The presented thesis discusses the issues of chronic inflammatory diseases of the digestive system and suggests the possibility of the alternative natural remedies improving the health conditions or prolonging the remission phase of IBD. The main goal is to design a probiotic supplement enriched by natural anti-inflammatory agents. Phytochemicals, concretely phenolic compounds, flavonoids, and carotenoids suppress harmful inflammatory processes by direct targeting the function of the immune cells or by inhibiting damaging oxidative stress in general. Therefore, there were several plants potentially rich for these biologically active substances selected. Concretely, blueberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), turmeric (Curcuma longa), peppermint (Mentha piperita), chamomile (Matricaria recutita), cinnamon (Cinnamomum zeylanicum), ginger (Zingiber officinale) and flax (Linum usitatissimum). The suitable parts of these plants were extracted by using a total of three different solvents – water, ethanol, and hexane for obtaining lipidic extracts. The concentration of total polyphenols, total flavonoids, total carotenoids, and the ascertainment of specific polyphenols was determined in the prepared extracts. These characteristics were enhanced by the measurement of antioxidant activity of the aqueous and ethanolic extracts. The interaction of individual samples with probiotic cultures (Bifidobacterium breve, Lactobacillus acidophilus) and the ability of extracts to influence the viability of probiotics in the model digestion process has been examined. The most interesting samples (aqueous extracts of blueberries, mint, chamomile, and cinnamon) were selected for further experiments. The possible cytotoxicity towards human intestinal epithelial cells was tested in vitro by the MTT tests utilizing the CaCo-2 cell line. Extracts showing the highest levels of beneficial phytochemicals and antioxidant activity, supporting the growth of probiotic cultures, and showing minimal cytotoxic effects on human intestinal cells were then co-encapsulated with probiotics into alginate particles of a diameter of 1 mm. Water extracts of mint, chamomile, and cinnamon have been chosen. The encapsulation efficiency of successfully entrapped probiotics and phenolic compounds was determined in prepared particles. Furthermore, the particles were studied during the process of model digestion, when the release of the desired substances in the various parts of the digestive system was observed and assessed whether the components would reach the crucial point of action – the colon. For the use of the proposed probiotic mixture as a dietary supplement, a recommended dose of 1 g has been determined. Lastly, possible adjustments such as lyophilization or sheathing by an extra protective polymerous layer, e.g. chitosan, were suggested to prolong the shelf life of the particles and volatile substances stability.

No description available.

PURPOSE OF REVIEW: This review article aims to explore the GI changes induced by SARS-CoV-2 and how gut microbial homeostasis can influence these changes and affect the lung-gut axis and its relationship with the induction of the cytokine release syndrome in severe COVID-19 patients. RECENT FINDINGS: Coronavirus disease 2019 (COVID-19) affects not only the respiratory system but can produce multi-systemic damage. The expression of angiotensin-converting enzyme 2 (ACE-2) receptors in the gastrointestinal (GI) tract, the high prevalence of GI symptoms in severely ill COVID-19 patients, and the abnormalities described in the gut microbiome in these patients have raised concerns about the influence of GI tract as a risk factor or as a potential modulator to reduce the severity of COVID-19. Understanding the mechanisms by which gut dysbiosis may influence viral transmission and disease progression in COVID-19 may help in shaping how accessible therapies, like diet modulation, can potentially help beat the devastating consequences of COVID-19.

