes tels que l’intestin ont fourni un moyen d’accéder à des informations génomique, transcriptomique et protéomique. La métaprotéomique, basée sur la puissance de la spectrométrie de masse à haute performance, permet d’effectuer la caractérisation à grande échelle de protéines issues du microbiote intestinal. Dans cette thèse nous nous sommes appesanti en premier sur les stratégies et applications de la métaprotéomique dans la recherche microbienne intestinale en présentant ses limites et défis dans l’ère des méta-omiques. Ensuite, nous avons montré dans la seconde partie qu’une intégration des données de culturomique, de métagénomique et de métaprotéomique permet d’avoir une vision exhaustive sur la cartographie taxonomique microbienne de l’intestin. L’utilisation de la culturomique dans cette partie a permis d’augmenter 19 nouvelles espèces bactériennes dans le répertoire du microbiote intestinal dont 7 sont décrites dans la dernière partie de ce travail
The human gut is colonized by many microorganisms that are closely associated to the human body. Culture-based approaches have provided a better understanding of the complexity of these microbial communities. However, the application of high-throughput sequencing technologies has identified large numbers of community members at both the phylogenetic and the metagenome level. The latter has allowed us to define a set of several millions of references genes, mainly bacterial genes and provided the basis for developing approaches that target the taxonomy, activity and function of the human microbiome. The availability of extensive genome information for many different microbes, including unculturable species in complex microbial ecosystems such as the gut, has provided a means of accessing genomic, transcriptomic and proteomic information. Therefore, the metaproteomics, based on the power of high-performance mass spectrometry, allow the large-scale characterization of intestinal microbiota proteins. Moreover, it becomes a complementary approach to metagenomic data.In this thesis, we first focused on the strategies and applications of metaproteomics in intestinal microbial research, presenting its limits and challenges in the era of meta-omics. Secondly, we showed that, an integration of the culturomics, metagenomics and metaproteomics data provides a comprehensive and complementary view of microbial taxonomic mapping of the intestine. The application of the culturomics in this part has allowed to increase 19 new bacterial species in the repertoire of gut microbiota of which, 7 are described in the last part of this work

Acteur clé de la symbiose hôte-microbiote, les IgA sécrétoires modulent le microbiote et participe à l'homéostasie intestinale. Chez la souris, les IgA sont polyréactives, en reconnaissent diverses bactéries, elles régulent la composition du microbiote et réduisent l'inflammation intestinale. Ces observations ouvrent des perspectives thérapeutiques intéressantes. Cependant, les caractéristiques des IgA et leurs spécificités restent mal connues chez l'homme. L'objectif de ce travail a donc été d'étudier la spécificité de la réponse IgA, en distinguant IgA1 et IgA2. La mise au point d'une technique de production in vitro d'IgA monoclonales 100% humaines issues de l'intestin nous a permis de montrer le profil de polyréactivité des IgA. Chaque IgA interagit avec un spectre large mais défini de bactéries commensales. Par ailleurs, les IgA1 et les IgA2 ciblent la même fraction du microbiote. Les réponses IgA1 et IgA2 convergent aussi au niveau des épitopes polysaccharidiques reconnus. Si les IgA sécrétoires contribuent largement à l’homéostasie intestinale, des IgG sériques anti-microbiote jouent un rôle dans la relation symbiotique hôte-microbiote. Leur présence n’ayant pas encore été démontrée chez l’homme en condition physiologique, ce travail a eu pour objectif secondaire de les explorer. Le développement d’une technique de cytométrie bactérienne nous a permis de détecter des IgG ciblant les bactéries commensales dans le sérum des individus sains. Ces IgG anti-microbiote sont dirigées vers les bactéries déjà reconnues par les IgA sécrétoires, elles présentent des spécificités propres à chaque individu
IgA, the dominant immunoglobulin produced in the gut, plays diverse roles ranging from toxin neutralization, immune functions regulation, intestinal homeostasis maintenance. It is now well established that polyreactive IgA, which target multiple bacteria, can modulate gut microbiota composition and have promising therapeutic effects. However, IgA features remain elusive in humans. We therefore determined the reactivity profile of native monoclonal antibodies from human colon and compared IgA1 and IgA2. We found that IgA are polyreactive and bind a diverse but restricted subset of gut commensals. Most commensals were dually coated by IgA1 and IgA2, yet IgA2 alone coated a distinct fraction of colonic bacteria. Besides their common microbial targets, IgA1 and IgA2 exhibited overlapping anti-carbohydrate repertoires. An essential link between IgA and IgG responses against microbiota has been recently demonstrated in mice. Nevertheless, it remains unclear whether symbiotic bacteria could induce systemic IgG under homeostatic conditions in humans. Hence, we characterized anti-microbiota IgG in serum of healthy donors by bacterial flow cytometry. We found that each individual harbored a diverse and private panel of anti-commensals IgG that converge with secretory IgA to cover a restricted fraction of the gut microbiota. Patients with IgA deficiency or common variable immunodeficiency (CVID) exhibited a distinct set of anti-commensals IgG suggesting that IgA replacement in addition to polyvalent IgG might be beneficial to treat gastro-intestinal symptoms in these patients

La microbiota intestinal es un factor determinante de la homeostasis intestinal, siendo por lo tanto un agente imprescindible del estado de salud. Su composición podría estar alterada como consecuencia de factores externos tales como tratamientos con antibióticos o causada por enfermedades intestinales como el síndrome del intestino irritable (SII). Esta tesis doctoral se centró en la comprensión de la alteración de esta ecología microbiana con respecto a una terapia con antibióticos y la presencia de movimientos intestinales modificados en personas que sufren de síndrome de intestino irritable. Nuestros resultados mostraron que la baja diversidad surgida después del tratamiento con antibióticos fue asociada, inesperadamente, con un aumento de la carga bacteriana total. Sin embargo, sujetos afectados con SII, particularmente del subtipo diarreico, tenían menor diversidad bacteriana acompañada de una reducción de las bacterias productoras de butirato y de las productoras de metano comparado con sujetos sanos. En general los hallazgos de este estudio son que una administración incontrolada de antibióticos puede causar cambios severos en la composición de la comunidad bacteriana intestinal, favorecer el sobrecrecimiento de microbios resistentes y esta observación puede explicar que las alteraciones microbianas en etapas iniciales de la vida podrían llegar a desarrollar enfermedades intestinales.
The intestinal microbiota is a key determinant of gut homeostasis, thereby being an imperative agent of health status. Its composition could be altered as a consequence of external factors such as antibiotic treatment or as a possible cause of intestinal disorders such as irritable bowel syndrome (IBS). In this doctoral thesis we focused on understanding to which extent antibiotic treatment could modify the gut microbiome and how an alteration of its composition could be associated with IBS. Our results showed that the lower diversity occurring after antibiotic treatment was unexpectedly associated with an increase of the overall microbial load. Furthermore, subjects suffering from IBS, particularly diarrhoea predominant subtype, had lower bacterial diversity accompanied by a reduced relative abundance of butyrate-producing and methanogenic microbes compared to healthy subjects. Altogether the findings of this study are that uncontrolled intake of antibiotics may cause tremendous changes in the gut microbial community composition, favouring the overgrowth of resistant microbes; and this observation may explain that alterations of the microbial composition earlier in life could lead to intestinal disorders.

El paper de determinats aliments o components de la dieta en la composició de la microbiota intestinal i el seu impacte en la salut humana representen una àrea d'interès creixent dins el marc de la nutrició de precisió. No obstant això, la comprensió del seu paper sota l’efecte d’un patró dietètic global com la dieta mediterrània (MedDiet) davant el consum d'un aliment potencialment prebiòtic com els fruits secs en un patró dietètic habitual queda encara per descobrir. El nostre primer objectiu va ser avaluar els efectes de la MedDiet sobre la composició de la microbiota intestinal i la seva funció en adults amb sobrepès/obesitat i síndrome metabòlic. En segon lloc, ens vam proposar analitzar el possible paper d'intermediari de la microbiota intestinal als beneficis induïts per la dieta sobre els paràmetres cardiometabòlics i sobre el metabolisme general de l'hoste, mitjançant l'avaluació del perfil metabolòmic fecal i plasmàtic de la nostra població. El present treball es duu a terme dins del marc d'estudi METADIET, un assaig clínic randomitzat i controlat amb un disseny creuat. Els resultats il·lustrats a aquesta tesi mostren que seguir un patró de dieta mediterrània es relaciona amb un enriquiment de Lachnospiraceae NK4A136 i de un membre de la família Ruminococcaceae. Al mateix temps, aquests canvis s’associen positivament amb les millores als paràmetres cardiometabòlics i es reflecteixen també als metabolomes fecals i plasmàtics de l'hoste
El papel de determinados alimentos o componentes de la dieta en la composición de la microbiota intestinal y su impacto en la salud humana representan un área de interés creciente en el marco de la nutrición de precisión. Sin embargo, la comprensión del potencial beneficioso de la microbiota bajo el efecto de un patrón dietético saludable como la dieta mediterránea (MedDiet) frente al consumo de un alimento prebiotico como los frutos secos en una dieta habitual, queda todavía por descubrir. Nuestro primer objetivo fue evaluar los efectos de la MedDiet sobre la composición de la microbiota intestinal y su función en comparación al consumo de frutos secos en adultos con sobrepeso/obesidad y síndrome metabólico. En segundo lugar, nos propusimos analizar el posible papel de intermediario de la microbiota intestinal en los beneficios inducidos por la dieta sobre los parámetros cardiometabólicos y sobre el metabolismo general del huésped. Para ello, evaluamos el perfil metabolómico fecal y plasmático de nuestra población. El presente trabajo se lleva a cabo en el marco del estudio METADIET, un ensayo clínico randomizado y controlado con un diseño cruzado. Los resultados ilustrados en esta tesis muestran que seguir un patrón de dieta mediterránea se relaciona con un enriquecimiento de Lachnospiraceae NK4A136 y de un miembro de la familia Ruminococcaceae
The role of specific foods or dietary components in shaping gut microbiota and their impact on human health represent a growing area of interest in the hallmark of precision nutrition. However, the effects of a global dietary pattern like Mediterranean diet (MedDiet) against consumption of a recognized potential prebiotic food like nuts into a habitual dietary pattern on the complex ecology of gut microbiome has not been fully elucidated. Our first aim was then to evaluate the effects of MedDiet on gut microbiota composition and function compared to the consumption of nuts in the context of a non-MedIDiet in adults with both overweight/obesity and metabolic syndrome. Secondly, we aimed to analyze the potential intermediatory role of gut microbiota in diet-induced benefits on cardiometabolic parameters and on the overall host metabolism, by evaluating the fecal and plasma metabolomics profile of our population. The present work is conducted in the framework of METADIET study, a controlled randomized clinical trial with a crossover design. The results illustrated in this thesis showed that consuming MedDiet is resulting in an enrichment of Lachnospiraceae NK4A136 and a member of Ruminococcaceae family. At the same time, these changes were positively associated with improvements in cardiometabolic parameters and reflected as well in the fecal and plasma metabolomes of the host.

L’accident de désaturation (ADD) est un accident de plongée lié à la charge en gaz diluants pendant la plongée, et à la formation de bulles dans l’organisme au cours de la décompression. Il est susceptible d’engendrer des séquelles neurologiques. Au cours de plongées utilisant l’hydrogène comme gaz diluant, la diminution de la charge tissulaire en hydrogène par l’inoculation au niveau de l’intestin de bactéries métabolisant ce gaz réduit le risque d’ADD.L’objectif de ce travail était d’évaluer si inversement : 1) la fermentation intestinale lors de la plongée peut favoriser la survenue d’un ADD, par l’intermédiaire de la production d’hydrogène endogène ; 2) la stimulation chronique de la fermentation avant plongée majore le risque d’ADD.Nos résultats sont en faveur d’un effet dual de la fermentation intestinale sur la décompression. Délétère à court terme lors de la plongée, la fermentation intestinale prolongée pourrait être favorable en dehors de la plongée en prévenant la survenue et la sévérité d’un ADD. L’hydrogène, molécule aux propriétés antioxydantes, et le butyrate, un acide gras à chaîne courte, sont en effet deux produits de la fermentation des hydrates de carbone qui ont des vertus neuroprotectrices.La prévention des accidents de désaturation pourrait passer par une exclusion des plongeurs présentant une fermentation importante le jour de la plongée, une élimination des gaz produits au niveau de l’intestin ou une modification de l’alimentation dans les 24 heures précédant une plongée. En revanche, tous les facteurs susceptibles de modifier le microbiote intestinal et d’augmenter la fermentation, en dehors de la plongée, pourraient être testés en prévention de l’ADD. En outre, l’hydrogène et le butyrate pourraient jouer un rôle bénéfique dans le cadre du traitement de l’ADD
Decompression sickness (DCS) is a diving accident related to the dissolution of diluent gas in blood and tissues during a dive, followed by bubble formation in the body during decompression. It can lead to neurological damage. In dives using hydrogen as the diluent gas, the concentration of hydrogen in the tissues can be reduced by the presence in the gut of bacteria capable of metabolising this gas and this reduces the risk of DCS.The aim of this work was conversely to check if: 1) fermentation in the gut at the time of diving could exacerbate DCS as a result of endogenous hydrogen generation; 2) long-term stimulation of fermentation before diving raises the risk of DCS.Our findings point to a two-edged effect of intestinal fermentation on decompression: although deleterious in the short term, i.e. at the time of diving, longer-term intestinal fermentation between dives might have a positive effect by preventing the occurrence of DCS and limiting its severity. Indeed, hydrogen which has antioxidant properties and butyrate, a short-chain fatty acid, are both by-products of the fermentation of carbohydrate and both have neuroprotective activity.DCS prevention could be promoted by excluding divers exhibiting strong fermentation on the day of a dive, by the elimination of gases being produced in gut or by modification of diet in the 24 hours before a dive. On the other hand, any factor that might affect the gut microbiota and stimulate fermentation between dives could be tested to investigate its potential in protecting against DCS. Furthermore, hydrogen and butyrate could play a positive role when it comes to treating DCS

La microbiota comensal del intestino se considera un factor clave en la homeostasis gastrointestinal. Las alteraciones funcionales gastrointestinales (síndrome del intestino irritable, SII) y las enfermedades inflamatorias intestinales (EII) se han relacionado con alteraciones de la microbiota comensal (disbiosis). Estos pacientes muestran una activación inmune local anormal, con respuestas motoras y sensoriales alteradas, que en el SII se traducen en estados de hipersensibilidad visceral. El papel causal de la microbiota no se conoce con exactitud, pero la presencia de disbiosis y los efectos positivos asociados al tratamiento con antibióticos o ciertos probióticos sugieren un papel destacado. Este trabajo explora la importancia de la microbiota intestinal modulando los sistemas sensoriales intestinales relacionados con el dolor visceral y sus efectos en respuestas nociceptivas viscerales. Para ello, se ha trabajado con ratas y ratones en los cuales se ha inducido un estado real (cambios adaptativos espontáneos, cambios inducidos por tratamiento con antibióticos) o simulado (estimulación directa de sistemas de interacción hospedador-microbiota) de disbiosis cólica. La microbiota (luminal y adherida al epitelio) se caracterizó usando hibridación in situ con sondas fluorescentes (FISH) y qPCR. La respuesta inmune local y los mecanismos de interacción hospedador-microbiota se valoraron determinando cambios en la expresión génica (RT-qPCR) de citoquinas, péptidos antimicrobianos, integrinas y receptores de tipo Toll (TLR), la producción de IgA secretora, alteraciones histopatológicas y el estado de la barrera de moco. Simultáneamente, se evaluaron cambios en la expresión de marcadores sensoriales. La sensibilidad visceral se valoró mediante el test de Writhing o la administración intracólica de capsaicina. La disbiosis cólica inducida por antibióticos, pero no la observada durante un proceso de adaptación espontánea al ambiente, se asoció a un estado de activación inmune local caracterizado por una regulación selectiva (tanto al alza como a la baja) de citoquinas pro-inflamatorias y de marcadores de interacción hospedador-microbiota y por cambios en los niveles luminales de IgA. Respuestas similares se observaron cuando se simuló al simular un estado de disbiosis mediante la estimulación local del TLR4 (lipopolisacárido) o del TLR7 (imiquimod). En ningún caso se observaron signos macroscópicos o microscópicos de colitis. Tanto en la rata como en el ratón, los estados de disbiosis cólica, real o simulada, se asociaron a una modulación local de la expresión de marcadores sensoriales (sistemas endocanabinoide, serotonérgico, opioide y vaniloide). Se observaron tanto regulaciones al alza como a la baja (RT-qPCR/inmunohistoquímica) dependiendo del modelo de disbiosis y del marcador sensorial considerado. Estos cambios moleculares se tradujeron en cambios funcionales relacionados con respuestas nociceptivas viscerales. Así, en ratones con disbiosis cólica inducida con antibióticos, las repuestas de dolor visceral determinadas con el test de Writhing o tras la administración intracólica de capsaicina mostraron una atenuación significativa con respecto a las observadas en animales control, sugiriendo un estado de hipoalgesia. Estos animales mostraron además una contractilidad cólica alterada (baño de órganos), indicativa de un estado de hipermotilidad. Estos resultados muestran que en estados de disbiosis intestinal se produce una activación inmune local, dirigida, probablemente, a la restauración de la composición de la microbiota. Se observa que la microbiota es capaz de modular la actividad de los sistemas sensoriales intestinales, generando cambios funcionales que se traducen, en las condiciones experimentales presentes, en una modificación de las respuestas de dolor visceral compatible con un estado de tipo analgésico. Mecanismos similares podrían explicar los efectos beneficiosos asociados al tratamiento con antibióticos o al uso de probióticos observados en pacientes con SII o EII. Estudios posteriores deberían centrarse en la caracterización de los grupos bacterianos específicamente responsables de estos efectos. Estos resultados muestran la importancia de la microbiota como factor patogénico en las alteraciones gastrointestinales y su interés como aproximación terapéutica para las mismas.
Gut commensal microbiota (GCM) is a key component of gastrointestinal homeostasis. Functional gastrointestinal disorders (mainly irritable bowel syndrome, IBS) and inflammatory bowel diseases (IBD) have been related to states of altered GCM (dysbiosis). Simultaneously, IBS and IBD patients show local states of abnormal immune activation with altered motor and sensory responses. In particular, in IBS patients sensory alterations lead to characteristic states of visceral hypersensitivity. The exact causal role of GCM remains unclear, but the presence of dysbiosis and the positive effects of antibiotics and some probiotics suggest a key role for the microbiota. The present work explores the potential role of gut microbiota affecting visceral pain-related sensory systems within the gut and the effects on nociceptive responses. For this purpose, states of real (spontaneous adaptive microbial changes, antibiotic treatment-derived microbial changes) or simulated (direct stimulation of host-bacterial interaction systems) colonic dysbiosis were generated in rats and mice. Colonic microbiota (luminal and wall-adhered) was characterized by fluorescent in situ hybridization (FISH) and qPCR. The immune status of the colon and bacterial-host interactions were determined assessing the expression (RT-qPCR) of pro- and anti-inflammatory cytokines; antimicrobial peptides, integrins and Toll-like receptors (TLRs), the production of secretory IgA (s-IgA), the presence of histopathological alterations and the state of the mucous barrier. Simultaneously, changes in sensory related markers were also assessed. Changes in viscerosensitivity were determined in conscious mice using the Writhing test or following the intracolonic administration of capsaicin. Overall, antibiotics-induced alterations of the GCM, but not spontaneous changes associated to environmental adaptation, generated a state of local immune activation within the colon. This state was characterized by selective up- and down-regulation of pro- and anti-inflammatory cytokines and host-bacterial interaction markers and changes in the amounts of s-IgA. Similar immune response was observed when a dysbiotic state was simulated in rats by the direct stimulation of colonic TLR4 with bacterial lipopolysaccharides (LPS) or TLR7 with the selective agonist imiquimod. Although these changes, and regardless the model considered, no macroscopical or microscopical signs of colonic inflammation were detected. In both, mice and rats, real or simulated colonic dysbiotic states were also associated to a local modulation of sensory-related markers (endocannabinoid, serotonergic, opioid and vanilloid systems), with specific treatment-related up- and down-regulatory responses (RT- qPCR and immunohistochemistry). These variations at the molecular level translated in functional changes as it relates to visceral pain-related responses. In mice with antibiotic-induced dysbiosis, visceral pain responses assessed using the Writhing test or the intracolonic administration of capsaicin were significantly attenuated when compared to non-dysbiotic animals; thus suggesting a hypoalgesic state. Moreover, colonic contractility assessed in vitro (organ bath) was also altered in dysbiotic mice, indicating a state of increased colonic motility. Generally, results obtained show that during states of dysbiosis of the GCM there is a complex host response that implies a local immune activation, probably directed towards the reshaping of the microbiota. Data obtained shows that the microbiota is able to influence gut sensory systems and that these changes translate at a functional level in the modulation of visceral pain, eliciting, at least in the present experimental conditions, analgesic-like responses. Similar underlying mechanisms might be responsible for the beneficial effects observed in IBD and, particularly, in IBS patients during antibiotic treatments or during the use of certain bacterial strains as probiotics. Further studies should address the characterization of the specific bacterial groups implicated in these effects. These results highlight the importance of the microbiota as pathogenic factor in gastrointestinal disorders and its potential as a therapeutic approach.

