Dissertations / Theses on the topic 'Host-microbiome interaction'

Chlamydia trachomatis (CT) est une bactérie sexuellement transmissible responsable d’infections génitales hautes pouvant conduire à une infertilité tubaire ou à des grossesses extra-utérines. C’est l’infection sexuellement transmissible la plus fréquente dans le monde, y compris en France. Les données épidémiologiques indiquent que l’incidence de cette infection est en augmentation malgré les mesures de contrôle mises en place, ce qui motive la révision des recommandations actuelles de dépistage de l’infection à CT. Le microbiote vaginal pourrait jouer un rôle majeur dans la prévention des IST via la compétition écologique et la production de métabolites, dont l’acide lactique. Le microbiote vaginal correspond à un équilibre dynamique fragile et susceptible d’être modifié par un ensemble d’expositions, parmi lesquelles les pratiques sexuelles et d’hygiène intime, l’exposition aux antibiotiques mais aussi la présence de pathogènes. L’objectif général de cette thèse est d’étudier ce triangle d’associations entre expositions, microbiote vaginal et infection par CT, à travers l’étude de la composition et de la dynamique du microbiote vaginal. Nous avons cherché à répondre aux questions suivantes : existe-t-il des marqueurs de l’infection par CT au niveau du microbiote vaginal ? La composition et la structure du microbiote vaginal sont-elles modifiées par l’infection par CT et la prise d’antibiotiques ? Quels sont les expositions associées à des perturbations du microbiote vaginal ? Une première étape a consisté à réaliser un état de l’art et d'estimer l’association entre microbiote vaginal et infection par CT dans la littérature, ainsi que pour trois autres IST d’importance clinique, et à évaluer le rôle de plusieurs facteurs dans l’hétérogénéité des mesures d’association observées. Dans un second temps, nous avons estimé cette association en s'appuyant sur la caractérisation moléculaire du microbiote vaginal, dans deux études en France et aux Etats-Unis. Nous avons montré qu’il y avait une surreprésentation des communautés bactériennes dominées par Lactobacillus iners (CST III) et de celles dépourvues de Lactobacillus spp. (CST IV) chez les femmes infectées par CT. En étudiant l’évolution du microbiote vaginal dans l’étude américaine, après traitement par azithromycine et clairance de CT, nous avons montré que le microbiote vaginal ne parvenait pas à évoluer vers un état optimal. Ce résultat laisse supposer qu’il persiste après traitement un risque vis-à-vis des réinfections. Enfin, dans deux études longitudinales à échantillonnage fréquent aux Etats-Unis, nous avons étudié les expositions associées à l’incidence et à la clairance d’un CST IV. Nous avons montré que lorsque le microbiote vaginal n’était pas dominé par L. iners, les facteurs associés à l’incidence d’un CST IV et à sa clairance étaient essentiellement les menstruations, tandis que chez les femmes dont le microbiote vaginal est dominé par L. iners, les menstruations mais aussi l’usage de lubrifiant, les douches vaginales, l’origine ethnique, l’âge et les rapports sexuels non protégés étaient associés à l’incidence d’un CST IV ou à sa clairance. Ainsi, ce travail de thèse a permis d'une part de confirmer l’association entre microbiote vaginal dépourvu de Lactobacillus et infection par CT en population en s'appuyant sur le séquençage génomique, et d'autre part de distinguer l’espèce L. iners des autres espèces de Lactobacillus et d’évaluer le risque associé au CST III. En permettant une meilleure compréhension de l’histoire naturelle de CT et des dynamiques du microbiote vaginal, nous espérons proposer des pistes pour améliorer les stratégies de contrôle de l’infection par CT et d’autres IST. Le potentiel innovant du projet réside dans l’usage de méthodes moléculaires nous permettant d’affiner notre approche de la santé en intégrant la prédisposition individuelle aux infections sexuellement transmissibles, ainsi ouvrant la voie vers la médecine personnalisée
Chlamydia trachomatis (CT) is a sexually transmitted bacteria responsible for cervicitis, urethritis, and pelvic inflammatory diseases leading to subsequent tubal infertility and ectopic pregnancies. It is the most frequent sexually transmitted infection worldwide, including in France. Epidemiological data indicate that the incidence rate is increasing despite the implementation of control measures, which motivates the revision of current screening strategies. The vaginal microbiota could play a major role in preventing sexually transmitted infections through ecological competition and metabolites, such as lactic acid production. The vaginal microbiota corresponds to a fine-tuned equilibrium likely to be modified by exposures such as sexual practices, hygiene practices, antibiotics but also presence of pathogens. The overall objective of this thesis is to study the association in this triangle composed of external exposures, vaginal microbiota and CT infection, through the study of the vaginal microbiota composition and dynamics. We aimed at answering these questions: are there biomarkers of CT infection in the vaginal microbiota? Are the vaginal microbiota composition and structure modified by CT infection and antibiotic consumption? What are the exposures associated with perturbations of the vaginal microbiota? To answer these questions, the first step consisted of a state of the art to estimate the association between vaginal microbiota and CT infection in the literature, as well as three other clinically relevant sexually transmitted infections, and to evaluate the role of several factors in the observed heterogeneity between studies. In a second step, we estimated this association using molecular characterization of the vaginal microbiota in two studies in France and in the United States. We showed that Lactobacillus iners-dominated communities (CST III) and Lactobacillus-deprived communities (CST IV) were over-represented among CT-positive women. By studying the vaginal microbiota after azithromycin treatment and CT clearance in the American study, we showed that the vaginal microbiota did not evolve towards an optimal state, suggesting that women may stay at risk of CT reinfections. Finally, in two longitudinal studies using frequent sampling in the United States, we studied exposures associated with incidence and clearance of a CST IV. We showed that when the vaginal microbiota was not dominated by L. iners, menses was the main factor associated with incidence and clearance of a CST IV, while for women whose vaginal microbiota is dominated by L. iners, menses but also lubricant use, douching, ethnic origins, age and condomless vaginal sex were associated with CST IV incidence and/or clearance. Therefore, this thesis allowed on the one hand to confirm the association between Lactobacillus-deprived vaginal microbiota and CT infection using genome sequencing, and on the other hand to single out L. iners from other Lactobacillus spp. and to evaluated the risk associated with CST III. By enabling a better understanding of the natural history of CT and of the vaginal microbiota dynamics, we hope to contribute to improving strategies for the control of CT infection and other STIs. The innovative potential of the project lies in the use of molecular methods, which allows refining of our approach of health management by integrating individual predisposition to sexually transmitted infections, thus paving the way for personalized medicine