Le microbiote intestinal joue un rôle important dans la santé des chiens. Les changements dans la composition du microbiote conduisent au déséquilibre de ces micro-organismes qui est appelé dysbiose. Les objectifs de cette étude étaient d’évaluer l’impact de l’administration orale de Saccharomyces cerevisiae sur le microbiote fécal des chiens en bonne santé et d’évaluer le potentiel de Saccharomyces cerevisiae dans la prévention de la dysbiose induite par les antibiotiques. Les chiens ont été divisés en un groupe témoin (n=10) et un groupe probiotique (n=10). Le groupe probiotique a reçu 1 g/kg de S. cerevisiae par jour de D0 à D31. Les deux groupes ont reçu 15 mg/kg de métronidazole par voie orale toutes les 12h, de D11 à D17. Des écouvillons fécaux ont été prélevés sur les échantillons D0, 3, 11, 17, 20, 24 et 31 pour analyse du microbiote. Du sérum sanguin a été prélevé sur D0 et D24 pour des cytokines IL-2, IL-6, IL-8, IL-10, IL-12, IFN-Υ et TNF-α. Le séquençage de l’ADN pour l’analyse du microbiote a été effectué à l’aide de la plateforme Illumina MiSeq et les données ont été analysées à l’aide du logiciel Mothur. La supplémentation en S. cerevisiae a été associée à des changements dans la composition du microbiote après 3 jours (p-value=0,002). Comme on pouvait s’y attendre, le métronidazole a considérablement modifié la composition du microbiote des deux groupes à partir de D11 jusqu’à D17 (valeur p < 0,001). Même si les deux groupes ont changé de façon marquée de D11 à D17, il y avait une différence significative entre les groupes de D17 (p-value=0,012) et de D20 (p-value=0,036), suggérant que le probiotique utilisé a la capacité de moduler le microbiote des chiens confrontés à la dysbiose. Il n’y avait pas de différence significative entre les groupes pour le D24 (p-value=0,388), mais pour le D31, les chiens du groupe probiotique ressemblaient à leur microbiote de référence, tandis que certains animaux du groupe témoin demeuraient dans l’état dysbiotique (p-value=0,002). Le TNF-α avait considérablement diminué dans le groupe des probiotiques à partir de D0 jusqu’à D24 (p-value=0,002). La quantité de TNF-α observée dans le groupe témoin par rapport au groupe probiotique de D24 était également significativement plus élevée (p-value=0,04). Il n’y avait aucune différence significative entre les autres cytokines mesurées dans le sang. On a également observé qu’un sous-ensemble des échantillons de référence (avant la supplémentation en probiotiques) présentait une abondance plus élevée de pathobiogènes (bactéries potentiellement pathogènes comme Escherichia, Helicobacter et Pseudomonadaceae)., tandis que les autres chiens avaient une plus grande abondance d’organismes bénéfiques (tels que Fusobacteriaceae, Bacteroides, Faecalibacillus, Bacterioidaceae, et Ruminococcaceae). Trois jours après la supplémentation en probiotiques, les chiens transportant plus de pathobiogènes se sont rapprochés d’un profil microbiote plus sain. En conclusion, on a observé que l’utilisation de S. cerevisiae était associée à des changements dans la composition du microbiote chez un groupe de chiens malades. Il a également été observé que la supplémentation avec S. cerevisiae a été en mesure de moduler les changements dans le microbiote intestinal pendant la dysbiose induite par les antibiotiques chez les chiens. Mots clés : Saccharomyces cerevisiae, manipulation du microbiote, microbiote intestinal du chien, antibiotiques. / The gut microbiota plays an important role in the health of dogs. The changes in the microbiota composition lead to the imbalance of these microorganisms which is called dysbiosis. The objectives of this study were to evaluate the impact of oral administration of Saccharomyces cerevisiae on the fecal microbiota of healthy dogs and to evaluate the potential of S. cerevisiae in preventing dysbiosis induced by antibiotics. Dogs were divided in a control (n=10) and a probiotic group (n=10). The probiotic group received 1 g/kg of S. cerevisiae per day from D0 to D31. Both groups were given oral metronidazole 15 mg/kg every 12h from D11 to D17. Fecal swabs were collected on D0, 3, 11, 17, 20, 24, and 31 for microbiota analysis. Blood serum was collected on D0 and D24 for measurements of cytokines IL-2, IL-6, IL-8, IL-10, IL-12, IFN-Υ, and TNF-α. DNA sequencing for microbiota analysis was performed using the Illumina MiSeq platform and data was analyzed using the software Mothur. Supplementation with S. cerevisiae was associated with changes in the microbiota composition after 3 days (p-value=0.002). As expected, metronidazole markedly changed the microbiota composition of both groups from D11 to D17 (p-value<0.001). Even though both groups changed markedly from D11 to D17, there was a significant difference between groups on D17 (p-value=0.012) and on D20 (p-value=0.036), suggesting that the probiotic used has the capacity to modulate the microbiota of dogs facing dysbiosis. There was no significant difference between groups on D24 (p-value=0.388) but on D31, dogs from the probiotic group resembled their baseline microbiota while some animals from the control group remained in the dysbiotic state (p-value=0.002). TNF-α was significantly decreased in the probiotic group from D0 to D24 (p-value=0.002). The amount of observed TNF-α in the control group compared to the probiotic group on D24 was also significantly higher (p-value=0.04). There were no significant differences in the other measured cytokines from the blood. It was also observed that a subset of the dogs at baseline (before probiotic supplementation) carried higher abundances of pathobionts (potentially pathogenic bacteria such as Escherichia, Helicobacter, and Pseudomonadaceae), while the other dogs had higher abundances of beneficial organisms (such as Fusobacteriaceae, Bacteroides, Faecalibacillus, Bacterioidaceae, and Ruminococcaceae). Three days after probiotic supplementation, dogs carrying more pathobionts converted into a healthier microbiota profile. In conclusion, the use of S. cerevisiae was associated with beneficial shifts in the microbiota in a group of heathy dogs and the supplementation was able to modulate the dysbiosis caused by the use of antibiotics. Keywords: Probiotics, microbiota manipulation, intestinal dysbiosis, canine microbiome, antibiotics.

