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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Impact of the maternal diet and the intervention with fructooligosaccharide on thehuman milk microbiota / Impacto da dieta materna e da intervenção com fruto-oligossacarídeo sobre a microbiota do leite humano

Padilha, Marina 17 April 2018 (has links)
Human milk is recognized as the main component for growth, metabolism, and immune development in infants. Furthermore, during lactation, human milk is an important source of microorganisms for the intestinal colonization of newborns. Mother-related factors have been associated with the human milk microbiota composition. Nevertheless, apparently, there has not been any study in which the maternal diet was evaluated as a modulator of the human milk microbiota. Therefore, the aim of this study was to investigate the impact of the maternal diet on the human milk microbiota composition of healthy women, and subsequently, to evaluate the effect of fructooligosaccharides supplementation on the human milk microbiota. This study consisted of two parts; the first was a cross-sectional study, including 94 lactating women recruited at the University Hospital of the University of São Paulo (HU/USP), to investigate the association between the maternal nutrient intake during pregnancy and lactation over the first month and the human milk microbiota. The second part consisted of a randomized, placebo-controlled clinical trial with 53 lactating, classified as FOS group (n = 28), which received 4.5 g of fructooligosaccharides + 2 g of maltodextrin or placebo group (n = 25), which received 2 g of maltodextrin, over a period of 20 days. The DNA was isolated and used as template for amplification and sequencing by the Illumina MiSeq® System. Overall, the maternal diet during lactation (\"short-term\" food intake) influenced specific bacterial groups, including positive correlations between polyunsaturated fatty acids/linoleic fatty acids and Bifidobacterium. However, only the maternal diet during pregnancy (\"long-term\" food intake) was statistically significant (p = 0.02) for the clustering analyzes (community structure analyzes), in which higher levels of vitamin C intake during pregnancy was related to cluster 2, driven by the Staphylococcus genus. After the intervention period on the maternal diet, no differences were found for relative abundance of genera between the placebo and the FOS groups. However, the distances of the trajectories covered by the samples from the beginning to the end of the supplementation was higher for the FOS group (p = 0.0007). According to our results, the maternal age affects the response for FOS supplementation (p = 0.02), though no patterns in the differences of relative abundances were found between the groups. Our results suggest that the maternal diet may influence the human milk microbiota, and the diet during pregnancy is a stronger factor over the bacterial community structure. Minor changes were found by the maternal short-term food intake or the maternal intervention with the prebiotic, and the changes seem to be individual-dependent and influenced by the maternal age, particularly in the intervention study. / O leite humano é, reconhecidamente, o principal componente para o crescimento e o desenvolvimento metabólico e imunológico de lactentes. Adicionalmente, durante a lactação, o leite humano consiste em uma importante fonte de micro-organismos para a formação da microbiota intestinal de neonatos. Fatores relacionados à mãe têm sido associados à composição da microbiota do leite humano. Entretanto, poucos estudos avaliaram a dieta materna como componente modulador da microbiota do leite humano. Os objetivos deste estudo foram investigar o impacto da dieta materna sobre a composição da microbiota do leite humano de mães saudáveis e, posteriormente, avaliar a influência da intervenção com fruto-oligossacarídeo na microbiota do leite humano, durante 20 dias de lactação. O estudo foi dividido em duas partes; a primeira parte consistiu de um estudo transversal, com 94 lactantes atendidas no Hospital Universitário da Universidade de São Paulo (HU/USP), a fim de investigar a associação entre o consumo materno de nutrientes durante a gestação e durante o primeiro mês de lactação e a microbiota do leite humano. A segunda parte consistiu em um ensaio clínico, aleatorizado, placebo-controlado, com 53 lactantes, classificadas em grupo FOS, que recebeu 4.5 g de fruto-oligossacarídeo + 2 g de maltodextrina (n = 28) ou grupo placebo, que recebeu 2 g de maltodextrina (n = 25), suplementados por 20 dias. O DNA das amostras de leite foi isolado e utilizado como molde para amplificação e sequenciamento em Illumina MiSeq® System. Em geral, a dieta materna durante a lactação (consumo a curto prazo) apresentou influência pontual sobre diversos grupos de micro-organismos, incluindo correlações positivas