The intestinal tract of virtually any metazoan, including mammals, is colonized with a complex microbial community to which we refer as intestinal or gut microbiota. One of the roles of the healthy intestinal microbiota is to protect the host against gut colonization with pathogenic bacteria through a phenomenon known as colonization resistance (CR). Dysbiosis of the intestinal microbiota, usually as a result of an antibiotic treatment, may lead to the disruption of the CR, and subsequent colonization with bacterial pathogens. However, and despite the importance, the role of the microbiota dysbiosis on the gut colonization by many bacterial pathogens, such as multidrug resistant Enterobacteriaceae, has not been elucidated: the members of the microbiota that confer CR and factors that promote colonization remain mostly unknown. The general aim of this thesis has been to improve the understanding of the role of the microbiota dysbiosis in gut colonization by bacterial pathogens. For this purpose, 3 projects have been established. In the first project we tried to elucidate the role of the microbiota dysbiosis on colonization by multidrug resistant Enterobacteriaceae (MRE) in mice. In the second project we investigated the risk factors and members of the microbiota associated with the MRE colonization in hospitalized patients. MRE infections represent a great threat for hospitalized patients. Specifically, acute leukemia patients are often colonized with MRE, probably due to the impaired CR as a result of intensive antibiotic treatments these patients receive. In the third project we studied the role of the microbiota dysbiosis on the development of Epizootic Rabbit Enteropathy (ERE). ERE is a severe gastrointestinal disease with a high percentage of mortality that occurs in young rabbits during first weeks post-weaning. ERE rabbits have been shown to suffer microbiota dysbiosis during the development of the disease. Moreover, the disease could be reproduced by contact between healthy and sick animals and by administration of cecal contents from ERE rabbits to healthy rabbits, suggesting that a pathogenic agent may be involved in the development of this intestinal pathology, although no causative agent has been identified until now.
El tracto intestinal de prácticamente cualquier metazoo, incluidos los mamíferos, está colonizado por una compleja comunidad microbiana a la que nos referimos como microbiota intestinal. Uno de los papeles de la microbiota intestinal es proteger al huésped contra la colonización intestinal con bacterias patógenas a través de un fenómeno conocido como resistencia a la colonización (RC). La disbiosis de la microbiota intestinal, a menudo como resultado de un tratamiento antibiótico, puede conducir a la alteración de la RC y posterior colonización por patógenos bacterianos. Sin embargo, y pese a su importancia, el papel de la disbiosis de la microbiota en la colonización intestinal por muchos patógenos bacterianos, como son las Enterobacterias multirresistentes, no se ha esclarecido: los miembros de la microbiota que confieren RC y los factores que promueven la colonización siguen siendo desconocidos. El objetivo general de esta tesis ha sido mejorar la comprensión del papel de disbiosis de la microbiota en la colonización intestinal por patógenos bacterianos. Para ello se han establecido tres proyectos. En el primer proyecto investigamos el papel de disbiosis de la microbiota intestinal en la colonización por Enterobacterias multiresistentes (MRE) en ratones. En el segundo proyecto investigamos los factores de riesgo y los miembros de la microbiota asociados con la colonización por MRE en pacientes hospitalizados. Las infecciones por MRE representan una gran amenaza para los pacientes hospitalizados. Específicamente, MRE a menudo colonizan los pacientes con leucemia aguda, probablemente debido a que la RC está alterada como resultado de tratamientos antibióticos intensivos recibidos por estos pacientes. En el tercer proyecto investigamos el papel de la disbiosis microbiana en desarollo de Enteropatía Epizoótica de Conejo (ERE). ERE es una enfermedad gastrointestinal severa con un alto porcentaje de mortalidad que ocurre en conejos jóvenes durante las primeras semanas después del destete. Se ha demostrado que los conejos con ERE sufren disbiosis microbiana después del inicio de la enfermedad, aunque no está claro el papel de la disbiosis en el desarollo de la enfermedad. Además, la enfermedad puede ser reproducida por contacto entre animales sanos y enfermos y por la administración del contenido cecal de conejos con ERE a conejos sanos, lo que sugiere que un agente patógeno podría estar implicado en el desarrollo de esta patología intestinal, aunque hasta ahora no se ha logrado identificar ningún agente causal.
El tracte intestinal de pràcticament qualsevol metazoo, inclosos els mamífers, està colonitzat per una complexa comunitat microbiana a la qual ens referim com microbiota intestinal. Un dels papers de la microbiota intestinal és protegir a l'hoste contra la colonització intestinal amb bacteris patògens a través d'un fenomen conegut com a resistència a la colonització (RC). La disbiosis de la microbiota intestinal, frecuentment com a resultat d'un tractament antibiòtic, pot conduir a l'alteració de la RC i posterior colonització per patògens bacterians. No obstant això, i malgrat la seva importància, el paper de la disbiosis de la microbiota en la colonització intestinal per molts patògens bacterians, com són les Enterobacteries multirresistentes, no s'ha esclarit: els membres de la microbiota que confereixen RC i els factors que promouen la colonització segueixen sent desconeguts. L'objectiu general d'aquesta tesi ha estat millorar la comprensió del paper de la disbiosis de la microbiota en la colonització intestinal per patògens bacterians. Per a això s'han establert tres projectes. En el primer projecte vam investigar el paper de la disbiosis de la microbiota intestinal en la colonització per Enterobacteries multiresistentes (MRE) en ratolins. En el segon projecte, investiguem els factors de risc i els membres de la microbiota associats amb la colonització per MRE en pacients hospitalitzats. Les infeccions per MRE representen una gran amenaça per als pacients hospitalitzats. Específicament, MRE sovint colonitza els pacients amb leucèmia aguda, probablement a causa de que la RC està alterada com a resultat de tractaments antibiòtics intensius rebuts per aquests pacients. En el tercer projecte, vam investigar el paper de la disbiosis microbiana en desenvolupament de l'Enteropatía Epizoótica de Conill (ERE). ERE és una malaltia gastrointestinal severa amb un alt percentatge de mortalitat que ocorre en conills joves durant les primeres setmanes després del deslleti. S'ha demostrat que els conills amb ERE sofreixen disbiosis microbiana després de l'inici de la malaltia, encara que no és clar el paper de la disbiosis en el desenvolupament de la malaltia. A més, la malaltia pot ser reproduïda per contacte entre animals sans i malalts i per l'administració del contingut cecal de conills amb ERE a conills sans, la qual cosa suggereix que un agent patogen podria estar implicat en el desenvolupament d'aquesta patologia intestinal, encara que fins ara no s'ha aconseguit identificar cap agent causal.
Djukovic, A. (2017). Role of intestinal dysbiosis on gut colonization by bacterial pathogens [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90415
TESIS

L’esclerosi múltiple (EM) és una malaltia crònica i degenerativa, i el trastorn inflamatori desmielinitzant del sistema nerviós central (SNC) més comú arreu del món. Tot i que la seva etiologia autoimmune encara es desconeix, en la seva patogènia participen tant la resposta immune innata com l’adaptativa. Recentment, la microbiota intestinal ha sorgit com un possible factor ambiental de risc per a desenvolupar EM. Estudis en models experimentals, com l’encefalomielitis autoimmune experimental (EAE), han mostrat que la microbiota intestinal és una peça clau per a desencadenar la desmielinització autoimmune. Tot i això, les dades experimentals demostren que algunes soques bacterianes tenen un efecte beneficiós en el curs clínic de l’EAE. L’administració de probiòtics s’està convertint en una estratègia terapèutica important que involucra a la microbiota intestinal. En el present estudi hem investigat l’efecte terapèutic de dos probiòtics comercials, Lactibiane iki y Vivomixx, en el curs clínic de l’EAE. L’administració de Lactibiane iki va millorar el curs clínic de la malaltia experimental de forma dosi dependent, i el tractament amb Vivomixx va mostrar una tendència a disminuir la gravetat de l’EAE. El tractament amb Lactibiane iki va reduir la resposta immune proinflamatòria en el SNC i va augmentar les poblacions immunoreguladores en la perifèria. Per altra banda, en els ratolins tractats amb Vivomixx es van detectar menys molècules coestimuladores en cèl·lules T helper (Th), el que indicaria que aquestes estarien menys activades en la perifèria. Aquests efectes immunològics haurien contribuït a la reducció en la desmielinització i la infiltració de cèl·lules T en el SNC en ambdós grups de tractament. L’administració dels dos probiòtics va resultar en la variació del nombre i del fenotip de les cèl·lules dendrítiques mieloides (mDCs), les quals podrien ser les responsables dels canvis observats en les poblacions de cèl·lules T en la perifèria. Concretament, l’administració de Lactibiane iki va afavorir que les mDCs presentessin un fenotip immadur i tolerogènic, pel que podrien induir tolerància immunològica i promoure poblacions immunoreguladores en la perifèria, mentre que l’administració de Vivomixx va resultar en la disminució de mDCs que expressaven molècules coestimuladores. Finalment tant la condició clínica com la progressió de la malaltia provocaven la modificació de la composició del microbioma intestinal. També es va estudiar l’efecte terapèutic de l’administració de 17 soques de Clostridia en el curs clínic de l’EAE. La millora clínica es va relacionar amb menys desmielinització i astrocitosi reactiva així com amb una tendència a presentar menys reactivitat microglial i dany axonal en el SNC. Aquesta millora clínica també es va associar amb una major resposta immunoreguladora de les cèl·lules T reguladores en la perifèria. En els estudis de transcriptòmica es va veure un augment de la resposta antiinflamatòria relacionada amb l’interferó beta en la perifèria i una menor activació, diferenciació i proliferació de les cèl·lules immunes en el SNC. En últim lloc, els alts nivells de butirat, un dels àcids grassos de cadena curta (AGCCs) amb propietats immunomoduladores, en el sèrum dels ratolins tractats amb les soques de Clostridia podrien ser els causants de l’augment de la resposta immunoreguladora. Finalment, el butirat es va provar en el model crònic d’EAE. Aquest producte bacterià es va seleccionar perquè té més capacitat immunoreguladora tant in vitro com in vivo en comparació amb la resta d’AGGC. A més a més, els AGCCs s’han identificat com la vertadera font de les respostes immunes antiinflamatòries que promouen alguns bacteris probiòtics. L’administració oral de butirat va demostrar un efecte preventiu i un lleu efecte terapèutic sobre el curs clínic de l’EAE.
La esclerosis múltiple (EM) es una enfermedad crónica y degenerativa y el trastorno inflamatorio desmielinizante del sistema nervioso central (SNC) más común en todo el mundo. Aunque su etiología autoinmune todavía no está clara, en su patogenia participan tanto la respuesta inmune innata como la adaptativa. Recientemente, la microbiota intestinal ha surgido como un posible factor ambiental de riesgo para desarrollar EM. Estudios en modelos experimentales, tales como la encefalomielitis autoinmune experimental (EAE), han mostrado que la microbiota intestinal es una pieza clave para desencadenar la desmielinización autoinmune. Sin embargo, los datos experimentales respaldan la idea de que algunas cepas de bacterias tienen un efecto beneficioso en el curso clínico de la EAE. Así, la administración de probióticos se está convirtiendo en una estrategia terapéutica importante que involucra a la microbiota intestinal. En este estudio, investigamos el efecto terapéutico de la administración de dos probióticos comerciales: Lactibiane iki y Vivomixx, en el curso clínico de la EAE. Lactibiane iki mejoró el curso clínico de la enfermedad experimental de forma dosis dependiente y el tratamiento con Vivomixx mostró una tendencia a disminuir la gravedad de la EAE. Lactibiane iki redujo la respuesta inmune proinflamatoria en el SNC y aumentó las poblaciones inmunoreguladoras en la periferia. Por otro lado, los ratones tratados con Vivomixx mostraron una menor presencia de moleculas co-estimuladoras en las células T helper (Th), lo que estaría indicando una menor activación celular de las células T en la periferia. Estos efectos inmunológicos pudieron haber contribuido a la reducción en la desmielinización y la infiltración de células T en el SNC en ambos grupos de tratamiento. La administración de cualquiera de los dos probióticos moduló el número y el fenotipo de las células dendríticas mieloides (mDCs) y estas, a su vez, podrían haber sido las responsables de los diferentes cambios observados en las poblaciones de células T en la periferia. Concretamente, Lactibiane iki promovió un fenotipo inmaduro y tolerogénico de las mDCs las cuales pudieron inducir tolerancia inmunológica y poblaciones inmunoreguladoras en la periferia, mientras que Vivomixx disminuyó el porcentaje de mDCs que expresaban moléculas co-estimuladoras. Finalmente, se describió que tanto la condición clínica como la progresión de la enfermedad modificaron la composición del microbioma intestinal. En segundo lugar, estudiamos el efecto terapéutico de la administración de 17 cepas de la clase Clostridia en el curso clínico de la EAE. La mejoría clínica se relacionó con una menor desmielinización y astrocitosis reactiva así como con una tendencia hacia una menor reactividad microglial y daño axonal en el SNC. Esta mejoría clínica también se asoció con una mayor respuesta inmunoreguladora de las células T reguladoras en la periferia. Los estudios de transcriptómica destacaron un aumento de la respuesta antiinflamatoria relacionada con el interferón beta en la periferia y una menor activación, diferenciación y proliferación de las células inmunes en el SNC. En último lugar, los altos niveles de butirato, uno de los ácidos grasos de cadena corta (AGCCs) con propiedades inmunomoduladoras, en el suero de los ratones tratados con las cepas de Clostridia podrían haber contribuido a la mayor respuesta inmunoreguladora en la periferia. Finalmente, el AGCC butirato se testó en el modelo crónico de EAE. Este producto bacteriano se seleccionó dada su mayor capacidad inmunoreguladora tanto in vitro como in vivo en comparación con el resto de AGCCs. Además, los AGCCs se han identificado como la verdadera fuente de las respuestas inmunes antiinflamatorias llevadas a cabo por algunas bacterias probióticas. Así, la administración oral del AGCC butirato demostró su efecto preventivo sobre la autoinmunidad en el SNC y un leve efecto terapéutico sobre el curso clínico de la EAE.
Multiple sclerosis (MS) is a chronic and degenerative disease and the most common inflammatory demyelinating disorder of the central nervous system (CNS) worldwide. Although its autoimmune aetiology is still unclear, both innate and adaptive immune responses participate in MS pathogenesis. Recently, the gut microbiota has emerged as a putative environmental risk factor for MS. Studies in experimental autoimmune encephalomyelitis (EAE) models have shown that the gut microbiota is an essential player in triggering autoimmune demyelination. However, experimental data support the idea that some bacterial strains, far from being harmful, have a beneficial impact on the outcome of EAE. Thus, the promotion of beneficial microorganisms via probiotics is being developed as an important therapeutic strategy involving the gut microbiota in EAE. In the present study, we investigated the therapeutic impact of two commercially available probiotics—Lactibiane iki and Vivomixx—on the clinical outcome of EAE. Lactibiane iki improved the clinical outcome of EAE mice in a dose-dependent manner and Vivomixx tended to ameliorate the clinical course of the experimental disease. Regarding the probiotics’ immunological effects, Lactibiane iki diminished the proinflammatory immune response in the CNS and increased immunoregulatory populations in the periphery. On the other hand, Vivomixx-treated mice showed a lower presence of costimulatory molecules in the T helper (Th) cell populations, which would be indicating an inefficient T cell activation profile in the periphery. These immunological processes may have contributed to the reduction in the demyelination and T cell infiltration in the CNS in both experimental groups. The administration of either probiotic modulated the number and phenotype of myeloid dendritic cells (mDCs) and these, in turn, could have been the responsible for the observed changes in the T cell populations in the periphery. Specifically, Lactibiane iki promoted an immature, tolerogenic phenotype of mDCs that could directly induce immune tolerance and immunoregulatory populations in the periphery, while Vivomixx decreased the percentage of mDCs expressing co-stimulatory molecules. Finally, it was described that both the clinical condition and disease progression altered the gut microbiome composition. Secondly, we studied the therapeutic effect of a previously selected mixture of human gut-derived 17-Clostridia strains on the clinical outcome of EAE. The observed clinical improvement was related to lower demyelination and astrocyte reactivity in addition to a trend to lower microglia reactivity and axonal damage in the CNS. An enhanced immunoregulatory response of regulatory T cells in the periphery was also associated to this clinical improvement. Furthermore, transcriptome studies highlighted increased antiinflammatory responses related to interferon beta in the periphery and lower activation, differentiation, and proliferation of immune cells in the CNS. Lastly, higher levels of the immunomodulatory short-chain fatty acid (SCFA) butyrate in the serum of Clostridia-treated mice might have contributed to the greater immunoregulatory response in the periphery. Finally, the SCFA butyrate was also tested on the chronic EAE model. This bacterial product—mainly produced from the fermentation of digestion-resistant oligosaccharides and dietary fibre—was selected since it has been described as the SCFA with the largest capability to generate regulatory immune responses in vitro and in vivo. Moreover, SCFAs have been stood out as the real sources of the antiinflammatory immune responses exerted by some probiotic bacteria. Thus, the oral administration of the SCFA butyrate proved its preventive effect on CNS autoimmunity and its slight therapeutic clinical impact on EAE clinical course. Our results emphasise that gut microbiota can be a potential therapeutic target in MS.

L’étude du microbiote digestif est un enjeu important de recherche en microbiologie. La première partie de cette thèse porte sur la recherche au sein du microbiote digestif de nouveaux antibactériens, qui apparait comme une des pistes clés dans la lutte contre la résistance aux antibiotiques. Les trois quarts des antibiotiques sont des produits naturels, ou dérivés, sécrétés par des microorganismes de l’environnement. Comme lui, le microbiote digestif représente un écosystème complexe où règne une grande compétition. Nous avons recherché des antagonismes de culture dans le microbiote digestif contre les bactéries les plus pathogènes pour l’homme. Nous avons trouvé une inhibition de S. aureus par P. avidum, de E. cloacae par B. fragilis, E. dispar, L. delbruckii, P. acidipropionici, S. equinus, S. gallolyticus, et enfin de E. aerogenes par B. vulgatus et E. dispar. Nous avons également trouvé des clusters de gène de métabolites secondaires dans le génome de ces bactéries. Ce travail préliminaire confirme que le microbiote digestif est une source potentielle de nouveau antibactériens. En dépit de l’explosion du nombre d’espèces isolées dans le microbiote digestif grâce à la culturomics, certaines restent fastidieuses à cultiver. Nous avons analysé par métagénomique et culturomics une selle avant et après incubation anaérobie en présence de 5% de rumen et 5% de sang de mouton. Ce travail montre une dynamique de croissance des bactéries très hétérogène. Le milieu d’enrichissement utilisé était efficace et permettait la culture d’un plus grand nombre d’espèces bactériennes. Ce travail apporte des éléments nouveaux permettant l’optimisation de cette étape de culturomics
Gut microbiota is a major health concern for microbiologists. Its alterations were previously related to diseases. In the first step of this thesis, we have searched for new antimicrobials within the gut microbiota. Indeed, antibiotic resistance is a global health concern and research for new antibiotics is a cornerstone for fight against it, according to the WHO. Three quarter of all current antibiotics are natural products, or derived from them, synthesised by bacteria and fungi from soil. Gut microbiota is another complex ecosystem with strong competition. We have searched for antagonism in the gut microbiota species against most human pathogenic species. We found an inhibition of growth of S. aureus by P. avidum, of E.cloacae by B. fragilis, E. dispar, L. delbruckii, P. acidipropionici, S. equinus, S. gallolyticus,and an inhibition of E. aerogenes by B. vulgatus and E. dispar. We also found BGCs for all these species. This preliminary work confirm that gut microbiota is a potential source for new antibiotics. Despite the explosion of bacterial species isolated from gut, some fastidious species remains difficult to grow. We performed a metagenomic and culturomics analysis of a fresh stool sample before and after incubation into an anaerobic blood bottle supplemented with sheep blood and rumen fluid. This medium used in culturomics for enrichment was effective, allowing the isolation of higher number of species. This work show that the dynamic growth of bacteria is very variable. This work brings some precisions in the dynamic of bacterial growth that could improve the culturomics process

Le système immunitaire muqueux, et plus particulièrement les réponses intestinales IgA sont essentielles non seulement à la défense contre les agents pathogènes, mais aussi au façonnement de la flore intestinale commensale. Dans les modèles murins de déficit en IgA, on observe une dysbiose intestinale majeure associée à une inflammation muqueuse, réversibles après restauration des IgA. Le but de ce travail est de décrire l'impact de l'absence d'IgA chez l'homme sur la composition du microbiote intestinal ainsi que ses conséquences locales et systémiques. L'étude comparative par analyse métagénomique des selles de 17 sujets déficitaires en IgA et de 34 donneurs sains retrouve l'absence de différence majeure en termes de répartition des phyla dominants, de diversité et de richesse génique bactériennes entre les deux groupes. En revanche, en analysant à l'échelon des espèces, on observe dans le déficit en IgA une surreprésentation d'espèces pro-inflammatoires et une sous-représentation d'espèces anti-inflammatoires. En outre, en l'absence d'IgA, nous observons la présence de réponses IgM qui opsonisent partiellement les genres ciblés par l'IgA, mais semblent maintenir la diversité au sein des Actinobactéries. Les patients présentent un biais phénotypique lymphocytaire T circulant (TH17) associé à des stigmates de translocation bactérienne. Enfin, l'absence d'IgA s'associe à une perturbation du réseau bactérien minimal "obligatoire". Ces résultats suggèrent que le déficit en IgA humain s'accompagne d'une dysbiose modérée associée à une altération de l'architecture du réseau bactérien induisant une hyperactivation du système immunitaire, malgré la présence de réponses IgM
IgA responses play a key role in gut mucosa, defending host against pathogens but also shaping the commensal flora. In order to get insights into the specific contributions of IgA to host/microbial symbiosis in humans, we explored patients that lack only IgA, using gut microbial metagenomics and systems immunology. Microbiota composition was compared between 34 healthy controls and 17 selective IgA deficiency (sIgAd) patients. Contrary to what was observed in murine models of IgA deficiency, we show that human sIgAd is not associated with massive perturbations of gut microbial ecology, regarding phyla distribution, bacterial diversity and gene richness. A clear gut microbial signature is however associated to sIgAd: we found 19 over-represented MGS mainly described to be pro-inflammatory, but also 14 under-represented MGS, mainly known to be beneficial. We also explored local consequences of IgA deficiency, particularly whether IgM could replace IgA at host/bacterial interface. Using a combination of bacterial flow sorting and DNA sequencing, we therefore analysed the composition of IgM-coated microbiomes observed in sIgAd. We show that IgM only partially supply IgA deficiency, as not all typical IgA targets can also be opsonized by IgM, but nevertheless contribute to maintain Actinobacteria diversity. IgA deficiency is associated with a skewed circulating CD4+ T cell profile towards TH17, as well as markers of bacterial translocation. Finally, sIgAd is associated with a perturbation of the minimal bacterial network. Altogether our results suggest that human IgA deficiency is associated with a mild dysbiosis associated to systemic inflammation despite the presence of IgM