Orientadores: Paulo César Giraldo, José Eleutério Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-29T00:05:07Z (GMT). No. of bitstreams: 1 Ferreira_JozianiBeghiniJunqueiradeCarvalho_D.pdf: 2154487 bytes, checksum: 796b71982b0f22714f56dabb13789296 (MD5) Previous issue date: 2015 / Resumo: Introdução: Os mecanismos de defesa da mucosa vaginal e a fisiopatologia das vulvovaginites têm se mostrado complexos e requerem a interação entre diversas substâncias. O papel da acidez vaginal, bem como a expressão de mediadores bioquímicos na luz vaginal necessitam ser melhor investigados para esclarecer as altas prevalências e recorrências das vulvovaginites. Objetivo: Avaliar as concentrações vaginais dos isômeros L e D do ácido lático, EMMPRIN (indutor de metaloproteinase da matriz extracelular), MMP-8 (metaloproteinase da matriz extracelular-8), NGAL (lipocalina associada à gelatinase dos neutrófilos), HA (hialurona), Hyal-1 (hialuronidase-1) e hBD2 (?-defensina) em mulheres hígidas e com vulvovaginites, correlacioná-los e verificar as associações com os leucócitos presentes na cavidade vaginal. Sujeitos e Métodos: Este estudo de corte transversal envolveu 233 mulheres atendidas no Ambulatório de Infecções Genitais da Unicamp no período de maio à novembro de 2013. Foram incluídas mulheres no menacme com e sem infecções genitais. Durante o exame ginecológico, coletou-se swab vaginal para análise microbiológica (bacterioscopia vaginal e cultura para fungos), além de material endocervical para pesquisa de Chlamydia trachomatis, Neisseria Gonorrhoeae e HPV por técnica de PCR (polymerase chain reaction). Outro swab da parede vaginal lateral foi diluído em 1 ml de PBS (phosphate buffer saline), processado e congelado para posterior análise dos mediadores imunes (ácidos láticos L e D, EMMPRIN, MMP-8, NGAL, HA, Hyal-1 e hBD2) por técnica de ELISA (enzyme-linked immunosorbent assay). Para analisar as diferenças entre os mediadores nos grupos de pesquisa foram utilizados testes não paramétricos: Kruskall-Wallis seguido de Mann-Whitney, devido à distribuição assimétrica dos dados. O estudo foi aprovado pela Comissão de Ética e Pesquisa da Unicamp e obteve financiamento FAPESP. Resultados: Dos 233 casos coletados foram diagnosticados: 52 casos de candidíase vulvovaginal (22%), 43 de vaginose bacteriana (18%), 21 de vaginose citolítica (9%), outros diagnósticos (n=36) e 77 controles sem infecção (33%). Quatro casos foram excluídos por diagnóstico incompleto. Além disso, foi avaliada a magnitude do processo inflamatório representada pela contagem de leucócitos presentes nos esfregaços vaginais. Consideraram-se os grupos: inflamação ausente (0 leucócitos/campo), inflamação moderada (1-4 leucócitos/campo) e inflamação intensa (>4 leucócitos por campo). Não se observaram diferenças significativas entre os grupos quanto à idade, raça, anos de estudo, método contraceptivo, número de parceiros sexuais, média de relações sexuais/mês e tabagismo. As concentrações de ácido lático D e L foram menores no grupo com vaginose bacteriana em comparação à candidíase e grupo controle. Na candidíase, os níveis de EMMPRIN e MMP-8 foram mais altos que nos grupos vaginose bacteriana e controle. O EMMPRIN esteve fortemente correlacionado ao ácido lático L, à razão ácido lático L/D e ao MMP-8 no grupo controle e à HA em toda a amostra estudada. Os níveis de NGAL foram maiores na candidíase e menores na vaginose bacteriana em relação aos controles. Os níveis de ácido lático-L e NGAL foram correlacionados. As concentrações vaginais de EMMPRIN, MMP-8, HA, NGAL e hBD2, exceto Hyal-1, foram maiores nos grupos com inflamação comparado ao grupo sem inflamação vaginal. Todos estes resultados foram estatisticamente significativos. Conclusões: Os isômeros do ácido lático apresentam maiores concentrações em pacientes cuja flora é dominada por lactobacilos (controles, candidíase e vaginose citolítica) e mais baixos em mulheres com flora anormal (vaginose bacteriana). As concentrações vaginais adequadas de ácido lático e suas correlações com NGAL, EMMPRIN e MMP-8, são relevantes nas defesas do trato genital inferior. Os níveis de EMMPRIN, MMP-8, HA, NGAL e hBD2 sofrem alteração com a inflamação vaginal, sendo que EMMPRIN, MMP-8 e HA são fundamentais na sua modulação. Mulheres com desequilíbrios nas concentrações destes mediadores podem estar mais sujeitas a desenvolver disbioses vaginais / Abstract: Introduction: The defense mechanisms of the vaginal mucosa and the pathophysiology of vulvovaginitis are complex and require interaction between several substances. The role of vaginal acidity, as well as the expression of biochemical mediators in vaginal lumen need to be better investigated to clarify the recurrences and high prevalence of VV. Objective: To evaluate the concentrations of L and D lactic acid, EMMPRIN (extracellular matrix metalloproteinase inducer), MMP-8 (metalloproteinase-8), NGAL (neutrophil gelatinase-associated lipocalin), HA (hyaluronan), Hyal-1 (hyaluronidase-1) and hBD2 (human ?-defensin-2) from vaginal fluid of healthy women and women with vulvovaginitis, correlate them and verify their associations with leukocytes present in the vaginal cavity. Subjects and Methods: This cross-sectional study involved 233 women who were seen at Genital Infections Clinic at Unicamp from May to November 2013. Inclusion criteria: women of reproductive age with or without genital infections. During gynecological examination, vaginal swab was collected for microbiological analysis (Gram stain and culture for fungi) and, endocervical material was analysed for Chlamydia trachomatis, Neisseria gonorrhoeae and HPV by PCR (polymerase chain reaction) technique. Another swab from lateral vaginal wall was diluted in 1 ml PBS (phosphate buffered saline), processed and frozen for later analysis of immune mediators (L and D lactic acid, EMMPRIN, MMP-8, NGAL, HA, Hyal-1 and hBD2) by ELISA (enzyme-linked immunosorbent assay). To analyze the mediators¿ differences between research groups, Kruskal-Wallis followed by Mann-Whitney were used due to the asymmetric distribution of data. The study was approved by the Ethics and Research Committee at the Campinas University and received funding from FAPESP. Results: After microbiological analysis of 233 cases, 52 vulvovaginal candidiasis cases were diagnosed (22%), 43 bacterial vaginosis (18%), 21 cytolytic vaginosis (9%), other diagnoses (n=36) and 77 uninfected controls (33%). Four cases were excluded due to incomplete diagnosis. In addition, the magnitude of the inflammatory process represented by the leukocyte count present in the vaginal smears was evaluated. It was considered to be in three groups: absent inflammation (0 leukocytes per field), moderate inflammation (1-4 leukocytes per field) and intense inflammation (> 4 white cells per field). There were no significant statistical differences among groups considering age, race, years of education, contraception, number of sexual partners, average of intercourse per month and smoking. The D and L lactic acid concentrations were lower in the bacterial vaginosis group compared to candidiasis and control groups. Levels of EMMPRIN and MMP-8 were higher in candidiasis than bacterial vaginosis and control groups. The EMMPRIN was strongly correlated to L lactic acid, L / D lactic acid ratio and MMP-8 in control group, and to HA across the sample. NGAL levels were higher in candidiasis and smaller in bacterial vaginosis compared to controls. The L-lactic acid and NGAL levels were correlated. Vaginal concentrations of EMMPRIN, MMP-8, HA, NGAL and hBD2, except Hyal-1, were higher in the groups with vaginal inflammation compared to the group without inflammation. All these