entre ácidos graxos poli-insaturados/linoleico e o gênero Bifidobacterium. No entanto, somente a dieta materna durante a gestação (consumo a longo prazo) foi estatisticamente significante (p = 0.02) para as análises de agrupamento das amostras (análises de estrutura de comunidade), sendo o maior teor de vitamina C consumido durante a gestação relacionado ao agrupamento 2, direcionado por maiores populações do gênero Staphylococcus. Após o período de intervenção na dieta materna, não foram encontradas diferenças entre a abundância relativa de gêneros entre os grupos placebo e FOS. No entanto, as distâncias do percurso das amostras do início até o final da suplementação foram maiores para o grupo FOS (p = 0.0007). De acordo com os resultados, a idade materna influencia essa resposta à suplementação por FOS (p = 0.02), embora, não tenham sido encontrados padrões nítidos nas diferenças de abundância relativa entre os grupos. Os resultados obtidos sugerem que a dieta materna consiste em um fator de modulação da microbiota do leite humano, sendo a dieta durante a gestação um fator mais intenso sobre a estrutura da comunidade bacteriana do leite humano. No entanto, o consumo a curto prazo ou a intervenção alimentar com prebiótico sobre a dieta materna apresentou influência pontual sobre a dinâmica da microbiota do leite, ainda que mudanças observadas sejam indivíduo-dependentes e influenciadas pela idade materna, como no caso do estudo de intervenção.
2

Detecting a Probiotic Product Within the Gut of Broiler Chickens

Pisula, Anneka 01 August 2018 (has links) (PDF)
As of January 2017, the U.S. poultry industry banned the use of antibiotics and now relies on alternatives such as probiotics to help protect animal health. Although probiotic use is not a new concept in the poultry industry, identifying the best combination of bacterial strains to generate an effective probiotic formula requires further investigation. This study aimed to detect a probiotic product of four bacterial strains (Pedioccoccus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, and Bacillus subtilis) in a feeding trial with broiler chickens. Birds given the probiotic were predicted to show an improved growth performance with the probiotics colonizing the gut. Ninety-six broiler chickens were equally divided into 3 treatment and 3 control pens. During the 25-day experiment, birds were fed a starter diet (days 0-11) and a grower diet (days 12-25). Experimental birds were administered the probiotic product via the drinking water at a concentration of 3.1×104 CFU/ml. Control birds had an equivalent amount of dextrose filler added to their water supply. Feces were collected hourly on day one and daily thereafter. On days 1, 22, and 25 of the experiment, 2 birds from each pen were euthanized for gut sampling. Lumen and mucosa samples were collected from the duodenum, jejunum, ileum, and ceca. Species-specific and strain specific PCR primers were employed for probiotic detection. Wild strains of P. acidilactici, P. pentosaceus, and L. plantarum were detected in the feeds, inhibiting detection of the probiotic strains when using species-specific PCR primers. Strain-specific primers were used to detect the probiotic Pedioccoccus acidilactici and Lactobacillus plantarum strains. B. subtilis was detected in feces within one hour of probiotic administration and was predominantly detected in experimental birds only. Both P. acidilactici and L. plantarum probiotic strains were initially detected in the feces of treated birds within two hours of probiotic administration and again ten days later. Both L. plantarum and B. subtilis were seen only in treated bird gut samples. L. plantarum was predominantly detected in the ceca near the end of the small intestine. P. pentosaceus was observed more often in treated gut samples and P. acidilactici was the least commonly detected probiotic strain. All administered bacteria were rarely seen in mucosa samples. Feed-endogenous P. acidilactici and L. plantarum strains became progressively more detectable in the mucosa along the gastrointestinal tract suggesting gut colonization, however, probiotic strains did not appear to colonize the mucosa of treated birds. Although probiotic strains were no longer detected after product removal, all probiotic strains were detected in feces and gut samples during probiotic administration, suggesting the bacteria can colonize the gut. Probiotic supplementation did not result in significant differences in body weight gain, feed intake, or feed conversion ratio. However, birds growing in a more stressful environment than the carefully controlled experimental set up used here may show probiotic-related effects. This study identified that the probiotic bacteria appeared to survive the gastrointestinal tract, exhibited a transit time of 1-2 hours, could possibly colonize chickens, and localized near the end of the chicken gut.