Les interactions entre les hôtes et leur microbiome sont probablement responsables de l'évolution de divers traits d'histoire de vie des hôtes. Pourtant, des approches expérimentales supportant cette hypothèse manquent encore. Dans cette thèse, nous avons étudié expérimentalement les effets du microbiome sur les coûts de la reproduction, le développement des jeunes, la communication et les défenses antimicrobiennes chez la mésange charbonnière (Parus major). Tout d'abord, nous montrons que le microbiome est impliqué dans la production par l'hôte des dommages oxydants inhérents à l'investissement dans la reproduction. Deuxièmement, nous avons trouvé que les femelles ajustent leur investissement en caroténoïdes dans leurs œufs en fonction de leur microbiome, et que l'exposition microbienne affecte la croissance des jeunes et leur condition à l'envol. Troisièmement, nous résultats suggèrent que les microorganismes ne dégradent pas la coloration du plumage chez des oiseaux sauvages, mais que l'exposition à des microorganismes lors de la reproduction influence l'investissement dans de nouvelles plumes durant la mue suivante. Finalement, nous avons montré que les mésanges modifient leur investissement dans leur glande uropygienne, un organe externe impliqué dans la régulation des microorganismes du plumage, en fonction de leur microbiome. Cette stratégie antimicrobienne qui diffère entre sexes pourrait alors permettre de réguler le microbiome du plumage. Nos résultats fournissent donc la première démonstration expérimentale de l'hypothèse stipulant que le microbiome joue un rôle dans l'évolution de traits d'histoire de vie des hôtes
Host-microbiome interactions have been pointed out to be potentially responsible for the evolution of numerous life history traits throughout the animal kingdom. However, experimental approaches are still lacking to support this hypothesis. In this thesis, we experimentally investigated the effects of the microbiome on the costs of reproduction, nestling development, communication and antimicrobial defences in Great tits (Parus major). Firstly, we showed that the microbiome mediated host oxidative damages inherent to investment in reproduction. Secondly, we found that females adjusted their investment of carotenoids in eggs depending on their microbiome, and that microbial exposure affected nestling growth and condition at fledging. Thirdly, our results suggest that microorganisms did not degrade feather colouration in wild birds, but microbial exposure during reproduction affected investment in new feathers during the following moult. Finally, we found that birds modified their investment in the uropygial gland, an external organ involved in the regulation of feather microbial communities, according to their microbiome. This antimicrobial strategy that differs between sexes could thus allowed regulation of feather microbiome. Our results consequently provide the first experimental evidence for the hypothesis that the microbiome plays a role in the evolution of host life history traits

The metabolic phenotype of any complex organism is dependent on a complex series of host and gut microbial gene (microbiome) interactions with diet. The microbiome itself can be affected by environmental factors such as stress, exposure to xenobiotics, lifestyle, and alterations in the mammalian-microbial-metabolic axis are associated with changes in disease factors. To understand further the impact of the commensal microbiota on the host metabolism, germ-free (GF) animals, inoculated with individual bacterial strains or complex microbiota, and conventional mice were characterised using 1H NMR spectroscopy and UPLC-MS-based metabolic profiling approaches, and by recording the physiological and immunological parameters. Higher systemic level of (D)-3-hydroxybutyrate and lower levels of circulating VLDL were observed in GF compared to conventional animals, indicating that the absence of gut microbiota stimulated lipolysis while it inhibited hepatic lipogenesis. Subsequently, the best inoculation procedure was obtained by inoculating single bacterial strains into individual animals followed by allowing the animals to exchange their microbes. Metabolic fingerprints showed that a 9 bacteria community is more able to regulate lipoprotein and circulating lipid levels compared to a 3 bacteria community. The origin of the inocula (mouse or human) impacts differently on the host metabolism since humanised mice were strongly disturbed (higher plasma triglyceride level) and displayed metabolic profiles similar to GF mice. Conversely, mouse-associated animals were physiologically, immunologically and metabolically similar to conventional animals. Finally, metabolic profiles and gut microbiota composition were statistically regressed and helped to identify specific bacteria, such as Ruminococcus lactaris and Faecalibacterium prausnitzii, and their putative role in the host homeostasis. To conclude, these results confirmed the influence of gut microbes on the host physiology and metabolism. This PhD thesis provides new insights into the role of gut bacteria to understand the microbial-related mechanisms that participate in the host lipid metabolism, potentially leading to development of obesity.

Obesity is known to contribute to the risk of developing type 2 diabetes mellitus, non-alcoholic fatty liver diseases, cancer, osteoarthritis and cardiovascular diseases. Obesity results from an imbalance between energy intake and energy expenditure but its aetiology is complex and not completely understood. Genetic and environmental factors, (high calorie diet, lack of physical activity) strongly influence the onset of obesity and metabolic diseases. Modifications of gut microbial-host metabolic interactions by dietary interventions also contribute to obesity and are associated with metabolic disturbances. Noticeably, some obese individuals seem protected from the development of metabolic diseases which highlight individual predispositions to obesity associated metabolic disturbances. It is therefore important to underpin the molecular mechanisms linked to obesity and the role of gut microbiota in the degradation of a healthy phenotype in an appropriate biological model. 1H nuclear magnetic spectroscopy and mass spectrometry analysis were employed to measure the abundant metabolites in C57BL/6 mouse urine, feces, and plasma. Metabolic profiles of mice subjected to dietary interventions were compared using multivariate statistics. Alterations of gut microbial ecosystem by dietary interventions were also assessed by 16s rRNA pyrosequencing of mouse fecal microbiota. When given a high fat diet, the obesity prone and obesity resistant mice showed differences in leucine catabolism and tricarboxylic cycle. This may indicate specific regulations of mitochondrial oxidative pathways during diet induced obesity (DIO), influencing obesity development and metabolic disturbances. Possible roles of gut microbiota in the regulation of mitochondrial oxidative pathways were also envisaged. Gut microbial remodelling by the supplementation of different prebiotic blends (galactosyl-oligosaccharides, fructosyl-oligosaccharides and inulin or galactosyl-oligosaccharides and cow milk oligosaccharides) during a high fat challenge also decreased obesity and low grade inflammation and affect mouse metabolism. Decreased gut bacterial amino acids fermentation by prebiotic supplementation may contribute to the improvement of mouse physiology during DIO.