La carne de cerdo es una de las carnes más consumidas en el mundo, cuyo valor se ve afectado por su calidad y las preferencias del consumidor. La composición de los ácidos grasos (AGs) en músculo y tejido adiposo modifica la calidad de la carne. Del mismo modo, la microbiota intestinal, a través de la producción de metabolitos como los ácidos grasos volátiles, puede también afectar su calidad. Sin embargo, la relación entre el genoma del cerdo y su microbiota intestinal no está bien estudiada. En la presente tesis se han realizado una serie de trabajos con el fin de profundizar en los mecanismos genéticos implicados en la determinación de la composición de los AGs. Además, se ha estudiado la composición de la microbiota a lo largo del intestino y su interacción con el genoma porcino. Se realizaron estudios de asociación del genoma completo (GWAS) entre 38.424 Single Nucleotide Polymorphisms (SNPs) y 60 caracteres fenotípicos relacionados con la composición de los AGs en músculo y grasa dorsal de 441 cerdos pertenecientes a tres retrocruces de la población experimental IBMAP: BC1_LD (25% Ibérico y 75% Landrace), BC1_PI (25% Ibérico y 75% Pietrain), y BC1_DU (25% Ibérico y 75% Duroc). El GWAS reveló nueve regiones del genoma porcino asociadas con doce caracteres de la grasa dorsal y seis regiones asociadas con seis medidas de la grasa intramuscular. Dentro de estas regiones, se identificaron 50 genes como candidatos funcionales a explicar la variación de estos caracteres. Los genes más relevantes fueron ELOVL3, ELOVL6, ELOVL7, FADS2, FASN y SCD. Además, el polimorfismo ELOVL6:c.‑394G>A fue el más asociado con los porcentajes de C14:0, C16:0, y C16:1(n-7) en grasa dorsal. Para estudiar otras variantes genéticas aparte de los SNPs, se detectaron 1.928.746 indels con tres programas (Dindel, SAMtools mpileup, y GATK) mediante los datos de secuenciación del genoma completo de siete fundadores (dos machos Ibéricos y cinco hembras Landrace) del material IBMAP. Se genotiparon diez indels localizados en genes relacionados con el metabolismo lipídico en los 441 cerdos de los tres retrocruces, encontrándose a distintas frecuencias alélicas. En la grasa intramuscular, el indel C1QTNF12:c.557_559delCCG presentó una asociación significativa con el porcentaje de ácido eicosadienoico (C20:2(n-6)). Para describir la composición de la microbiota a lo largo del intestino, se recogió el contenido luminal de cinco regiones (duodeno, yeyuno, íleo, colon proximal y distal) de trece cerdos Ibéricos. Posteriormente, mediante el método de amplificación y secuenciación del gen 16S rRNA, se identificaron 1.669 operational taxonomic units (OTUs) agrupados en 179 géneros, siendo los más abundantes Lactobacillus, Clostridium y Prevotella. Las muestras de colon eran más ricas en especies y se parecían más entre cerdos que las muestras del intestino delgado. Además, las predicciones funcionales del metagenoma a lo largo del intestino mostraron que sus rutas energéticas eran distintas. Finalmente, se estudió la asociación entre el genoma del cerdo y su microbiota intestinal. Se obtuvo la composición de la microbiota del recto de 285 cerdos Ibérico × Duroc mediante la amplificación y secuenciación del gen del 16S rRNA, identificándose un total de 1.257 OTUs agrupados en 101 géneros y 18 filos, siendo los filos más abundantes Firmicutes y Bacteroidetes. El GWAS reveló 17 regiones del genoma porcino asociadas con la abundancia relativa de los géneros Akkermansia, CF231, Phascolarctobacterium, Prevotella, SMB53 y Streptococcus. Dentro de estas regiones, se identificaron 38 genes como candidatos a modular la composición de la microbiota intestinal por su relación con el sistema inmunitario y el metabolismo de los mucopolisacáridos y los ácidos biliares.
Pork is one of the most consumed meats worldwide and it is subjected to consumer’s preferences. Meat quality is affected by fatty acid (FA) composition in muscle and adipose tissues. Gut microbiota composition can also affect meat quality through the production of metabolites such as short-chain fatty acids. However, the relationship between pig genome and gut microbiota is not fully understood. In the current thesis, several studies have been performed to improve our knowledge about the genetic determinism of FA composition. In addition, the composition of the microbiota along the pig gut and its interaction with the host genome has been also analysed. Genome-wide association studies (GWAS) were performed among 38,424 single nucleotide polymorphisms (SNPs) and 60 phenotypic traits related to FA composition in backfat and muscle. This analysis was performed in 441 pigs from three different backcrosses: BC1_LD (25% Iberian and 75% Landrace), BC1_PI (25% Iberian and 75% Pietrain), and BC1_DU (25% Iberian and 75% Duroc) belonging to the IBMAP experimental population. Nine regions of the pig genome were associated with twelve backfat traits, while six regions were associated with six intramuscular fat (IMF) traits. A total of 50 candidate genes were proposed to explain the variation in these traits. The most promising candidate genes were ELOVL3, ELOVL6, ELOVL7, FADS2, FASN and SCD. Furthermore, ELOVL6:c.‑394G>A was the most associated SNP with the percentages of C14:0, C16:0, and C16:1(n-7) in backfat. With the aim of detecting other variants apart from SNPs, we performed an indel detection with the whole genome sequencing data from seven founders (two Iberian boars and five Landrace sows) of the IBMAP pigs. A total of 1,928,746 indels were found in common among the three programs used (Dindel, SAMtools mpileup, and GATK). Ten indels inside genes related with lipid metabolism (ASPH, C1QTNF12, CAPN9, CCR7, CRP, GZMA, JMJD1C, LYST, PEX19 and SAMD4B) were genotyped in pigs belonging to the three IBMAP backcrosses, obtaining different allelic frequencies. The C1QTNF12:c.557_559delCCG indel was associated with the percentage of eicosadienoic acid (C20:2(n-6)) in IMF. To describe the microbiota composition along the pig gut, luminal contents of five gut sections (duodenum, jejunum, ileum, and proximal and distal colon) were collected in thirteen Iberian pigs. A total of 1,669 operational taxonomic units (OTUs) grouped in 179 genera were found using the 16S rRNA gene sequencing method. Lactobacillus, Clostridium and Prevotella were the three most abundant genera. Colon samples were more similar among pigs and richer in species than small intestine samples were. The metagenome predictions showed that the energy pathways were different along gut sections. Finally, to reveal the association between host genome and gut microbiota in pigs, the microbiota composition of the rectum of 285 Iberian × Duroc pigs was obtained using the 16S rRNA gene sequencing method, finding 1,257 OTUs distributed in 101 genera and 18 phyla. Firmicutes and Bacteroidetes were the most abundant phyla. GWAS identified 17 genomic regions of the pig genome associated with the relative abundance of six genera (Akkermansia, CF231, Phascolarctobacterium, Prevotella, SMB53 and Streptococcus). A total of 38 candidate genes, related with the host defence system and the metabolism of mucopolysaccharides and bile acids, were suggested to be modulators of the gut microbiota composition.

L'obesitat i la síndrome metabòlica són problemes importants de salut pública que estan en augment a tot el món. S'ha establert que la microbiota intestinal juga un paper important en l'obesitat, el metabolisme energètic de l'hoste i és essencial comprendre el seu paper en el context de la salut. Com a objectiu principal d'aquesta tesi, avaluem l'efecte que va tenir 1 any d'intervenció intensiva de pèrdua de pes, en el context de l'estudi PREDIMED-Plus, en la composició de la microbiota intestinal. Vàrem observar que la pèrdua de pes mediada per la intervenció indueix canvis en la microbiota intestinal i alguns d’aquests gèneres microbians es van associar amb canvis en els paràmetres d'adipositat. En segon lloc, vàrem explorar les diferències en la composició microbiana pel que fa a diverses fonts d'ingesta de proteïnes, i vàrem observar que el consum de proteïnes d'origen animal pot tenir una influència superior a la de les proteïnes d'origen vegetal. Finalment, a partir de la revisió narrativa realitzada sobre els greixos d'origen vegetal i la microbiota intestinal, es conclou que el reemplaçament de greixos saturats per fonts vegetals de greixos insaturats podria ajudar en la modulació positiva de la microbiota intestinal. Remarquem que es necessiten més estudis en humans per comprendre els efectes de diferents fonts de greixos i proteïnes sobre la microbiota intestinal i, en conseqüència, sobre la salut. En conclusió, la dieta mediterrània hipocalòrica, juntament amb l'activitat física i canvis de comportament, poden tenir efectes beneficiosos en l'hoste, potencialment modulats a través de la microbiota intestinal. Les fonts de proteïnes o greixos vegetals poden tenir efectes positius sobre la composició i funcionalitat microbiana intestinal. Ens esperen en el futur una gran quantitat de desafiaments i oportunitats per a comprendre millor les interaccions dieta-hoste-microbioma.
La obesidad y el síndrome metabólico son importantes problemas de salud pública que están en aumento en todo el mundo. Se ha establecido que la microbiota intestinal juega un papel importante en la obesidad, el metabolismo energético del huésped y es esencial comprender su papel en el contexto de la salud. Como objetivo principal de esta tesis, evaluamos el efecto que tuvo 1 año de intervención intensiva de pérdida de peso, en el contexto del estudio PREDIMED-Plus, en la composición de la microbiota intestinal. Observamos que la pérdida de peso mediada por la intervención induce cambios en la microbiota intestinal y que estos géneros microbianos se asociaron con cambios en los parámetros de adiposidad. En segundo lugar, exploramos las diferencias en la composición microbiana con respecto a varias fuentes de ingesta de proteínas y observamos que el consumo de proteínas de origen animal puede tener una influencia superior que la de las proteínas de origen vegetal. Por último, a partir de la revisión descriptiva realizada sobre las grasas de origen vegetal y la microbiota intestinal, se concluye que el reemplazo de grasas saturadas por fuentes vegetales de grasas insaturadas podría ayudar en la modulación positiva de la microbiota intestinal. Remarcamos que existe una gran necesidad de estudios en humanos para comprender los efectos de diferentes fuentes de grasa y proteínas sobre la microbiota intestinal y, en consecuencia, sobre la salud. En conclusión, una dieta mediterránea hipocalórica, junto con la actividad física y cambios comportamentales, puede tener efectos beneficiosos en el huésped, potencialmente modulados a través del microbiota intestinal. Las fuentes de proteínas o grasas vegetales pueden tener efectos positivos sobre la composición y funcionalidad microbiana intestinal. Nos esperan en el futuro una gran cantidad de desafíos y oportunidades para comprender mejor las interacciones dieta-huésped-microbioma.
Obesity and metabolic syndrome are major public health issues increasing worldwide. Gut microbiota has established to play an important role in obesity, host energy metabolism and understanding its role in the context of health is essential. As the primary objective of this thesis, we evaluated the effect of 1-year intensive weight-loss intervention in the context of PREDIMED-Plus study on gut microbiota composition. We observed that weight loss mediated by the intervention induces changes in gut microbiota and some of these microbial genera were associated with changes in adiposity parameters. Secondly, we explored the differences in microbial composition with respect to various sources of protein intake, we observed that consuming animal-based proteins may have a stronger influence than plant-based proteins on gut microbiota. Finally, from the narrative review conducted on plant-based fats and gut microbiota, it can be concluded that replacement of saturated fats with plant sources of unsaturated fats could help in positive modulation of gut microbiota. We remark that there is a great need for human studies in the context of understanding the effects of different fat and protein sources on gut microbiota and consequently on health. Overall, we conclude from this Doctoral thesis that hypocaloric Mediterranean diet, along with physical activity and behavioral changes can have beneficial effects on the host, potentially modulated via gut microbiota. Plant based protein or fat sources may have positive effects on gut microbial composition and functionality. Huge amount of challenges and opportunities awaits in front of us to better understand diet-host-microbiome interactions.

En élevage porcin, le sevrage est une période critique caractérisée par un stress nutritionnel, environnemental et social, avec une forte sensibilité des animaux à la diarrhée. Le microbiote intestinal doit s'adapter à un changement alimentaire, avec le passage d'une alimentation lactée à un aliment plus complexe à base de céréales, et les animaux sont soumis à la pression exercée par les agents infectieux environnementaux. Les bactéries entérotoxiques Escherichia coli (ETEC) sont les principaux agents pathogènes responsables de la diarrhée post-sevrage et peuvent entrainer des pertes économiques considérables. Le rôle de la génétique de l’hôte dans la sensibilité à l'infection est bien établi, le polymorphisme des gènes Mucine 4 (MUC4) et Fucosyltransférase 1 (FUT1) étant associé à la sensibilité à ETEC F4 et F18, respectivement. Nous avons réalisé deux études afin d’analyser l’effet de facteurs pouvant influer sur la sensibilité des porcelets à la diarrhée au sevrage. Dans une première étude, nous avons évalué l'impact de l'âge au sevrage sur la diversification du microbiote intestinal, par comparaison du microbiote d’animaux sevrés à différents âges. Quarante-huit porcelets de race Large White ont été répartis en quatre groupes de 12 animaux sevrés à 14 jours (sevrage précoce), 21 ou 28 jours (âge au sevrage courant en élevage intensif) et 42 jours (sevrage tardif). La composition bactérienne du microbiote a été établie par séquençage du gène de l'ARNr 16S d’ADN fécal extrait de selles prélevées le jour du sevrage, sept jours après et à l'âge de 60 jours. Nous avons montré que le sevrage tardif augmente la diversité du microbiote, avec une plus grande abondance de Faecalibacterium prausnitzii identifiée comme bénéfique chez l'homme. Ces résultats suggèrent que la composition du microbiote intestinal pré-sevrage conférée par un sevrage à 42 jours pourrait améliorer la santé intestinale des porcelets, en leur permettant d’acquérir un microbiote plus diversifié avec des bactéries potentiellement bénéfiques lors du sevrage. La seconde étude a eu comme objectif d’évaluer, chez des porcelets sevrés, les effets du génotype des gènes MUC4 et FUT1 et des voies d'administration de l’amoxicilline sur la présence de diarrhée et la composition du microbiote fécal, lors d'une infection naturelle par des souches d'ETEC multirésistantes. Soixante et onze porcelets ont été répartis en trois groupes: deux groupes se différenciant par la voie d'administration de l'amoxicilline, parentérale (P) ou orale (O), et un groupe témoin sans antibiotiques (C). Nous avons confirmé que MUC4 et FUT1 sont des marqueurs génétiques de l’hôte pour la sensibilité aux infections à ETEC et montré que le traitement à l'amoxicilline pouvait avoir des effets néfastes sur la santé du porc au cours d'une infection à ETEC multirésistante, accentués lors d’une administration par voie orale. Les deux études ont mis en évidence l’importance de considérer des méthodes alternatives de conduite d’élevage. Avec la nécessité de limiter l'utilisation d'antibiotiques, la sélection de génotypes résistants, la supplémentation en next-generation probiotics dans l’alimentation et une meilleure optimisation de l'âge au sevrage devraient être prises en compte dans les pratiques, afin de favoriser un microbiote intestinal diversifié, capable de réduire les diarrhées au sevrage
In pig production systems, weaning is a crucial period characterized by nutritional, environmental and social stress. During this process, piglets are susceptible to diarrhoea and the gut ecosystem needs to adapt to dietary changes, from a milk-based diet to a solid and more complex cereal-based feed, and to environmental pathogen pressure. One of the most important etiological agent of the post-weaning diarrhoea (PWD) is the Enterotoxigenic Escherichia coli (ETEC) able to cause severe outcomes and considerable economic losses to farmers worldwide. A role of host genetics in infection appearance is well-established, the SNPs located on the Mucine 4 (MUC4) and Fucosyltransferase 1 (FUT1) genes being associated with the susceptibility to ETEC F4 and ETEC F18, respectively.To investigate aspects related to weaning diarrhoea, two studies have been performed. The aim of the first study was to evaluate the impact of weaning age on gut microbiota diversification in piglets comparing animals at different weaning ages. Forty-eight Large White piglets were divided into four groups of 12 animals weaned at 14 days old (early weaning), 21 or 28 days old (main weaning ages in pig intensive farming) and 42 days old (late weaning). In each group, faecal bacteria composition was assessed by sequencing the 16S rRNA gene of faecal DNA on the weaning day, 7 days post-weaning and at 60 days of age. Our results showed that late weaning increases the gut microbiota diversity including a higher abundance of Faecalibacterium prausnitzii, reported as beneficial in humans. Our results suggest than the pre-weaning gut microbiota composition conferred by a late weaning at 42 days of age could enhance gut health in piglets. This would provide a competitive advantage to piglets accumulating a higher diversity of potentially beneficial microbes prior to the stressful and risky weaning transition.The aim of the second study was to evaluate the effects of the host-genotype and different routes of amoxicillin administration on the presence of diarrhoea and the microbiota composition, during a natural infection by multi-resistant ETEC strains in weaned piglets. For this purpose, seventy-one piglets were divided into three groups: two groups differing by amoxicillin administration routes – parenteral (P) or oral (O) and a control group without antibiotics (C). Our results confirmed the MUC4 and FUT1 as host genetic markers for the susceptibility to ETEC infections. Moreover, our data highlighted that amoxicillin treatment may produce adverse outcomes on pig health in course of multi-resistant ETEC infection and this effect is stronger when the antibiotic is orally administered than parenterally.Both studies highlighted the importance of alternative control measures related to farm management in controlling weaning related diarrhoea. With a need to limit the use of antibiotics, selection of resistant genotypes, next-generation probiotics supplementation in feed, and correct procedures of weaning age, should be considered in farm management practices in order to preserve a balanced and stable gut microbiota and consequently reduce occurrence of diarrhoea at weaning
Lo svezzamento rappresenta un momento cruciale nell’allevamento suinicolo ed è caratterizzato da stress nutrizionale, ambientale e sociale. In questa fase, i suinetti risultano a maggior rischio di insorgenza di diarrea in quanto la microflora intestinale deve adattarsi ai cambiamenti alimentari legati al passaggio da una dieta a base lattea ad un alimento solido a base di cereali e più complesso e all’elevata pressione infettiva ambientale. Uno dei più importanti agenti eziologici responsabili della diarrea post-svezzamento (PWD) è Escherichia coli Enterotossigeno (ETEC) in grado di provocare gravi quadri clinici nonché ingenti perdite economiche per gli allevatori. Che ci sia una componente genetica nell'evoluzione di queste infezioni è stato ben definito attraverso l’individuazione degli SNP situati sui geni Mucine 4 (MUC4) e Fucosyltransferase 1 (FUT1) associati rispettivamente alla suscettibilità nei confronti di ETEC F4 e ETEC F18. Nella presente tesi sono illustrati due studi che hanno avuto l’obiettivo di approfondire alcuni aspetti legati alla comparsa di diarrea durante lo svezzamento. Lo scopo del primo studio è stato quello di valutare l'impatto dell'età di svezzamento sulla diversità del microbiota intestinale, confrontandone la composizione in suinetti svezzati a diverse età. Quarantotto suinetti di razza Large-White sono stati suddivisi in quattro gruppi da 12 soggetti, svezzati rispettivamente a 14 giorni di età (svezzamento precoce), a 21 o 28 giorni (età di svezzamento principale nell'allevamento intensivo) e a 42 giorni (svezzamento tardivo). In ogni gruppo è stata valutata la composizione batterica fecale il giorno dello svezzamento, 7 giorni post-svezzamento e a 60 giorni di età, sequenziando il gene 16S rRNA dal DNA batterico fecale. I risultati ottenuti hanno evidenziato come lo svezzamento tardivo aumenti il grado di diversificazione del microbiota intestinale, aumentando l’abbondanza di Faecalibacterium prausnitzii, già considerato benefico per l'uomo. Emerge, inoltre, come la composizione del microbiota intestinale nel pre-svezzamento associata allo svezzamento tardivo incrementi il livello di salute intestinale nei suinetti. Tale condizione, comporterebbe un notevole vantaggio per gli animali che acquisiscono una maggiore differenziazione del microbiota intestinale, incrementando l’abbondanza di batteri beneficiali prima di affrontare lo stress dello svezzamento. Lo scopo del secondo studio è stato quello di valutare gli effetti del genotipo dell’ospite e le vie di somministrazione dell’amoxicillina sulla comparsa della diarrea e sulla composizione del microbiota intestinale, durante un'infezione naturale causata da ETEC multi-resistente, in suinetti svezzati. A tale scopo, settantuno suinetti sono stati divisi in tre gruppi: due gruppi diversificati dalla via di somministrazione dell’amoxicillina - parenterale (P) o orale (O), e un terzo gruppo di controllo in cui non sono stati somministrati antibiotici (C). I risultati ottenuti hanno confermato il ruolo di MUC4 e FUT1 quali marcatori genetici di suscettibilità alle infezioni da ETEC. Inoltre, i nostri dati hanno evidenziato come la somministrazione di amoxicillina possa influenzare negativamente lo stato di salute dei suini in corso di infezione da ETEC, effetti ancora più evidenti quando la somministrazione antibiotica avviene per via orale. Entrambi gli studi hanno sottolineato l'importanza di adottare misure alternative legate al management aziendale per il controllo della diarrea post-svezzamento. Nell’ottica di limitare l'utilizzo di antibiotici, azioni quali la selezione di genotipi resistenti, l'integrazione di probiotici di nuova generazione nei mangimi ed una corretta gestione dell’età di svezzamento, dovrebbero essere prese in considerazione nelle pratiche gestionali aziendali al fine di preservare un microbiota intestinale equilibrato e stabile e di conseguenza ridurre l'insorgenza di diarrea allo svezzamento

A microbiota intestinal é um ecossistema complexo que desempenha um importante papel na gênese da obesidade. A ocorrência e participação dos Mollicutes na microbiota intestinal é praticamente desconhecida. Deste modo, o objetivo do presente estudo foi analisar a participação dos Mollicutes e dos Filos Firmicutes e Bacteroidetes na microbiota intestinal de mulheres obesas e eutróficas. A casuística foi de 20 mulheres obesas e 20 mulheres em eutrofia. Foram obtidas amostras de fezes, sangue e aplicado questionário semiestruturado sobre fatores relacionados com obesidade, microbiota intestinal e ambiente, além de Bioimpedância e questionário de frequência alimentar. Constatou-se uma associação positiva estatisticamente significante entre a presença de Mollicutes e mulheres obesas. Foi observada maior proporção de Firmicutes/Bacteroidetes na microbiota intestinal das mulheres obesas. Os resultados obtidos permitiram obter evidências importantes da participação dos micro-organismos da classe Mollicutes. As alterações da microbiota intestinal também contribuíram na definição de subconjuntos de indivíduos com diferentes perfis de risco metabólico e a da heterogeneidade associada a fenótipos humanos relacionados com a adiposidade.
The gut microbiota is a complex ecosystem that plays an important role in the pathogenesis of obesity. The occurrence and participation of Mollicutes in the gut microbiota is pratically unknown. The aim of this study was to analyze the participation of Mollicutes and Firmicutes and Bacteroidetes phylos in the gut microbiota of obese and normal weight women. For the study, it was collected samples of 20 women with obesity and 20 women of normal weight. It was collected stool samples, blood, semi-structured questionnaire on factors associated with obesity, gut microbiota and the environment, and anthropometric measurements using bioelectrical impedance and food frequency questionnaire. It was detected a statistically significant positive association between the presence of Mollicutes and obese women, and there was a higher proportion of Firmicutes/Bacteroidetes in the gut microbiota of obese women. The results provide important evidence about the participation of Mollicutes class in the gut microbiota of the population studied and interactions in intestinal microbiota can define subsets of individuals with different metabolic risk profiles and thus contribute to investigation of the heterogeneity associated phenotypes related to adiposity.