3

Conséquences d'un stress chronique sur la barrière de mucus intestinal chez le rat : effet du probiotique Lactobacillus farciminis / Consequences of a chronic stress on intestinal mucus barrier in rat : effect of a probiotic Lactobacillus farciminis

Da Silva, Stéphanie 07 November 2013 (has links)
Si les modifications dans l’expression et les propriétés des mucines ont été largement décrites dans la physiopathologie des maladies inflammatoires chroniques de l’intestin, la caractérisation structurale et fonctionnelle de la barrière de mucus reste parcellaire dans le contexte micro-inflammatoire du syndrome de l’intestin irritable (SII). Par ailleurs, certains traitements probiotiques préviennent la rupture de l’intégrité de la barrière épithéliale intestinale, provoquée en conditions de stress mais peu de travaux décrivent leur influence sur les modifications de la structure du mucus, induites par le stress. Cette étude a comme objectifs d’évaluer chez le rat (i) si un stress chronique modifie le nombre de cellules à mucus et l’expression de la mucine Muc2, ainsi que la nature biochimique des mucines secrétées au niveau de l’iléon et du côlon, et plus particulièrement les O-glycanes, (ii) si un traitement probiotique (Lactobacillus farciminis) prévient les modifications du mucus potentiellement induites, (iii) si les effets observés sont en lien avec la capacité de colonisation in vivo de L. farciminis.Méthodes. Des rats Wistar mâles ont reçu L. farciminis ou une solution saline. Les animaux ont été soumis au stress d’évitement passif de l’eau (WAS) pendant 1h/jour ou à un stress fictif (contrôle), pendant les 4 derniers jours des traitements. Différents prélèvements ont été effectués au niveau de l’iléon et du côlon pour (i) des analyses sur coupes (immuno-marquage, nombre de cellules à mucus) et (ii) la détermination du profil de O-glycosylation des mucines. La morphologie de la couche de mucus a été évaluée par microscopie à force atomique (AFM). L. farciminis a été visualisé par "Fluorescence in situ Hybridization", confirmée par qPCR et son adhésion à la muqueuse a été déterminée par une méthode ex situ. La perméabilité paracellulaire et la sensibilité viscérale ont été évaluées.Résultats. Le WAS ne modifie pas le nombre de cellules à mucus ni l’expression de la mucine Muc2 aux niveaux iléal et colique. L’analyse par spectrométrie de masse a révélé que le stress induit des altérations de la O-glycosylation des mucines. Une augmentation marquée du degré de complexité des structures glycaniques a été observée, se traduisant par l’apparition de chaînes polylactosaminiques, sans modification toutefois du taux de sialylation et de sulfatation des mucines. La couche de mucus en condition de stress, observée par AFM, présente une morphologie aplatie et moins cohésive. L'hypothèse serait que les modifications structurales des O-glycanes influencent les interactions physico-chimiques entre les fibres de mucines, impactant de manière négative l’intégrité de la barrière de mucus. Cette altération de la couche de mucus s'accompagne d'un défaut de la barrière épithéliale et d'une hypersensibilité viscérale. L’administration de la souche probiotique prévient ces changements provoqués par le WAS. L. farciminis a été retrouvé au sein de l’iléon et du côlon. Par ailleurs, la présence de "Segmented Filamentous Bacteria" (SFB) a été mise en évidence, par FISH et microscopie électronique, au niveau de l’iléon sur l'ensemble des animaux testés. Le traitement par L. farciminis induit une diminution de la population des SFB aussi bien chez les animaux contrôles que stressés. Conclusion. Nous avons montré qu’un stress chronique chez le rat, outre les modifications fonctionnelles de l'épithélium intestinal (hyperperméabilité intestinale et hypersensibilité viscérale), altère la structure O-glycanique des mucines sans affecter l’expression de Muc2. Ces altérations se traduisent par une perte des propriétés cohésives de la couche de mucus. La souche probiotique L. farciminis, en prévenant l’ensemble des modifications induites par le