results were statistically significant. Conclusions: The isomers of lactic acid have higher concentrations in patients whose microflora is dominated by lactobacilli (controls, candidiasis and cytolytic vaginosis) and, lower in women with abnormal flora (bacterial vaginosis). Appropriate vaginal concentrations of lactic acid and its correlations with NGAL, EMMPRIN and MMP-8 are relevant in the lower genital tract defenses. The levels of EMMPRIN, MMP-8, HA, NGAL and hBD2 are changed by vaginal inflammation, and EMMPRIN, MMP-8 and HA are critical in modulating vaginal inflammation. Women with imbalances in the concentrations of these mediators may be more likely to develop vaginal disbioses / Doutorado / Fisiopatologia Ginecológica / Doutora em Ciências da Saúde

Le développement des méthodes de séquençage de nouvelle génération a permis d’approfondir les connaissances sur le rôle des communautés bactériennes commensales pour la santé de leur hôte, et l’impact négatif de la perturbation de leur équilibre. Les antibiotiques sont les principaux perturbateurs de cet équilibre, mais leur impact n’a pas été quantifié précisément.Nous avons quantifié la relation entre les concentrations fécales d’antibiotiques et la perturbation de la diversité bactérienne au sein du microbiote intestinal, et modélisé le lien entre la perte de diversité bactérienne et la probabilité de décès dans un modèle animal de colite à Clostridium difficile induite par les antibiotiques. Nous avons montré que l’indice de diversité de Shannon et la distance UniFac non pondérée étaient les indices de diversité qui étaient le plus prédictif du décès dans ce modèle d’infection.Chez des volontaires sains, nous avons développé un modèle mathématique semimécanistique de l’évolution de la diversité au sein du microbiote, mesurée par deux indices de diversité, après perturbation antibiotique, et quantifié la relation entre l’exposition individuelle plasmatique et fécale à un antibiotique, et son effet sur la perturbation de la diversité bactérienne au cours du temps. Nous avons également analysé le rôle de la voie d’élimination des antibiotiques pour la limitation de l’impact d’un antibiotique sur le microbiote. Ces travaux nous ont permis de montrer que le microbiote intestinal présente une grande sensibilité aux antibiotiques, et que la voie d’élimination ne semble de ce fait pas jouer un rôle prépondérant dans la perspective de limiter l’impact des antibiotiques sur le microbiote intestinal. / The development of next generation sequencing broadened our knowledge on the role of commensal bacterial communities on their host’s health, and the negative impact of their disruption. Antibiotics are the main disrupting factor, but their impact has not been precisely quantified.We quantified the relationship between antibiotic fecal concentrations and the loss of bacterial diversity in the intestinal microbiota, and modelled the link between the loss of diversity and mortality in a hamster model of antibiotic-induced Clostridium difficile infection. We showed that the Shannon diversity index and the unweighted UniFrac distance are the 2 indices that best predict mortality in this model. In healthy volunteers, we developed a semi-mechanistic model of the evolution over time of bacterial diversity – measured by two indices – after an antibiotic perturbation, and quantified the relationship between antibiotic concentrations in plasma and feces and the loss of bacterial diversity in the intestinal microbiota. We also analyzed the role of the antibiotic elimination pathway in the reduction of their impact on the microbiota. In this work, we showed that the intestinal microbiota is highly susceptible to antibiotics, and that the elimination route doesn’t have a major role, in the perspective of limiting antibiotics’ impact on the intestinal microbiota.