Orientadores: Jaime Amaya-Farfan, Fernanda de Pace
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
Made available in DSpace on 2018-08-27T12:05:55Z (GMT). No. of bitstreams: 1 Roquetto_AlineRissetti_M.pdf: 2148135 bytes, checksum: 6a59bf58e9dbc6285574497bb9558141 (MD5) Previous issue date: 2015
Resumo: A obesidade é um dos maiores problemas de saúde pública no mundo, sendo associada a diversas doenças metabólicas como inflamação, resistência insulínica, dislipidemia, esteatose hepática, entre outras. Recentemente, tem sido demonstrado que alterações nas proporções dos filos que compõem a microbiota intestinal repercutem negativamente sobre o metabolismo e processos fisiológicos do hospedeiro. A dieta moderna é apontada como um dos fatores capazes de modular as bactérias intestinais e desencadear respostas inflamatórias. Diante deste cenário e tendo conhecimento de que a própolis, resina produzida por abelhas que possui ação anti-inflamatória e antimicrobiana, a presente pesquisa teve como objetivo avaliar o efeito da suplementação da própolis em camundongos tratados com dieta hiperlipídica sobre a microbiota intestinal e biomarcadores inflamatórios. Quarenta camundongos da linhagem C57BL/6 foram divididos em 4 grupos (n=10) aleatoriamente: grupo controle ¿ dieta baseada na AIN-93G; grupo hiperlipídico (HF) ¿ dieta com 37% de gordura; e grupos HFP2 e HFP5 tratados com dieta hiperlipídica, seguida de suplementação com própolis 0,2% nas duas e cinco semanas que antecederam ao sacrifício respectivamente. Foram coletadas amostras de sangue e músculo para determinações bioquímicas e indicadores de inflamação, o conteúdo cecal foi extraído para sequenciamento do DNA da microbiota intestinal. Os resultados não mostraram diferenças no ganho de peso entre os grupos experimentais, mas o tratamento com própolis por 5 semanas foi efetivo em reverter a disbiose causada pela dieta HF, com relação aos filos Firmicutes, e Proteobacteria. Os níveis de lipopolissacarídeos (LPS) no soro, bem como a expressão de toll-like receptor-4 (TLR4) e de citocinas pró-inflamatórias no músculo foram reduzidos pelo tratamento prolongado com própolis. Além disso, esta intervenção melhorou os níveis séricos de glicose e triacilgliceróis. Estes resultados sugerem a possibilidade de que a própolis exerça ação benéfica modificando o microbioma que limita a permeabilização da parede intestinal, regulando a translocação de componentes bacterianos para a corrente sanguínea e, consequentemente, conduzindo a uma menor expressão de citocinas inflamatórias
Abstract: Obesity is a major world-wide public health problem and is associated with metabolic disorders as generalized inflammation, insulin resistance, dyslipidemia, hepatic steatosis, among others. Recently, it has been demonstrated that changes in the proportions of phyla that make up the gut microbiota have a profound effect on the metabolism and physiology of the host. The modern diet has been identified as one of the factors that modulate the intestinal bacteria and trigger inflammatory responses. Considering this state of affairs and knowing that propolis, a resin present in bee honey, has anti-inflammatory and anti-microbial action, the present study was designed to evaluate the effect of propolis supplementation on the intestinal microbiome and inflammatory biomarkers of mice pre-conditioned with a high-fat diet. Forty mice of the C57BL/6 strain were randomly divided into four groups (n = 10): control group ¿ diet based on the AIN 93-G; high-fat group ¿ diet with 37% fat; and two other groups treated with high-fat, HFP2 and HFP5, that were supplemented with 0.2% propolis during two and five weeks preceding sacrifice, respectively. Blood and muscle samples were collected for biochemical analyses and inflammation markers, the cecal contents were extracted for DNA sequencing of the intestinal microbiota¿s genome. The results showed no differences in weight gain among the experimental groups, but treatment with propolis for 5 weeks effectively reverted the dysbiosis caused by the HF diet with respect to the Firmicutes and Proteobacteria phyla. The levels of serum lipopolysaccharide (LPS), and Toll-like receptor-4 (TLR4) expression, and proinflammatory cytokines in muscle were reduced by the longer propolis treatment. In addition, this intervention improved serum glucose and serum triacylglycerol levels. The present results suggest that ingested propolis exerts its beneficial action, first modifying the intestinal microbiota, which limits intestinal wall permeability and controls the translocation of bacterial components into the bloodstream and thus averting inflammatory cytokine overexpression
Mestrado
Nutrição Experimental e de Alimentos
Mestra em Alimentos e Nutrição

Les lipides polaires (LP) laitiers (~2% des lipides du lait) présentent un potentiel bioactif élevé, notamment lié à leur richesse en sphingomyéline (SM, ~25% des LP). Nos hypothèses sont que les LP laitiers peuvent exercer certains de leurs effets bénéfiques par l'intermédiaire de la SM, notamment sur l'intégrité de la barrière intestinale et le microbiote, ce qui pourrait contribuer à réduire l'inflammation métabolique. Nous avons testé à long terme in vivo l'impact de régimes hyperlipidiques (HF) supplémentés en LP laitiers. In vitro, nous avons étudié l'effet des LP laitiers et de la SM (laitière ou d'oeuf) sur l'expression génique des protéines de jonctions serrées. Nos travaux in vitro ont également permis de tester que l'interleurkine-8 (IL-8), impliquée dans la maturation de l'épithélium intestinal, serait un acteur des modifications intestinales en réponse aux LP laitiers et/ou à la SM. L'impact à court terme d'un gavage chez la souris avec des LP laitiers ou de la SM laitière a également été étudié. Après 8 semaines de régime HF supplémenté en LP laitiers (1,6%) les souris présentent un moindre gain de poids en comparaison au régime HF. Nous observons une augmentation de Bifidobacterium animalis pour le groupe contenant 1,1% de LP laitiers. Le groupe nourri avec une supplémentation de 1,6% de LP laitiers présente une diminution de Lactobacillus reuteri et des cryptes coliques plus profondes. Nous retrouvons également une plus forte teneur en acide gras spécifiques des LP laitiers (C23:0, C24:0 et C24:1, présents dans la SM laitière) dans les lipides fécaux. Ces acides gras sont corrélés à la teneur en Lactobacillus spp. Parmi les protéines de jonctions serrées impliquées dans la perméabilité paracellulaire, seule l'expression de ZO-1 tend à être augmentée dans le duodénum. In vitro, lorsque les cellules Caco-2/TC7 sont incubées avec des micelles mixtes supplémentées en SM pure, une augmentation de l'expression génique des protéines de jonctions serrées, ainsi qu'une augmentation de la concentration d'IL-8 en apicale et en basolatérale, sont observées. Ces effets sont également retrouvés avec la SM d'oeuf, contrairement aux LP laitiers totaux. L'incubation d'IL-8 recombinante humaine conduit à une augmentation de l'expression génique des protéines de jonctions serrées. Un gavage avec de la SM laitière pure chez la souris induit une augmentation de l'expression des homologues murins de l'IL-8 (KC et Mip-2). Cette étude suggère que les LP laitiers peuvent limiter la prise de poids induite par un régime HF et moduler le microbiote intestinal. La présence de produits d'hydrolyse spécifiques de la SM pourrait expliquer les effets sur le côlon et le microbiote intestinal. Les résultats in vitro, suggèrent un impact spécifique de la SM sur la barrière intestinale. L'IL-8 semble impliquée dans la régulation de l'expression des protéines de jonctions serrées. Ces résultats contribuent à expliquer les effets bénéfiques démontrés des LP laitiers. L'exploration mécanistique des effets directs et/ou indirects de la SM et de l'IL-8 sur la barrière intestinale reste à élucider
Interest is growing for the metabolic impact of milk polar lipids (MPL, ~2% of dairy lipids), which present a high bioactive potential, particularly related to their content in sphingomyelin (SM, ~ 25% of MPL). Our hypotheses are that MPL can exert some of their beneficial effects through SM, including the integrity of the intestinal barrier and the microbiota, which could contribute to reduce metabolic inflammation. We tested the metabolic impact of the addition of MPL in a high-fat (HF) diet in mice on the modulation of the intestinal barrier. In vitro, we studied the effect of SM (milk or egg) on tight junction protein We also tested in vitro, that interleurkin-8 (IL-8), which is involved in the maturation of the intestinal epithelium, is an actor of intestinal changes in response to MPL and/or MSM. The short-term impact in mice of MPL or milk SM was also studied. After 8 weeks of diet, the supplementation with 1.6% of MPL prevented the HF-diet-induced body weight gain. In caecal microbiota, addition of 1.1% of MPL induced a specific increase in Bifidobacterium spp., in particular B. animalis. The group fed with a 1.6% MPL-supplementation showed a specific decrease in Lactobacteria reuteri and colonic crypt depth were greatest. We also found a higher content of fatty acids specific of MPL (C23:0, C24:0 and C24:1, found in milk SM) in fecal lipids of mice. These fatty acids are correlated with Lactobacillus spp. Among the tight junction proteins involved in paracellular permeability, only the expression of ZO-1 tended to be increased in the duodenum. In vitro, when Caco-2/TC7 cells were incubated with mixed micelles supplemented with pure SM, an increase in the gene expression of tight junction proteins (ZO-1, occludin, JAM-1, claudin-1) and an increase in apical and basolateral IL-8 concentration were observed. These effects were also found with egg SM, unlike total MPL. Incubation of recombinant human IL-8 led to an increase in gene expression of tight junction proteins. Gavage with pure milk- SM in mice induced an increase in the expression of murine homologs of IL-8 (KC and Mip-2). Our results show that MPL can limit HF-induced body weight gain and modulate the abundance of beneficial bacteria of the gut microbiota. The presence of SM-specific hydrolysis products may explain the effects on the colon and gut microbiota. In vitro results suggest a specific impact of pure SM on the intestinal barrier. IL-8 appears to be involved in the regulation of tight junction protein expression. This can contribute to explain reported beneficial effects of MPL in mice regarding HF induced metabolic disorders. The mechanistic exploration of direct and / or indirect effects of SM and IL-8 on the intestinal barrier remains to be elucidated

The study of the gut microbiota of fish began in the 1930’s and since that time a considerable amount of information has been collated on its composition and diversity. These studies have revealed that the microbial communities of the fish gastrointestinal tract are generally difficult to culture on bacteriological media and mainly consist of bacteria, archaea, viruses, yeasts and protists. The bacteria appear to be the most abundant of these microbial groups and their activity may have major implications for host health, development, immunity and nutrition. Therefore, much of the most recent published research has focused on developing improved methods of identifying the extent of the bacterial diversity within the fish gut and unravelling the potential influence of these microorganisms on the health of farmed fish species. However, whilst such studies have improved our knowledge of the dominant bacterial groups present in the rainbow trout gastrointestinal tract, the limited resolution capacity of many of the methods used has meant that our understanding of their baseline composition in healthy fish remains poorly understood. In this study, the bacterial communities that inhabit the intestine, now commonly referred to as the ‘microbiome’, of farmed Rainbow trout (Oncorhynchus mykiss) were characterized using a culture independent high-throughput molecular sequencing method. The microbiome of the intestinal lumen and mucosa was investigated to ascertain the true extent of the bacterial diversity present in this fish species prior to further experiments. It was found that the diversity of the intestinal microbiome was greater than previous studies had reported with a total of 90 and 159 bacterial genera being identified in both the lumen and mucosal regions respectively. The dominant bacterial phyla identified in both of the regions investigated were Proteobacteria, Firmicutes, Fusobacteria, Bacteroidetes and Actinobacteria. Furthermore, the data collected suggested that the intestinal microbiome may be similar in structure between individual fish, and illustrate the utility of next generation molecular methods in the investigation of the fish gut microbiome. A study was conducted to examine the effect of diet on the composition of the intestinal microbiome of rainbow trout. Two diets, one control and one treatment, were prepared which were identical apart from that the treatment diet contained a microalgal component at 5% of the total formulation. These diets were fed to rainbow trout for a total of 15 weeks. At the end of the trial period a total of 12 fish, three from each of four tanks, were sacrificed from each of the control and treatment groups and their intestinal tissue was sampled in order to compare the composition of the microbiome of both groups. The results revealed that both groups of fish shared similar microbiome compositions, with the Tenericutes being by far the most dominant phylum observed. The structure of the intestinal microbiome was not significantly different between both populations of trout tested. An increased level of bacterial diversity was noted in the treatment fish, however, this was not found to be statistically significant. A limited number of bacterial taxa were discriminatory between diets and were significantly elevated in the treatment group. These taxa were predominantly lactic acid bacteria of the genera Streptococcus, Leuconstoc, Lactobacillus, Lactococcus and Weissella. The results of this study suggested that the minor difference in the diets fed resulted in a correspondingly minor alteration in the intestinal microbiome of the tested rainbow trout. This may indicate that diet composition can modify the composition of the intestinal microbiome of these fish. A further study was conducted to investigate the structure of the intestinal microbiome from groups of fish reared in both freshwater cages and aquarium systems, in order to assess whether or not fish raised in different environments share similar microbiomes. This study also employed a novel computational tool, PICRUSt, to analyse the predicted functional capacity of the microbial communities of individual fish sampled from both environments. The data collected suggested that the structure of the intestinal microbiome was similar regardless of where the fish were raised, with the Tenericutes, Firmicutes, Proteobacteria, Spirochaetae and Bacteroidetes representing the dominant bacterial phyla recorded in the rainbow trout intestine. This suggests that the host may regulate the formation of the intestinal microbiome. A significant difference was however noted in community membership between the fish populations tested, which may point to an environmental influence on the intestinal microbiome. These data suggest that both deterministic host factors and stochastic environmental influences play important roles in shaping the composition of the bacterial communities in the intestine of these fish. The PICRUSt analysis revealed that gene pathways relating to metabolism, transport and cellular processes were enhanced in all of the fish studied, which may signal an involvement of these communities in the digestive processes of rainbow trout. In conclusion, this study used high-throughput sequencing methods in order to improve our understanding of the intestinal microbiome of farmed rainbow trout, and the effect of dietary and environmental factors on its composition. This research has generated scientific information relating to baseline bacterial community compositions in healthy fish, which may be used in future experiments including screening these baselines against the effects of novel aquafeed formulations, environmental perturbations or pathogenic challenges.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A microbiota intestinal apresenta impacto direto na saúde do hospedeiro sendo fortemente influenciada pela dieta. O consumo de suco de laranja vem sendo associado à redução do risco de desenvolvimento de doenças crônicas, principalmente devido à presença de compostos bioativos. Os compostos bioativos presentes no suco de laranja, especialmente os polifenóis, também podem estar relacionados com a composição e o metabolismo da microbiota intestinal. O objetivo desse trabalho foi avaliar a influência do suco de laranja fresco e pasteurizado sobre a microbiota intestinal usando o Simulador do Ecossistema Microbiano Humano (SEMH®). O SEMH® foi utilizado para investigar a fermentação do suco de laranja ao longo do cólon e para avaliar as alterações na composição e no metabolismo microbiano. A atividade antioxidante dos sucos e das amostras dos compartimentos do SEMH® também foi avaliada. Foi observado no tratamento com suco de laranja fresco aumento (p≤0,05) das populações de Lactobacillus spp., Enterococcus spp., Bifidobacterium spp. e Clostridium spp. e diminuição (p≤0,05) de enterobactérias, enquanto no tratamento com suco de laranja pasteurizado houve aumento (p≤0,05) da população de Lactobacillus spp. e diminuição (p≤0,05) de enterobactérias. A análise de PCR-DGGE mostrou redução dos valores de riqueza da população de bactérias totais para ambos os sucos. Em relação ao metabolismo microbiano, foi observado aumento (p≤0,05) da produção de ácidos graxos de cadeia curta (AGCC) e diminuição (p≤0,05) do conteúdo de íons amônio no tratamento com os sucos de laranja fresco e pasteurizado. A atividade antioxidante das amostras dos compartimentos do SEMH® no tratamento com os sucos de laranja foi elevada, com ligeira redução em comparação àquela do suco fresco e do suco pasteurizado. A Análise de Componentes Principais (ACP) permitiu diferenciar o tratamento com os sucos dos períodos controle e washout, mostrando que os sucos de laranja fresco e pasteurizado apresentaram impacto sobre a microbiota intestinal. Os sucos mostraram efeito prebiótico e seletivo sobre a microbiota intestinal com aumento de AGCC e bactérias comensais e diminuição de íons amônio, embora com redução dos valores de riqueza da população de bactérias totais.
The gut microbiota has a direct impact on host's health being strongly influenced by diet. Orange juice consumption has been associated with a reduced risk of chronic diseases, largely because of the presence of bioactive compounds. The bioactive compounds present in orange juice, particularly polyphenols, may also be associated with the composition and metabolism of gut microbiota. The aim of this work was to evaluate the influence of fresh orange juice and pasteurized orange juice on gut microbiota using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). SHIME® was used to investigate orange juice fermentation throughout the colon and to assess changes in microbial composition and microbial metabolism. Antioxidant activity of the SHIME® vessels and juice was also evaluated. An increase (p≤0.05) in Lactobacillus spp., Enterococcus spp., Bifidobacterium spp. and Clostridium spp. population was observed in fresh orange juice treatment, as well as a reduction (p≤0.05) in enterobacteria. Regarding pasteurized orange juice treatment, an increase (p≤0.05) in Lactobacillus spp. population and a decrease (p≤0.05) in enterobacteria was observed. The PCR-DGGE analysis showed a reduction in total bacteria population richness values on both juices. According to microbial metabolism, an increasing (p≤0.05) of short-chain fatty acids (SCFA) production and decreasing (p≤0.05) of ammonium was observed for two juices treatments evaluated. The antioxidant activity of the samples from the SHIME® vessels in the orange juice treatments was high, with a slight reduction compared to that of fresh juice and pasteurized juice. Both fresh and pasteurized orange juice influenced on gut microbiota according to Principal Component Analysis (PCA), which enabled to differentiate the orange juice treatments from control and washout periods. Both juices showed a prebiotic and selective effect on gut microbiota which is in agreement with increases in both SCFAs and commensal bacteria, as well as with decreases in ammonium levels, though total bacteria richness values were reduced.