stress, contribue au renforcement de la fonction barrière de l'intestin. Cette étude fournit un argumentaire complémentaire pour l’utilisation de cette souche dans le traitement du SII. / Background. Despite a large body of literature incriminating mucus alterations in the pathogenesis of Intestinal Bowel Diseases (IBD), structural and physical changes in the mucus layer remain poorly understood in the micro-inflammatory context of Irritable Bowel Syndrome (IBS). Moreover, some probiotic treatments prevent stress-induced intestinal epithelial barrier impairment but little is known about their influence on intestinal mucin structural modifications and mucus properties induced by stress. Thereby, this study aimed at evaluating whether (i) a chronic stress modified the number of gut goblet cells and Muc2 expression, nature of secreted mucins in both ileum and colon and more particularly mucin O-glycosylation, (ii) L. farciminis treatment prevented these alterations and (iii) observed effects were related to the in vivo colonization capacity of L. farciminis.Methods. Wistar rats received orally L. farciminis (1011 UFC/day) or vehicle (NaCl 0.9% (w/v)) for 14 days. From day 10 to day 14, they were submitted either to sham (control) or 4-day Water Avoidance Stress (WAS) during 1 hour per day. After sacrifice, different samples (tissues, mucosa) were collected in both ileal and colonic regions for (i) histological analyses (Muc 2 immunohistochemistry, number of goblet cells by Periodic Acid Schiff/Hemalun) and (ii) O-glycosylation profile by mass spectrometry after mucin extraction and purification. In parallel, the morphology of the mucus layer was evaluated by atomic force microscopy. Spatial localization of L. farciminis was assessed by Fluorescence In Situ Hybridization (FISH), confirmed by qPCR. Mucosal adhesion of L. farciminis was determined by an ex situ method. In complement, intestinal paracellular permeability and visceral sensitivity were measured.Results. WAS did not modify neither the number of intestinal goblet cells nor Muc2 expression in both ileum and colon. In contrast, the mass spectrometry analysis demonstrated that O-glycosylation of mucins was strongly affected by WAS. Indeed, a strongly increase in the complexity degree of O-glycan structures was observed in both ileum and colon, with the appearance of elongated polylactosaminic chains (repetition of the disaccharidic unit composed of galactose and N-acetylglucosamine), without modifications of mucin sialylation and sulfation. Under stress conditions, the mucus layer, observed by atomic force microscopy, showed a flattened morphology, probably indicative of a loss in its cohesive properties. We hypothesized that O-glycan structural modifications influence physico-chemical interactions between mucins fibers. Stress also induced intestinal hyperpermeability and visceral hypersensitivity. The mucus layer alteration was, thus, in relation with epithelial barrier impairment and visceral hypersensitivity. L. farciminis administration prevented WAS-induced functional, biochemical and physical changes of mucus. The presence of L. farciminis in the ileum and colon was detected by FISH and qPCR, albeit with quantitative and qualitative differences in the colonization capacity within these two intestinal compartments. Furthermore, the presence of Segmented Filamentous Bacteria (SFB) was shown in the ileum whatever the conditions under study. L. farciminis reduced the SFB population level in both control and stressed animals.Conclusion. Chronic stress induced functional changes (intestinal hyperpermeability and visceral hypersensitivity) in rats, as well as a shift in mucin O-glycosylation rather than changes in mucin expression. Intestinal mucin O-glycan modifications resulted in a loss of mucus layer cohesive properties. L. farciminis treatment prevented impairment of both intestinal epithelial and mucus barriers, reflecting an enhancement of the protective barrier function. These results confirm that L. farciminis is a valuable probiotic in the IBS management.