Food-borne infections are a major source of mortality and morbidity. Salmonella causes the highest number of Food-borne bacterial infections in the US. This work contributes towards developing strategies to control Salmonella by (a) defining receptors used by Salmonella to adhere to and invade the host epithelium; (b) developing a host receptor based rapid detection method for the pathogen in food matrix; (C) and defining mechanisms of how probiotics can help alleviate Salmonella-induced cell death in the host epithelium. We developed a cell-cell crosslinking method to discover host-microbe receptors, and discovered three new receptor-ligand interactions. Interaction of Salmonella Ef-Tu with Hsp90 from epithelial cells mediated adhesion, while interaction of Salmonella Ef-Tu with two host proteins that negatively regulate membrane ruffling (myosin phosphatase and alpha catenin) mediated adhesion and invasion. We also showed the role of host ganglioside GM1 in mediating invasion of epithelial cells by Salmonella. Further we exploited pathogen affinity for immobilized gangliosides to concentrate them out of solution and from complex food matrices for detection by qPCR. A sensitivity of 4 CFU/ml (3 hours) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml. Next we screened several probiotic strains for pathogen exclusion potential and found that Bifidobacterium longum subspp. infantis showed the highest potential for Salmonella enterica subspp. enterica ser. Typhimurium exclusion in a caco-2 cell culture model. B. infantis shared its binding specificity to ganglioside GM1 with S. ser. Typhimurium. Further, B. infantis completely inhibited Salmonella-induced caspase 8 and caspase 9 activity in intestinal epithelial cells. B. infantis also reduced the basal caspase 9 and caspase 3/7 activity in epithelial cells in absence of the pathogen. Western blots and gene expression profiling of epithelial cells revealed that the decreased caspase activation was concomitant with increased phosphorylation of pro-survival protein kinase Akt, increased expression of caspase inhibiting protein cIAP, and decreased expression of genes involved in mitochondrion organization, biogenesis and reactive oxygen species metabolic processes. Hence, B. infantis exerted its protective effects by repression of mitochondrial cell death pathway which was induced in the presence of S. ser. Typhimurium.

Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium's ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells.
Master of Science in Life Sciences

The last half-century has seen a dramatic and alarming rise in the incidence of autoimmune disease in industrialized nations too rapid to be accounted for by genetics alone. Among those, Inflammatory Bowel Disease (IBD) has risen from a western disease affecting industrialized populations to an emerging global threat affecting diverse populations around the world. IBD is a complex disease that combines genetic susceptibility and environmental exposure, but one aspect appears to be clear: the involvement of the gut microbiome. Current thought holds that IBD is an autoimmune attack on commensal microbiota, causing extensive collateral damage to the host intestinal tissues in the process. However, it has remained unclear in the field whether the changes observed in the IBD microbiome are causative in nature or whether the microbiome is responding to already-underway inflammatory processes within the host. This dissertation investigates one host factor in particular with regard to the microbiome and the development of inflammation: sodium-hydrogen exchange at the brush border, mediated by sodium hydrogen exchanger 3 (NHE3). NHE3 is inhibited during active IBD, but its loss in knockout animals is also enough to promote spontaneous colitis in a microbiome-dependent fashion. This dissertation investigates the specific contribution of the microbiome in NHE3 knockout animals to determine whether loss of NHE3 may be mediating the onset of colitis through pro-inflammatory changes in the microbiome. Our results suggest that the microbiome fostered in an NHE3-deficient environment may accelerate the onset and severity of experimental colitis, though likely in concert with additional host factors.

Les populations de bivalves exploités subissent régulièrement des épizooties qui affaiblissent voire déciment les stocks, et qui peuvent avoir des conséquences majeures pour l’aquaculture. Ces maladies, dues à des virus, bactéries, ou parasites, se développent particulièrement au printemps et en été. Ces périodes de l’année offrent également des conditions propices aux efflorescences de micro-algues toxiques, dont des dinoflagellés du genre Alexandrium. Ainsi, le risque de co-occurrence d’efflorescences d’Alexandrium sp. et de maladies infectieuses chez les bivalves est élevé. Or, ces micro-algues synthétisent et excrètent des neurotoxines et des composés cytotoxiques responsables d’altérations physiologiques chez les bivalves. L’objectif de cette thèse est d’évaluer les effets combinés d’une exposition à Alexandrium sp. et d’une infection par des agents pathogènes sur la physiologie des bivalves, à travers l’étude de différentes interactions tripartites bivalve – pathogène – Alexandrium sp. Les résultats de ce travail indiquent que différents profils de réponse existent en fonction des espèces impliquées dans ces interactions. Ainsi, une exposition à Alexandrium sp. peut augmenter le taux d’infection par des agents pathogènes chez des bivalves ou au contraire le diminuer. Les réponses hémocytaires associées peuvent traduire l’implication des défenses immunitaires dans ces modulations hôte-pathogène. De plus, l’exposition à des agents pathogènes peut interférer avec le processus d’accumulation de toxines algales dans les tissus des bivalves, illustrant la complexité de ces interactions. Ces résultats, associés à l’observation de lésions tissulaires chez les bivalves peuvent traduire l’altération des activités de nutrition (filtration, digestion…). Ce travail de thèse apporte une meilleure compréhension de l’implication des efflorescences toxiques dans le développement des maladies touchant les bivalves d’intérêt commercial, mais également de l’implication de l’environnement biotique des bivalves sur l’accumulation de phycotoxines réglementées
Bivalve populations undergo regular epidemics that weaken or decimate exploited stocks and thus limit aquaculture. These diseases are caused mainly by viruses, bacteria or parasites, and occur primarily during spring and summer. This period of the year also provides favorable conditions for toxic dinoflagellate blooms, including species of the genus Alexandrium. Thus, the risk of Alexandrium sp. blooms and infectious diseases co-occurring in bivalves is high. However, these micro-algae synthesize and excrete toxins and cytotoxic compounds responsible for physiological changes in bivalves and could lead to an immuno-compromised status.The objective of this thesis is to evaluate the combined effects on bivalve physiology of exposure to the toxic dinoflagellate, Alexandrium sp., and infection by pathogens, through the study of different bivalve - pathogen - Alexandrium sp. tripartite interactions. The results of this work highlight the species-specific nature of these impacts.Thus, exposure to Alexandrium catenella reduces the herpesviruses infection in oyster Crassostrea gigas, whereas the dinoflagellate A. fundyense increases the susceptibility of C. virginica oyster to the parasite Perkinsus marinus, probably via immuno-suppression, as suggested by the partial inhibition of hemocyte responses. Additionally, the effect of a toxic algal bloom on oyster susceptibility to opportunistic diseases when exposed to a new microbial environment (simulating a transfer) was evaluated. Hemocyte responses to a changing microbial environment were suppressed by exposure to A. catenella, although no new bacterial infection was detected.Finally, exposure to pathogens or to a new microbial environment interferes with the processes by which oysters exposed to A. catenella accumulate algal toxins, illustrating the complexity of these interactions. These results provide a better understanding of the involvement of toxic algal blooms in the development of diseases affecting commercial bivalve species, but also of the involvement of the bivalve biotic environment in the accumulation of regulated toxins