博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

Le microbiote gastro-intestinal joue un rôle important dans le maintien de la santé de l’hôte. Il est composé de nombreux micro-organismes tels que des bactéries, des virus, des champignons et des archées. Cependant, la majorité de ces cellules microbiennes sont des cellules bactériennes et, pour cette raison, de nombreuses études se concentrent sur l’exploration des communautés bactériennes en particulier dans le tube gastro-digestif. Un déséquilibre de cette microbiote, appelé dysbiose, a été observé dans plusieurs pathologies telles que la diarrhée, la pneumonie, après l'administration d'antibiotiques ou une modification du régime alimentaire. L’objectif de cette étude était de caractériser la dynamique du microbiote fécal des veaux entrant dans une unité d’élevage. Cinquante veaux Holstein âgés de 8 à 14 jours et arrivant dans une unité de veaux ont été inscrits à cette étude. Des échantillons fécaux ont été collectés à l'arrivée et les jours 4, 10 et 24 après l'arrivée. Les scores fécaux, le poids des veaux et l’administration d’antibiotiques ont été enregistrés au cours de l’étude. Le séquençage a été réalisé à l'aide de la plateforme Illumina MiSeq et les données analysées à l'aide du logiciel Mothur. Contrairement aux attentes, la richesse et la diversité étaient plus élevées lorsque la proportion d'animaux diarrhéiques était plus élevée (p < 0,001) et, comme prévu, la composition et la structure du microbiote changeaient au fil des jours de collecte (p > 0,001), mais les changements n'étaient pas associés à présence ou non de diarrhée et de traitement antibiotique comme prévu, ils sont associés aux jours de prélèvement. La proportion de diarrhée (nombre de veaux diarrhéiques par jour) était numériquement plus élevée les jours 4, 10 et 24 après l'arrivée. Comme prévu, les abondances relatives de bactéries associées à la santé (par example : Bifidobacterium, Lactobacillus et Faecalibacterium) ont diminué chez les veaux diarrhéiques. Bien que l'analyse de la diarrhée et de l'utilisation d'antibiotiques ne fît pas partie des objectifs de cette étude, il y avait une tendance (p=0,09) dans le poids des animaux ayant eu la diarrhée et ayant reçu des antibiotiques. Le poids final des veaux malades ayant reçu des antibiotiques avant l'abattage étaient inférieurs par rapport au poids final des animaux qui n'étaient pas malades et n'avaient pas reçu d'antibiotiques (p=0,072). La principale limite de cette étude est le manque d'information sur l'origine des veaux avant leur arrivée à l'unité d'élevage. Cette étude contribue à une meilleure compréhension des changements microbiens liés au stress auquel sont confrontés les veaux de boucherie et pourrait servir de base à d’autres études visant à proposer des méthodes alternatives de manipulation du microbiote pour prévenir les maladies et rétablir la santé des veaux. / The gastrointestinal microbiota plays an important role in maintaining the health of the host. It is composed of many microorganisms such as bacteria, viruses, fungi, and archaea. However, the majority of these microbial cells are bacterial cells, and for that reason, many studies focus on exploring especially bacterial communities in the GIT. Imbalance of the GIT microbiota, termed dysbiosis, has been observed in several conditions such as diarrhea, pneumonia, after antibiotic administration, or diet modification. The objective of this study was to characterize the dynamics of the fecal microbiota of veal calves entering a rearing unit. Fifty Holstein calves ranging from 8-14 days of life and arriving in a veal unit were enrolled in this study. Fecal samples were collected on arrival and on days 4 ,10 and 24 after arrival. Fecal scores, calves’ weight and antibiotic administration were recorded during the study. Sequencing was performed using the Illumina MiSeq platform and data analysed using the software Mothur. Contrary to expectations, richness and diversity were higher when the proportion of diarrheic animals were higher (p<0.001), and as expected, the microbiota composition and structure changed among days of collection (p>0.001), but the changes were not associated to presence or absence of diarrhea and antibiotic treatment as expected, they are associated to the sampling days. Diarrhea proportion (number of diarrheic calves per day) were numerically higher on days 4, 10 and 24 after arrival (As expected, the relative abundances of bacteria associated to health (i.e., Bifidobacterium, Lactobacillus and Faecalibacterium) were decreased in the diarrheic calves. Although analyzing diarrhea and antibiotic usage was not one of the objectives of this study, there was a tendence (p=0.09) in the weigh of the animals that had diarrhea and received antibiotics. The final weight of the sick calves that received antibiotics before slaughter were lower when compared to the final weigh of the animals that were not sick and did not received antibiotics (p=0.072). The main limitation of this study is the lack of information about calves’ origin before arrival at the rearing unit. This study contributes to the better understanding of the microbial changes related to the stress faced by veal calves and might be the basis for further studies to propose alternative methods of microbiota manipulation to prevent disease and restore health in calves.