4

Characterization of Genes and Functions Required by Multidrug-resistant Enterococci to Colonize the Intestine

Flor Duro, Alejandra 14 May 2021 (has links)
[ES] Las bacterias resistentes a múltiples antibióticos, como el Enterococo resistente a vancomicina (ERV), son un problema creciente en los pacientes hospitalizados, por lo que se necesita estrategias alternativas para combatir estos patógenos. Las infecciones causadas por ERV suelen comenzar con la colonización del tracto intestinal, un paso crucial que se afectado por la presencia de la microbiota. Sin embargo, los antibióticos alteran la microbiota y esto promueve la colonización de ERV. Una vez que el patógeno ha colonizado el intestino, alcanza niveles muy altos pudiendo diseminar a otros órganos y pacientes. A pesar de su importancia, se sabe muy poco sobre los genes que codifica para colonizar el intestino y sobre el mecanismo por el cual la microbiota suprime su colonización intestinal, siendo los dos objetivos principales. En primer lugar hemos utilizado una metodología previamente descrita (Zhang et al., 2017, BMC Genomics), basada en la generación de una librería de mutantes por transposición junto a secuenciación masiva, con el fin de identificar los genes codificados por ERV necesarios para la colonización del intestino en ratones. Además, hemos realizado análisis metatranscriptómicos para identificar aquellos genes más expresados. El análisis ha identificado genes cuya interrupción reduce significativamente la colonización intestinal en el intestino grueso. Los genes que más afectaron a la colonización codifican proteínas relacionadas con la absorción o el transporte de diversos nutrientes como los carbohidratos (subunidad EIIAB del transportador PTS de manosa, el regulador transcripcional de la familia LacI, ácido N-acetilmurámico 6-fosfato eterasa) o iones (proteína transportadora dependiente de ATP (ABC) y proteínas del grupo [Fe-S]). El papel de estos genes en la colonización se ha confirmado mediante experimentos de mutagénesis directa y de competición con la cepa salvaje. Además, estos genes afectan a la colonización intestinal con diferentes antibióticos (clindamicina y vancomicina). Para identificar el mecanismo molecular por el cual cada gen afecta a la colonización, hemos realizado experimentos in vitro y ex vivo además del análisis transcriptómico. Los experimentos in vitro confirman que las proteínas del grupo [Fe-S] están involucradas en el transporte iones de hierro, principalmente Fe3+. Por otra parte, los genes de la subunidad EIIAB del transportador de manosa y del ácido N-acetilmurámico 6-fosfato eterasa son necesarios para la utilización de la manosa y el ácido N-acetilmurámico, respectivamente, azúcares que suelen estar presentes en el intestino. También confirmamos que el regulador transcripcional de la familia LacI es un represor que afecta a proteínas transportadoras ABC, probablemente implicadas en la absorción de carbohidratos. Además, algunos de estos genes están codificados principalmente por cepas clínicas de E. faecium y en menor medida por cepas comensales. En segundo lugar, estudiamos los mecanismos de protección de un consorcio de cinco bacterias comensales, que anteriormente se había demostrado que disminuían la colonización intestinal por ERV en ratones. Mediante transcriptómica, metabolómica y los ensayos in vivo observamos que el consorcio bacteriano inhibe el crecimiento de ERV mediante la reducción de nutrientes, concretamente fructosa. Por último, el análisis ARN-Seq in vivo de cada aislado en combinación con los ensayos ex vivo e in vivo demostraron que una sola bacteria (Olsenella sp.) proporciona protección. En conjunto, los resultados obtenidos han identificado la función de genes específicos requeridos por ERV para colonizar el intestino. Además, hemos identificado un mecanismo mediante el cual la microbiota confiere protección. Estos resultados podrían conducir a nuevos enfoques terapéuticos para prevenir las infecciones causadas por este patógeno multiresistente