Colorectal cancer (CRC) is one of the most treatable cancers, with a 5-year survival rate of similar to 64%, yet over 50,000 deaths occur yearly in the United States. In 15% of cases, deficiency in mismatch repair leads to null mutations in transforming growth factor beta (TGF-beta) type II receptor, yet genotype alone is not responsible for tumorigenesis. Previous work in mice shows that disruptions in TGF-beta signaling combined with Helicobacter hepaticus cause tumorigenesis, indicating a synergistic effect between genotype and microbial environment. Here, we examine functional shifts in the gut microbiome in CRC using integrated - omics approaches to untangle the role of host genotype, inflammation, and microbial ecology. We profile the gut microbiome of 40 mice with/without deficiency in TGF-beta signaling from a Smad3 (mothers against decapentaplegic homolog-3) knockout and with/without inoculation with H. hepaticus. Clear functional differences in the microbiome tied to specific bacterial species emerge from four pathways related to human colon cancer: lipopolysaccharide (LPS) production, polyamine synthesis, butyrate metabolism, and oxidative phosphorylation (OXPHOS). Specifically, an increase in Mucispirillum schaedleri drives LPS production, which is associated with an inflammatory response. We observe a commensurate decrease in butyrate production from Lachnospiraceae bacterium A4, which could promote tumor formation. H. hepaticus causes an increase in OXPHOS that may increase DNA-damaging free radicals. Finally, multiple bacterial species increase polyamines that are associated with colon cancer, implicating not just diet but also the microbiome in polyamine levels. These insights into cross talk between the microbiome, host genotype, and inflammation could promote the development of diagnostics and therapies for CRC. IMPORTANCE Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

L'avènement des technologies de séquençage ADN à haut-debit a initié une tendance grandissante dans l'assemblage de génomes. La qualité de ces génomes est un prérequis essentiel pour comprendre les interactions au sein de et entre ces chromosomes. Nos méthodes se basent principalement sur les technologies de capture de conformation de chromosomes comme le Hi-C. Lors d'un protocole de Hi-C, les molécules d'ADN sont réticulées avec les protéines environnantes pour former un complexe protéine-ADN statique et volumineux. Ceci permet de capturer la conformation spatiale en piégeant les molécules physiquement proches dans l'espace. Ainsi, le Hi-C est très approprié pour l'analyse de la structure 3D des génomes, ce qui permet d'obtenir un certain nombre d'informations sur le génome. Il a été ainsi montré que sa structure tridimensionnelle peut être reliée directement à sa structure 1D grâce aux propriétés physiques des polymères d'ADN. De plus, une telle proximité en 3D donne également accès à des informations de compartimentation, ce qui a ouvert la voie à une nouvelle approche de binning métagénomique, connue sous le nom de meta3C. Au cours de ce travail, nous étendons ces méthodes à des études de cas présentant une complexité grandissante. Tout d'abord, nous améliorons les outils d'assemblage de génomes et démontrons leur validité avec l'assemblage de Ectocarpus sp., puis nous mettons en évidence des réarrangements chromosomiques au sein d'assemblages joints de Trichoderma reesei et Cataglyphis hispanica. Enfin, nous utilisons la même approche avec le binning métagénomique sur des échantillons de souris in vivo afin de reconstruire des centaines de génomes
The advent of high-throughput DNA sequencing technologies has set off an expanding trend in genome assembling and scaffolding. Such genome quality is an essential preliminary to understand interactions between and among chromosomes. We built upon a computational and technological framework that let us tackle genome assembly problems of increasing complexity. Our methods are mainly based on chromosome conformation capture technologies such as Hi-C. In a Hi-C experiment, DNA molecules are cross-linked with the surrounding proteins and form a large, static protein-DNA complex. This captures the spatial conformation by trapping together molecules that are physically close to each other. Therefore, Hi-C is very suitable for 3D genome structure analysis, which lets us infer a wealth of information about the genome. It was indeed shown that the tridimensional structure of the genome can be unambiguously linked to its 1D structure thanks to the physical properties of DNA polymers. Moreover, such 3D proximity also gives access to cell compartment information, thus opening the way for an additional approach for metagenomic binning, known as meta3C. In this work, we expand upon these methods and apply them to use cases with more and more complexity. We first improve on tools for genome assembly and demonstrate their validity with the scaffolding of Ectocarpus sp., then unveil rearrangements in joint scaffoldings of Trichoderma reesei and Cataglyphis hispanica. Lastly, we use the same approach with metagenomic binning on live mouse microbiome samples to reconstruct hundreds of genomes