Au cours des dernières années, l’immunothérapie a révolutionné le paysage en oncologie. L’anti-CTLA-4 a montré son efficacité sur la survie globale des patients atteints de mélanome métastatique. Cependant, ce traitement présente des limites à son utilisation telles que l'efficacité clinique obtenu chez seulement 20% des patients et la survenue fréquente de colites pouvant être sévères. La recherche de biomarqueurs prédictifs de réponse clinique et/ou de développement de toxicité devient maintenant un enjeu majeur pour sélectionner les patients pouvant avoir un bénéfice à l’utilisation de ces traitements. En partant de l’observation que les colites induites par l’anti-CTLA-4 présentent des similitudes avec les maladies inflammatoires chroniques de l'intestin, nous avons émis l’hypothèse de l’existence d’un microbiote intestinal associé à une dysrégulation du système immunitaire pouvant prédire la réponse clinique et/ou la survenue d’une colite induite par l’anti-CTLA-4. Nous avons montré dans une cohorte de patients atteints de mélanome métastatique et traités par ipilimumab, qu'un microbiote intestinal enrichi en Faecalibacterium et autres Firmicutes est associé à une meilleure survie globale et sans progression ainsi qu'un risque accru de développer une colite. Les patients avec une flore enrichie en Firmicutes présentent également après traitement par ipilimumab, une activation lymphocytaire plus efficace. Par la suite, nous nous sommes intéressés aux métabolites issus du microbiote fécal et leur implication dans la réponse à l'anti-CTLA-4. Le butyrate est le principal métabolite produit par les Firmicutes. Nous avons observé chez la souris, une inhibition de l'efficacité anti-tumorale de l'anti-CTLA-4 lorsqu'elles étaient supplémentées en butyrate. In vivo, nous avons montré que le butyrate inhibe la surexpression sur les cellules dendritiques, des molécules CD80 et CD86 (molécules B7) induite par l'anti-CTLA-4. Cette immaturité des cellules dendritiques entraine un défaut d'activation des lymphocytes T spécifiques d'antigènes dépendant de l'axe CD28/B7 réduisant ainsi l'efficacité anti-tumorale. Chez l'Homme, nous avons valider cette hypothèse en montrant qu'une concentration sérique élevée en butyrate est associée à une diminution de la survie globale et sans progression comparativement aux patients avec un faible niveau de butyrate sérique.Ces travaux mettent en évidence le lien entre la composition du microbiote et les réponses immunologiques au blocage du CTLA-4. Ils apportent une explication sur un lien indirect via le butyrate entre la composition du microbiote intestinal et la réponse anti-tumorale aux immunothérapies
In the last years, immunotherapy has revolutionized the landscape in oncology. The efficacy of anti-CTLA-4 has been demonstrated by improving overall survival of patients with metastatic melanoma. However, this treatment has limitations to its use such as the clinical efficacy obtained in only 20% of patients and the high incidence of severe colitis. Predictive biomarkers of clinical response and / or toxicity development are mandatory for a better selection of patients who will benefit from this treatment. Based on the observation that anti-CTLA-4-induced colitis has similarities with inflammatory bowel disease, we hypothesized that the gut microbiota associated with dysregulation of the immune system may predict the clinical response and / or occurrence of anti-CTLA-4-induced colitis.In a cohort of patients with metastatic melanoma treated with ipilimumab, we have shown that a gut microbiota enriched with Faecalibacterium and other Firmicutes is associated with a better of overall and progression-free survival as well as an increased risk of developing colitis. Firmicutes-driven microbiota is also associated with an improvement in lymphocyte T activation after ipilimumab treatment. Subsequently, we were interested in microbial metabolites and their involvement in the clinical response to anti-CTLA-4. Butyrate is the main metabolite produced by the Firmicutes. In mice, we observed an inhibition of anti-tumor effect of anti-CTLA-4 in butyrate-supplemented mice. In vivo, we have shown that butyrate inhibits the overexpression on dendritic cells, of CD80 and CD86 molecules (B7molecules) induced by anti-CTLA-4. This immaturity of the dendritic cells leads to a poor signaling of CD28 / B7 axis and activation of antigen-specific T-cells, thereby reducing anti-tumor efficacy. In humans, we validated this hypothesis by showing that a high serum concentration of butyrate is associated with decreased overall and progression-free survival compared to patients with low serum butyrate levels.This studie highlights the link between the composition of gut microbiota and the immunological responses to CTLA-4 blockade. They provide an explanation of an indirect link via butyrate, between the composition of the gut microbiota and the anti-tumor response to immunotherapies

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in the United States. Aldo-keto reductase 1 B10 (AKR1B10) is highly expressed in colon and small intestine of normal humans, but its expression is lost or markedly down-regulated in tissues of patients with ulcerative colitis (UC) and CRC. AKR1B10 is a monomeric cytosolic enzyme with strong enzymatic activity to α, β-unsaturated carbonyl compounds, protecting cells from carbonyl lesions; AKR1B10 also mediates de novo synthesis of long chain fatty acids and membrane lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2). To study the etiopathogenic role of AKR1B10 in UC and CRC, our lab generated AKR1B8 deficient (AKR1B8 -/-) mice. AKR1 B8 is the orthologue in mice of human AKR1B10,

L'Entérocolite Ulcéro-Nécrosante (ECUN) est la pathologie intestinale la plus grave chez les nouveau-nés prématurés. Aucun essai clinique ou étude de cohorte ne s’est intéressé uniquement au stade I de l’ECUN. Nous avons analysé des échantillons de selles prélevées chez des nourrissons de moins de 34 semaines d'âge gestationnel, dont 11 nourrissons qui ont développé une entéropathie (ECUN-I)) et 21 témoins appariés. Les communautés bactériennes des selles ont été profilées par séquençage du gène 16S de l'ARNr bactérien. Le métabolome fécal a été analysé par NMR. Au cours de la première décade de vie (J1 à 10), les échantillons de nourrissons entéropathes tendaient vers une diversité alpha plus faible que chez les témoins avec une différence significative dans la répartition des communautés bactériennes, mais pas au niveau des métabolites fécaux. Dans la deuxième décade (J11-J20), l’analyse du microbiome ne montre pas de différence entre entéropathes et témoins mais l’analyse métabolomique montre une diminution significative du taux de sérine dans le groupe des entéropathes. Entre J21 et J30, Bacillus, Staphylocococcus, Bacteroidetes et Streptococcus sont les groupes bactériens les plus abondants chez les entéropathes, alors que les genres Klebsiella et Raoultella sont plus abondants chez les témoins. Au-delà de 30 jours de vie, les témoins ont un microbiote plus riche en entérobactéries (Klebsiella et Enterobacter) que les entéropathes. Les divergences au niveau métabolomique sont plus importantes au cours du deuxième mois de vie. Les modifications structurelles et fonctionnelles du microbiote intestinal semblent plus reconnaissables à la troisième décade de la vie, comparativement aux enfants témoins. La fonctionnalité du microbiote intestinal est plus affectée par les facteurs maternels et infantiles alors que la structure du microbiote intestinal semble plus stable. Ces données pourraient fournir de nouvelles cibles microbiennes pour lutter contre la progression de l’ECUN dès le stade 1
Necrotizing enterocolitis (NEC) is the most serious intestinal pathology in preterm infants. No clinical trial or cohort studies included only stage I of NEC (NEC-1). We analysed stool samples collected from infants under 34 weeks of gestational age, including 11 infants who developed enteropathy (NEC-1 group) and 21 matched controls. Fecal Bacterial communities were profiled by sequencing the 16S rRNA bacterial gene. Faecal metabolomic profiles were analysed by NMR. During the first ten days of life (d1-10), samples from NEC-1 infants showed a lower alpha diversity than controls with a significant difference in the distribution of bacterial communities but not in faecal metabolome. Between d11-d20, microbiome did not differ between NEC-1 infants and controls, but metabolomic analysis showed a significant decrease in serine levels in NEC-1 infants. Between d21 and d30, Bacillus, Staphylocococcus, Bacteroidetes and Streptococcus bacterial groups were more abundant in NEC-1 infants than in controls, whereas Klebsiella and Raoultella species were more abundant in controls. Beyond 30 days of life, controls showed a microbiota richer in Klebsiella and Enterobacter than NEC-1 infants. Leucine levels in faeces were lower in NEC-1 infants. The modifications of gut microbiota and microbiome during NEC-1 development appear more distinguishable by the third decade of life, when compared to healthy children. These data may suggest new microbial targets to fight/blunt the progression of NEC as early as by stage 1

L’exposition aux polluants environnementaux a été associée à de nombreux désordres métaboliques, immunitaires et reproductifs ainsi qu’à divers cancers. De plus en plus de travaux, indiquent que le microbiote intestinal, qui joue un rôle majeur dans l’immunité et le métabolisme de l’hôte, interagit avec les xénobiotiques dont les polluants organiques persistants (POPs) et les contaminants néoformés dans les aliments. Cette interaction peut avoir des conséquences toxicologiques importantes via la modification des fonctions du microbiote intestinal mais également via la métabolisation des xénobiotiques, entraînant une potentielle altération de l'homéostasie de l'hôte. Dans le cadre de cette thèse, nous avons démontré, en modèle in vitro, qu’une exposition aigüe du microbiote intestinal humain au benzo[a]pyrène (hydrocarbure aromatique polycyclique) a entraîné une altération des fonctions du microbiote intestinal au niveau du volatolome et du métatranscriptome microbien. Cependant, dans nos conditions expérimentales, aucun impact sur la structure microbienne n'a été observé. L’Homme étant continuellement exposé à un panel de composés chimiques environnementaux, nous avons par la suite étudié l'impact de divers POPs et produits néoformés dans les aliments sur le microbiote intestinal humain. Des familles de gènes ainsi que des composés volatiles microbiens ont été identifiés comme altérés après l’exposition, conduisant à une perturbation de l'activité microbienne. Nous avons finalement démontré que l'interaction microbiote-polluant pourrait conduire à l'établissement d'un état pro-inflammatoire modéré dans l'intestin avec une libération de cytokine IL-8 par les cellules épithéliales intestinales. Ces résultats appuient le concept émergent selon lequel les contaminants alimentaires pourraient altérer les activités du microbiote intestinal
Exposure to environmental pollutants has been associated with various life-threatening disorders, including dysregulation of the immune and reproductive systems, metabolic diseases and various cancers. Growing evidences indicate that the gut microbiota, which plays major roles in host metabolic and immune functions, interacts with xenobiotics including persistent organic pollutants (POPs) and foodborne chemicals. The toxicological relevance of the gut microbiota-pollutant interplay is of great concern for the host since the chemicals may disrupt the gut microbiota functions leading to a potential impairment of the host homeostasis. During this PhD thesis, we demonstrated that in vitro acute exposure of the human gut microbiota with benzo[a]pyrene (polycyclic aromatic hydrocarbon) led to an impairment of the gut microbiota functions with a specific shift of the microbial volatolome and metatranscriptome. However, in our experimental conditions, no impact on the microbial structure was observed. Since humans are exposed to a wide range of environmental chemicals we investigated the impact of various POPs and foodborne chemicals on the human gut microbiota. We identified microbial volatiles and gene families that shifted after this exposure leading to an imbalance of the microbial activity. Furthermore, we demonstrated that the interaction between the pollutants and the gut microbiota lead to a significant release of pro-inflammatory IL-8 cytokine by the intestinal epithelial cells which may contribute to the establishment of a low-grade inflammatory state in the gut. All together, these data support the emerging concept that food pollutants could alter the gut microbiota activities

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Humans live in constant association with microorganims. The amount of microorganims present in the human body exceeds our own cell number. That community of microorganisms has deep influence in health and disease. The use of high-throughput DNA sequencing technologies and culture independent approaches have been enlarging the understanding concerning the communities of microorganisms and the association of these with the host. The human gastrointestinal tract contains one of the most complex bacterial communities. It was proposed recently that the microbiome can be classified in three enterotypes. In our study, we used data metagenomics quantitative search to identify phylogenetic patterns in the intestinal microbiome to develop prediction models for the enterotypes. To reach this aim, statistical tests were applied to the data regarding abundance of bacteria in level taxonomic corresponding to genus. We identified genus significantly with the abundance different and important correlations. Besides the ratio among genus to be used as parameter bioindicator of the respectives enterotypes. Through the logistic regression test we identified that the prediction model for ET1 was influenced significantly by the ratio of Bacteroides / (Prevotella + Ruminococcus). In the model for prediction of ET2, it was the ratio of Prevotella / Bacteroides with such as characteristic significance. And for the model of ET3, we identified the ratio of (Akkermansia + Alistipes) / (Bacteroides + Prevotella) as significant parameter. These models were assessed against two groups of independent data and associated with the value of cut-off 5%; 20% and 95% respectively. Besides the value of cutoff for each models, the crossed validation allowed the association of the model with the measures of PPV for ET1, specificity for ET2 and PNV for ET3. We propose the experimental validation of these models for the qPCR technique. And with that methodology established, it would be possible to do the diagnosis of the enterotype individually.
Humanos vivem em constante associação com microrganismos. A quantidade de microrganismos presentes no corpo humano ultrapassa o nosso próprio número de células. Essa comunidade de microrganismos tem profunda influência na saúde e doenças. As tecnologias de sequenciamento de DNA de alta capacidade e abordagens moleculares independente de cultura têm ampliado a compreensão acerca das comunidades de microrganismos e a associação destes com o hospedeiro. O trato gastrointestinal humano abriga uma das mais complexas comunidades bacterianas. Foi proposto recentemente que o microbioma pode ser categorizado em três enterotipos. No nosso estudo, utilizamos dados metagenômicos quantitativos buncando identificar padrões filogenéticos no microbioma intestinal para desenvolver modelos de predição para os enterotipos. Para alcançar este objetivo, testes estatísticos foram aplicados aos dados referente a abundância de bactérias em nível taxonômico correspondente a gênero. Identificamos gêneros com a abundância significativamente diferente e correlações importantes. Além da razão entre gêneros para ser utilizada como parâmetro bioindicativo dos respectivos enterotipos. Através do teste de regressão logística identificamos que o modelo de predição para o ET1 foi influenciado significativamente pela razão de Bacteroides / (Prevotella + Ruminococcus). No modelo para predição do ET2, foi a razão de Prevotella / Bacteroides que apresentou significância. E para o modelo do ET3, identificamos a razão de (Akkermansia + Alistipes) / (Bacteroides + Prevotella) como parâmetro significativo. Estes modelos foram avaliados contra dois conjuntos de dados independentes e associados com o valor de cut-off 5%; 20% e; 95% respectivamente. Além do valor de cut-off para cada modelos, a validação cruzada permitiu a associação do modelo com as medidas de PPV para o ET1, especificidade para o ET2 e PNV para o ET3. Propomos a validação experimental destes modelos pela técnica de qPCR. E com essa metodologia estabelecida, seria possível fazer o diagnóstico do enterotipo individualmente.

Les infections digestives à Clostridium difficile nécessitent une première étape de colonisation de l’écosystème intestinal. Avant l’âge de deux ans, la colonisation par C. difficile est fréquente mais paradoxalement le plus souvent asymptomatique. Nous avons montré que plus d’un tiers des enfants de 0-3 ans, et plus de 70% de ceux de 7 à 9 mois (15% pour les souches toxinogènes), étaient porteurs sains de C. difficile à l’hôpital et dans la communauté. Deux périodes d’acquisition de C. difficile ont été identifiées : néonatale ou 3-6 mois. Les souches infantiles de C. difficile étaient identiques aux souches isolées chez l’adulte, faisant du jeune enfant un réservoir potentiel de souches infectieuses. Nous avons également montré par méthode moléculaire que des changements en espèces dominantes du microbiote étaient associés à la colonisation par C. difficile. Bifidobacterium longum caractérisait le microbiote des enfants non colonisés, et pourrait participer à la résistance à la colonisation par C. difficile
Gastrointestinal infections with Clostridium difficile require a first step of colonization of the intestinal ecosystem. Under the age of two years, C. difficile colonization is frequent but paradoxically most often asymptomatic. We have shown that more than a third of children 0-3 years and more than 70% of those from 7 to 9 months (15% for toxigenic strains) were healthy carriers of C. difficile in the hospital and in the community. Two C. difficile-acquisition periods were identified: neonatal or 3-6 months. The C. difficile strains from infants were identical to strains isolated from adults, making infants a potential reservoir of infectious strains. We also showed by molecular method that changes in dominant species of the microbiota were associated with colonization by C. difficile. Bifidobacterium longum characterized the microbiota of children not colonized by C. difficile, and could be involved in the colonization resistance process

Un nombre grandissant d’associations entre diverses pathologies humaines et dysbiose intestinale (définie ici comme altération de la composition du microbiote par rapport à sa composition habituelle) sont observées. Parmi les facteurs qui pourraient induire la dysbiose, les bactériophages (dit phages), sont des candidats pertinents par leur fonction prédatrice.L’objectif de la thèse a été de déterminer si les prophages de souches bactériennes du microbiote intestinal humain ont un impact négatif sur la stabilité de leur hôte dans l’intestin. Pour cela, nous avons utilisé des souris sans germes primo-colonisées avec la souche Escherichia coli LF82, puis inoculées soit avec Faecalibacterium prausntizii A2-165, soit avec Roseburia intestinalis L1-82, deux souches appartenant aux espèces dominantes du microbiote intestinal humain. Chacune de ces souches possède deux prophages dans son génome, Lagaffe et Mushu pour F. prausnitzii, Jekyll et Shimadzu pour R. intestinalis. L’impact des prophages a également été étudié lors d’une inflammation intestinale induite au DSS.Pour la combinaison F. prausnitzii/E. coli, aucun des deux prophages de F. prausnitzii n’a d’activité délétère pour son hôte bactérien chez la souris, même durant une inflammation induite au DSS. Afin de mieux caractériser l’ensemble des prophages présents chez cette espèce, une analyse bio-informatique effectuée sur 15 souches de F. prausnitzii a permis de constater que la prévalence de Mushu et Lagaffe était faible, mais aussi de découvrir une remarquable richesse phagique : au total, 18 espèces de prophages répartis en nouveaux 8 genres viraux ont été décrits. Une étude in silico de l’abondance de ces phages dans les viromes intestinaux humains a révélé que des phages du genre ‘Lugh’ et ‘Epona’ sont plus souvent présents et/ou abondants dans les viromes de patients des maladies inflammatoires chroniques de l’intestin (MICI) que chez les individus sains. Sachant que les patients atteints de MICI ont une population appauvrie de F. prausnitzii dans leur microbiote, ces observations suggèrent une activité accrue de ces phages pendant la maladie : ils pourraient déclencher ou aggraver la baisse de population de F. prausnitzii dans les patients, participant ainsi à l’aggravation des symptômes des MICI.Avec la combinaison R. intestinalis/E. coli., aucune variation de population ou effet délétère du phage Jekyll n’a pu être observé. En revanche, la population du phage Shimadzu est loin d’être stable. Dans toutes les souris, et même en l’absence d’un traitement au DSS, un mutant virulent de Shimadzu émerge, appelé Shi-vir. Ce mutant lyse massivement la population intestinale de R. intestinalis, menant à un effondrement de la population hôte. La population bactérienne remonte ensuite à son niveau initial grâce à l’émergence de mutants bactériens résistants à l’infection. Cette résistance a essentiellement pour origine l’acquisition d’un espaceur associé au système CRISPR-Cas de type IIC de R. intestinalis, et dirigé contre le phage Shimadzu. Cependant, l’acquisition de cet espaceur ne peut se faire sans qu’une sous-population de R. intestinalis soit préalablement guérie du prophage Shimadzu, sans quoi un tel espaceur tuerait la bactérie.J’ai ainsi démontré qu’un prophage peut déstabiliser sa population hôte dans l’environnement intestinal et créer des dysbioses intestinales transitoires. La pression de sélection qui résulte de l’infection par le phage Shi-vir a permis l’accélération de l’évolution de l’hôte bactérien.En conclusion, une fraction des phages tempérés du microbiote intestinal pourrait avoir un impact négatif sur la stabilité de sa population hôte dans l’environnement intestinal, soit parce le ratio phage/bactérie augmente dans cet environnement (cas des phages Lugh et Epona de F. prausnitzii), soit parce qu’il évolue vers la virulence (cas de Shi-vir chez R. intestinalis) et induit une dysbiose transitoire
A growing number of associations is observed between various human pathologies and intestinal dysbiosis, here defined as an alteration of the microbiota composition. Among the potential factors inducing dysbiosis, bacteriophages, called phages, are relevant candidates by their predatory function.The aim of the thesis was to determine whether prophages of bacterial strains from the human gut microbiota have a negative impact on the stability of their host in the gut environment. We studied this question by using germ-free mice colonized first with Escherichia coli strain LF82, then inoculated with two bacterial strains belonging to dominant species of the human intestinal microbiota, Faecalibacterium prausnitzii strain A2-165 or Roseburia intestinalis strain L1-82. Each of these strains has two prophages in its genome, Lagaffe and Mushu for F. prausnitzii, Jekyll and Shimadzu for R. intestinalis. The impact of these prophages was also studied during intestinal inflammation using DSS (Dextran Sulfate Sodium)-induced colitis in mice.In mice colonized with F. prausnitzii and E. coli , prophages of F. prausnitzii did not have any deleterious activity for the bacterial host, even during DSS-induced inflammation. In order to better characterize prophages of the F. prausnitzii species, a bioinformatic analysis carried out on 15 strains of F. prausnitzii highlighted that the prevalence of Mushu and Lagaffe was low. However, this analysis revealed also an enormous diversity of phages and we described 18 species of prophages divided into 8 new proposed genera. An in silico study of their abundance in 173 human intestinal viromes revealed that the phage genera 'Lugh' and 'Epona' were more present and/or abundant in viromes of Inflammatory Bowel Disease (IBD) patients compared to healthy subjects. Given that IBD patients have lower populations of F. prausnitzii in their microbiota compared to healthy subjects, our observations suggest an increased activity of these phages during disease. They may trigger or worsen population decline of F. prausnitzii in patients, participating thus to the aggravation of IBD symptomsIn mice colonized with R. intestinalis and E. coli, we did not observe variation of Jekyll population or deleterious effect of this phage on its host. In contrast, the Shimadzu population was not stable. Indeed, even in the absence of DSS treatment we observed in all mice the emergence of a virulent mutant of Shimadzu, called Shi-vir. This mutant massively lysed R. intestinalis, leading to a collapse of the bacterial host population. Then this population rose back to its original level thanks to the emergence of bacterial mutants resistant to the viral infection. This resistance was mainly due to the acquisition of a spacer associated with the CRISPR-Cas type IIC system of R. intestinalis, directed against the Shimadzu phage. However, acquisition of this spacer could not be observed unless the Shimadzu prophage was cured from the strain, showing that this spacer would kill the Shimadzu lysogen.I have shown therefore that a prophage can destabilize its host population in the intestinal environment and create transient intestinal dysbiosis. I have also highlighted that the selection pressure imposed by an ex-temperate phage infection, the Shi-vir phage, has allowed an acceleration of its host evolution.Overall, this work establishes that a fraction of the temperate phages present in intestinal microbiota may impact negatively bacterial population stability, either because the phage/bacteria ratio increases (for the Lugh and Epona phages de F. prausnitzii), or because the temperate phage evolves towards virulence (case of the Shi-vir mutant on R. intestinalis), and induces a transient dysbiosis