a los antibióticos. / [CA] Els bacteris resistents a múltiples antibiòtics, com el Enterococo resistent a vancomicina (ERV), són un problema creixent en els pacients hospitalitzats, que són resistents a la majoria d'antibiòtics disponibles per la qual cosa es necessita estratègies alternatives per a combatre aquests patògens. Les infeccions causades per ERV solen començar amb la colonització del tracte intestinal, un pas crucial que es veu afectat per la presència de la microbiota. No obstant això, els antibiòtics alteren la microbiota i això promou la colonització de ERV. Una vegada que el patogen ha colonitzat l'intestí, aconsegueix nivells molt alts podent disseminar a altres òrgans i pacients. Malgrat la seua importància, se sap molt poc sobre els gens que codifica ERV per a colonitzar l'intestí i sobre el mecanisme pel qual la microbiota suprimeix la seua colonització intestinal. En primer lloc hem utilitzat una metodologia prèviament descrita (Zhang et al., 2017, BMC Genomics), basada en la generació d'una llibreria de mutants per transposició junt amb seqüenciació massiva, amb la finalitat d'identificar els gens codificats per ERV necessaris per a la colonització de l'intestí en ratolins. A més a més, hem realitzat anàlisi metatranscriptòmics per a identificar aquells gens més expressats. L'anàlisi ha identificat gens quina interrupció redueix significativament la colonització intestinal en l'intestí gros. Els gens que més van afectar la colonització codifiquen proteïnes relacionades amb l'absorció o el transport de diversos nutrients com els carbohidrats (subunitat EIIAB del transportador PTS de manosa, el regulador transcripcional de la família LacI, àcid N-acetilmuràmic 6-fosfat eterasa) o ions (proteïna transportadora dependent d'ATP (ABC) i proteïnes del grup [Fe-S]). El paper d'aquests gens en la colonització s'ha confirmat mitjançant experiments de mutagènesis directa i de competició amb el cep salvatge. A més, aquests gens afecten la colonització intestinal amb diferents antibiòtics (clindamicina i vancomicina). Per a identificar el mecanisme molecular pel qual cada gen afecta a la colonització, hem realitzat experiments in vitro i ex viu a més de l'anàlisi transcriptòmic. Els experiments in vitro confirmen que les proteïnes del grup [Fe-S] estan involucrades en el transport d'ions de ferro, principalment Fe3+. D'altra banda, els gens de la subunitat EIIAB del transportador PTS de manosa i de l'àcid N-acetilmuràmic 6-fosfat eterasa són necessaris per a la utilització de la manosa i l'àcid N-acetilmuràmic, respectivament, sucres que solen estar presents en l'intestí. També confirmem que el regulador transcripcional de la família LacI és un repressor que afecta proteïnes transportadores ABC, probablement implicades en l'absorció de carbohidrats. A més a més, alguns d'aquests gens estan codificats principalment per ceps clínics de E. faecium i en menor mesura per ceps comensals. En segon lloc, estudiem els mecanismes de protecció d'un consorci de cinc bacteris comensals, que adès s'havia demostrat que disminuïen la colonització intestinal per ERV en ratolins. Amb l'ús de transcriptòmica, metabolòmica i els assajos in vivo observem que el consorci bacterià inhibeix el creixement de ERV mitjançant la reducció de nutrients, concretament fructosa. Finalment, l'anàlisi ARN-Seq in vivo de cada aïllat en combinació amb els assajos ex viu i in vivo van demostrar que un sol bacteri (Olsenella sp.) proporciona protecció. En conjunt, els resultats obtinguts han identificat la funció de gens específics requerits per ERV per a colonitzar l'intestí. A més, hem identificat un mecanisme mitjançant el qual la microbiota confereix protecció. Aquests resultats podrien conduir a nous enfocaments terapèutics per a previndre les infeccions causades per aquest patogen multiresistent als antibiòtics. / [EN] Multidrug-resistant bacteria, such as vancomycin-resistant-Enterococcus (VRE), are an increasing problem in hospitalized patients. Some VRE strains can be resistant to most available