Les amibes libres sont des microorganismes unicellulaires eucaryotes dont l'écologie au sein des réseaux d'eau potable est mal connue. Les amibes libres représentent un enjeu de santé publique, du fait de leur capacité à favoriser la présence de bactéries potentiellement pathogènes, parmi lesquelles des mycobactéries.Une campagne de prélèvement menée sur le réseau d'eau potable de Paris a permis d'évaluer la diversité des amibes libres et de leur microbiome bactérien, par pyroséquençage ciblant les gènes ribosomaux (16S et 18S). Ces analyses ont suggéré la prédominance des genres Acanthamoeba, Vermamoeba, Echinamoeba et Protacanthamoeba. Le microbiome des amibes a révélé une grande diversité bactérienne, dominée par Pseudomonas, Stenotrophomonas, Bradyrhizobium, Sphingomonas et Pseudoxanthomonas. L'intégration des paramètres physicochimiques a permis de suggérer l'importance de l'origine de l'eau, la température, le pH et la concentration en chlore dans la dynamique des populations amibiennes. Une endosymbiose originale entre V. vermiformis et des bactéries du phylum TM6 a également été mise en évidence.Les amibes ont été fréquemment co-isolées avec des mycobactéries dans le réseau, principalement les espèces M. llatzerense et M. chelonae. Des expériences d'infection chez A. castellanii ont permis d'observer la capacité de ces mycobactéries à survivre et croitre en présence d'amibes. Par génomique comparative et analyses transcriptomiques, plusieurs facteurs de virulence, conservés entre M. llatzerense, M. chelonae et M. tuberculosis, ont été identifiés et sont surexprimés au cours de l'infection. Ces données suggérent leur implication dans la résistance à la prédation amibienne.L'ensemble de ces travaux a permis d'améliorer la connaissance des populations amibiennes et de leur microbiome au sein du réseau d'eau potable, apportant des éléments supplémentaires concernant leur implication dans la survie et la persistance des mycobactéries
Free-living amoebae are unicellular eukaryotes whose ecology in drinking water networks remains poorly understood. They may represent a public health concern, because of their ability to favour the presence of potentially pathogenic bacteria, among which are mycobacteria.A sampling scheme based on Paris drinking water network allowed identifying the diversity of both freeliving amoebae and their bacterial microbiome, using ribosomal RNA targeted pyrosequencing. These analyses indicated the major presence of Acanthamoeba, Vermamoeba, Echinamoeba and Protacanthamoeba genera. The microbiome was highly diverse and dominated by Pseudomonas, Stenotrophomonas, Bradyrhizobium, Sphingomonas and Pseudoxanthomonas. The coupling of physicochemical parameters to this analysis allowed underlining the importance of water origin, temperature, pH and chlorine concentration in shaping amoebal populations. Also an original endosymbiosis between V. vermiformis and a bacterium of the TM6 phylum was described. Free-living amoebae were frequently co-isolated with mycobacteria in the water network, mainly M. llatzerense and M. chelonae species. Infection experiments on A. castellanii illustrated the capacity of these species to resist and grow in presence of amoebae. Through genomics and transcriptomics approaches, several virulence factors, conserved between M. llatzerense, M. chelonae and M. tuberculosis were identified, and found to be upregulated during infection experiments. These results suggest their involvement in mycobacterial resistance to amoebal predation.Altogether, this work helped to better understand the ecology of free-living amoebae and their microbiome in drinking water networks, as well as the role of free-living amoebae in the survival and persistence of mycobacteria in such environments

Il est désormais admis que le microbiote intestinal joue un rôle prépondérant dans la santé de son hôte humain. Le co-métabolisme hôte-microbiote produit un très grand nombre de biomolécules intégrées au sein d’axes métaboliques complexes. De ce fait, le microbiote intestinal est considéré comme un organe endocrine à part entière. Bien que de nombreuses études se soient attachées à la caractérisation fonctionnelle spécifique de certaines molécules, les études envisageant plus globalement les relations métaboliques entre l’hôte et son microbiote intestinal restent rares. Parmi les nombreux facteurs influençant la composition et l’activité métabolique du microbiote intestinal, l’alimentation joue un rôle prépondérant. Toutefois, les relations entre l’alimentation usuelle et le microbiote intestinal n’ont pas été complètement élucidées. La compréhension des facteurs modulant le microbiote intestinal est un enjeu majeur des recherches actuelles, car des liens entre le microbiote intestinal et de nombreuses pathologies (troubles gastro-intestinaux, cardio-métaboliques, neuropsychiatriques, etc.) ont été suggérés. Dans ce contexte, nous avons utilisé une approche épidémiologique pour caractériser les associations entre la composition du microbiote intestinal d’une part et le métabolisme systémique et l’alimentation usuelle de l’hôte d’autre part, au sein de la population Milieu Intérieur (N=1 000). Enfin, dans la cohorte prospective NutriNet-Santé (N≈160 000), nous avons analysé les associations entre la consommation de fibres d’une part et le risque de maladies chroniques et le microbiote intestinal d’autre part.Nos résultats décrivent des associations spécifiques entre les caractéristiques du microbiote intestinal et certaines composantes du métabolisme de l’hôte, et suggèrent un rôle important de l’axe intestin-rein. De plus, des associations inverses entre la diversité du microbiote intestinal et la consommation d’aliments caractéristiques du régime occidental ont été détectées. Enfin, nos travaux confirment que la consommation de fibres est associée à une réduction du risque de maladies chroniques, dans un contexte où un nombre croissant d’études suggère une implication du microbiote intestinal dans de tels effets
It is now admitted that the gut microbiota plays a key role in the health status of its human host. It is indeed fully recognized as an endocrine organ producing biologically active molecules which are integrated within human metabolism. However, comprehensive studies characterizing host-gut microbial metabolic relationships remain scarce. Numerous factors have been shown to exert a modulatory impact on the gut microbiota. Notably, diet is supposed to be a major driver, but the relationships between usual diet and the gut microbiota are not fully elucidated yet. Furthermore, many studies have suggested the implication of the gut microbiota in a wide range of disease states, such as gastrointestinal, cardio-metabolic, neuropsychiatric, etc. disorders. Thus, understanding the factors influencing the gut microbiota constitutes an active area of research. In this context, we adopted an epidemiological approach to investigate one of the largest population-based samples so far (Milieu Intérieur population, N=1,000). We notably assessed the associations between gut microbiota composition on one hand and the systemic metabolism and the usual diet of the host on the other. Finally, in the NutriNet-Santé cohort (N≈160,000), we investigated the associations between the intake of dietary fibers and the risk of a variety of chronic diseases, and described how dietary fibers are associated with the gut microbiota.Overall, our results suggest that gut bacterial features are specifically associated with certain components of the systemic metabolism of the host, and we hypothesize a substantial role of the gut-kidney axis. Besides, negative associations between food items for which a limited consumption is generally recommended (i.e. processed foods) and gut microbial features were detected. Additionally, we confirm robust inverse associations between the consumption of dietary fibers and several major chronic diseases. Mounting evidence suggests that such effects could be mediated by the gut microbiota