Le microbiote intestinal joue un rôle primordial dans l’homéostasie du tractus gastro-intestinal, et plus généralement dans celle de son hôte. A ce titre, de nombreuses pathologies humaines sont associées à une dysbiose de ce microbiote intestinal, tels que les cancers colorectaux, les maladies inflammatoires chroniques de l’intestin (MICI), les troubles du métabolisme ou encore les maladies auto-immunes. Ces pathologies ont une étiologie mal connue et multifactorielle dans laquelle l’environnement semble jouer un rôle clé. Des études récentes ont ainsi mis en évidence un lien entre la pollution atmosphérique et des pathologies humaines telles que les MICI. Parmi les différentes substances polluantes répertoriées, le benzo[a]pyrène (BaP), qui fait partie de la famille des hydrocarbures aromatiques polycycliques, est soumis à une surveillance accrue en raison de ses effets toxiques sur la santé humaine. De par ses propriétés pro-inflammatoires et mutagènes, le BaP pourrait modifier la composition du microbiote intestinal, induisant alors à une réponse inflammatoire et à une altération des fonctions intestinales. Dans le cadre de ce travail de thèse, une surexposition orale et chronique au BaP en modèle murin a conduit à une inflammation modérée principalement au niveau de la muqueuse iléale. L’analyse des amplicons du gène codant l’ARNr 16S a mis en évidence des modifications de la composition et de l’abondance relative des communautés bactériennes fécales et associées à la muqueuse intestinale avec notamment une augmentation et une diminution des taxa pro et anti-inflammatoires respectivement. Ainsi, dans des conditions de susceptibilité génétique et/ou en association avec d’autres facteurs environnementaux, l’exposition à ce polluant pourrait déclencher et/ou accélérer le développement de pathologies inflammatoires. L’identification des potentialités métaboliques des différentes populations bactériennes caractérisées précédemment et impactées par le polluant revêt donc un caractère primordial. La reconstruction de génomes directement à partir de l’écosystème microbien peut permettre d’établir ce lien entre structure et fonction. C’est également dans ce contexte, qu’une approche innovante de capture de gènes en solution a été développée. En effet, cette technique d’enrichissement permet de reconstruire de larges portions génomiques pouvant relier un biomarqueur phylogénétique à des gènes fonctionnels, y compris pour des populations bactériennes présentes en très faible abondance dans l’écosystème
Gut microbiota plays a primordial role in gastro-intestinal tract and host homeostasis. Numerous pathologies are associated with a gut microbiota dysbioses, such as colorectal cancer, inflammatory bowel diseases (IBD), metabolism disorders or autoimmune diseases. The physiopathology of these diseases has multifactorial aetiology in which environmental factors seem to play a crucial role. Recent evidences have highlighted a link between air pollution and human diseases such as IBD. Among the different pollutant listed, benzo[a]pyrene (BaP), which belong to the family of polycyclic aromatic hydrocarbons, is subject to an increase surveillance due to its toxic effects on human health. By its pro-inflammatory and mutagenic proprieties, BaP could lead to modifications of gut microbiota composition, then inducing an inflammatory response and an alteration of intestinal functions. As part of this thesis, BaP subchronic oral exposure in murine model has led to a moderate inflammation mostly in ileal mucosa. The analysis of ARNr 16S amplicons has highlighted composition and abundance alterations of faecal and mucosa-associated microbiota, especially with increase and decrease of pro and anti- inflammatory taxa respectively. Thus, under conditions of genetic susceptibility and/or in association with other environmental factors, exposure to this pollutant could trigger and/or accelerate the development of inflammatory pathologies. Metabolic potential identification of different bacterial populations previously characterized and affected by the pollutant appears therefore primordial. Genome reconstruction directly from microbial ecosystem could allow to establish this link between structure and function. Also in this context, an innovative approach of gene capture in solution was developed. Indeed, this enrichment technique allows to reconstruct large genomic portions that could link phylogenetic biomarker and functional genes, including for bacterial populations present at very low abundance in the ecosystem

Les N-acyl homosérine lactones sont des molécules du quorum sensing impliquées dans la communication interbactérienne mais elles sont également capables d'intéragir avec les cellules eucaryotes. Rechercher ces molécules dans le contexte des maladies inflammatoires chroniques intestinlaes (MICI) et plus particulièrement dans le cadre de l'étude des conséquences de la dysbiose sur les voies de l'inflammation intestinale est séduisant. En utilisant la spectrométrie de mase, nous avons mis en évidence pour la première fois des AHLs dans l'écosystème intestinal humain, et plus particulièrement une nouvelle AHL, 3-oxo-C12 :2, qui est prédominante. Cette AHL est corrélée à la normobiose, est perdue au cours des MICI et exerce un effet protecteur sur les cellules épithéliales intestinales. En effet, la 3-oxo-C12 :2 exerce un effet anti-inflammatoire in vitro sur les cellules Caco-2 sans augmenter la perméabilité paracellulaire. De plus, les premiers résultats in vivo montrent que la 3-oxo-C12 est également capable d'influencer la composition du microbiote intestinal des souris. Ces résultats ouvrent de nombreuses perspectives notamment dans la recherche de traitements écologiques au cours des MICI
Quorum sensing molecules N-acyl-homoserine lactones (AHLs) involved in bacterial communication network are also able to interact with eukaryotic cells. Searching for these molecules in the context of inflammatory bowel diseases (IBD) and more precisely when studying consequences of dysbiosis on gut inflammation pathways is appealing. Using mass spectrometry, we identified for the first time AHLs in human intestinal ecosystem, and among them a new AHL, 3-oxo-C12:2 which is prominent. This AHL correlates with normobiosis, is lost IBD and exerts protective effect on gut epithelial cells. In fact, 3-oxo-C12:2 exerts anti-inflammatory effect in vitro on Caco-2 cells without increased paracellular permeability. Furthermore, first results from in vivo experiments show that 3-oxo-C12:2 is also able to influence mice gut microbiota composition. These results open multiple perspectives especially on new ecological treatments in IBD

Les relations entre bactéries intestinales, aliments et hôte jouent un rôle crucial dans lemaintien de la santé humaine. La caractérisation fonctionnelle d’Uhgb_MP, une enzyme dela famille 130 des glycoside hydrolases découverte par métagénomique fonctionnelle, arévélé une nouvelle fonction de dégradation par phosphorolyse des polysaccharides de laparoi végétale et des glycanes de l'hôte tapissant l'épithélium intestinal. Les déterminantsmoléculaires de la spécificité d’Uhgb_MP vis-à-vis des mannosides ont été identifiés grâce àla résolution de sa structure cristallographique, sous forme apo et en complexe avec sesligands. Un nouveau procédé de synthèse par phosphorolyse inverse d'oligosaccharidesmannosylés à haute valeur ajoutée, a aussi été développé. Enfin, la caractérisationfonctionnelle de la protéine BACOVA_03624 issue de Bacteroides ovatus ATCC 8483, unebactérie intestinale hautement prévalente, a révélé que la famille GH130 comprend à la foisdes glycoside-hydrolases et des glycoside-phosphorylases capables de dégrader lesmannosides et les galactosides, et de les synthétiser par phosphorolyse inverse et/outransglycosylation. L’ensemble de ces résultats, ainsi que l’identification d’inhibiteurs desenzymes de la famille GH130, ouvrent de nouvelles perspectives pour l'étude et le contrôledes interactions microbiote-hôte
The interplay between gut bacteria, food and host play a key role in human health. Thefunctional characterization of Uhgb_MP, an enzyme belonging to the family 130 of glycosidehydrolases, discovered by functional metagenomics, revealed novel functions of plant cellwall polysaccharide and host glycan degradation by phosphorolysis. The moleculardeterminants of Uhgb_MP specificity towards mannosides were identified by solving itscrystal structure, in apo form and in complex with its ligands. A new process of high addedvalue mannosylated oligosaccharide synthesis by reverse-phosphorolysis was alsodeveloped. Finally, the functional characterization of the BACOVA_03624 protein fromBacteroides ovatus ATCC 8483, a highly prevalent gut bacterium, revealed that GH130 familyboth contains glycoside phosphorylases and glycoside hydrolases, which are able to degrademannosides and galactosides, and to synthesize them by reverse-phosphorolysis and/ortransglycosylation. All these results, together with the identification of GH130 enzymeinhibitors, open new perspectives for studying, and potentially also for controlling,interactions between host and gut microbes

Background Con il termine microbiota intestinale, si definisce la comunità microbica del tratto enterico, composta da trilioni di microorganismi in grado di svolgere funzioni essenziali per l’ospite: metaboliche, strutturali e di protezione. Il suo ruolo, nei meccanismi di salute e di prevenzione, è al centro dell’interesse medico. Ma le modalità con cui il microbiota può essere alterato a nostro favore, non sono ancora del tutto chiare. Evidenze sulla modulazione da parte della dieta sono sempre più attendibili, mentre il ruolo dell’attività fisica, è ancora da approfondire, con una letteratura basata principalmente sull’effetto dell’esercizio fisico negli animali. Scopo della ricerca Indagare la letteratura a supporto dell’efficacia dell’attività fisica come fattore in grado di alterare la composizione del microbiota intestinale nell’adulto sano. Materiali e metodi La ricerca è stata condotta nel periodo di tempo che va da maggio 2020 a settembre 2020, consultando i database Pubmed, PEDro e Cochrane. Le parole chiave utilizzate sono state Physical execise, Aerobic exercise, Physical activity, Cardiorespiratory fitness, Gut microbiota, Microbiome, Adult, Human, Probiotics e Diet, combinate con l’operatore booleano AND, OR e NOT. Sono stati inclusi studi primari e secondari, in lingua italiana o inglese, che valutassero l’alterazione della comunità microbiotica, in adulti sani, dopo aver svolto esercizio fisico. Risultati La ricerca in letteratura e l’analisi degli studi, attraverso i criteri di eleggibilità, hanno condotto all’individuazione di un RCT, un clinical trial e due case report. Conclusioni Gli studi analizzati riportano risultati incoraggianti, per quanto riguarda l’effetto dell’attività fisica nel modificare la comunità microbiotica. I limiti riscontrati in questa revisione, rendono però difficile interpretare i risultati in maniera oggettiva, in quanto appare impossibile escludere altri cofattori.

The increasing prevalence of allergic diseases in affluent societies has been associated with changes in microbial exposure early in life and a less diverse gut flora. The objective of this thesis was to assess the development of allergic sensitisation and symptoms during the first four years of life in a non-selected birth cohort in relation to environmental factors, family history, gut microbiota and salivary IgA antibodies. The cohort comprised all 1,228 infants living in a Swedish county who were born over a one-year period. The parents replied to questionnaires, and 817 children (67 %) were skin prick tested both at 1 and 4 years of age. Saliva (n=279), faecal (n=139) and blood (n=253) samples were collected at 1 year of age from children with a positive skin prick test at 1 year and from a sample of children with a negative skin prick test. Faecal samples were also obtained from 53 children at 4 years of age. Dog keeping during infancy was associated with a decreased risk of sensitisation to pollen and late-onset wheezing at age 4, and the reduced odds ratios persisted after adjustment for heredity and avoidance measures, OR 0.3, 95% CI 0.1-0.9 and OR 0.5, 95% CI 0.2-1.0, respectively. In contrast, early dog keeping was associated with an increased risk of earlyonset transient wheezing but only in children with parental asthma (OR 2,8, 95% CI 1.3-6.4). Levels of short chain fatty acids (SCFAs) in faeces were assessed both at 1 and 4 years of age and related to the development of sensitisation and symptoms. The levels of acetic (p<.01) and propionic (p<.01) acids decreased from one to four years of age, whereas valeric acid (p<.001) increased which is in line with a more complex gut microbiota with age. Allergic children, compared with non-allergic children, had lower levels of i-butyric, i-valeric and valeric acid in faeces both at 1 and 4 years of age. Low levels of secretory IgA (SIgA) in saliva were associated with wheezing but only in sensitised children. In children with positive SPT to at least one allergen both at 1 and 4 years of age and in children with circulating IgE antibodies to egg or cat at one year of age, those who developed late-onset wheezing had lower levels of SIgA than those who did not, p=.04 and p=.02 respectively. Of 9 children with levels of SIgA in the upper quartile and persistent sensitisation, none developed wheezing, compared with 10/20 children with lower levels, (p=. 01). Having older siblings, more than three infections during infancy, at least one smoking parent and male gender were all associated with high levels (in the upper quartile) of total IgA and SIgA. The findings in this thesis indicate that the microbial load early in life could affect the development of allergy. A functional assessment of the gut flora demonstrated differences between allergic and non-allergic children both at 1 and 4 years of age. Salivary IgA was associated with infections during infancy, and high levels of secretory IgA protected from symptoms in sensitised children. Finally, dog keeping in infancy may offer protection from allergy, but the mechanism is uncertain.

Les maladies psychiatriques présentent de fortes comorbidités avec des désordres gastrointestinaux, ce qui suggère l’existence de bases physiopathologiques communes. Une littérature abondante démontre que l’adversité précoce (infection, stress) augmente la vulnérabilité aux désordres psychiatriques à l’âge adulte. Chez le rongeur, le modèle de séparation maternelle induit chez la descendance adulte des comportements hyperanxieux associés à une hypersensibilité au stress, ainsi que des dysfonctionnements de la sphère gastrointestinale. De plus, des études récentes rapportent une hyperperméabilité de la barrière intestinale chez les ratons soumis au stress de séparation, un effet conduisant potentiellement à une dysbiose et une perturbation de la communication intestin-cerveau. Le but de ma thèse était donc d’étudier le rôle de l’axe intestin-cerveau dans la mise en place des effets à long terme du stress précoce. Nos travaux récents ont montré que certains effets à long-terme de la séparation maternelle peuvent être atténués par l’exposition des mères à un régime hyperlipidique. Dans un premier temps, nous avons testé les effets du régime hyperlipidique maternel sur le cerveau et l’intestin de ratons soumis à la séparation maternelle. Nos résultats montrent que le régime maternel hyperlipidique protège de l’augmentation de la permeabilité intestinale induite par le stress. Nous avons ensuite testé le rôle causal de la perméabilité intestinale sur les comportements émotionnels à travers une approche pharmacologique et une approche génétique. Nous rapportons 1) que la restauration de la fonction barrière de l’intestin atténue certains effets de la séparation maternelle et 2) qu’une hyperperméabilité intestinale chez des souris transgéniques non soumises à un stress produit des effets similaires à ceux de la séparation maternelle. Enfin, nous avons examiné les effets d’une adversité précoce multifactorielle sur le cerveau et l’intestin (perméabilité et microbiote) chez la descendance adulte mâle et femelle dans un modèle combinant infection prénatale et séparation maternelle. Nos résultats mettent en évidence un effet sexe très marqué sur les phénotypes comportements et intestinaux. D’autres études sont nécessaires pour identifier les mécanismes sous-tendant les effets de la perméabilité et la dysbiose intestinale sur la vulnérabilité émotionnelle associée au stress précoce
Early-life adversity is a main risk factor for psychiatric disorders at adulthood; however the mechanisms underlying the programming effect of stress during development are still unknown. In rodents, chronic maternal separation has long lasting effects in adult offspring, including hyper-anxiety and hyper-responsiveness to a novel stress, along with gastrointestinal dysfunctions. Moreover, recent studies report gut barrier hyper-permeability in rat pups submitted to maternal separation, an effect that could potentially lead to dysbiosis and altered gut-brain communication. Therefore, the aim of my PhD was to unravel the role of the gut-brain axis in the neurobehavioral effects of early-life stress. We recently reported that some neural, behavioral and endocrine alterations associated with maternal separation in rats could be prevented by maternal exposure to a high-fat diet. We first addressed the effects of maternal high-fat diet on brain and gut during development in the maternal separation model. We show that maternal high-fat diet prevents the stress-induced decrease in spine density and altered dendritic morphology in the medial prefrontal cortex. Moreover, maternal high-fat diet also attenuates the exacerbated intestinal permeability associated with maternal separation. To explore a potential causal impact of gut leakiness on brain functions, we then examined the impact of pharmacological and genetic manipulations of intestinal permeability on brain and behavior. We report 1) that restoration of gut barrier function attenuates some of the behavioral alterations associated with maternal separation and 2) that chronic gut leakiness in naive adult transgenic mice recapitulates the effects of maternal separation. Finally, we examined the effects of multifactorial early-life adversity on behavior, gut function and microbiota composition in males and females using a combination of prenatal inflammation and maternal separation in mice. At adulthood, offspring exposed to early adversity displayed sex-specific behavioral (social behavior deficits in males and increased anxiety in females) and intestinal phenotypes. In conclusion, our work demonstrates an impact of gut dysfunctions, in particular gut leakiness, on the emergence of emotional alterations. Further studies are needed to unravel the role of the gut dysbiosis in the expression of the behavioral phenotypes associated with early-life adversity