antibiotics, thus, alternative strategies to antibiotics are urgently needed to combat these challenging pathogens. Infections caused by VRE frequently start by colonization of the intestinal tract, a crucial step that is impaired by the presence of the intestinal microbiota. Administration of antibiotics disrupts the microbiota, which promotes VRE intestinal colonization. Once VRE has colonized the gut, it reaches very high levels, which promotes its dissemination to other organs and its transfer to other patients. Despite the relevance of VRE gut colonization, very little is known about the genes encoded by this pathogen to colonize the gut and about the mechanisms by which the microbiota suppresses VRE gut colonization. In this thesis, we have utilized a previously described methodology (Zhang et al., 2017, BMC Genomics), based on the generation of a transposon mutant library coupled with high-throughput sequencing, in order to identify VRE encoded genes required for colonization of the mouse intestinal tract. In addition, we have performed metatranscriptomic analysis in mice to identify VRE genes specifically expressed in the gut. Our analysis has identified genes whose disruption significantly reduces VRE gut colonization in the large intestine. The genes that most affected VRE gut colonization encoded for proteins related to the uptake or transport of diverse nutrients such as carbohydrates (PTS mannose transporter subunit EIIAB, LacI family DNA-binding transcriptional regulator, N-acetylmuramic acid 6-phosphate etherase) or ions (phosphate ABC transporter ATP-binding protein and proteins from [Fe-S] cluster). The role of these genes in gut colonization has been confirmed through targeted mutagenesis and competition experiments against a wild type strain. Moreover, these genes affect gut colonization under different antibiotic treatments (clindamycin and vancomycin). To elucidate the mechanism by which each gene influences gut colonization, we have performed in vitro and ex vivo experiments besides transcriptomic analysis. In vitro experiments confirm that proteins from [Fe-S] cluster are involved in the transport of different forms of iron ions, mostly Fe3+. On the other hand, the PTS mannose transporter subunit EIIAB and N-acetylmuramic acid 6-phosphate etherase genes are required for the utilization of mannose and N-acetyl-muramic acid, respectively, sugars that are usually present in the intestinal environment. We have also confirmed that LacI family DNA-binding transcriptional regulator is a repressor that affects the expression of genes encoding for an ABC transporter probably involved in the uptake of carbohydrates. Furthermore, we have confirmed that some of these genes are encoded mainly by E. faecium clinical strains but not or to a lower extent by commensal strains. Secondly, we studied the mechanisms of protection of a consortium of five commensals bacteria, previously shown to restrict VRE gut colonization in mice. Functional transcriptomics in combination with targeted metabolomics and in vivo assays performed in this thesis indicated that the bacterial consortium inhibits VRE growth through nutrient depletion, specifically by reducing the levels of fructose. Finally, in vivo RNA-Seq analysis of each bacterial isolate of the consortium in combination with ex vivo and in vivo assays demonstrated that a single bacterium (Olsenella sp.) could recapitulate the protective effect. Altogether, the results obtained have identified the function of specific genes required by VRE to colonize the gut. In addition, we have identified a specific mechanism by which the microbiota confers protection against VRE colonization. These results could lead to novel therapeutic approaches to prevent infections caused by this pathogen. / Flor Duro, A. (2021). Characterization of Genes and Functions Required by Multidrug-resistant Enterococci to Colonize the Intestine [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166494 / TESIS

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