Host-microbe interactions are now considered essential for maintaining host health. It is known that short and long term dietary interventions influences the structure and activity of gut bacterial communities. However, our understanding of the forces shaping the gut microbiota is still limited and controversial, and most of the studies of the gut microbiota use the microbiota from faeces as a proxy for the intestinal tract populations. As such, the overarching aim of this thesis is to contribute to the understanding of host-microbiome interactions using an animal model. In this thesis I describe the effect of diet changes on microbial community structure and host-microbiome interactions following 14 weeks on one of the three experimental diets. The diets consisted of a basal diet low in fibre (LF); the basal diet together with 26 % cellulose; a difficult to ferment fibre (HF); and the basal diet together with 50% dried cooked red kidney beans (B); a diet relatively high in easily fermentable fibre. These diets were fed to 45, 21 day old female Wistar rats originating from 6 litters for 14 weeks. Diet had little effect on rat growth rates or adult body mass. However, diet had profound effects on gastro-intestinal morphology and dynamics. Caecum size was smallest in animals fed the LF diet, and caecums were about 2x as large in animals fed the B diet, while animals on the HF diet had intermediate-sized caecums. Food transit times were slowest in animals on the B and LF diets and fastest in animals on the HF diets. At the end of the diet experiment, colon and caecum contents were collected when the animals were killed and short chain fatty acids, nitrogen, carbon, as well fibre concentrations were determined. These data showed that the ‘chemical’ environment of the hindgut varied substantially among animals fed the different diets. E. coli diversity and dynamics were described by characterizing more than three thousand isolates. E. coli diversity was low, and more than 97% of the isolates were represent by three strains: one phylogroup B2 strain and two phylogroup B1 strains. A decline of the frequency of the B2 strain in the animals fed on the bean diet was observed. The faecal microbiota was characterized when the animals were 21 days old, while faecal, caecal and rectal microbial communities characterized at the end of the experiment. 16S amplicon sequencing of the V4 region on the Ion Torrent platform was the approach used to characterize the microbiota. Members of 23 microbial families were detected in communities of the animals before and after 14 weeks on the experimental diets. At the start of the experiment there were significant litter membership effects on the structure of the faecal microbial communities. After 14 weeks on the experimental diets, both litter and diet explained a significant amount of the variation in microbial community structure. There were substantial differences in the microbial communities of the caecum and rectum and the extent of these differences depended on diet and on the time taken for material to move through the hindgut. The outcomes of the present study make a contribution to our understanding of the factors that shape gut microbial communities. Microbial characterization of faecal samples is frequently used as proxy of gut microbiota. However, stool samples are probably most likely representative of the microbial communities in the rectum than other parts of the gastrointestinal tract. Indeed, the findings also throw doubt on the value of faecal community characterization as a means to understand community structure and function in the gastro-intestinal tract. Further, the results of these experiments suggest that efforts attempting to achieve positive health outcomes through diet manipulation may have limited success in general due to among individual differences in microbial community composition, and in how these different communities respond to dietary manipulation.

Le choléra est une infection diarrhéique aiguë qui représente encore aujourd’hui un grave problème de santé publique dans les pays où l’accès à l’eau potable et un système d’assainissement adéquat ne peut pas être garanti. Vibrio cholerae, le pathogène bactérien responsable de cette maladie, peut provoquer toute une série de symptômes chez les individus infectés, allant d’une diarrhée intense conduisant à une déshydratation sévère, au portage asymptomatique de la bactérie. Bien que notre compréhension du choléra à une échelle macro-épidémiologique a considérablement été améliorée par le développement des techniques de séquençage à haut débit et par les avancées dans le domaine de la génomique bactérienne, aucune étude n’a encore été menée pour caractériser son évolution à l’échelle des individus infectés. De plus, le rôle des porteurs asymptomatiques au sein d’une épidémie et la raison derrière l’absence de symptômes chez ces individus infectés sont encore méconnus. L’objectif principal de cette thèse est donc de (1) caractériser la diversité génomique de V. cholerae au niveau des individus et des cercles familiaux, mais aussi (2) d’évaluer le rôle potentiel du microbiome intestinal dans la susceptibilité de contracter cette maladie entérique aiguë et de présenter des symptômes sévères. Dans un premier temps, nous caractérisons la diversité génomique de colonies isolées à partir de patients symptomatiques. Le séquençage de génomes entiers de souches provenant de patients du Bangladesh et d’Haïti révèle que cette diversité sous la forme de mutations ponctuelles reste limitée, mais détectable au sein des hôtes. Une grande partie de la variation du contenu génétique semble être surtout due au gain et à la perte de phages et de plasmides au sein de la population de V. cholerae, avec des échanges occasionnels entre le pathogène et d’autres membres commensaux du microbiote intestinal. Cela contredit l’hypothèse couramment acceptée que les infections par V. cholerae sont majoritairement clonales, et confirme que le transfert horizontal de gènes est un facteur important dans l’évolution de V. cholerae. De plus, nos résultats montrent que certains de ces variants peuvent avoir un effet phénotypique, impactant par exemple la formation de biofilms, et peuvent être sélectionnés au sein des individus infectés. Par la suite, nous appliquons une association de méthodes de séquençage de génomes entiers et de méthodes métagénomiques afin d’améliorer la détection des variants intra-hôte, à la fois chez des patients symptomatiques, mais aussi chez des porteurs asymptomatiques. Notre étude montre que l’approche métagénomique offre une meilleure résolution dans la détection de la diversité dans la population microbienne, mais reste difficile à appliquer chez des patients asymptomatiques, en raison du faible nombre de cellules de V. cholerae chez ces patients. Dans l’ensemble, nous constatons que le niveau de diversité au sein de la population bactérienne intra-hôte est similaire entre les patients symptomatiques et asymptomatiques. Nous détectons aussi la présence de souches hypermutantes chez certains patients. De plus, alors que les mutations chez les patients porteurs de phénotypes d’hypermutations ne semblent pas sous l’effet de la sélection, des signes d'évolution parallèle sont détectés chez les patients présentant un plus faible nombre de mutations, suggérant des mécanismes d’adaptation au sein de l’hôte. Nos résultats soulignent la puissance de la métagénomique combinée au séquençage de génomes entiers pour caractériser la diversité intra-hôte dans le cas d’une infection aiguë du choléra, mais aussi dans le cas de portage asymptomatique, tout en identifiant pour la première fois le phénotype d’hypermutation chez des patients infectés. Finalement, nous nous intéressons aux facteurs liés à la susceptibilité à la maladie et à la sévérité des symptômes. Basée sur une étude récente utilisant le séquençage 16S pour montrer le lien potentiel entre le microbiome intestinal et la susceptibilité à l’infection par V. cholerae, nos analyses utilisent les méthodes de séquençage métagénomique sur les mêmes échantillons de cette précédente étude afin de caractériser les profils taxonomiques et fonctionnels du microbiome intestinal de contacts familiaux exposés à V. cholerae. Les échantillons sont prélevés avant l’infection de ces contacts familiaux et l’apparition ou non de symptômes, et sont analysés pour identifier des prédicteurs à la maladie symptomatique. Grâce à un algorithme d’apprentissage machine, nous pouvons identifier des espèces, des familles de gènes et des voies métaboliques du microbiome au moment de l'exposition à V. cholerae pour détecter des biomarqueurs potentiels corrélés avec les risques d'infection et la gravité des symptômes. Nos résultats montrent que l’utilisation du séquençage métagénomique améliore la précision et l’exactitude des prévisions par rapport au séquençage 16S. Nos analyses permettent aussi de prédire la gravité de la maladie, bien qu’avec une plus grande incertitude que la prédiction de l’infection. Des taxons bactériens des genres Prevotella et Bifidobacterium ont été identifiées comme des marqueurs potentiels de protection contre l’infection, tout comme gènes impliqués dans le métabolisme du fer. Nos résultats soulignent le pouvoir de la métagénomique pour prédire l’évolution des maladies et identifient des espèces et des gènes spécifiques pouvant être impliqués dans des tests expérimentaux afin d’étudier les mécanismes liés au microbiome intestinal expliquant la potentielle protection contre le choléra.
Cholera is an acute diarrhoeal disease that remains a global threat to public health in countries where access to safe water and adequate sanitation cannot be guaranteed. Vibrio cholerae, the bacterial pathogen responsible for this disease, can cause a range of symptoms in infected individuals, from intense diarrhea leading to severe dehydration, to asymptomatic carriage of the bacteria. Although our understanding of cholera on a macro-epidemiological scale has been considerably improved by the development of high-throughput sequencing techniques and by advances in bacterial genomics, no studies have yet been conducted to characterize its evolution at the scale of infected individuals. Furthermore, the role of asymptomatic carriers in an epidemic and the reason behind the absence of symptoms in these infected individuals remains unknown. The main objective of this thesis is therefore to characterize the genomic diversity of V. cholerae at the level of individuals and households, but also to evaluate the potential role of the gut microbiome in the susceptibility to contract this acute enteric disease and to present severe symptoms. First, we characterize the genomic diversity of colonies isolated from symptomatic patients. The whole genome sequencing of strains from patients in Bangladesh and Haiti reveals that this diversity is detectable in the form of point mutations within hosts, but remains limited. Much of the variation detected within patients appears to be due to the gain and loss of phages and plasmids within the V. cholerae population, with occasional exchanges between the pathogen and other commensal members of the gut microbiota. These results challenge the commonly accepted assumption that V. cholerae infections are predominantly clonal, and confirm that horizontal gene transfer is an important factor in the evolution of V. cholerae. In addition, our results show that some of these variants may also have a phenotypic effect, for example by impacting biofilm formation, and can be selected within infected individuals. Next, we apply a combination of whole genome sequencing and metagenomic approaches to improve the detection of intra-host variants, both in symptomatic patients and in asymptomatic carriers. Our study shows that the metagenomic approach offers a better resolution in the detection of the diversity in the microbial population, but remains difficult to apply in asymptomatic patients, due to the low number of V. cholerae cells in these individuals. Overall, we find that the level of diversity within the intra-host bacterial population is similar between symptomatic and asymptomatic patients. We also detect the presence of hypermutator strains in some patients. In addition, while mutations in patients with hypermutator phenotypes did not appear to be driven by selection, signs of parallel evolution are detected in patients with fewer mutations, suggesting adaptive mechanisms within the host. Our results underline the power of metagenomics combined with whole genome sequencing to characterize intra-host diversity in acute cholera infection, but also in asymptomatic carriers, while identifying for the first time an hypermutator phenotype in infected patients. Finally, we are interested in factors related to susceptibility to the disease and related to the severity of symptoms. Based on a recent study using 16S rRNA amplicon sequencing to show the potential link between the intestinal microbiome and susceptibility to V. cholerae infection, our study uses metagenomic sequencing methods on the same samples from this previous study to characterize the taxonomic and functional profiles of the gut microbiome of household contacts exposed to V. cholerae. Samples are collected prior to infection of these household contacts, and used to identify predictors of symptomatic disease. Using a machine learning algorithm, we can identify species, gene families and metabolic pathways in the microbiome at the time of exposure to V. cholerae to detect potential biomarkers correlated with risk of infection and symptom severity. Our results show that the use of metagenomic sequencing improves the precision and accuracy of predictions compared to 16S rRNA amplicon sequencing. Our analyses also predict disease severity, although with greater uncertainty than the prediction of infection. Bacterial taxa from the genera Prevotella and Bifidobacterium have been identified as potential markers of protection against infection, as well as genes involved in iron metabolism. Our results highlight the power of metagenomics to predict disease progression and identify specific species and genes that could be involved in experimental tests to study the mechanisms related to the microbiome explaining potential protection against cholera.