La dysbiose est une cause importante dans la survenue de maladies, favorisant la prolifération de pathogènes ou induisant l’inflammation. L’étude de ce phénomène est devenue possible grâce aux approches d’analyse génomique (AG) associé avec d’autres techniques. L’entérocolite nécrosante (ECN) et l’infection du pied diabétique (IPD) demeurent deux maladies associées à la dysbiose dans lesquels différents bactéries ont été décrites, notamment C. butyricum dans l’ECN, E. coli et S. aureus dans l’IPD. Dans le cadre de l’ECN, C. butyricum demeure l’espèce la plus fréquente chez les ECN. L’identification clusters liés géographiquement et en fonction du temps. Le portage asymptomatique est suggéré par une similarité génomique des souches patients et contrôles. La prédiction d’un gène de β-hémolysine ainsi leur effet cytotoxique sur les cellules Jurkat avait été observé. De même, sur les cellules Caco-2 malgré le KO du gène de β-hémolysine. En se basant sur l’analyse physico-chimique du surnageant bactérien, nous avons suggéré que la fraction cytotoxique est protéique. La purification de la fraction cytotoxique a permis de trouver une protéine codant pour PspC family possédant un domain conservé commun avec celui de la toxine A/B. L’inactivation du gène codant pour cette protéine n’a pas supprimé l’effet cytotoxique, suggérant la présence d’une combinaison gènes. En parallèle, nous avons ciblé l’impact de C. neonatale par qRPC spécifique rpoB. Cette espèce était plus fréquente chez les patients, ainsi de clones géographiques ont été identifiées. Enfin, des SNPs ont été observés dans des gènes de virulence dans le cas des E. coli et S. aureus isolés de l’IPD. / Dysbiosis remains a main cause during the establishment of several diseases, by promoting bacterial translocation, leading to inflammation process. Specific microorganisms were involved in the pathogenesis of dysbiosis-associated diseases, notably necrotizing enterocolitis (NEC) and diabetic foot (DF). This was possible by the implication of whole-genome analysis (WGA) in association with other techniques. In case of NEC, C. butyricum was significantly associated with in NEC; tested on a South-East French cohort. Geographical and/or temporal clusters were identified, thus genomic relationship between NEC-associated isolates and controls, suggesting the presence of asymptomatic carriage. Genes encoding for β-hemolysin was detected and C. butyricum supernatant exhibited cytotoxic effect on Jurkat cells. Cytotoxic effect was also presented on Caco-2 cells. Supernatant of β-hemolysin-mutant C. butyricum showed enterotoxic effect. Basing on physico-chemical data, we assumed that the evaluated fraction was a protein. Proteomics analysis revealed that PspC family was the cytotoxic protein. This protein owned a glucan-binding domain, shared by C. difficile toxin A/B. The KO of PspC gene was enterotoxic, suggesting by this the existence of a combination of genes. In parallel, a specific rpoB-based qPCR was developed to identify C. neonatale. We found that, C. neonatale was more prevalent in NEC than in controls. Although co-identified in association with C. butyricum. C. neonatale clones were distinguished especially in strains isolated from the same hospital. Regarding to DF infection, SNPs were identified within S. aureus and E. coli genomes, especially in virulent genes.

Le microbiote intestinal est constitué d’un écosystème complexe de microorganismes appartenant à différents règnes. Cependant, la majorité de ces microorganismes sont d’origine bactérienne. Par conséquent, de nombreuses études, y compris la présente, se concentrent sur l’étude des communautés bactériennes. Les microorganismes ont développé une relation mutualiste avec le corps humain et agissent de plusieurs manières sur sa santé. Une perturbation du microbiote intestinal, nommée dysbiose, est reliée au développement d’une multitude de problèmes de santé chez diverses espèces animales. La transplantation de microbiote fécal suscite l’intérêt dans le domaine de la médecine vétérinaire. La préparation et l’entreposage affectent la composition et la viabilité bactérienne des fèces destinées à la transplantation de microbiote fécal (TMF). Jusqu’à présent, il demeure l’absence d’un protocole vétérinaire pour effectuer la préparation et l’entreposage des transplants fécaux canins. Par conséquent, l’objectif de cette étude était de comparer la viabilité bactérienne d’échantillons fécaux en présence et en absence d’oxygène et d’effectuer la congélation à l’aide de deux cryoprotecteurs différents. Les hypothèses de ce projet étaient les suivantes : la préparation des échantillons en absence d’oxygène préservera la viabilité bactérienne, l’utilisation d’un cryoprotecteur contenant des antioxydants pour la congélation générera le meilleur taux de viabilité, et le microbiote de chaque individu n’aura pas la même capacité à résister aux effets de la préparation et de l’entreposage. Les fèces de 10 chiens en santé ont été collectées et immédiatement transférées à l’intérieur d’une chambre anaérobique. Des aliquotes