La colitis ulcerosa (CU) és una malaltia inflamatòria idiopàtica crònica que afecta l’intestí gros. És un dels dos trastorns principals que engloba el terme Malaltia Inflamatòria Intestinal (MII). L’etiologia de la CU involucra una resposta immune a una microbiota intestinal desequilibrada en individus genèticament susceptibles, després d’esdeveniments desencadenants desconeguts. Els estudis de la microbiota han trobat una menor riquesa i diversitat en la microbiota intestinal en els pacients amb MII que en els controls sans, i aquesta descans de la diversitat es manifesta especialment durant i després dels brots de la malaltia. El microbioma intestinal és essencial per a mantenir la salut; els factors ambientals i de l’hoste influeixen en la composició de la microbiota intestinal. La remissió clínica, endoscòpica i histològica impacta positivament en la història natural de la CU. Aquesta tesi té com a objectiu determinar si els pacients amb CU que aconsegueixen la remissió clínica, endoscòpica i histològica durant un període prolongat presenten una composició de microbiota intestinal significativament diferent a la dels pacients amb CU en períodes de remissió més curts o amb malaltia activa. És a dir si els pacients amb CU en remissió perllongada mostren una microbiota intestinal similar a la dels individus sans. Per a això, hem analitzat mostres fecals de pacients amb CU en remissió llarga, pacients amb CU en remissió curta i pacients amb CU en brot i la hem comparat amb les mostres d’individus sans. Hem utilitzat dos mètodes diferents: seqüenciació del gen 16S ARNr i qPCR per a bacteris específics. També hem explorat els components no bacterians de la microbiota intestinal mitjançant la determinació de la càrrega fúngica mitjançant qPCR. Com es preveia, observem la presència de disbiosis en la CU amb una reducció en la diversitat i riquesa bacterianes, sub-representació de bacteris beneficiosos i l’increment de bacteris potencialment nocius. Aquesta disbiosis és més pronunciada en pacients amb CU en brot de la malaltia, però també és present en pacients amb CU en remissió curta. Els pacients amb CU que van aconseguir una remissió profunda i histològica estable i a llarg termini de la seva malaltia, presenten una composició bacteriana intestinal més pròxima a la dels controls sans, per tant menys “disbiótica”. Més enllà del component bacterià, també trobem un vincle entre l’abundància de fongs i l’estat inflamatori en la CU. Els pacients en brot de la malaltia presenten una major abundància de càrrega fúngica que els pacients en remissió, però aquests resultats han d’interpretar-se amb cautela i avaluar-se més a fons en estudis longitudinals més amplis. Creiem que una microbiota en els pacients amb CU semblant a la que trobem en individus “sans”, pot ser d’utilitat per a definir amb més precisió la remissió de la malaltia.
La colitis ulcerosa (CU) es una enfermedad inflamatoria idiopática crónica que afecta el colon. Es uno de los dos trastornos principales que engloba el término Enfermedad Inflamatoria Intestinal (EII). La etiología de la CU involucra una respuesta inmune a una microbiota intestinal desequilibrada en individuos genéticamente susceptibles, después de eventos desencadenantes desconocidos. Los estudios de la microbiota han encontrado una menor riqueza y diversidad en la microbiota intestinal en los pacientes con EII que en los individuos sanos, y estas diferencias se manifiestan especialmente durante y después de los brotes de la enfermedad. El microbioma intestinal es esencial para mantener la salud; los factores ambientales y del huésped influyen en la composición de la microbiota intestinal. La remisión clínica, endoscópica e histológica impacta positivamente en la historia natural de la CU. Esta tesis tiene como objetivo determinar si los pacientes con CU que alcanzan la remisión clínica, endoscópica e histológica durante un período prolongado de tiempo, presentan una composición de la microbiota intestinal significativamente diferente a la de los pacientes con CU en periodos de remisión más cortos o con enfermedad activa. Para ello, hemos analizado muestras fecales de pacientes con CU en remisión larga, pacientes con CU en remisión corta y pacientes con CU en brote y hemos comparado con las muestras fecales obtenidas de individuos sanos. Hemos utilizado dos métodos diferentes: secuenciación del gen 16S ARNr y qPCR para bacterias específicas. También hemos explorado los componentes no bacterianos de la microbiota intestinal mediante la determinación de la carga fúngica mediante qPCR. Como se preveía, observamos la presencia de disbiosis en la CU con una reducción en la diversidad y riqueza bacterianas, sub-representación de bacterias beneficiosas y el incremento de bacterias potencialmente nocivas. Esta disbiosis es más pronunciada en pacientes con CU en brote de la enfermedad, pero también está presente en pacientes con CU en remisión corta. Los pacientes con CU que lograron una remisión profunda e histológica estable y a largo plazo de su enfermedad, presentan una composición bacteriana intestinal más próxima a la de los controles sanos, por lo tanto menos “disbiótica”. Más allá del componente bacteriano, también encontramos un vínculo entre la abundancia de hongos y el estado inflamatorio en la CU. Los pacientes en brote de la enfermedad presentan una mayor abundancia de carga fúngica que los pacientes en remisión, pero estos resultados deben interpretarse con cautela y evaluar más a fondo en estudios longitudinales más amplios. Creemos que una microbiota en los pacientes con CU similar a la que encontramos en individuos “sanos”, puede ser de utilidad para definir con mayor precisión la remisión de la enfermedad.
Ulcerative colitis (UC) is a chronic idiopathic inflammatory disease that affects the large bowel. It is one of the two major disorders under the broad term of Inflammatory Bowel Disease (IBD). The etiology of UC involves an immune response to an imbalanced gut microbiota in genetically susceptible individuals, following unknown triggering events. Microbiome studies have found a lower microbial richness and diversity in the gut microbiota of IBD patients than healthy controls, and the drop is especially manifested during and after flares of disease. The gut microbiome is essential in maintaining health and mediating illness, and environmental and host factors influence its composition. Clinical, endoscopic, and histological remission positively impacts the natural history of UC. This thesis aimed to determine if UC patients who reach clinical, endoscopic, and histological remission for an extended period would present a different gut microbiota composition than UC patients with shorter remission lengths or active disease. UC patients in long remission will show a gut microbiota that is similar to healthy individuals. For this purpose, we analyzed fecal samples of UC patients in long remission, UC patients in short remission and UC patients in flare and compared it to healthy individuals. We used two different methods: 16S rRNA gene sequencing and qPCR for specific bacteria. We also sought to explore non-bacterial constituents of the gut microbiota by determining the fungal load using qPCR. As expected, we observed dysbiosis in UC with a reduction in diversity and richness, underrepresentation of beneficial bacteria, and the gain of potentially harmful microbes. This dysbiosis was greater in UC patients in disease-flare but was also present in UC patients in short remission. UC patients who were able to achieve a long-term, stable, deep, and histological remission of their disease presented a gut bacterial composition closer to health, thus less dysbiotic. Beyond the bacterial component, we found a link between fungi abundance and inflammatory status in UC. Patients in disease-flare present a greater abundance of the fungal load than patients in remission, but these results must be interpreted cautiously and further evaluated in larger, longitudinal studies. We believe that a ‘healthier’ gut microbiota in UC patients could potentially be a goal to pursue in order to define disease remission further.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina

The presence of microbial commensals in the gut requires the establishment of a complex network of reciprocal interactions between the microbiota and the host immune system to allow nutrient absorption while preventing undesired mucosal immune responses. Despite these homeostatic mechanisms, during intestinal inflammation alterations of the microbiota composition, namely dysbiosis, trigger abnormal immune responses. Here, we aimed at investigating the functional crosstalk between gut microbiota and the mucosal immune system during inflammation and upon induction of microbial dysbiosis. We observed that inflammation-induced and antibiotic-driven types of dysbiosis are phenotypically and functionally modifying CD4+ T and iNKT cells activity. Moreover, during intestinal inflammation, the experimental manipulation of the microbiota community through Faecal Microbiota Transplantation (FMT) reduces colonic inflammation and initiates the restoration of intestinal homeostasis through the induction of IL-10 production by immune cells. Further, we performed a comprehensive analysis on intestinal iNKT cells isolated from surgical specimens of active Inflammatory Bowel Disease (IBD) patients and non-IBD donors. We report that the exposure to mucosa-associated microbiota drives iNKT cell pro-inflammatory activation, inducing direct pathogenicity against the intestinal epithelium. Collectively, we provided solid evidence that a strict crosstalk between the gut microbiota and the intestinal conventional and non-conventional T cells exists. Antibiotic-associated dysbiosis has immunostimulatory functions. Moreover, FMT can therapeutically control intestinal experimental colitis and this poses FMT as a valuable therapeutic option in immune-related pathologies. In addition, we generated fundamental knowledge about the pathogenic functions exerted by human intestinal iNKT cells upon the interaction with mucosa-associated microbiota communities.

Il termine malattia renale cronica (Chronic Kideny Disease: CKD) si riferisce a differenti condizioni caratterizzate da un progressivo declino della funzione renale. Le linee guida internazionali hanno definito la CKD come una condizione in cui siano presenti marcatori di danno renale e/o la velocità di filtrazione glomerulare stimata (Estimated Glomerular Filtration Rtae: eGFR) sia inferiore a 60 ml/min/1.73 m2 per almeno 3 mesi. L’insufficienza renale in stadio terminale è associata ad un alto rischio di malattia cardiovascolare (Cardiovascular Disease: CVD), la più frequente causa di morte in questi pazienti. Fattori di rischio “non-tradizionali” come: infiammazione cronica, stress ossidativo, deplezione proteico-energetica, disordini del metabolismo minerale e deficit di inibitori della calcificazione, partecipano alla patogenesi della CVD. L’infiammazione gioca un ruolo cruciale nella risposta fisiologica all’infezione e al danno renale e partecipa anche nell’evoluzione del danno renale irreversibile con la produzione di diverse molecole infiammatorie a partire da acidi grassi polinsaturi a lunga catena (Long Chain PolyuUsaturated Fatty Acids: LCPUFA) della serie Omega-6. La supplementazione di Omega-3, con effetto antinfiammatorio, nei pazienti affetti da CKD è stata ed è oggetto di molti studi, nonostante ciò, l’effetto sul danno renale è ancora poco chiaro. Comunque, è ampiamente riconosciuto che un alterato profilo lipidico possa determinare la progressione della patologia, inducendo lo stato infiammatorio. Inoltre, elevati/normali livelli di Omega-3 potrebbero essere associati al miglioramento della funzionalità renale, diminuendo quindi il rischio di peggioramento della malattia. Le concentrazioni e il rapporto di Omega-3 e Omega-6 sono strettamente associati alla salute del rene, poiché svolgono ruoli importanti in differenti vie metaboliche. Un altro aspetto, preso poco in considerazione, è l’effetto dei livelli di acidi grassi circolanti e dei loro metaboliti sullo stato infiammatorio e sulla sua modulazione. Il primo scopo di questo studio è stato quello di analizzare il profilo degli acidi grassi in soggetti anziani affetti da CKD. Sono stati arruolati 57 pazienti afferenti agli ambulatori di Nefrologia dell’Ospedale Maggiore Policlinico di Milano e sono stati raccolti campioni di sangue su cui è stata effettuata l’analisi del profilo lipidico. Negli ultimi anni, diversi studi hanno sottolineato la stretta associazione tra infiammazione a livello intestinale e peggioramento del quadro in pazienti con CKD. Il mantenimento di un ottimo stato del tratto gastrointestinale è fondamentale per assicurare lo stato di salute dell’ospite, contribuendo ai processi metabolici, fisiologici e immunologici. Le comunità batteriche instaurano un rapporto mutualistico con l’individuo che colonizzano, giocando un ruolo importante negli stati di salute e malattia. Un’anomala colonizzazione o cambiamenti nella composizione del microbiota intestinale, determina disbiosi, uno squilibrio associato a diverse condizioni patologiche come obesità, diabete di tipo II, malattia intestinale cronica, CVD e anche CKD. Il rapporto tra intestino e rene è bidirezionale, nei pazienti affetti da malattia renale cronica, la composizione del microbiota intestinale risulta essere modificata rispetto a quella del soggetto sano. Alti livelli di urea che si riversano facilmente nel tratto intestinale modificano il microambiente chimico con conseguente innalzamento del pH del colon che esercita una pressione selettiva a favore di specie ureasi-positive, responsabili della conversione dell’urea in ammoniaca. Lo strato protettivo di muco viene degradato e la permeabilità della barriera intestinale viene compromessa. In conseguenza di ciò si ha il passaggio di materiale batterico attraverso la mucosa e l’attivazione di un meccanismo infiammatorio. Nei pazienti con funzionalità renale compromessa, il rene perde progressivamente la capacità di eliminare sia le sostanze provenienti dal metabolismo umano, sia quelle della comunità microbica intestinale. Alcune di queste sostanze sono rappresentate dalle tossine uremiche, tra quelle di derivazione intestinale le principali e più studiate sono p-cresil solfato (PCS) e indossile solfato (IS). IS e p-CS, strettamente legate all’albumina sierica (Human Serum Albumin: HSA), non vengono eliminate facilmente ma rimangono nel torrente ematico. HSA è la più abbondante proteina sierica ed è la principale trasportatrice di composti esogeni ed endogeni, inclusi gli acidi grassi che sembrano rappresentare il maggior ligando endogeno della proteina. Multipli siti di legame vengono utilizzati per gli acidi grassi monoinsaturi (MonoUnsaturated Fatty Acids: MUFA) e PUFA. Acidi grassi e tossine uremiche competono quindi per gli stessi siti di legame sulla proteina. Il potenziale ruolo degli acidi grassi nel contrastare l’accumulo di tossine uremiche derivate dalla comunità batterica intestinale ne giustifica l’importanza della valutazione dei loro livelli ematici. Secondo scopo di questa tesi di dottorato è stato quello di valutare la possibile correlazione tra i livelli di acidi grassi circolanti e la composizione del microbiota intestinale in soggetti affetti da CKD. Sono stati arruolati nello studio 64 pazienti anziani con CKD non dializzati e 15 soggetti anziani con normale funzionalità renale. La composizione del microbiota intestinale è stata precedentemente caratterizzata attraverso l’impiego delle tecniche di elezione: PCR-DGGE e la PCR quantitativa (qPCR). In accordo con la letteratura scientifica, è stata evidenziata una riduzione di batteri saccarolitici e produttori di butirrato nei pazienti con CKD rispetto al gruppo di controllo. Il butirrato sembra giocare un ruolo cruciale nel mantenimento delle ottimali condizioni della barriera intestinale. Tenendo ciò in considerazione è stato deciso di approfondire lo studio e valutare l’associazione tra la comunità microbica intestinale e i livelli di acidi grassi basali in tali pazienti. Come risultato più importante ottenuto, è stata osservata una correlazione positiva statisticamente significativa tra la specie batterica Faecalibacterium Prausnitzii e i livelli totali di Omega-3 entrambi associati a proprietà antinfiammatorie. La presente tesi di dottorato evidenzia la necessità di sostenere ulteriori ricerche per supportare i risultati qui presentati. Studi futuri potrebbero essere utili per migliorare la comprensione del ruolo degli acidi grassi circolanti e i loro metaboliti sulla composizione del microbiota intestinale, sullo stato infiammatorio e sulla malattia renale cronica.
The aim of this thesis was to explore the possible associations between fatty acids (FA) profile and gut microbiota (gMb) with several conditions throughout the lifespan, from infancy to old age. In particular, we focused our attention on elderly subjects with Chronic Kidney Disease (CKD) and children with Acute Otitis Media (AOM). The terms “Chronic Kidney Disease” refers to several disorders with a progressive kidney function decline. International guidelines approved the definition of CKD as a condition with the presence of markers of kidney damage or with the estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73 m2 or both, for at least three months. End-stage renal disease is associated with a high cardiovascular disease (CVD) risk, the major cause of death in these patients. Chronic inflammation, oxidative stress, protein-energy wasting, disordered mineral metabolism, and deficiency of endogenous calcification inhibitors, known as non-traditional risks factor, take part in cardiovascular pathology in CKD. Inflammatory processes influence the physiological response to renal infection and injury but also participate in the development of potentially irreversible kidney damage with the production of various inflammatory molecular species, among whom eicosanoids and cytokines, from parental omega-6 long-chain polyunsaturated fatty acids (LCPUFA). Several studies focused their attention on the potential role of omega-3 (n-3) LCPUFA supplementation in subjects with CKD. Despite this, their effect on kidney damage is still not clear. However, it is widely agreed that a modified FA profile in CKD can determine a progression of the disease, inducing the inflammatory state. Moreover, high/normal n-3 LCPUFA levels decrease the risk of a decline of the disease. Omega-3 and omega-6 (n-6) LCPUFA concentrations and their ratios are tightly associated with renal health, because of their important roles in different pathways. Another aspect not very considered in the field of CKD is the role of circulating FA levels and their metabolites on the modulation of inflammation. The first aim of this study is to analyze the FA profile in elderly subjects with CKD. Blood samples have been collected from 57 subjects enrolled in the study, and FA analysis has been performed. During the last years, several studies underlined the strong relationship between intestinal inflammation and adverse outcomes in CKD. The health of gastrointestinal tract is fundamental to ensure the well being of the host contributing to its nutrition, metabolism, physiology, and immune function. The bacterial communities colonizing humans have been seen in terms of mutualistic symbiosis with their hosts, a mutually beneficial coexistence, playing an important role in health and disease. Abnormal colonization or changes in the gut microbial composition determine dysbiosis, a state associated with different illnesses, such as obesity, type 2 diabetes, inflammatory bowel disease, cardiovascular disease, and also chronic kidney disease. The relationship between gut and kidney is a bi-directional relation with a mutual influence. Chronic kidney disease influences gMB characteristics, especially through high levels of urea that easily spread in the intestinal fluid where bacterial urease enzymes degrade it, then it is hydrolyzed in ammonium hydroxide that increases fecal pH with a consequent alteration of intestinal cellular junctions. Besides, high levels of urea change intestinal microbiota composition damaging permeability of intestinal barrier and promoting proteolysis with production and absorption of uremic toxins, such as indoxyl sulfate (IS) and p-cresol sulfate (p-CS). These toxins induce an inflammatory process associated with CKD. Under physiologic conditions, the kidney through the urine eliminates these compounds, but CKD patients have a compromised renal clearance. Therefore, these solutes tend to accumulate in the organs. IS and p-CS are tightly bound to human serum albumin (HSA), the most abundant plasma protein in the bloodstream. HSA is recognized as the main means of transport for endogenous and exogenous compounds, including fatty acids that seem to be the main endogenous ligand of HSA, multiple binding sites are used for monounsaturated fatty acids (MUFA) and PUFA. Thus, free fatty acids and uremic toxins compete for the same binding sites on HSA. It is important to assess fatty acid (FA) levels in patients with CKD because of the potential role to contrast the accumulation of uremic toxins derived from the intestinal bacterial community. As a consequence of this bi-directional relation between gut and kidney and the possible involvement of some compounds as metabolites of FA in the inflammatory response, we investigate the correlation between circulating FA levels and the gMB composition in the same subjects with CKD, as the second aim of this thesis. 64 old CKD non-dialysis patients (eGFR 15-45 ml/min/1.73 m2) and 15 elderly subjects (>65 years) with normal renal function (eGFR >60 ml/min/1.73 m2, CKD-EPI) are enrolled. Bacterial composition was studied in a previous observational study by denaturating gel gradient electrophoresis (DGGE), high-throughput sequencing (16S ribosomal RNA), and quantitative real-time PCR (qPCR). This study described an increased abundance of some bacteria associated with pathological conditions. In agreement with the literature, the author found a reduced abundance of saccharolytic and butyrate-producing bacteria (Prevotella, Faecalibacterium prausnitzii, Roseburia) in CKD patients respect to the control group. Butyrate plays a crucial role in the maintenance of the gut barrier function. Taking that into account, we decided to investigate the correlation between gMB composition and FA profile in these subjects. The main result of the study was the significant positive correlation between Faecalibacterium prausnitzii and total n-3 levels, both associated with the antiinflammatory action. The present doctoral thesis underlines the need to perform further investigations in order to support evidence presented. Future studies may be useful to improve understanding of the effect of circulating fatty acids levels and their metabolites on gut microbial composition, inflammation process, and pathological conditions such as kidney disease. Our results showed that CKD patients with previous cardiovascular events had lower total and specific n-3 levels comparing with patients without them. Moreover, higher docosahexaenoic acid (DHA) levels and having had previous cardiovascular events seemed to have protective effects against further cardiovascular events. Moreover, we observed a significant reduction of the genera Roseburia and Faecalibacterium in CKD patients compared to C group and a significant lower abundance of F. prausnitzii and Roseburia spp. in CKD patients. Thus, our results seem in accordance with anti-inflammatory actions of total n-3, DHA, and saccharolytic and butyrateproducing bacteria. Many gMB changes seem to be related both to CKD and CVD. If the different gMB composition might play a causal role in cardiovascular events by an unbalanced production of some toxic substances, or if the gMB changes are merely a consequence of different dietary and lifestyle behaviours of these patients, it cannot be explained by the present study and all the yet available data. Further studies, possibly utilizing new high-throughput tools, will be required to understand the potential correlations between the gMB composition and other inflammation and oxidative stress markers in these patients. Other two studies have been performed during the doctoral course, to reach a better comprehension of fatty acids, gut microbial community and inflammatory states. A prospective pilot clinical study has been performed to to explore possible changes of gMB composition in children with AOM treated with amoxicillin with or without clavulanic acid. AOM is one of the most common bacterial infections in children and is normally treated with antibiotic therapies that lead to increasing antimicrobial resistance rates among otopathogens and may impair the correct development of the microbiota in early life. No significant differences were shown in the gMB composition of the overall cohort at different time intervals of the samples collection and in subjects treated with amoxicillin with or without clavulanic acid at different time intervals (T0, T1 and T2). A literature revision on lipids in infant formulae has been performed to better understanding quality and quality of dietary lipids because of their significant impact on health outcomes, especially when fat storing and/or absorption are limited (e.g., preterm birth and short bowel disease) or when fat byproducts may help to prevent some pathologies. The lipid composition of infant formulae varies according to the different fat sources used, and the potential biological effects are related to the variety of saturated and unsaturated FAs. Instead, ruminant-derived trans FAs and metabolites of n-3 LCPUFA with their anti-inflammatory properties can modulate immune function. Furthermore, dietary fats may influence the nutrient profile of formulae, improving the acceptance of these products and the compliance with dietary schedules. During the doctoral course, I spent a period abroad at Dell Pediatric Research Institute (DPRI), The University of Texas at Austin. In particular, I attended the laboratory of Doctor Brenna. I focused my research activity on a specific regulatory insertion-deletion polymorphism in the FADS gene cluster for better understanding its influence on PUFA and lipid profile.

Il termine malattia renale cronica (Chronic Kideny Disease: CKD) si riferisce a differenti condizioni caratterizzate da un progressivo declino della funzione renale. Le linee guida internazionali hanno definito la CKD come una condizione in cui siano presenti marcatori di danno renale e/o la velocità di filtrazione glomerulare stimata (Estimated Glomerular Filtration Rtae: eGFR) sia inferiore a 60 ml/min/1.73 m2 per almeno 3 mesi. L’insufficienza renale in stadio terminale è associata ad un alto rischio di malattia cardiovascolare (Cardiovascular Disease: CVD), la più frequente causa di morte in questi pazienti. Fattori di rischio “non-tradizionali” come: infiammazione cronica, stress ossidativo, deplezione proteico-energetica, disordini del metabolismo minerale e deficit di inibitori della calcificazione, partecipano alla patogenesi della CVD. L’infiammazione gioca un ruolo cruciale nella risposta fisiologica all’infezione e al danno renale e partecipa anche nell’evoluzione del danno renale irreversibile con la produzione di diverse molecole infiammatorie a partire da acidi grassi polinsaturi a lunga catena (Long Chain PolyuUsaturated Fatty Acids: LCPUFA) della serie Omega-6. La supplementazione di Omega-3, con effetto antinfiammatorio, nei pazienti affetti da CKD è stata ed è oggetto di molti studi, nonostante ciò, l’effetto sul danno renale è ancora poco chiaro. Comunque, è ampiamente riconosciuto che un alterato profilo lipidico possa determinare la progressione della patologia, inducendo lo stato infiammatorio. Inoltre, elevati/normali livelli di Omega-3 potrebbero essere associati al miglioramento della funzionalità renale, diminuendo quindi il rischio di peggioramento della malattia. Le concentrazioni e il rapporto di Omega-3 e Omega-6 sono strettamente associati alla salute del rene, poiché svolgono ruoli importanti in differenti vie metaboliche. Un altro aspetto, preso poco in considerazione, è l’effetto dei livelli di acidi grassi circolanti e dei loro metaboliti sullo stato infiammatorio e sulla sua modulazione. Il primo scopo di questo studio è stato quello di analizzare il profilo degli acidi grassi in soggetti anziani affetti da CKD. Sono stati arruolati 57 pazienti afferenti agli ambulatori di Nefrologia dell’Ospedale Maggiore Policlinico di Milano e sono stati raccolti campioni di sangue su cui è stata effettuata l’analisi del profilo lipidico. Negli ultimi anni, diversi studi hanno sottolineato la stretta associazione tra infiammazione a livello intestinale e peggioramento del quadro in pazienti con CKD. Il mantenimento di un ottimo stato del tratto gastrointestinale è fondamentale per assicurare lo stato di salute dell’ospite, contribuendo ai processi metabolici, fisiologici e immunologici. Le comunità batteriche instaurano un rapporto mutualistico con l’individuo che colonizzano, giocando un ruolo importante negli stati di salute e malattia. Un’anomala colonizzazione o cambiamenti nella composizione del microbiota intestinale, determina disbiosi, uno squilibrio associato a diverse condizioni patologiche come obesità, diabete di tipo II, malattia intestinale cronica, CVD e anche CKD. Il rapporto tra intestino e rene è bidirezionale, nei pazienti affetti da malattia renale cronica, la composizione del microbiota intestinale risulta essere modificata rispetto a quella del soggetto sano. Alti livelli di urea che si riversano facilmente nel tratto intestinale modificano il microambiente chimico con conseguente innalzamento del pH del colon che esercita una pressione selettiva a favore di specie ureasi-positive, responsabili della conversione dell’urea in ammoniaca. Lo strato protettivo di muco viene degradato e la permeabilità della barriera intestinale viene compromessa. In conseguenza di ciò si ha il passaggio di materiale batterico attraverso la mucosa e l’attivazione di un meccanismo infiammatorio. Nei pazienti con funzionalità renale compromessa, il rene perde progressivamente la capacità di eliminare sia le sostanze provenienti dal metabolismo umano, sia quelle della comunità microbica intestinale. Alcune di queste sostanze sono rappresentate dalle tossine uremiche, tra quelle di derivazione intestinale le principali e più studiate sono p-cresil solfato (PCS) e indossile solfato (IS). IS e p-CS, strettamente legate all’albumina sierica (Human Serum Albumin: HSA), non vengono eliminate facilmente ma rimangono nel torrente ematico. HSA è la più abbondante proteina sierica ed è la principale trasportatrice di composti esogeni ed endogeni, inclusi gli acidi grassi che sembrano rappresentare il maggior ligando endogeno della proteina. Multipli siti di legame vengono utilizzati per gli acidi grassi monoinsaturi (MonoUnsaturated Fatty Acids: MUFA) e PUFA. Acidi grassi e tossine uremiche competono quindi per gli stessi siti di legame sulla proteina. Il potenziale ruolo degli acidi grassi nel contrastare l’accumulo di tossine uremiche derivate dalla comunità batterica intestinale ne giustifica l’importanza della valutazione dei loro livelli ematici. Secondo scopo di questa tesi di dottorato è stato quello di valutare la possibile correlazione tra i livelli di acidi grassi circolanti e la composizione del microbiota intestinale in soggetti affetti da CKD. Sono stati arruolati nello studio 64 pazienti anziani con CKD non dializzati e 15 soggetti anziani con normale funzionalità renale. La composizione del microbiota intestinale è stata precedentemente caratterizzata attraverso l’impiego delle tecniche di elezione: PCR-DGGE e la PCR quantitativa (qPCR). In accordo con la letteratura scientifica, è stata evidenziata una riduzione di batteri saccarolitici e produttori di butirrato nei pazienti con CKD rispetto al gruppo di controllo. Il butirrato sembra giocare un ruolo cruciale nel mantenimento delle ottimali condizioni della barriera intestinale. Tenendo ciò in considerazione è stato deciso di approfondire lo studio e valutare l’associazione tra la comunità microbica intestinale e i livelli di acidi grassi basali in tali pazienti. Come risultato più importante ottenuto, è stata osservata una correlazione positiva statisticamente significativa tra la specie batterica Faecalibacterium Prausnitzii e i livelli totali di Omega-3 entrambi associati a proprietà antinfiammatorie. La presente tesi di dottorato evidenzia la necessità di sostenere ulteriori ricerche per supportare i risultati qui presentati. Studi futuri potrebbero essere utili per migliorare la comprensione del ruolo degli acidi grassi circolanti e i loro metaboliti sulla composizione del microbiota intestinale, sullo stato infiammatorio e sulla malattia renale cronica.
The aim of this thesis was to explore the possible associations between fatty acids (FA) profile and gut microbiota (gMb) with several conditions throughout the lifespan, from infancy to old age. In particular, we focused our attention on elderly subjects with Chronic Kidney Disease (CKD) and children with Acute Otitis Media (AOM). The terms “Chronic Kidney Disease” refers to several disorders with a progressive kidney function decline. International guidelines approved the definition of CKD as a condition with the presence of markers of kidney damage or with the estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73 m2 or both, for at least three months. End-stage renal disease is associated with a high cardiovascular disease (CVD) risk, the major cause of death in these patients. Chronic inflammation, oxidative stress, protein-energy wasting, disordered mineral metabolism, and deficiency of endogenous calcification inhibitors, known as non-traditional risks factor, take part in cardiovascular pathology in CKD. Inflammatory processes influence the physiological response to renal infection and injury but also participate in the development of potentially irreversible kidney damage with the production of various inflammatory molecular species, among whom eicosanoids and cytokines, from parental omega-6 long-chain polyunsaturated fatty acids (LCPUFA). Several studies focused their attention on the potential role of omega-3 (n-3) LCPUFA supplementation in subjects with CKD. Despite this, their effect on kidney damage is still not clear. However, it is widely agreed that a modified FA profile in CKD can determine a progression of the disease, inducing the inflammatory state. Moreover, high/normal n-3 LCPUFA levels decrease the risk of a decline of the disease. Omega-3 and omega-6 (n-6) LCPUFA concentrations and their ratios are tightly associated with renal health, because of their important roles in different pathways. Another aspect not very considered in the field of CKD is the role of circulating FA levels and their metabolites on the modulation of inflammation. The first aim of this study is to analyze the FA profile in elderly subjects with CKD. Blood samples have been collected from 57 subjects enrolled in the study, and FA analysis has been performed. During the last years, several studies underlined the strong relationship between intestinal inflammation and adverse outcomes in CKD. The health of gastrointestinal tract is fundamental to ensure the well being of the host contributing to its nutrition, metabolism, physiology, and immune function. The bacterial communities colonizing humans have been seen in terms of mutualistic symbiosis with their hosts, a mutually beneficial coexistence, playing an important role in health and disease. Abnormal colonization or changes in the gut microbial composition determine dysbiosis, a state associated with different illnesses, such as obesity, type 2 diabetes, inflammatory bowel disease, cardiovascular disease, and also chronic kidney disease. The relationship between gut and kidney is a bi-directional relation with a mutual influence. Chronic kidney disease influences gMB characteristics, especially through high levels of urea that easily spread in the intestinal fluid where bacterial urease enzymes degrade it, then it is hydrolyzed in ammonium hydroxide that increases fecal pH with a consequent alteration of intestinal cellular junctions. Besides, high levels of urea change intestinal microbiota composition damaging permeability of intestinal barrier and promoting proteolysis with production and absorption of uremic toxins, such as indoxyl sulfate (IS) and p-cresol sulfate (p-CS). These toxins induce an inflammatory process associated with CKD. Under physiologic conditions, the kidney through the urine eliminates these compounds, but CKD patients have a compromised renal clearance. Therefore, these solutes tend to accumulate in the organs. IS and p-CS are tightly bound to human serum albumin (HSA), the most abundant plasma protein in the bloodstream. HSA is recognized as the main means of transport for endogenous and exogenous compounds, including fatty acids that seem to be the main endogenous ligand of HSA, multiple binding sites are used for monounsaturated fatty acids (MUFA) and PUFA. Thus, free fatty acids and uremic toxins compete for the same binding sites on HSA. It is important to assess fatty acid (FA) levels in patients with CKD because of the potential role to contrast the accumulation of uremic toxins derived from the intestinal bacterial community. As a consequence of this bi-directional relation between gut and kidney and the possible involvement of some compounds as metabolites of FA in the inflammatory response, we investigate the correlation between circulating FA levels and the gMB composition in the same subjects with CKD, as the second aim of this thesis. 64 old CKD non-dialysis patients (eGFR 15-45 ml/min/1.73 m2) and 15 elderly subjects (>65 years) with normal renal function (eGFR >60 ml/min/1.73 m2, CKD-EPI) are enrolled. Bacterial composition was studied in a previous observational study by denaturating gel gradient electrophoresis (DGGE), high-throughput sequencing (16S ribosomal RNA), and quantitative real-time PCR (qPCR). This study described an increased abundance of some bacteria associated with pathological conditions. In agreement with the literature, the author found a reduced abundance of saccharolytic and butyrate-producing bacteria (Prevotella, Faecalibacterium prausnitzii, Roseburia) in CKD patients respect to the control group. Butyrate plays a crucial role in the maintenance of the gut barrier function. Taking that into account, we decided to investigate the correlation between gMB composition and FA profile in these subjects. The main result of the study was the significant positive correlation between Faecalibacterium prausnitzii and total n-3 levels, both associated with the antiinflammatory action. The present doctoral thesis underlines the need to perform further investigations in order to support evidence presented. Future studies may be useful to improve understanding of the effect of circulating fatty acids levels and their metabolites on gut microbial composition, inflammation process, and pathological conditions such as kidney disease. Our results showed that CKD patients with previous cardiovascular events had lower total and specific n-3 levels comparing with patients without them. Moreover, higher docosahexaenoic acid (DHA) levels and having had previous cardiovascular events seemed to have protective effects against further cardiovascular events. Moreover, we observed a significant reduction of the genera Roseburia and Faecalibacterium in CKD patients compared to C group and a significant lower abundance of F. prausnitzii and Roseburia spp. in CKD patients. Thus, our results seem in accordance with anti-inflammatory actions of total n-3, DHA, and saccharolytic and butyrateproducing bacteria. Many gMB changes seem to be related both to CKD and CVD. If the different gMB composition might play a causal role in cardiovascular events by an unbalanced production of some toxic substances, or if the gMB changes are merely a consequence of different dietary and lifestyle behaviours of these patients, it cannot be explained by the present study and all the yet available data. Further studies, possibly utilizing new high-throughput tools, will be required to understand the potential correlations between the gMB composition and other inflammation and oxidative stress markers in these patients. Other two studies have been performed during the doctoral course, to reach a better comprehension of fatty acids, gut microbial community and inflammatory states. A prospective pilot clinical study has been performed to to explore possible changes of gMB composition in children with AOM treated with amoxicillin with or without clavulanic acid. AOM is one of the most common bacterial infections in children and is normally treated with antibiotic therapies that lead to increasing antimicrobial resistance rates among otopathogens and may impair the correct development of the microbiota in early life. No significant differences were shown in the gMB composition of the overall cohort at different time intervals of the samples collection and in subjects treated with amoxicillin with or without clavulanic acid at different time intervals (T0, T1 and T2). A literature revision on lipids in infant formulae has been performed to better understanding quality and quality of dietary lipids because of their significant impact on health outcomes, especially when fat storing and/or absorption are limited (e.g., preterm birth and short bowel disease) or when fat byproducts may help to prevent some pathologies. The lipid composition of infant formulae varies according to the different fat sources used, and the potential biological effects are related to the variety of saturated and unsaturated FAs. Instead, ruminant-derived trans FAs and metabolites of n-3 LCPUFA with their anti-inflammatory properties can modulate immune function. Furthermore, dietary fats may influence the nutrient profile of formulae, improving the acceptance of these products and the compliance with dietary schedules. During the doctoral course, I spent a period abroad at Dell Pediatric Research Institute (DPRI), The University of Texas at Austin. In particular, I attended the laboratory of Doctor Brenna. I focused my research activity on a specific regulatory insertion-deletion polymorphism in the FADS gene cluster for better understanding its influence on PUFA and lipid profile.

Les malalties cardiovasculars (MCVs) són la principal causa de mort al mon, juntament amb les malalties cardiometabòliques (MCM). L’objectiu principal és avaluar els efectes dels probiòtics i postbiòtics, consumits sols o amb altres compostos bioactius, en forma de càpsula/pols o afegits a matrius alimentàries, sobre els factors de risc de MCMs, particularment l’obesitat. A més, dilucidar possibles mecanismes d’acció, revelar nous biomarcadors d’obesitat i determinar possibles associacions de la microbiota intestinal amb la ingesta dietètica i paràmetres clínics. Es van realitzar cinc estudis: una revisió sistemàtica i metaanàlisi i una revisió narrativa per integrar la literatura disponible sobre la relació i l'eficàcia dels probiòtics i productes lactis fermentats (PLF) sobre els factors de risc MCM; dos estudis d’intervenció nutricional en subjectes amb obesitat abdominal que van consumir Bifidobacterium animalis subsp. lactis CECT 8145 (Ba8145) per avaluar els efectes sobre els factors de risc de MCM i la microbiota intestinal; un estudi transversal amb subjectes prims i amb sobrepès/obesitat per diferenciar la microbiota intestinal i determinar associacions amb variables clíniques i la ingesta dietètica. En conclusió, el consum regular de PLF redueix el risc de MCM i la suplementació amb probiòtics millora els factors de risc de MCM. El Ba8145 consumit com a probiòtic o postbiòtic redueix els marcadors antropomètrics d’adipositat. El postbiòtic Ba8145 va millorar paràmetres glucèmics, la pressió arterial diastòlica i pressió del pols. Els efectes beneficiosos del Ba8145 es podrien explicar parcialment per canvis en la microbiota intestinal. A més, l’anàlisi metagenòmic va mostrar possibles biomarcadors d’obesitat i noves associacions entre la microbiota intestinal amb la ingesta dietètica i els paràmetres clínics. L’addició de probiòtics o postbiòtics a les estratègies dietètiques actuals podria conduir a noves perspectives pel que fa al maneig de les MCM en humans amb almenys un factor de risc de MCM.
Las enfermedades cardiovasculares (ECV) son la principal causa de muerte en el mundo, junto con las enfermedades cardiometabólicas (ECM). El objetivo principal es evaluar los efectos de los probióticos y postbióticos, consumidos solos o con otros compuestos bioactivos, en forma de cápsulas/polvo o añadidos a matrices alimenticias sobre los factores de riesgo de ECM, particularmente la obesidad. Además, dilucidar posibles mecanismos de acción, revelar nuevos biomarcadores de obesidad y determinar posibles asociaciones de la microbiota intestinal con la ingesta dietética y parámetros clínicos. Se realizaron cinco estudios: una revisión sistemática y metaanálisis y una revisión narrativa para integrar toda la literatura disponible sobre la relación y efectividad de los probióticos y productos lácteos fermentados (PLF) en los factores de riesgo de ECM; dos estudios de intervención nutricional en sujetos con obesidad abdominal que consumieron Bifidobacterium animalis subsp. lactis CECT 8145 (Ba8145) para evaluar los efectos sobre factores de riesgo de ECM y sobre la microbiota intestinal; un estudio transversal con sujetos delgados y con sobrepeso/obesidad para diferenciar la microbiota intestinal y determinar asociaciones con variables clínicas y ingesta dietética. En conclusión, el consumo regular de PLF reduce el riesgo de ECM, y la suplementación con probióticos mejora los factores de riesgo de ECM. El Ba8145 consumido como probiótico o postbiótico reduce los biomarcadores antropométricos de adiposidad. El postbiótico Ba8145 tuvo efectos beneficiosos sobre los parámetros glucémicos, presión arterial diastólica y presión del pulso. Los efectos beneficiosos del Ba8145 podrían explicarse parcialmente por cambios en la microbiota intestinal. Además, el análisis metagenómico mostró posibles biomarcadores de obesidad y nuevas asociaciones entre la microbiota intestinal con la ingesta dietética y parámetros clínicos. La adición de probióticos o postbióticos a las estrategias dietéticas actuales podría conducir a nuevas perspectivas para el manejo de las CMD en humanos con al menos un factor de riesgo de ECM.
Cardiovascular diseases (CVDs) are the leading cause of death globally, along with cardiometabolic diseases (CMDs). The main objective is to evaluate the effects of probiotics and postbiotics, consumed alone or with other bioactive compounds, and consumed in capsule/powder form or added into dietary matrices, on CMD risk factors, particularly obesity. Moreover, to elucidate possible mechanisms of action, reveal new obesity biomarkers and determine possible associations of the gut microbiota with dietary intake and clinical parameters. Five studies were carried out: a systematic review and meta-analysis and a narrative review to integrated all the available literature on the relationship and effectiveness of probiotics and fermented dairy products on CMDs risk factors; two randomized controlled trials in subjects with abdominal obesity who consumed Bifidobacterium animalis subsp. lactis CECT 8145 (Ba8145) to assess the effects on CMD risk factors and on the gut microbiota; a cross-sectional study with lean and overweight/obese subjects to differentiate gut microbiota and to determine its association with clinical variables and dietary intake. Therefore, the regular consumption of fermented dairy products is associated with a reduced risk of CMDs, and probiotic supplementation improves CMD risk factors. Ba8145 consumed as a probiotic or postbiotic reduces anthropometric adiposity biomarkers. Postbiotic Ba8145 had beneficial effects on glycemic parameters, diastolic blood pressure and pulse pressure. Beneficial effects of Ba8145 could be partially explained by changes in the gut microbiota. Furthermore, the metagenomic analysis showed possible obesity biomarkers, and new associations between gut microbiota with dietary intake and clinical parameters. The addition of probiotic or postbiotic consumption to the current recommended dietary strategies could lead to new perspectives regarding the management of CMDs in humans with at least one CMD risk factor.

La consommation d'aliments riches en ellagitannins (ETs) pourrait être associée principalement à la prévention des maladies cardiovasculaires et la régulation des cancers hormono-dépendants. Néanmoins, les ETs ne sont pas biodisponibles en tant que tel et, après avoir été partiellement transformés en acide ellagique (EA) dans le tractus gastro-intestinal (GI) supérieur, ils sont métabolisés dans le côlon par la flore intestinale en urolithines, un groupe de molécules plus biodisponibles et bioactives qui peuvent persister jusqu'à 4 jours à des concentrations relativement élevées dans le plasma et l'urine. La variabilité de l'excrétion des urolithines dans l'urine est importante et à partir d'un échantillon de population de 26 volontaires sains, trois groupes principaux d'individus ont pu être distingués : "faible ou non-excréteur d'urolithin », « Excréteur prédominant d'UA et dérivés» et « Excréteur prédominant d'UB et dérivés»". Ces groupes ont également été observés en considérant la cinétique totale d'excrétion sur une période de 4 jours après ingestion du jus et à des périodes différentes tout au long d'une année. Bien que les variabilités inter-et intra-individuelles soient relativement élevées, les individus conservent leur statut au cours des différentes périodes d'intervention même en modifiant les quantités d'ETs ingérées. L'analyse par UPLC-PDA/ESI-Q-TOF/MS2 a permis d'attribuer hypothétiquement une identité à 15 autres métabolites d'ETs dans l'urine, mais le profilage métabolomique n'a pas permis de discriminer d'autres composés exceptés les dérivés d'UA ou d'UB. La fermentation in-vitro des ETs et EA, par les matières fécales a montré une voie métabolique spécifique qui débouche sur la production d'UA. Néanmoins, les métabolites excrétés in vivo sont beaucoup plus complexes ce qui met en évidence de fortes interactions entre le système excréteur de l'hôte et la composition du microbiote intestinal. La recirculation hépatique suivie par une re-conversion des métabolites de phase II dans le côlon permettrait d'expliquer l'excrétion d'UB chez certains volontaires. L'écologie spécifique de la flore intestinale évaluée par la méthode des empreintes PCR-DGGE a permis d'identifier quelques microorganismes associés à une plus grande capacité de bioconversion des ETs en urolithins
Consumption of dietary ellagitannins (ETs) could be associated mainly with prevention of cardiovascular diseases and regulation of hormone-dependent cancers. Nonetheless, ETs are not bioavailable as such; therefore, after being partially converted into ellagic acid (EA) in the upper gastrointestinal (GI) tract, they undergo sequential bioconversion in the colon by gut microbiota into urolithins, a more bioavailable and bioactive group of molecules that persist up to 4 days at relatively high concentrations in urine. Variability of urolithin excretion in urine is high and three main groups, “no or low urolithin excreters,” “predominantly UA derivatives excreters” and “predominantly UB derivatives excreters,” were observed on a cohort of 26 healthy volunteers. These categories were also unambiguously observed following the total excretion of main ETs' metabolites over a 4 day period after ingesting one shot of juice, and at different periods of time along one year. Although relatively high inter- and intra-individual variabilities were observed, individuals preserved their status during various intervention periods with different amounts of ETs ingested. UPLC-PDA and ESI-Q-TOF/MS1 and MS2 allowed the tentative assignment of an identity to 15 other ETs metabolites in urine, but this profiling did not allow the discrimination of any other compounds aside from UA or UB derivatives. In-vitro fermentation of ETs and EA with fecal stools showed a specific metabolic pathway ending in the production of UA. Nonetheless, metabolites excreted in-vivo are much more complex, highlighting strong interactions between host excretory system and composition of gut microbiota. Hepatic recirculation and additional bioconversion of Phase II metabolites in the colon may explain predominant excretion of UB in some volunteers. Microbiota ecology assessed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE) fingerprint method allowed the association of some microorganism species to higher capacity of bioconversion of dietary ETs into urolithins.Key words: Ellagitannins, blackberry, urolithin, colonic metabolites, ETs degradation patterns, gut microbiota, gastrointestinal tract