Eastern black walnut (Juglans nigra L.) ranks among the most highly valued timber species in the central hardwood forest and across the world. This valuable tree fills a critical role in native ecosystems as a mast bearing pioneer on mesic sites. Along with other Juglans spp. (Juglandaceae), J. nigra is threatened by thousand cankers disease (TCD), an insect-vectored disease first described in 2009. TCD is caused by the bark beetle Pityophthorus juglandis Blackman (Corthylini) and the phytopathogenic fungus Geosmithia morbida Kol. Free. Ut. & Tiss. (Bionectriaceae). Together, the P. juglandis-G. morbida complex has expanded from its historical range in southwest North America throughout the western United States (U.S.) and Europe. This range expansion has led to widespread mortality among naïve hosts J. nigra and J. regia planted outside their native distributions.

The severity of TCD was previously observed to be highest in urban and plantation environments and outside of the host native range. Therefore, the objective of this work was to provide information on biotic and abiotic environmental factors that influence the severity and impact of TCD across the native and non-native range of J. nigra and across different climatic and management regimes. This knowledge would enable a better assessment of the risk posed by TCD and a basis for developing management activities that impart resilience to natural systems. Through a series of greenhouse-, laboratory- and field-based experiments, environmental factors that affect the pathogenicity and/or survival of G. morbida in J. nigra were identified, with a focus on the microbiome, climate, and opportunistic pathogens. A number of potentially important interactions among host, vector, pathogen and the rest of the holobiont of TCD were characterized. The holobiont is defined as the whole multitrophic community of organisms—including J. nigra, microinvertebrates, fungi and bacteria—that interact with one another and with the host.