de 1,8 g ont été diluées dans 7,2 ml d’un cryoprotecteur contenant du glycérol à 10% (Gly) ou des antioxydants (Cryo). Les échantillons ont été homogénéisés et filtrés en condition aérobique (Ae) et en condition anaérobique (An) à l’intérieur d’une chambre anaérobique, simulant la préparation de la TMF. Les échantillons ont été congelés à -20 °C durant 90 jours (F) pour l’évaluation des effets de l’entreposage. La viabilité bactérienne des échantillons a été déterminée à l’aide de la cytométrie de flux. L’analyse de la composition bactérienne chez les 10 donneurs de matières fécales a été réalisée par le séquençage de la région V4 du gène de l’ARNr 16S à l’aide de la plateforme Illumina MiSeq. Les échantillons non congelés, préparés en absence d’oxygène et dilués avec Cryo présentaient les plus grands taux de viabilité (66,78 %) par rapport aux autres groupes (p < 0,05). Les échantillons exposés à l’oxygène avaient une viabilité bactérienne inférieure (p < 0,01). Toutefois, les échantillons dilués avec Cryo présentaient une viabilité plus élevée (65,26 %) que les échantillons dilués dans Gly (55,20 % ; p < 0,001) en présence d’oxygène. La viabilité bactérienne a diminué en raison de la congélation des échantillons (p < 0,001). L’ensemble des échantillons frais avaient une viabilité médiane de 62,23 % et, à la suite de la congélation, elle était de 22,68 %. Cependant, les échantillons congelés à l’aide de glycérol avaient une viabilité plus élevée (30,61 % ; p < 0,001). Le genre Prevotella était fortement corrélé à la viabilité (R = 0,731 ; p < 0,05, R = 0,756 ; p < 0,05, R = 0,834 ; p < 0,01, R = 0,752 ; p < 0,05). Ces résultats indiquent que la viabilité bactérienne est optimale lors de l’utilisation de matières fécales en absence d’oxygène et lors d’une dilution à l’aide d’un cryoprotecteur contenant des antioxydants. La congélation a significativement réduit la viabilité bactérienne, mais le glycérol semble mieux préserver les bactéries. La présence de certaines espèces plus résistantes et l’impact de la composition du microbiote sur l’efficacité de la TMF nécessitent une enquête plus approfondie. / The intestinal microbiota is made up of a complex ecosystem of microorganisms belonging to different kingdoms. However, bacterial cells are much more numerous. Therefore, many studies, including the present one, focus on the study of bacterial communities. Microorganisms have developed a mutualistic relationship with the animal body and act in several ways on its health. A disturbance of the intestinal microbiota, called dysbiosis, is linked to the development of a multitude of health problems in various animal species. There is an emerging interest in the transplantation of fecal microbiota in veterinary medicine. Preparation and storage affect the quality of transplants intended for faecal microbiota transplantation (FMT). Considering the absence of a protocol in veterinary medicine, the objective of this study was to optimize bacterial viability during the preparation and storage of canine fecal transplants. The hypotheses of this project were that the preparation of samples in the absence of oxygen will preserve bacterial viability, that the use of a cryoprotectant containing antioxidants for freezing will yield the best viability rate and the microbiota of individuals does not have the same ability to withstand the effects of preparation and storage. Feces from ten healthy dogs were collected, and immediately transferred inside an anaerobic chamber. Aliquots of 1.8 g were diluted in 7.2 ml of a cryoprotectant containing glycerol (Gly) or antioxidants (Cryo). The samples were homogenized and filtered, simulating the TMF preparation. To evaluate the impact of oxygen on bacterial viability, the procedures were performed outside (Ae) and inside (An) the anaerobic chamber. Samples were frozen at -20°C for 90 days (F) to evaluate effect of freezing. The bacterial viability of samples was determined using flow cytometry. Analysis of the bacterial composition was performed by sequencing the V4 region of the 16S rRNA gene, using the Illumina MiSeq platform. Fresh samples prepared under anaerobiosis and diluted with Cryo had the highest viability (66.78%) compared to the other groups (p < 0.05). Bacterial viability was affected by oxygen (p < 0.01) but solutions prepared with Cryo had higher viability (65.26%) than samples diluted in Gly (55.20%; p < 0.001). Freezing decreased bacterial viability from 62.23% to 22.68% (p < 0.001). However, samples frozen using glycerol showed higher viability (30.61%; p < 0.001). The genus Prevotella was strongly correlated with viability (R = 0.731; p < 0.05, R = 0.756; p < 0.05, R = 0.834; p < 0.01, R = 0.752; p < 0.05). These results show that bacterial viability is optimal when preparing feces under anaerobic conditions and using a cryoprotectant containing antioxidants. If freezing is necessary, glycerol seems to preserve the bacteria better. The presence of some more resilient species and the impact of microbiota composition on the efficacy of TMF requires further investigation.