Our findings indicate that interactions among host, vector, pathogen, secondary pathogens, novel microbial communities, and novel abiotic environments modulate the severity of TCD in native, non-native, and managed and unmanaged contexts. Prevailing climatic conditions favor reproduction and spread of G. morbida in the western United States due to the effect of wood moisture content on fungal competition. The microbiome of soils, roots, and stems of trees and seedlings grown outside the host native range harbor distinct, lower-diversity communities of bacteria and fungi compared to the native range, including different communities of beneficial or pathogenic functional groups of fungi. The pathogen G. morbida was also associated with a distinct community of microbes in stems compared to G. morbida-negative trees. The soil microbiome from intensively-managed plantations facilitated positive feedback between G. morbida and a disease-promomting endophytic Fusarium solani species complex sp. in roots of J. nigra seedlings. Finally, the nematode species Bursaphelenchus juglandis associated with P. juglandis synergizes with G. morbida to cause foliar symptoms in seedlings in a shadehouse; conversely, experiments and observations indicated that the nematode species Panagrolaimus sp. and cf. Ektaphelenchus sp. could suppress WTB populations and/or TCD outbreaks.

In conclusion, the composition, function, and interactions within the P. juglandis and J. nigra holobiont play important roles in the TCD pathosystem. Managers and conservationists should be aware that novel associations outside the host native range, or in monocultures, intensive nursery production, and urban and low-humidity environments may favor progression of the disease through the effects of associated phytobiomes, nematodes, and climatic conditions on disease etiology. Trees in higher diversity, less intensively managed growing environments within their native range may be more resilient to disease. Moreover, expatriated, susceptible host species (i.e., J. nigra) growing in environments that are favorable to novel pests or pest complexes (i.e., the western U.S.) may provide connectivity between emergent forest health threats (i.e., TCD) and native host populations (i.e., J. nigra in its native range).

Dissertação de mestrado em Bioquímica Aplicada
Nowadays, there is a growing concern about the integrity and ecological sustainability of marine environment due to several anthropogenic impacts that affect directly and strongly ocean life. Numerous aquatic species become more susceptible to diseases and infections, leading sea animals to an endangered status. Accordingly, the evaluation and monitorization of ocean ecosystem is crucial to recognize the major contributors to this imbalance as well as the at-risk populations, providing relevant information to make ecologically appropriate management decisions for both population and environmental surveillance. Cetaceans are key animals in marine habitat due to their crucial sentinel role in both disturbances and wellness of sea life. Consequently, the assessment and monitorization of their health status provides insights about aquatic health status and potential risks to ocean. Given the relevance of host-microbiome interactions, the identification of bacterial communities associated to respective hosts in distinct health and environmental contexts is extremely important. This type of studies was potentiated with the development of metagenomics approaches and progress of sequencing technologies. In this context, this work was focused on the identification of bacterial community present across fortyfour oral cavity samples from animals that differ in cause of death, occurrence local and other parameters, through DNA extraction, 16S rRNA region metagenomics sequencing and bioinformatics analysis. The results suggested a higher dominance of pathogenic genera, some of which involved in marine and human diseases. However, a considerable variation in terms of relative abundances of taxa in the target samples were also detected. This coupled with the restrict core bacteria genera may indicate that the oral microbiome of animals in study varies widely according to the corresponding animal characteristics. The PLS-DA of samples that were grouped by different metadata (species, gender, development stage, cause of death and occurrence local) indicated that these characteristics may influence the microbiome of oral cavity. Some taxa (e.g. Photobacterium and Phocoenobacter) were appointed as the genera possible involved in the microbiome discrimination, for instance, in accidental captured and diseased animals. In a near future, increasing the number of oral cavity samples analyzed the microbiome discriminations based on meta-parameters will become more statistically robust. Additionally, as the oral cavity is an easily accessible non-invasive tissue, its study is appealing due to the application in live animals, allowing a possible bio-monitorization approach based on the microbiome profile.
Atualmente há uma crescente preocupação relativamente à integridade e sustentabilidade ecológica do ambiente marinho, devido aos diversos impactos antropogénicos que afetam diretamente e fortemente a vida do oceano. Enumeras espécies aquáticas tornam-se, deste modo, mais suscetíveis a doenças e infeções, conduzindo-as a um perigo de extinção. Neste sentido, a avaliação e monitorização do ecossistema marinho é crucial para reconhecer as principais causas para o desequilíbrio, bem como identificar as populações de risco, fornecendo informações relevantes para a implementação de medidas ecologicamente apropriadas tanto para a vigilância da população como do meio ambiente. Os cetáceos são animais chave no habitat aquático devido ao seu papel sentinela em relação aos distúrbios e bem-estar da vida marinha. Consequentemente, a avaliação e monitorização do seu estado de saúde fornece igualmente perspetivas sobre o estado de saúde e potenciais riscos para o oceano. Dada a relevância das interações hospedeiromicrobioma, a identificação das comunidades bacterianas associadas aos respetivos hospedeiros em diferentes contextos ambientais e de saúde é extremamente importante. Este tipo de estudos foi potenciado pelo desenvolvimento das abordagens de metagenómica e progresso das tecnologias de sequenciação. Neste contexto, o presente trabalho foca-se na identificação das comunidades bacterianas presentes em quarenta e quatro amostras de cavidade oral, de animais com diferente causa de morte, local de ocorrência e outros parâmetros, através da extração de DNA, metagenómica e sequenciação da região de rRNA 16S e análise bioinformática. Os resultados sugeriram uma elevada prevalência de géneros patogénicos, alguns deles envolvidos em doenças marinhas e humanas. No entanto, uma variação considerável em termos de abundância relativa dos taxa bacterianos foi também detetada. Isto acoplado a um restrito microbioma core, ao nível do género, poderá indicar que o microbioma oral dos animais em estudo varia amplamente, podendo esta variação estar relacionada com as diversas caraterísticas dos animais. A PLS-DA das amostras agrupadas pelos diferentes metadados (espécie, género, estágio de desenvolvimento, causa de morte e local de ocorrência) podem indicar que estas características influenciam o microbioma da cavidade oral. Alguns géneros, como Photobacterium e Phocoenobacter, foram apontados como os possíveis causadores da discriminação do microbioma em, por exemplo, animais acidentalmente capturados e doentes. Futuramente, com o aumento do número de amostras de cavidade oral estudadas, as discriminações do microbioma, baseadas nos diferentes meta-parâmetros, tornar-se-ão estatisticamente mais robustas. Adicionalmente, como a cavidade oral é um tecido não invasivo e de fácil acesso, o seu estudo é apelativo pela aplicação em animais vivos, podendo permitir uma possível bio-monitorização baseada no perfil do microbioma.
The work presented in this thesis was performed at Molecular and Environmental Biology Centre (CBMA) on Department of Biology, specifically in Molecular Biotechnology and Molecular Genetics Laboratories, School of Sciences, University of Minho, Braga, Portugal. This work was supported by CETSENTI project (FCT/RECI/AAG-GLO/0470/2012), funded by Portuguese Foundation for Science and Technology. The work was also supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI).