Dissertations / Theses on the topic 'Microbiota'

Trabalho Complementar apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de licenciada em Ciências da Nutrição
Os microrganismos presentes no microbioma humano coexistem em harmonia com o seu hospedeiro, mas podem, em determinadas circunstâncias, causar doença. O estudo do microbioma humano e, em particular, da microbiota intestinal está em franco desenvolvimento, tendo vindo a surgir novas evidências relativas à sua associação a diferentes patologias e ao seu papel na fisiologia humana. O microbioma humano é caracterizado pela sua complexa plasticidade e um aumento do seu conhecimento é visto como promissor para o entendimento de vários processos e doenças, incluindo cancro. A sua relação com a saúde é muito abrangente e ainda pouco conhecida. Inúmeros estudos são desenvolvidos como forma de explorar novas estratégias de tratamento. Além das intervenções já aplicadas, a manipulação do microbioma humano através do uso de probióticos e prebióticos, de uma combinação de ambos e do transplante de microbiota fecal (TMF), têm vindo a ser consideradas opções em relação e em complemento à antibioterapia para potenciar a eficácia dos tratamentos, reduzir a toxicidade e prevenir a carcinogénese. Nesta revisão, são apresentadas formas de manipulação do microbioma como adjuvantes ao tratamento do cancro.
Microorganisms present in the human microbiome coexist in harmony with their host but can be the origin of disease under certain circumstances. Study of the human microbiome and particularly of the intestinal microbiota is developing, with new evidence emerging regarding its association with different pathologies and its role in human physiology. Human microbiome is characterized by its complex plasticity and an increase in knowledge is seen as promising for the understanding of various processes and diseases, including cancer. Human microbiome relationship with health is very wide and still little known. Numerous studies are being carried out as a way to explore new treatment strategies. In addition to the interventions already applied: manipulation of the human microbiome using probiotics and prebiotics, a combination of both and the fecal microbiota transplantation (FMT), are being considered options to support antibiotic therapy to enhance effectiveness of treatments, reduce toxicity and prevent carcinogenesis. In this paper, ways of manipulating the microbiome as a component of cancer treatment are presented.
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Le microbiote intestinal humain a été fortement corrélé avec la santé humaine et les maladies et a montré un potentiel dans les développements thérapeutiques. La métagénomique a déjà montré qu'elle était capable de générer beaucoup de données, dont certaines sont dénuées de sens et constituaient la "matière noire". Alors culturomics a été développée pour compléter la métagénomique en ciblant des espèces bactériennes précédemment non cultivées. En utilisant la culturomics, nous avons décrit le microbiote intestinal humain des Pygmées et réussi à isoler un nombre significatif d'espèces bactériennes parmi lesquelles 38 étaient de nouvelles espèces. En comparant les résultats métagénomiques aux données culturomics, on constate que seulement 26% des espèces isolées ont été récupérées par métagénomique et que jusqu'à 59% des Operational taxonomic units détectées correspondaient à de nouvelles espèces bactériennes isolées par culturomique dans cette étude ou dans les précédentes
The human gut microbiota has been correlated in general health and diseases. Thus its description became mandatory to better understand its role and therapeutic potential. However, metagenomics has previously showed to be able to generate a lot of data, of which some are meaningless and constituted the “Dark matter”. Thus, culturomics was developed to complement metagenomics by targeting previously uncultured bacterial species. Using culturomics, we described the human gut microbiota of Pygmy people and succeeded in isolating a significant number of bacterial species out of which 38 were new species. Comparing metagenomics results to culturomics data, we see that only 26% of the isolated species were recovered by metagenomics and that up to 59% of the Operational taxonomic units detected corresponded to new bacterial species isolated by culturomics either in this study or in previous ones

Les anticorps bloquant les rétrocontrôles inhibiteurs du système immunitaire (ICB) ont révolutionné la prise en charge des patients atteints de certains cancers. Les ICB bloquent l’axe cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ou programmed cell death protein 1/PD-L1-PD-L2 et permettent une réactivation des cellules T stimulant l’immunité anti-tumorale. Toutefois, malgré cette avancée plus de 70 % des patients finiront par progresser et ces traitements peuvent entrainer des toxicités de type auto-immunes sévères. Il est donc fondamental d’identifier des biomarqueurs prédictifs de la réponse clinique et ainsi développer une nouvelle stratégie thérapeutique efficace pour augmenter l’index thérapeutique des ICB. Plusieurs groupes ont participé à décrire le lien étroit entre le microbiote intestinal et la réponse à la chimiothérapie (cyclophosphamide), à la greffe allogénique ainsi qu’aux les thérapies immunomodulatrices (anti-CTLA4 and PD1 Abs). Mon travail de thèse vient à la suite de ces travaux princeps et a permis de montrer que la composition du microbiote intestinal est en partie responsable de l’activité anti-tumorale des ICB dans plusieurs cancers. L’analyse de 249 patients atteints d’un cancer métastatique du poumon, du rein et cancer urothelial traités par l’anti-PD-1 ou l’anti-PD-L1 les antibiotiques (ATB) diminuaient la survie sans progression (PFS) de 3.5 vs 4.1 mois (p=0.017) et la survie globale de 11.5 vs 20.6 mois (p<0.001) par rapport aux patients n’ayant pas pris d’ATB avant de débuter les ICB. Nous avons par la suite analysé par métagénomique les selles de 153 patients atteints d’un cancer du poumon et du rein traités par l’anti-PD-1. Les résultats ont montré que Akkermmansia muciniphila est plus abondante chez les patients ayant une meilleure réponse clinique et une meilleure PFS. De plus, nous avons démontré que la présence d’une réponse mémoire spécifique des cellules CD4+ ou CD8+ envers A. muciniphila est associée à une plus longue PFS. Par la suite, des transplantations de microbiote fécal (FMT) avec les selles de ces patients chez des souris axéniques ou traitées par ATB montrent que les selles des répondeurs à l’anti-PD-1 entrainent une forte réponse immunitaire anti-tumorale post anti-PD-1 comparé aux selles de non-répondeurs. De plus, un gavage oral avec A. muciniphila après la FMT avec des selles de non-répondeurs restaure une forte réponse immunitaire post anti-PD-1 via la production d’IL-12 par les cellules dendritiques entrainant l’augmentation du recrutement des cellules T CD4+CXCR3+CCR9+ du ganglion mésentérique vers le site tumoral et une augmentation du ratio CD4/Treg. L’ensemble de ces résultats suggèrent que la découverte de bactérie immunogène capable de prédire le bénéfice clinique des ICB permettra de développer une stratégie thérapeutique efficace afin d’augmenter la survie des patients atteints de cancer.Mots clés : ICB, anti-PD-1, anti-PDL-1, cancer du poumon, cancer du rein, microbiote intestinal
In oncology, a novel therapeutic era based on immune checkpoint blockades (ICB) targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1) inhibitory T-cell receptors has come of age. Targeting CTLA-4 or PD-1/PDL-1/PDL-2 unleashes T cells and restores antitumor immunity. However, 70% of patients will eventually progress and drug-induced autoimmune toxicities are frequent. Therefore, predictors of clinical benefit and strategies to safely enhance ICB efficacy are urgently needed. Multiple lines of evidence have shown that conventional chemotherapy, allogeneic transplantation and immune-based therapies (IL-10R blockade, anti-CTLA4 and PD1 Abs) rely upon the composition of the gut microbiota to exert their bioactivity. During my PhD, I showed in a cohort of 249 patients with advanced NSCLC, RCC and urothelial cancer treated with anti-PD-1/PDL-1 mAb that antibiotic (ATB) prescription before ICB decreased PFS from 3.5 months vs 4.1 months (p=0.017) and OS from 11.5 months vs 20.6 months (p<0.001) compared to patients without ATB. Next, using quantitative metagenomics by shotgun sequencing, we explored the microbiota composition of 153 patients with advanced NSCLC and RCC amenable to anti-PD-1 mAb. Akkermansia muciniphila was found to be strongly associated with favorable objective response rate and longer PFS. To validate the relevance of these clinical findings, we brought up two major lines of evidence. First, we demonstrated that in NSCLC patient, the presence of specific IFNγ+ memory CD4+ and CD8+ T cells toward A. muciniphila predicted a longer PFS. Secondly, fecal microbiota transplantation (FMT) was performed using patient feces to recolonize germ-free or ATB-treated mice in two tumor models. Feces from patients with clinical response conveyed a stronger immune response against the tumor compared to feces from non-responders. Subsequently, oral supplementation with A. muciniphila post-FMT with non-responder feces restored the efficacy of PD-1 blockade. In this setting, dendritic cells secreted more IL-12, increasing the recruitment of CCR9+CXCR3+CD4+ T lymphocytes from the mesenteric lymph nodes into tumor beds as well as an increase of CD4+/Treg ratio within the tumor bed of mice co-treated with anti-PD1 mAb and A. muciniphila. The discovery of immunogenic bacteria capable of predicting and increasing clinical benefit of ICB will help for the development of novel biomarker tools and a future therapeutic concept, whereby treatment of cancer can be improved by the modulation of gut microbiota. Keywords: NSCLC, RCC, Immune checkpoint blockades (ICB), immunotherapies, Programmed Cell Death Protein-1 (PD-1), Microbiota

Nous avons collecté et comparé les microbiotes et les microbiomes intestinaux de plusieurs dizaines de lézards de l’espèce Podarcis sicula, vivant dans des populations continentales et insulaires croates. L’une de ces populations présentait la particularité d’avoir subi un changement de régime alimentaire récent, une transition d’un régime insectivore vers un régime omnivore (à 80% herbivores) sur une période de 46 ans. Les analyses de diversité menées sur la région V4 de l’ARN ribosomique 16S de ces communautés microbiennes ont révélé que la diversité spécifique (diversité alpha) des microbiotes de lézards omnivores (enrichis en archées méthanogènes) excède celle des microbiotes de lézards insectivores. Les communautés microbiennes des lézards apparaissent en outre faiblement structurées : 5 entérotypes peuvent être identifiés au niveau du phylum, et 3 phyla majoraires (les Bactéroidètes, les Firmicutes et les Protéobactéries) sont présents dans cette espèce. Cependant, ni le régime alimentaire, l’origine spatiale ou temporelle, et le sexe des lézards ne se traduisent par des différences significatives et majeures dans les microbiotes. Des analyses linéaires discriminantes avec effet de la taille des OTUs et des reads des microbiomes fonctionnellement annotés indiquent plutôt que le changement de régime alimentaire de Podarcis sicula est associé à des changements ciblés dans l’abondance de certains composants du microbiote et du microbiome de ces lézards, nous conduisant à formuler l’hypothèse de changements ciblés des communautés microbiennes dans cet holobionte non-modèle, par opposition à des transformations plus radicales. Sur un plan plus théorique, cette thèse propose également des modèles de réseaux (réseaux de similarité de reads et graphes bipartis) susceptibles d’aider à approfondir les analyses des microbiomes
We collected and compared intestinal microbiota and microbiomes from several Podarcis sicula lizards, which live in Croatian continental and insular populations. One of these populations has recently changed its diet over an 46 years timespan, switching from an insectivorous diet to an omnivorous one (up to 80% herbivorous). Diversity analyses of these microbial communities, based on the V4 region of their 16S rRNA, showed that the microbiota taxonomic diversity (or alpha diversity) is higher in omnivorous lizards (enrichment in methanogenic archaea) than in insectivorous ones. Besides, microbial communities seem weakly structured: 5 enterotypes are detected at the phylum level, and 3 major phyla (Bacteroidetes, Firmicutes and Proteobacteria) are present. However, neither diet, spatial or temporal origin, nor lizard gender correlate with significant differences in microbiota. Linear discriminant analyses with size effect, based on OTUs and functionally annotated reads from the microbiomes, suggest that Podarcis sicula diet change is associated to targeted changes of the abundance of some enzymes in the microbiomes. Such a result leads us to propose a hypothesis of targeted changes in the microbial communities of this non-model holobiont, instead of more radical transformations. On a more theoretical level, this thesis also proposes network models (Reads similarity networks and bipartite graphs) that can help improving microbiome analyses

Tesis por compendio
[ES] El análisis del control genético del hospedador sobre su microbiota ha sido señalado recientemente como un tema prometedor en diferentes campos de estudio. La relación entre el holobionte hospedador-microbioma y los fenotipos en el ganado lechero podría conducir a nuevos conocimientos en los programas de selección genética. Dentro de esta tesis doctoral, se realizó la estimación y análisis a través de diferentes enfoques estadísticos con el objetivo de desentrañar el control genético del hospedador sobre la microbiota en ganado lechero. Además, se analizó el rasgo de concentración de metano como un fenotipo potencial para ser incluido en el programa de mejora de ganado lechero español. Mayor abundancia relativa de la mayoría de los eucariotas (principalmente protozoos ciliados y hongos) y algunas arqueas (Methanobrevibacter spp. Methanothermus spp. y Methanosphaera spp.) fueron factores de riesgo para ser clasificadas en la categoría alta. Se propuso un conjunto de modelos de ecuaciones estructurales (SEM) de tipo recursivo dentro de un marco de Cadenas de Markov Monte Carlo (MCMC) para analizar conjuntamente la relación hospedador-metagenoma-fenotipo. Se estableció un modelo bivariado no-recursivo como punto de referencia. La heredabilidad de CH4 se estimó en 0,12 ± 0,01 en ambos modelos, recursivo y no recursivo. Asimismo, las estimaciones de heredabilidad para la abundancia relativa de los taxones se superpusieron entre los modelos y variaron entre 0.08 y 0.48. Las correlaciones genéticas entre la composición microbiana y el CH4 variaron de -0,76 a 0,65 en el modelo bivariado no recursivo y de -0,68 a 0,69 en el modelo recursivo. Doce matrices de relación de microbiota (K) fueron construidas a partir de diferentes métricas de distancia del microbioma, con el objetivo de comparar su desempeño dentro de un marco de estimación de componentes de varianza para CH4 y toda la microbiota. Análisis de simulación (n = 1000) y datos reales fueron desarrollados considerando cuatro modelos posibles: un modelo genómico aditivo (GBLUP), un modelo de microbioma (MBLUP), un modelo de efectos genéticos y microbioma (HBLUP) y un modelo de efectos de interacción genético, microbioma y genético × microbioma (HiBLUP). Un nuevo término "Holobiabilidad" fue definido para referirse a la proporción de la varianza atribuible a los efectos del holobionte hospedador-microbioma. Las estimaciones a partir de datos reales usando HiBLUP variaron dependiendo de la K utilizada y estuvieron entre 0.15-0.17, 0.15-0.21 y 0.42-0.59 para heredabilidad, microbiabilidad y holobiabilidad, respectivamente. El conjunto de datos de microbioma fue agregado a través de análisis de componentes principales (PCA), en pocos componentes principales (PCs) que fueron utilizados como aproximaciones del metagenoma central. Parte de la variabilidad condensada en estos PC está controlada por el genoma de la vaca, con estimaciones de heredabilidad para el primer PC (PC1) de ~ 0,30 en todos los niveles taxonómicos, con una gran probabilidad (> 83%) de que la distribución posterior sea > 0,20 y con un intervalo de mayor densidad posterior al 95% (95% HPD) no conteniendo cero. La mayoría de las estimaciones de correlación genética entre PC1 y metano fueron grandes (>0,70) en todos los niveles taxonómicos, con la mayor parte de la distribución posterior (> 82%) siendo > 0,50 y con su 95% HPD no conteniendo cero. Estos resultados sugieren que todo el metagenoma del rumen regula recursivamente las emisiones de metano en las vacas lecheras, y que tanto el CH4 como las composiciones de la microbiota están parcialmente controladas por el genotipo del hospedador. Las variables agregadas (PC) propuestas podrían ser usadas en programas de mejora de animales para reducir las emisiones de metano en las generaciones futuras.
[CA] L'anàlisi del control genètic de l'hoste sobre la seva microbiota s'ha assenyalat recentment com un tema prometedor en diferents camps d'estudi. La relació entre el holobiont hoste-microbioma i els fenotips en bovins de llet podria conduir a nous coneixements en els programes de cria. Dins d'aquest doctorat es van realitzar tesis, estimacions i anàlisis mitjançant diferents enfocaments estadístics amb l'objectiu de desentranyar el control genètic de l'hoste sobre la microbiota en bestiar lleter. A més, es va analitzar el tret de concentració de metà com a fenotip potencial a incloure en el programa espanyol de cria de bestiar lleter. La major abundància relativa de la majoria dels eucariotes (principalment protozous i fongs ciliats) i algunes arquees (Methanobrevibacter spp. Methanothermus spp i Methanosphera spp.) Van ser factors de risc per classificar-se en les categories altes. Es va proposar un conjunt de models d'equacions estructurals (SEM) de tipus recursiu dins d'un marc de cadena Markov Monte Carlo (MCMC) per analitzar conjuntament la relació hoste-metagenoma-fenotip. Es van establir models no recursius com a referència. L'heretabilitat del CH4 es va estimar en 0,12 ± 0,01 en ambdós models, recursius i no recursius. De la mateixa manera, les estimacions d'heretabilitat de l'abundància relativa dels tàxons es van superposar entre models i van oscil·lar entre 0,08 i 0,48. Les correlacions genètiques entre la composició microbiana i el CH4 van oscil·lar entre -0,76 i 0,65 en els models bivariables no recursius i de -0,68 a 0,69 en els models recursius. Dotze matrius de relació de microbiota (K) de diferents mètriques de distància de microbiomes, amb l'objectiu de comparar el seu rendiment dins d'un marc d'estimació de components de variància per CH4 i anàlisi de microbiomes sencers en simulació (n = 1000, 25 rèpliques) i es van realitzar dades reals , considerant quatre possibles models: un model genòmic additiu (GBLUP), un model de microbioma (MBLUP), un model d'efectes genètics i microbiomes (HBLUP) i un model d'efectes d'interacció genètics, microbiomes i genètics × microbiomes (HiBLUP). Es va definir un nou terme "Holobiabilitat" per referir-se a la proporció de la variància fenotípica atribuïble als efectes holobiont del microbioma host. Les estimacions de dades reals mitjançant HiBLUP van variar en funció de la K utilitzada i van oscil·lar entre 0,15-0,17, 0,15-0,21 i 0,42-0,59 per heretabilitat, microbiabilitat i holobiabilitat, respectivament. El conjunt de dades de microbiomes es va agregar mitjançant l'anàlisi de components principals (PCA) en pocs components principals (PC) que es van utilitzar com a proxies del metagenoma principal. Part de la variabilitat condensada en aquestes PC està controlada pel genoma de la vaca, amb estimacions d'heretabilitat per a la primera PC (PC1) de ~ 0,30 a tots els nivells taxonòmics, amb una gran probabilitat (> 83%) de la distribució posterior> 0,20 i amb un 95% més alt interval de densitat posterior (95% HPD) que no conté zero. La majoria de les estimacions de correlació genètica entre PC1 i metà eren grans (>0,70) en tots els nivells taxonòmics, amb una gran part de la distribució posterior (> 82%)> 0,50 i amb un 95% de HPD que no contenia zero. Aquests resultats suggereixen que tot el metagenoma del rumen regula recursivament les emissions de metà en vaques lleteres i que tant el CH4 com les composicions de microbiota estan parcialment controlades pel genotip de l'hoste. Les variables agregades proposades (PC) es podrien utilitzar en programes de cria d'animals per reduir les emissions de metà en les generacions futures.
[EN] The analysis of the host genetic control over its microbiota has recently been pointed out as a promising theme in different fields of study. The relationship between the host-microbiome holobiont and phenotypes in dairy cattle could lead to new insights in breeding programs. Within this Ph.D. thesis, estimation and analysis through different statistical approaches were performed aiming to unravel the host genetic control over the microbiota in dairy cattle. Besides, methane concentration trait was analyzed as a potential phenotype to be included in the Spanish dairy cattle breeding program. Higher relative abundance of most eukaryotes (mainly ciliate protozoa and fungi) and some archaea (Methanobrevibacter spp. Methanothermus spp and Methanosphera spp.) were risk factors for being classified in the high categories. a set of structural equation models (SEMs) of a recursive type within a Markov chain Monte Carlo (MCMC) framework was proposed to jointly analyze the host-metagenome-phenotype relationship. Non-recursive models were set as benchmark. Heritability of CH4 was estimated at 0.12 ± 0.01 in both, the recursive and non-recursive, models. Likewise, heritability estimates for the relative abundance of the taxa overlapped between models and ranged between 0.08 and 0.48. Genetic correlations between the microbial composition and CH4 ranged from -0.76 to 0.65 in the non-recursive bivariate models and from -0.68 to 0.69 in the recursive models. Regardless of the statistical model used, positive genetic correlations with methane were estimated consistently for the 7 genera pertaining to the Ciliophora phylum, as well as for those genera belonging to the Euryarchaeota (Methanobrevibacter sp.), Chytridiomycota (Neocallimastix sp.) and Fibrobacteres (Fibrobacter sp.) phyla. Twelve microbiota relationship matrices (K) from different microbiome distance metrics were built, aiming to compare its performance within a variance component estimation framework for CH4 and whole microbiome analysis on simulation (n = 1000, 25 replicates) and real data were performed, considering four possible models: an additive genomic model (GBLUP), a microbiome model (MBLUP), a genetic and microbiome effects model (HBLUP) and a genetic, microbiome and genetic × microbiome interaction effects model (HiBLUP). A new term "Holobiability" was defined to refer to the proportion of the phenotypic variance attributable to the host-microbiome holobiont effects. Estimates from real data using HiBLUP varied depending on the K used and ranged between 0.15-0.17, 0.15-0.21 and 0.42-0.59 for heritability, microbiability and holobiability, respectively. The microbiome dataset was aggregated through Principal Component Analysis (PCA) into few principal components (PCs) that were used as proxies of the core metagenome. Part of the variability condensed in these PCs is controlled by the cow genome, with heritability estimates for the first PC (PC1) of ~0.30 at all taxonomic levels, with a large probability (>83%) of the posterior distribution being > 0.20 and with the 95% highest posterior density interval (95%HPD) not containing zero. Most genetic correlation estimates between PC1 and methane were large (>0.70) at all taxonomic levels, with most of the posterior distribution (>82%) being >0.50 and with its 95%HPD not containing zero. These results suggest that rumen's whole metagenome recursively regulate methane emissions in dairy cows, and that both CH4 and the microbiota compositions are partially controlled by the host genotype. The purposed aggregated variables (PCs) could be used in animal breeding programs to reduce methane emissions in future generations.
This research was financed by RTA2015-00022-C03-02 (METALGEN) project from the national plan of research, development and innovation 2013-2020 and the Department of Economic Development and Competitiveness (Madrid, Spain). We thank the regional Holstein Associations and farmers collaborating in the project. Computational support from the High-Performance Computing Centre in Galicia (Spain) is acknowledged. Alejandro Saborío-Montero acknowledges the scholarship from Universidad de Costa Rica for his doctorate studies which partially conducted to the progress of this study.
Saborío Montero, A. (2021). Study of the Host Genetic Control over the Ruminal Microbiota and their Relationships with Methane Emissions in Dairy Cattle [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172633
TESIS
Compendio

INTRODUÇÃO: A doença de Chagas é causada pelo protozoário flagelado Trypanosoma cruzi (T. cruzi) e ainda hoje representa um grande problema de saúde pública tendo infectado mais de oito milhões de pessoas. A patogênese da cardiomiopatia chagásica ainda não é completamente compreendida. A inflamação no miocárdio é intensa em relação ao número de parasitos presentes e também se observa um dano progressivo em outros órgãos como esôfago e cólon em 30% a 40% dos casos em que se diagnosticou a doença. Alguns estudos começaram a mostrar que a resposta imunológica a um parasita pode depender da microbiota intestinal; porém, ainda não existem estudos com a tecnologia de sequenciamento de nova geração (NGS) que descrevam a microbiota intestinal em doença de Chagas. É possível que uma pequena alteração do peristaltismo intestinal, decorrente da infecção por T. cruzi possa alterar a colonização de algumas bactérias as quais podem causar mudanças na reatividade do sistema imune como aumentar a resposta autoimune, gerando maior dano ao coração. OBJETIVO: Este trabalho objetivou descrever a microbiota intestinal de acordo com a forma clínica da doença de Chagas, através da amplificação do gene 16s RNA ribossomal e avaliar seu papel na patogênese da doença. MÉTODO: Foram selecionados 114 indivíduos, sendo 30 portadores da forma cardíaca da doença, 11 com a forma digestiva (megacólon), 32 com a indeterminada e 31 indivíduos saudáveis (controles). De cada um deles, foram coletadas amostras de fezes para a análise da microbiota por meio de técnicas de sequenciamento de nova geração Ion Torrent. Os resultados obtidos foram analisados pelo software QIIME para determinar a população de bactérias presentes nas amostras. A análise estatística foi realizada utilizando-se os testes não paramétricos de Kruskal-Wallis e Mann-Whitney U-test. RESULTADOS: A frequência relativa do filo Verucomicrobia foi significantemente menor no grupo cardíaco em comparação ao grupo controle e as outras formas clínicas: indeterminada e digestiva. Apesar da abundância relativa desse filo ser menor do que 1%, a diferença observada se manteve significante, mesmo após a correção de Bonferroni. CONCLUSÕES: Nosso estudo sugere que uma menor proporção do filo Verrucomicrobia possa estar relacionada ao processo inflamatório na forma cardíaca; porém, ainda pouco se conhece sobre este grupo de bactérias e seus componentes, que nos permita afirmar o seu efetivo papel na forma cardíaca da doença de Chagas
INTRODUCTION: Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi (T.cruzi) and still represents a major public health problem with more than eight million people infected. Chagas cardiomyopathy pathogenesis is still not completely understood. Inflammation in the myocardium is intense in relation to the number of parasites present and progressive damage is also observed in other organs such as the esophagus and colon in 30% to 40% of the cases. Some studies are beginning to show that the immune response to a parasite may depend on the intestinal microbiota. However, there are no studies using NGS technology that describes the intestinal microbiota of Chagas disease. It is possible that a small change in intestinal peristalsis due to T cruzi infection may alter the colonization of some bacteria. These changes could cause changes in the reactivity of the immune system such as increasing the autoimmune response causing greater damage to the heart. OBJECTIVE: This study aimed to describe the intestinal microbiota according to the clinical form of Chagas disease, through amplification of the 16s ribosomal RNA gene and to evaluate its role in the pathogenesis of the disease. METHODS: A total of 114 individuals were selected, 30 of cardiac form of the disease, 11 with the digestive form (megacolon), 32 with indeterminate form and 31 healthy individuals (controls). Stool samples were collected and analysed for the microbiota using Ion Torrent sequencing technique. The results were analyzed by the QIIME software to determine the population of bacteria present in the samples. Statistical was performed using Kruskal-Wallis non-parametric test and Mann-Whitney U-test. RESULTS: The relative frequency of the Verrucomicrobia phylum was significantly lower among the cardiac group when compared to control, indeterminate and digestive form. Our study suggest that the phylum Verrucomicrobia may play a role in the miocardio inflammation process in Chagas disease, however little is known about these bacteria to infer the mechanism

Until recently it was assumed that the healthy mammalian lung did not harbour a microbiota, unlike other body sites. However, through the use of sequencing based technologies this has been shown to not be the case. Low biomass communities of microbes can be identified in the healthy lung and the lung microbiota in various diseases states has been shown to differ form these 'healthy' communities. The sheep respiratory microbiota is of interest from both an animal health perspective and due to the potential use of the sheep as a large animal model for studying the lung microbiota. In this thesis I seek to characterise the composition and variability of the sheep lung microbiota; the differences between the sheep upper and lower respiratory tract bacterial communities and to assess whether exhaled breath condensate collection can be used as a non-invasive lung microbiota sampling method. To study the bacterial communities present in samples I have used 16S rRNA gene sequencing and analysis. In Chapter 3 I examine the inter-individual and spatial variability present within the sheep lung microbiota. Protected specimen brushings were collected from three lung segments in six animals at three time-points. In a separate sheep a greater number of brushings was taken (n=16) in order to examine the amount of variability over a smaller spatial scale. I find that there can be large differences between the bacterial communities isolated from different locations within the lung, even over short distances. Samples also cluster by the sheep from which they were taken, indicating a host specific influence on the lung microbiota. In Chapter 4 I compare whole lung washes and oropharyngeal swabs from 40 lambs in order to examine the differences between the upper and lower respiratory tract microbiotas. I find that oropharyngeal swabs separate into rumen-like or upper respiratory tract-like bacterial communities. Despite the fact that in humans the upper and lower respiratory microbiotas have been shown to have similar compositions, the sheep lung microbiota samples in this study do not resemble either oropharyngeal samples or reagent only controls. In my first two results chapters, lung sampling methods were used which involved either anaesthesia combined with a bronchoscopic procedure (Chapter 3) or samples being taken from dead animals (Chapter 4). In Chapter 5 I assess whether there is a less invasive way of taking lung microbiota samples from a living individual, both to minimise the procedural stress on animals used as models and to increase the pool of potential volunteers for human lung microbiota studies. I compared samples taken via protected specimen brushings to samples taken via exhaled breath condensate collection, a less invasive sampling technique. I find that condensate samples contain less bacterial DNA and different bacteria than brushing samples, indicating that it is unlikely they could be used as a replacement for invasive sampling methods. In my final results chapter I compare the results across Chapters 3, 4 and 5 to identify bacteria which occur consistently in the sheep lung and could therefore potentially be described as core lung microbiota members. In conclusion, while I have found that there are large differences between the sheep lung microbiota and that which has previously been described in humans, the sheep can still be of use as a model in studies where these differences would not have a significant impact, such as in Chapter 5 of this thesis. I have identified several bacterial members of the core sheep lung microbiota which in future it would be interesting to better characterise and to assess whether they play a role in sheep health.

Thesis advisor: Catherine Y. Read
Background. Clostridium difficile infections (CDI) account for 20-30% of healthcare-acquired infections, resulting in serious patient and economic burdens. CDI incidence has grown rapidly due to overuse of antibiotics and an aging population, posing a significant public health threat. Fecal microbiota transplantation (FMT) using donor stool has demonstrated clinical efficacy rates up to 94% and long-term restoration of a healthy intestinal microbiome. Challenges with donor screening, lack of research about optimal stool donor characteristics and intestinal microbiome composition, and a poorly fit screening model, create barriers to the availability of FMT. Purpose. This study aimed to generate essential information about FMT donor characteristics predictive of passing the screening and donor intestinal microbiome compositions associated with FMT clinical efficacy. The primary aims were to 1) identify previously unstudied characteristics of prospective FMT donors that are predictive of passing a stool bank’s screening process; and 2) determine whether donor intestinal microbial diversity is related to FMT clinical efficacy in preventing recurrent CDI. Methods. This study was conducted as a secondary analysis on a cohort of previously screened donors (n=770). Aim 1 was tested through a logistic regression of donor characteristics (gender, age, body mass index, frequency of bowel movements, diet, tobacco and alcohol use, and seasonality) with screening outcomes. Aim 2 was tested through a simple regression evaluating donor intestinal microbial diversity and rates of FMT clinical efficacy. Results. One donor characteristic in the logistic regression, frequency of bowel movements (p = 0.018), was significantly predictive of whether a donor passed the screening. Specifically, donors who had fewer than two bowel movements per day were more likely to pass. All other characteristics were not predictive. Similarly, the linear regression evaluating alpha diversity and FMT clinical efficacy was not significantly predictive of clinical efficacy (p = 0.140). Conclusion. Findings were used to support recommendations for improving prospective donor screening that nurses and other clinicians can implement to decrease challenging logistics, reduce costs and barriers, and potentially increase FMT clinical efficacy
Thesis (PhD) — Boston College, 2019
Submitted to: Boston College. Connell School of Nursing
Discipline: Nursing

The human gut harbours a vast diversity of microbial cells, collectively known as the gut microbiota, that are crucial for human health and dysfunctional in many of the most prevalent chronic diseases. Until recently culture dependent methods limited our ability to study the microbiota in depth including the collective genomes of the microbiota, the microbiome. Advances in culture independent metagenomic sequencing technologies have since provided new insights into the microbiome and lead to a rapid expansion of data rich resources for microbiome research. These high throughput sequencing methods and large datasets provide new opportunities for research with an emphasis on bioinformatics analyses and a novel field for drug discovery through data mining. In this thesis I explore a range of metagenomics analyses to extract insights from metagenomics data and inform drug discovery in the microbiota. Firstly I survey the existing technologies and data sources available for data mining therapeutic targets. Then I analyse 16S metagenomics data combined with metabolite data from mice to investigate the treatment model of a proposed antibiotic treatment targetting the microbiota. Then I investigate the occurence frequency and diversity of proteases in metagenomics data in order to inform understanding of host-microbiota-diet interactions through protein and peptide associated glycan degradation by the gut microbiota. Finally I develop a system to facilitate the process of integrating metagenomics data for gene annotations. One of the main challenges in leveraging the scale of data availability in microbiome research is managing the data resources from microbiome studies. Through a series of analytical studies I used metagenomics data to identify community trends, to demonstrate therapeutic interventions and to do a wide scale screen for proteases that are central to human-microbiota interactions. These studies articulated the requirement for a computational framework to integrate and access metagenomics data in a reproducible way using a scalable data store. The thesis concludes explaining how data integration in microbiome research is needed to provide the insights into metagenomics data that are required for drug discovery.

A estase de secreção salivar e alimentos deglutidos na luz esofágica de pacientes com megaesôfago chagásico traz como consequências: (1) supercrescimento bacteriano na luz do órgão, (2) episódios de aspiração pulmonar e infecções respiratórias de repetição, (3) aumento do risco dos procedimentos terapêuticos cirúrgicos ou endoscópicos em caso de perfuração pela maior possibilidade de contaminação, (4) desenvolvimento de processos inflamatórios crônicos na mucosa esofágica, que podem predispor ao aparecimento de displasia e câncer. Apesar disto, a microbita esofágica no megaesôfago nunca foi estudado. Esse estudo teve o objetivo de analisar qualitativa e quantitativamente a microbiota presente no líquido de estase esofágico de pacientes portadores de megaesôfago chagásico, comparando-a com a existente em indivíduos sadios. Foram estudados prospectivamente 25 pacientes (10 homens e 15 mulheres) com idades variando de 24 a 74 anos (  = 49,1a). Quinze pacientes eram portadores de esofagopatia chagásica, sendo 5 portadores de mega grau I (MG1), 5 portadores de mega grau II (MG2) e 5 portadores de mega grau III (MG3), segundo a classificação de Rezende; e 10 indivíduos sadios, agrupados no Grupo Controle (GC). Utilizou-se método de coleta que permitia aspiração de líquido através de sonda de Levine diretamente da luz esofágica, evitando-se a contaminação com microrganismos da orofaringe. Após análise microbiológica qualitativa e quantitativa, foi feita a descrição dos microrganismos encontrados nos vários grupos e sua classificação em aeróbios Gram positivos, aeróbios Gram negativos, anaeróbios e fungos. A análise estatística visou avaliar diferenças quantitativas entre os microrganismos nos diferentes grupos, sendo para tanto utilizado o teste não paramétrico de Kruskal-Wallis, com nível de rejeição menor 0,05 (5%). A positividade das culturas no Grupo Controle foi 40%, com predomínio do gênero Streptococcus sp, em concentrações que variaram de 101 a 102 ufc/ml. No Grupo Megaesôfago 93,3% da culturas foram positivas, com grande variedade de bactérias, mas predomínio de aeróbios Gram positivos (Streptococcus sp. foi o mais comum) e anaeróbios (Veillonella sp foi a mais freqüente) em concentrações que variaram de 101 a 105 ufc/ml. As concentrações foram geralmente mais elevadas em MG3, quando comparado com MG1, MG2 e GC (p<0,05). Concluiu-se que no megoesôfago, diferentemente dos indivíduos sadios, existe a presença de rica microflora bacteriana, constituída principalmente por aeróbios Gram positivos e anaeróbios, em concentrações tanto maiores quanto maior o seu grau de dilatação. Parte desta microbiota tem capacidade de metabolizar nitratos, etapa importante na formação de nitrosaminas.
The stasis of saliva and swallowed food in the esophageal lumen of patients with chagasic megaesophagus causes: (1) bacterial overgrowth in the esophageal lumen, (2) recurring pulmonary aspirations and respiratory infections, (3) increased risk of surgical or endoscopic procedures if perforation occurs by the major possibility of contamination, and (4) the development of chronic inflammatory process in esophageal mucosa, that can predispose to the development of dysplasia and cancer. In spite of this, esophageal microbiota in the megaesophagus has never been studied. The aim of this study was to analyze qualitatively and quantitatively the microbiota in chagasic megaesophagus in comparison to the normal esophagus. Twenty-five patients (10 men and 15 women) were prospectively studied, with ages varying from 24 to 74 years (=49,1), from March to September 2000. Fifteen patients with chagasic megaesophagus (MG), were divided into three sub- groups according to the grade of esophageal dilation: MG1 – 5 patients with megaesophagus grade I; MG2- 5 patients with megaesophagus grade II; MG3- 5 patients with megaesophagus grade III. Another group of ten patients without any esophageal disease was constituted in the Control Group (CG). The sample collection was performed using a method specially developed to avoid contamination with microorganisms of the oral cavity and oropharynx. After qualitative and quantitative analysis, the microorganisms found were described and classified as Gram positive aerobes, Gram negative aerobes, anaerobes and fungus. Statistical analysis using Kruskal-Wallis non-parametric test was performed in order to find quantitative differences of microorganisms in the different groups. In CG 40% of the cultures were positive with predominance of the genus Streptococcus sp, in concentrations that varied from 101 to 102 cfu/ml. In MG, 93,3% of the cultures were positive, with great bacterial variability and predominance of a variety of aerobic Gram-positive (Streptococcus sp was the most common) and anaerobic bacteria (Veillonella sp was the most frequent), in concentrations that varied from 101 to 105 cfu/ml. The bacterial concentrations were generally more elevated in MG3 in comparison to MG1, MG2 and CG (p<0,05). It was concluded that patients with megaesophagus present a varied microbiota constituted mostly of aerobic Gram positive and anaerobic bacteria, in concentrations that vary with the megaesophagus dilatation degree. Some of the bacteria found in MG are able to metabolize nitrates intro nitrites, an important step in the formation of nitrosamines.

Studying the microbiome in natural populations could improve our understanding of the biological factors that influence microbiome variation. If host genetic variation is important in microbiota assembly, then understanding genetic divergence among natural populations could be informative. Despite advances in sequencing technology, we have not yet taken full advantage of this technology in natural populations. Here we integrate genome-wide population genomic and microbiome analyses in wild threespine stickleback (Gasterosteus aculeatus) fish distributed throughout western Oregon, USA. We found that gut microbiome varied in diversity and composition more among than within wild host populations. Furthermore, this among population variation was better explained by host population genetic divergence than by environment and geography. We also identified a subset of gut microbial taxa that were most strongly sorted both across environments and across genetically divergent populations. We believe this study contributes generalizable methods and findings in host systems. This thesis includes supplemental materials.
2021-04-30

The human gut microbiota plays a crucial role in the functioning of the gastrointestinal tract and its alteration can lead to gastrointestinal abnormalities and inflammation. Additionally, the gut microbiota modulates central nervous system (CNS) activities affecting several aspect of host physiology. Motivated by the increasing evidences of the role of the gut microbiota in the complex set of interactions connecting the gut and the CNS, known as gut-brain axis, in this Ph.D. thesis we asked whether the gastrointestinal abnormalities and inflammation commonly associated with neurological disorders such as Rett syndrome (RTT) and Autism could be related to alterations of the bacterial and fungal intestinal microbiota. First, since only few reports have explored the fungal component of the gut microbiota in health and disease, we characterized the gut mycobiota in a cohort of healthy individuals, in order to reduce the gap of knowledge concerning factors influencing the intestinal microbial communities. Next, we compared the gut microbiota of three cohorts of healthy, RTT and autistic subjects to investigate if these neurological disorders harbour alterations of the gut microbiota. Culture-based and metataxonomics analysis of the faecal fungal populations of healthy volunteers revealed that the gut mycobiota differs in function of individuals’ life stage in a gender-related fashion. Different fungal species were isolated showing phenotypic adaptation to the intestinal environment. High frequency of azoles resistance was also found, with potential clinical significance. It was further observed that autistic subjects are characterized by a reduced incidence of Bacteroidetes and that Collinsella, Corynebacterium, Dorea and Lactobacillus were the taxa predominating in the gut microbiota of autistic subjects. Constipation has been associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by higher levels of Escherichia/Shigella and Clostridium cluster XVIII than constipated neurotypical subjects. RTT is a neurological disorder caused by loss-of-function mutations of MeCP2 and it is commonly associated with gastrointestinal dysfunctions and constipation. We showed that RTT subjects harbour bacterial and fungal microbiota altered from those of healthy controls, with a reduced microbial richness and dominated by Bifidobacterium, different Clostridia and Candida. The alterations of the gut microbiota observed did not depend on the constipation status of RTT subjects while this microbiota produced altered SCFAs profiles potentially contributing to the constipation itself. Phenotypical and immunological characterizations of faecal fungal isolates from RTT subjects showed Candida parapsilosis as the most abundant species isolated in RTT, genetically unrelated to healthy controls’ isolates and with elevated resistance to azoles. Furthermore these isolates induced high levels of IL-10 suggesting increased tolerance and persistence within the host. Finally, the importance of multiple sequence alignment (MSA) accuracy in microbiome research was investigated comparing three implementations of the widely used NAST algorithm. By now, different implementations of NAST have been developed but no one tested the performances and the accuracy of the MSAs generated with these implementations. We showed that micca, a new bioinformatics pipeline for metataxonomics data improves the quality of NAST alignments by using a fast and memory efficient reimplementation of the NAST algorithm.

The human gut microbiota plays a crucial role in the functioning of the gastrointestinal tract and its alteration can lead to gastrointestinal abnormalities and inflammation. Additionally, the gut microbiota modulates central nervous system (CNS) activities affecting several aspect of host physiology. Motivated by the increasing evidences of the role of the gut microbiota in the complex set of interactions connecting the gut and the CNS, known as gut-brain axis, in this Ph.D. thesis we asked whether the gastrointestinal abnormalities and inflammation commonly associated with neurological disorders such as Rett syndrome (RTT) and Autism could be related to alterations of the bacterial and fungal intestinal microbiota. First, since only few reports have explored the fungal component of the gut microbiota in health and disease, we characterized the gut mycobiota in a cohort of healthy individuals, in order to reduce the gap of knowledge concerning factors influencing the intestinal microbial communities. Next, we compared the gut microbiota of three cohorts of healthy, RTT and autistic subjects to investigate if these neurological disorders harbour alterations of the gut microbiota. Culture-based and metataxonomics analysis of the faecal fungal populations of healthy volunteers revealed that the gut mycobiota differs in function of individuals’ life stage in a gender-related fashion. Different fungal species were isolated showing phenotypic adaptation to the intestinal environment. High frequency of azoles resistance was also found, with potential clinical significance. It was further observed that autistic subjects are characterized by a reduced incidence of Bacteroidetes and that Collinsella, Corynebacterium, Dorea and Lactobacillus were the taxa predominating in the gut microbiota of autistic subjects. Constipation has been associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by higher levels of Escherichia/Shigella and Clostridium cluster XVIII than constipated neurotypical subjects. RTT is a neurological disorder caused by loss-of-function mutations of MeCP2 and it is commonly associated with gastrointestinal dysfunctions and constipation. We showed that RTT subjects harbour bacterial and fungal microbiota altered from those of healthy controls, with a reduced microbial richness and dominated by Bifidobacterium, different Clostridia and Candida. The alterations of the gut microbiota observed did not depend on the constipation status of RTT subjects while this microbiota produced altered SCFAs profiles potentially contributing to the constipation itself. Phenotypical and immunological characterizations of faecal fungal isolates from RTT subjects showed Candida parapsilosis as the most abundant species isolated in RTT, genetically unrelated to healthy controls’ isolates and with elevated resistance to azoles. Furthermore these isolates induced high levels of IL-10 suggesting increased tolerance and persistence within the host. Finally, the importance of multiple sequence alignment (MSA) accuracy in microbiome research was investigated comparing three implementations of the widely used NAST algorithm. By now, different implementations of NAST have been developed but no one tested the performances and the accuracy of the MSAs generated with these implementations. We showed that micca, a new bioinformatics pipeline for metataxonomics data improves the quality of NAST alignments by using a fast and memory efficient reimplementation of the NAST algorithm.

The human gut microbiota plays a crucial role in the functioning of the gastrointestinal tract and its alteration can lead to gastrointestinal abnormalities and inflammation. Additionally, the gut microbiota modulates central nervous system (CNS) activities affecting several aspect of host physiology. Motivated by the increasing evidences of the role of the gut microbiota in the complex set of interactions connecting the gut and the CNS, known as gut-brain axis, in this Ph.D. thesis we asked whether the gastrointestinal abnormalities and inflammation commonly associated with neurological disorders such as Rett syndrome (RTT) and Autism could be related to alterations of the bacterial and fungal intestinal microbiota. First, since only few reports have explored the fungal component of the gut microbiota in health and disease, we characterized the gut mycobiota in a cohort of healthy individuals, in order to reduce the gap of knowledge concerning factors influencing the intestinal microbial communities. Next, we compared the gut microbiota of three cohorts of healthy, RTT and autistic subjects to investigate if these neurological disorders harbour alterations of the gut microbiota. Culture-based and metataxonomics analysis of the faecal fungal populations of healthy volunteers revealed that the gut mycobiota differs in function of individuals’ life stage in a gender-related fashion. Different fungal species were isolated showing phenotypic adaptation to the intestinal environment. High frequency of azoles resistance was also found, with potential clinical significance. It was further observed that autistic subjects are characterized by a reduced incidence of Bacteroidetes and that Collinsella, Corynebacterium, Dorea and Lactobacillus were the taxa predominating in the gut microbiota of autistic subjects. Constipation has been associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by higher levels of Escherichia/Shigella and Clostridium cluster XVIII than constipated neurotypical subjects. RTT is a neurological disorder caused by loss-of-function mutations of MeCP2 and it is commonly associated with gastrointestinal dysfunctions and constipation. We showed that RTT subjects harbour bacterial and fungal microbiota altered from those of healthy controls, with a reduced microbial richness and dominated by Bifidobacterium, different Clostridia and Candida. The alterations of the gut microbiota observed did not depend on the constipation status of RTT subjects while this microbiota produced altered SCFAs profiles potentially contributing to the constipation itself. Phenotypical and immunological characterizations of faecal fungal isolates from RTT subjects showed Candida parapsilosis as the most abundant species isolated in RTT, genetically unrelated to healthy controls’ isolates and with elevated resistance to azoles. Furthermore these isolates induced high levels of IL-10 suggesting increased tolerance and persistence within the host. Finally, the importance of multiple sequence alignment (MSA) accuracy in microbiome research was investigated comparing three implementations of the widely used NAST algorithm. By now, different implementations of NAST have been developed but no one tested the performances and the accuracy of the MSAs generated with these implementations. We showed that micca, a new bioinformatics pipeline for metataxonomics data improves the quality of NAST alignments by using a fast and memory efficient reimplementation of the NAST algorithm.

This study investigated the microbial colonization and maternal influences on the neonatal calf gut microbiome. Microbiome samples were collected from dams (n = 6) and calves (n = 6) using sterile flocked swabs. The vaginal, oral, and fecal bacterial communities were examined from the dam and the fecal community of calves was examined from birth to 60 d of age. Microbial communities varied by anatomical location and age of the calf. Metagenomic analysis 16s ribosomal DNA revealed ten phyla associated with microbiomes of the dam and the same ten phyla associated with calf feces at various time points: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, Verrucomicrobia, Spirochaetes, Tenericutes, Fibrobacteres, and Lentisphaerae. Overall, the calf meconium and fecal microbiome is influenced by a combination of the maternal vagina, oral, and fecal microbiomes. Further studies will be needed to identify the transference mechanisms of maternal microbes to offspring and the associated host-microbial interactions.
Master of Science in Life Sciences
This study investigated the microbial colonization and maternal influences on the neonatal calf gut microbiome. Microbiome samples were collected from dams (n = 6) and calves (n = 6) using sterile flocked swabs. The vaginal, oral, and fecal bacterial communities were examined from the dam and the fecal community of calves was examined from birth to 60 d of age. Microbial communities varied by anatomical location and age of the calf. Metagenomic analysis 16s ribosomal DNA revealed ten phyla associated with microbiomes of the dam and the same ten phyla associated with calf feces at various time points: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, Verrucomicrobia, Spirochaetes, Tenericutes, Fibrobacteres, and Lentisphaerae. Overall, the calf meconium and fecal microbiome is influenced by a combination of the maternal vagina, oral, and fecal microbiomes. Further studies will be needed to identify the transference mechanisms of maternal microbes to offspring and the associated host-microbial interactions.

Remyelination describes the regeneration of myelin sheaths, and is considered one of the most promising strategies for improving the prognosis of demyelinating diseases such as multiple sclerosis. Data from animal models and human studies have shown that remyelination can occur extensively in the central nervous system (CNS), leading to functional recovery and axonal protection. However, remyelination does not always proceed to completion, and its failure is associated with progressive neurological disability. Thus, there is clinical need for interventions that can optimise the conditions for remyelination. Recent advances in genomics and animal husbandry have kindled an interest in the microbiome as a means to influence processes throughout the body. Our commensal microbes communicate with host cells at epithelial barriers, stimulate neural and endocrine axes and directly produce a plethora of long-range signalling molecules. Critically, the development and maintenance of the immune system depend on signals from the microbiota, and we know that a well-coordinated immune response is a key determinant of the success of remyelination. This thesis explores how the microbiome can influence CNS remyelination. To do so, I have studied remyelination in three murine models of microbiome alteration. Firstly, long-term oral administration of an antibiotic cocktail was used to deplete the microbiota of adult mice. Following focal demyelination, these mice had deficits in their inflammatory response, clearance of myelin debris and differentiation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs). Faecal microbial transplant was able to rescue aspects of the inflammatory response and phagocytosis, but not OPC differentiation. Secondly, I looked at remyelination in germ-free (GF) mice following cuprizone-induced demyelina- tion. As with the antibiotics-treated mice, there were deficits in inflammation following demyelination, which tended to peak later than in control mice. Finally, I investigated the potential of a therapeutic probiotic (VSL#3) to improve remyelination in aged mice. In contrast to antibiotic treatment, probiotic administration caused a slight enhancement in the onset of inflammation following focal demyelination. However, there was no significant improvement in OPC differentiation or toluidine blue rank analysis, suggesting these changes in inflammation were not sufficient to positively modulate remyelination. The results from these three studies introduce a significant but previously unconsidered environmental influence on remyelination in the CNS. Whilst the effects are subtle relative to more direct interventions, the microbiome can be manipulated simply and non-invasively, which may provide a useful adjunct to other strategies for optimising remyelination.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
The human gastrointestinal tract is home to a dense and dynamic microbial community. Recent advancements in sequencing technology have revealed numerous relationships between the composition of these communities and human and health and disease. In some cases, researchers have shown causal relationships between the presence or absence of particular microorganisms and disease. These findings offer promise for using microorganisms or microbial communities to modulate health and disease, but to date, we lack tools and mechanistic insight needed for rational engineering of these communities. Understanding how microorganisms enter, colonize, grow, and disperse to new hosts present key considerations for rational engineering of the human gastrointestinal tract. In this thesis, I use experimental studies of the human and murine gastrointestinal tract to address these considerations. In the first study, I examined endospores and other resistant cell types in the gastrointestinal communities of unrelated humans to identify the ecological role of these states in the distribution of bacterial populations in healthy people. I used this information to infer shared roles for these organisms in successional states in the human gut, and identify host- and diet-derived metabolites as environmental signals mediating the growth and colonization of these organisms. In the second study, I examined the potential for using targeted manipulations of diet to favor selective growth and colonization by an introduced bacterium in the murine gastrointestinal tract. I showed that resource exclusivity of this bacterium permits its selective expansion in this environment, and negatively impacts the growth of other commensals. Central to the goal of rational engineering of the gut microbiota, these studies reveal ecological considerations that may promote or inhibit colonization by introduced commensals in this complex ecosystem. This work invites provides a conceptual framework for integrating systems microbial ecology with engineering design to manipulate the composition of the gastrointestinal microbiota.
by Sean M. Kearney.
Ph. D.

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

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

For the majority of patients with end-stage kidney disease, kidney transplantation remains the optimal treatment modality, offering superior survival and quality of life at a fraction of the cost of dialysis. However, the requirement for long-term immunosuppression increases the susceptibility of transplant recipients to metabolic complications, infection, cancer, and cardiovascular disease, meaning that a “normal” lifespan is rarely, if ever achieved. Innovative strategies are needed to reduce the burden of current immunosuppressive regimens and to maintain the balance between immunosuppression and the maintenance of protective immunity. The gut microbiome is a critical determinant of human health and modulates host immunity and metabolism through a number of recognised mechanisms, and likely others as yet unrecognised. In transplantation, the gut microbiome offers a novel target to alleviate the deleterious immune and metabolic responses that result from exposure to alloantigen and long-term immunosuppression. Herein, we show for the first time, that dietary modification of the gut microbiome can prevent allograft rejection in a murine model of kidney transplantation through signalling via GPR43 and mediated by the action of T regulatory cells. The aims of this thesis were to investigate the role of the gut microbiome in mediating immune responses in kidney transplantation, and to assess strategies to measure glycaemic risk (pre-transplant) and status (post-transplant). This thesis comprises a pre-clinical model of kidney transplantation and translational studies in the clinic, and is presented as a collection of published and submitted works.

Orientador: Márcio Garcia Ribeiro
Resumo: O estreitamento da relação entre tutores e animais de companhia, nas últimas décadas, aumentou consideravelmente o risco de transmissão de patógenos dos animais para os humanos. A microbiota da cavidade oral de animais de companhia é polimicrobiana e estes agentes podem potencialmente infectar humanos pelas mordeduras ou contato direto com mucosas ou feridas de pele. No entanto, são escassas as informações sobre a identificação destes micro-organismos por técnicas moleculares (microbioma, proteômica). Ainda, o perfil de sensibilidade microbiana in vitro da microbiota bacteriana bucal de cães e a etiologia dos agentes envolvidos em mordeduras em humanos não são completamente elucidados, posto que muitos cães são errantes e evadem após a agressão. Com efeito, o presente estudo investigou a presença de agentes de origem bacteriana e fúngica na cavidade oral de 100 cães hígidos por técnicas de cultivo microbiano convencional, sequenciamento genético em larga escala (microbioma) e espectrometria de massas (MALDI-TOF MS), bem como investigou o perfil de sensibilidade/resistência in vitro dos isolados. Foram identificados 213 micro-organismos de origem bacteriana e 20 de origem fúngica. Os agentes bacterianos mais prevalentes no diagnóstico microbiológico e espectrometria de massas foram Staphylococcus pseudintermedius (40/100=40%), Streptococcus α-hemolítico (37/100=37%) e Pasteurella stomatis (22/100=22%). O gênero de fungo mais prevalente foi Aspergillus (10/100=10%). Imipenem (2... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The close relationship between humans and companion animals in recent decades has strongly increased the risk of transmission of pathogens from pets-to-humans. The microbiota of the oral cavity from companion animals is polymicrobial and these agents may potentially infect humans through bites or by direct contact with mucous membranes or cutaneous lesions. Nonetheless, the identification of these microorganisms by molecular techniques (microbiome, proteomics) is scarce. Besides, the in vitro microbial susceptibility pattern of oral bacterial microbiota from dogs and the etiology of agents involved in human bites are not fully understood, since many dogs are homeless and/or evade after aggression. The present study investigated the presence of bacterial and fungal agents in the oral cavity of 100 healthy dogs based on conventional microbiological culture, large-scale DNA sequencing (microbiome), and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In vitro antimicrobial susceptibility profile of the isolates was assessed as well. A total of 213 bacterial and 20 fungal microorganisms were identified. The most prevalent bacterial agents diagnosed by microbiological culture and mass spectrometry were Staphylococcus pseudintermedius (40/100=40%), α-hemolytic Streptococcus (37/100=37%), and Pasteurella stomatis (22/100=22%), whereas the most common genus of fungi was Aspergillus (10/100=10%). Imipenem (207/213=97.2%), ceftiofur (196/213=... (Complete abstract click electronic access below)
Mestre

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Funep
Monsanto
Avaliou-se o efeito de oito herbicidas em duas concentrações (2 e 10 vezes a dose media recomendada por hectare) sobre a microbiota de solo. Os herbicidas (bentazon, metolachlor, trifluralin, imazethapyr, imazethapyr+lactofen, haloxyfop-methyl, glyphosate e chlorimuron-ethyl) foram selecionados em função dos resultados de um estudo prévio. Como bioindicadores de atividade se utilizou: respiração microbiana, quantificando-se a emissão de CO2 aos 2, 4, 8, 12, 16, 20, e 24 dias de incubação, mineralização de nitrogênio, atividade da enzima desidrogenase e a hidrólise do diacetato de fluoresceína (FDA), aos 8 e 28 dias. Também se estimou diversidade microbiana. Bentazon e a mistura de imazethapyr+lactofen, na maior concentração, e o haloxyfop-methyl nas duas concentrações, apresentaram efeitos inibitórios na respiração microbiana, embora diferentes em época e duração do efeito. Na mineralização de nitrogênio não foi possível detectar efeitos dos tratamentos. Nenhum dos tratamentos herbicidas afetou a hidrólise do FDA. A atividade da desidrogenase mostrou comportamento variável aos 8 e 28 dias, com resultados de inibição e de estimulo. Somente o herbicida metolachlor 10x causou inibição na quantidade de fungos, os restantes efeitos detectados resultaram em incrementos dos microrganismos. A única correlação significativa encontrada foi entre a atividade da desidrogenase e a respiração basal aos oito dias de incubação.
The effects of eight herbicides on soil microorganism activity were evaluated at two concentrations: 2x and 10x the recommended doses for each product in soybean. These herbicides were selected through the results of a prior experiment were 17 herbicides were tested. The selected herbicides were: bentazon, metolachlor, trifluralin, imazethapyr, imazethapyr+lactofen, haloxyfop-methyl, glyphosate e chlorimuron-ethyl. To study the microorganism activity the following parameters were measured: CO2 soil production until 28 days of incubation, nitrogen mineralization rate, dehydrogenase and FDA activities, at 8 and 28 days of incubation. The functional microbe diversity also was investigated. Bentazon (10x) and the mix imazethapyr+lactofen (10x) and haloxyfopmethyl at both concentrations, reduced the CO2 production, although with differences in timing and duration. No effects of the herbicides could be detected on nitrogen mineralization or FDA hydrolyses. Variable effects involving inhibition or stimulation were detected in the dehydrogenase activity according on herbicide, concentration and period of incubation. Only metolachlor (10x) had inhibition effects on the soil fungi population; the other herbicides promoted increases on microorganism populations. Only dehydrogenase activity and soil respiration at 8 days correlated significatively.

This thesis has addressed both the possibility of a potential pathogenic organism, or a shift in the mucosal microbial community, associated with UC. Firstly by developing a dual molecular technique to detect Helicobacteraceae species in colonoscopic biopsies from the human colon alongside attempting to culture these organisms and secondly by assessing the mucosal colonic microbiota in both detail by the construction of phylogenetic clone libraries and broadly by comparison of DGGE profiles between inflamed and uninvolved mucosa during active disease. The dual molecular approach demonstrated that enterohepatic Helicobacter prevalence was significantly higher in the UC cohort compared to controls (30 of 77 versus 2 of 59, p<0.0001). These findings suggest that these species, for which there is significant evidence from animal models for initiation of colonic inflammation, warrant consideration as potential pathogenic entities in UC. Detailed assessment of the colonic mucosal microbial community by the construction of clone libraries in this thesis revealed a wide variation in the constituents of the microbiota between individuals making comparisons between groups difficult with the small numbers of subjects. Comparison of DGGE profiles between inflamed and uninvolved mucosa indicated differences in only 12 of the 36 subjects. Analysis to identify factors associated with this showed a statistically significantly relationship with no bowel preparation prior to the colonoscopy, 5ASA therapy, and antibiotic treatment but not with mucosal state or extent of disease, suggesting that it is not the inflammation itself that results in the microbial shifts seen between the inflamed and uninvolved mucosa in active UC.

Quaternary amines such as choline and carnitine are essential nutrients for humans supplied from daily food; however, quaternary amines metabolism by gut microbiota can lead to the development of various diseases, including non-alcoholic fatty liver disease and cardiovascular disease. It is hypothesized that both diseases are promoted by microbial catabolism of choline and carnitine to trimethylamine (TMA). Proteus mirabilis is a Gram-negative gut proteobacterium, which can metabolize choline anaerobically to form TMA. I demonstrated that the identified cutC gene is essential for choline degradation and subsequent TMA production in this bacterium. Using P. mirabilis as the model, I investigated the physiological role of quaternary amine metabolism from the bacterial perspective and demonstrated that P. mirabilis can rapidly uptake and degrade choline to enhance growth rate, cell yield and swarming speed under anaerobic and microaerophilic conditions. I also provide the first evidence of a novel choline-metabolizing microcompartment, which is present in both vegetative and swarming cells supplemented with choline. Another important dietary source of TMA in human gut is carnitine. I used two model proteobacteria Acinetobacter baumannii and Escherichia coli in this project to investigate the role of carnitine metabolism to TMA in health and disease. A. baumannii and E. coli can use carnitine as a growth substrate to produce TMA. To better understand the role of quaternary amine metabolism in host health and disease, I used Caenorhabditis elegans model to investigate carnitine metabolism on the life span of the worm. My data suggest that malate, the degradation product of carnitine, extends the life span of C. elegans fed on A. baumannii or E. coli. Together, my study reveals that choline and carnitine metabolism as an adaptation strategy for gut proteobacteria and contributes to better understand the ecology of these TMA-forming gut bacteria in health and disease.

Orientador: Vanderlei Perez Canhos
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Para o levantamento da microbiota em amostras de diferentes etapas da produção de polvilho azedo, foram utilizados os meios Plate Count Agar (PCA) e Man Rogosa e Sharpe Agar (MRSA) em condições de aerobiose e anaerobiose. As contagens totais dos microrganismos aeróbios e anaeróbios não foram alteradas durante a fermentação da fécula, demonstrando que ocorre uma mudança no tipo de microbiota, devido ao decréscimo do pH. Foram selecionadas 590 culturas das quais 23 (3,9%) perderam a viabilidade de crescimento, sendo portanto identificados 567 isolados do quais 18 (3,1 %) eram bactérias gram-negativo; 13 (2,2%) leveduras; 46 (7,8%) bactérias dos gêneros Bacillus e Corynebacterium; 14 (2,4%) Staphylococcus e Micrococcus como contaminantes do processo, e 476 (80,6%) como bactérias ácido-láticas, mostrando predominância deste último grupo. A identificação dos isolados foi feita através de matrizes de similaridade colocadas em um programa de computação. A água utilizada no processo também foi analisada, onde o número de aeróbios mesófilos foi de 1,1 x 103/ml; 9,5 x 103/l00ml de coliformes totais e 2,5 x 103/100ml de coliformes fecais. O gênero Lactobacillus foi predominante no processo, com 189 isolados (32,0%), seguido por Leuconostoc (21,0%) e cocos homofermentativos como Lactococcus (12,6%), Enterococcus (8,3%), Pediococcus (5,9%) e Streptococcus (0,8%). As linhagens de Lactobacillus foram classificadas como homofermentativos: L.farciminis (0,84%); L crispatus (1,19%); L sharpae (2,71%); L. vitulinus (0,34%); como heterofermentativos facultativos: L agilis (0,50%); L maltaromicus (3,56%); L. murinus (6,95%), L. plantarum (2,54%); L. sake (2,71%); L casei rhamnosus (4,58%); L. coryniformis torquens (0,51%); L. coryniformis coryniformis (0,34%); como heterofermentativos obrigatórios: L fermentum (0,50%),; L. bifermentum (0,17%); L. halotolerans (0,50%); L v;r;descens (0,17%) e Lactobacillus sp (3,89%). As linhagens de Leuconostoc foram classificadas como Leuconostoc mesenteroides mesenteroides (3,05%); Leuconostoc mesenteroides dextranicum (14,91%); Leuconostoc oenos (1,35%); Leuconostoc paramesenteroides (0,17%) e Leuconostoc sp (1,52%). As linhagens de Lactococcus foram classificadas como: Lactococcus lactis cremoris (1,02%); Lactococcus raffino/actis (4,41%); Lactococcus lactis lactis (7,0%), Lacotococcus sp (0,17%); de Pediococcus como Pediococcus inopinatus (5,90%); de Enterococcus como: E. faecium (2,38%); E. faecalis (2,38%); E. avium (1,35%); E. pseudoavium (1,35%) e Enterococcus sp (0,84%); e de Streptococcus como S. hansenii (0,17%) e Streptococcus sp (0,67%)
Abstract: To survey the bacterial microflora of samples collected in differents steps of the production of "polvilho azedo", it was utilized the Plate Count Agar (PCA) and Man Rogosa & Sharpe Agar(MR.SA) in both aerobic and anaerobic conditions. The total counting of both aerobic and anaerobic microorganisms was not altered during fermentation of the cassava starch, shown that a shift of the bacterial microflora occurs, due to a pH decreasing. It was selected 590 cultures of which 23 (3,9%) were lost , it was therefore identified 567 isolateds of which 18 (3,1%) gram-negative bacteria, 13 (2,2%) yeast; 46 (7,8%) bacteria of the genera Bacillus and Corynebacterium; 14 (2,4%) Staphylococcus and Micrococcus as a contaminant of the process, and 476 (80,6%) as acid lactic bacteria, show predominance of the later group. The isolated identification was carried out by the use of probability matrices using a computer program. The water utilized in the process was also analyzed. The number of aerobie mesophilics of this water was 1,1 x 103/ml~ 9,5 x 103/l00ml of total coliforms, and 2,5 x 103/100ml fecal coliforms. The Lactobacillus sp. was prevailent in the process, with 189 strains (32,0%), followed fermentum (0,50%); L. bifermentum (0,17%); L. halotolerans (0,50%); L. viridescens (0,17%) Lactobacillus sp (3,89%). by strains of the Leuconostoc (21,0%) and homofermentative cocei such as Lactococcus (12,6%), Enterococcus (8,3%), Pediococcus (5,9%) and Streptococcus (0,8%). The Lactobacillus strains were classified as L. farciminis (0,84%); L. crispatus (1,19%); L. sharpae (2,71%); L. vitulinus (0,34%); L. agilis (0,50%); L maltaromiclls (3,56%); L. murinus (6,95%); L. plantarum (2,54%); L. sake (2,71%); L. casei rhamnosus ( 4,58%); L. coryniformis torquens (0,51%); L. coryniformis coryniformis (0,34%); L.
Doutorado
Doutor em Ciência de Alimentos

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Yogurt is a fermented milk resulting from a mutual interaction between the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. For yoghurt quality assurance, the cell numbers of each microorganism, should not be less than 1x107per gram, therefore a relative ratio lactobacilli and streptococci should be 1:1, although a 1:2 ratio is also accepted. During storage of yoghurt exposed for sale, post-acidification may occur, changing the ratio of the two microorganisms. The objectives of this research were to evaluate yoghurts of four different manufacturers named A, B, C and D, in relation to the number and balance between streptococii and lactobacilli during the storage and its relation with acidity and pH. In this way, yoghurts with up to 20 days of manufacturing (band I) and more than 20 days (band II) were evaluated. Better recovery of L. delbruckii subsp. bulgaricus and S. thermophilus was observed with LB and M17 media respectively, which were used to determine the relative ratio of the two microorganisms. An imbalance in the lactobacilli numbers which were lower than that of streptococci was verified and considered inadequate, in two out of four commercial brands. In yoghurts from the manufacturer A, there was a significant reduction in the number of lactobacilli from band I to band II leading to an increase in the relative ratio of streptococii to lactobacilli. There was no significant difference in the counts of streptococci or bacilli from one band to another with the other three brands of yoghurts. The acidity of yoghurts from manufacturer D revealed significantly higher (P< 0.05) than the others, although it did not result in an increased pH reduction. All samples attended the legislation in relation to total lactic acid bacteria counts, acidity, pH and mould and yeast count
Iogurte ? um leite fermentado resultante de uma intera??o microbiana mutualista entre as bact?rias l?cticas Streptococcus thermophilus e Lactobacillus delbrueckii subsp. bulgaricus. Para que a qualidade do iogurte seja garantida o n?mero de c?lulas destes dois microrganismos individualmente, n?o deve ser inferior a 1x107 por grama, portanto uma propor??o relativa de lactobacilos e estreptococos de 1:1 sendo a propor??o 1:2 tamb?m aceita. Durante o armazenamento dos iogurtes expostos para venda podem ocorrer p?s-acidifica??o e modifica??o na propor??o dos dois microrganismos. Os objetivos desta pesquisa foram avaliar iogurtes de quatro diferentes fabricantes as quais foram denominados A, B, C, D quanto ao n?mero e equil?brio entre as duas bact?rias l?cticas durante o armazenamento, e ao mesmo tempo determinar as caracter?sticas f?sico-quimicas (pH e acidez) e contagem de bolores e leveduras. Para tanto, adotou-se duas faixas de avalia??o, faixa I (at? 20 dias de fabrica??o) e faixa II (ap?s 20 dias). Foi observada melhor recupera??o L. delbruckii subsp. bulgaricus e S. thermophilus, respectivamente com os meios LB e M17 que foram usados para determina??o da propor??o relativa dos dois microrganismos. Foi verificado um desequil?brio no n?mero de lactobacilos que foi inferior ao de cocos e, considerado inadequado, em duas das quatro marcas comerciais. Nos iogurtes da marca A houve redu??o significativa no n?mero de lactobacilos da faixa I para faixa II levando a um aumento na propor??o de cocos relativa ao de bacilos. Nos iogurtes dos tr?s outros fabricantes n?o houve diferen?a significativa nas contagens de cocos ou de lactobacilos de uma faixa para outra. Tamb?m n?o foi observada diferen?a significativa de acidez e pH relacionados ao tempo de prazo comercial nas faixas I e II nas quatro marcas de iogurte analisadas. A acidez dos iogurtes do fabricante D foi significativamente mais elevada (P<0,05) que a dos demais, embora n?o tenha resultado em maior redu??o de pH nestes produtos. Todas as amostras analisadas estavam em conformidade com a legisla??o vigente no que se refere ao m?nimo exigido de bact?rias l?ticas totais que ? de 1x107 UFC/mL. Tamb?m estavam em conformidade com rela??o ? acidez, pH e contagem de bolores e leveduras. Palavras-chave: leite

L’étude du microbiote digestif a connu un regain d’intérêt au début des années 2000, avec l’avènement des techniques moléculaires. La culturomics a démontré sa complémentarité depuis 2010 en réduisant une partie des biais des méthodes moléculaires. Une revue sur les techniques d’étude du microbiote digestif et l’analyse du microbiote des sujets africains. Les études de métagénomique en Afrique ont révélé une augmentation de la biodiversité, en particulier des Spirochaetes et des Prevotella chez les africains par rapport aux occidentaux. Sur les 1162 bactéries isolées par culturomics, 476 n'étaient pas africaines, 445 étaient communes et 241 étaient d’origine africaine dont 68 nouvelles espèces. Pour ma participation au travail de culturomics, 102750 colonies testées par MALDI-TOF,ont permis d'identifier 377 espèces incluant 40 nouvelles espèces,17 nouveaux genres et 2 nouvelles familles.Ces nouvelles espèces ont été décrites par taxonogenomics ou new species announcement.Les archaea méthanogènes ont une prévalence de 97,4% pour M. smithii et associés à des pathologies comme l’abcès du cerveau,les parodontites etc. La culture est fastidieuse et nécessitait une source extérieure d’hydrogène. Sous enceinte anaérobie, nous avons cultivé avec succès M. smithii à partir d’un milieu de culture liquide inoculé d’échantillon de selle. L’isolement en culture pure a été un succès sur milieu gélosé en réalisant une coculture avec Bacteroides thetaiotaomicron. Nous avons aussi testé avec succès la coculture de M. smithii avec d’autres bactéries productrices d’hydrogène connues. Les tests de chromatographie en phase gazeuse montraient que ces souches produisaient de l’hydrogène
The study of the digestive microbiota was a renewed interest in the early 2000s, with the advent of molecular techniques. The culturomics has demonstrated its complementarity since 2010 by reducing some of the biases of molecular methods. A review on the techniques of studying the digestive microbiota and the analysis of the microbiota of African subjects. Metagenomic studies in Africa have revealed an increase in biodiversity, especially Spirochaetes and Prevotella among Africans compared to Westerners. Of the 1162 bacteria isolated by culturomics, 476 were non-African, 445 were common, and 241 were of African origin, including 68 new species. For my participation in the work of culturomics, 102750 colonies tested by MALDI-TOF, identified 377 species including 40 new species, 17 new genera and 2 new families. These new species have been described by taxonogenomics or new species announcement.Methanogenic archaea have a prevalence of 97.4% for M. smithii and associated with pathologies such as brain abscess, periodontitis and so on. The cultivation is tedious and required an external source of hydrogen. Under anaerobic enclosure, we successfully cultivated M. smithii from a liquid culture medium inoculated with a stool sample. The isolation in pure culture was a success on agar medium by performing a coculture with Bacteroides thetaiotaomicron. We have also successfully tested the co-culture of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen

LIMA, José Vinícius Leite. Populações microbianas e antagonismo de actinobactérias sobre rizóbios em solos do semiárido. 2017. 81 f. Tese (Doutorado em Ecologia e Recursos Naturais)-Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2017.
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The structure of the communities living in the soil is driven by various mechanisms and the ecological interactions established between the living organisms. In the same way, the components of the microbiota present harmonic or non-harmonic relationships, for example, between actinobacteria and rhizobia. Interspecific competition among microorganisms occurs when many species require the same resources, and the negative effect on the availability of common resources adversely affects the others. In this context, the objective of this study was to quantify the microbial population in the soil and leaf litter in the vegetation of the caatinga and carrasco in the Brazilian semiarid region (Chapter I) and to characterize and evaluate the antagonistic effect of actinobacteria on rhizobia (Chapter II). The soil samples were collected at the Ecological Station of Aiuaba (Aiuaba, Ceará), characterized by the vegetation of caatinga and carrasco. In chapter I, the population density of the microbial community was evaluated by sampling of soil and litter in the vegetation of caatinga and carrasco at the Aiuaba Ecological Station. The microorganisms were isolated from the soil samples in flasks containing 0.8% saline solution and cultured, after consecutive dilutions, in culturing media specific to cultivable populations of total bacteria, actinobacteria, cellulolytic bacteria, phosphate solubilizing bacteria and fungi. Population estimation was then performed by standard counting on plates and the values were expressed in CFU.g-1. The microbial populations from the soil and leaf litter differed each other quantitatively and in the two vegetations, but in general were greater in the leaf litter. Thus, the knowledge of the population structure of the microbial community can be extended to the semiarid soils. In Chapter II, the strains of actinobacteria isolated in the first study were characterized and tested for “in vitro” inhibitory effect on strains of rhizobia also isolated from semiarid soils. The actinobacteria were purified in their respective culture medium (CDA medium). The strains were characterized for their color and morphology of the colonies, tolerance to pH levels, production of melanin and use of carbon sources. The “in vitro” antagonism of actinobacteria on rhizobia was evaluated in Petri dish containing yeast mannitol agar (YMA) medium by the formation of inhibition zone. The actinobacteria and rhizobia that had greater antagonistic effect or did not presented inhibition zones were molecularly identified. Sixty strains were identified in seven genera of actinobacteria in which had tolerance to variations in pH, low melanin production and generalist use of carbon sources. It was also observed “in vitro” antagonism of actinobacteria species, standing out the genus Streptomyces, on strains of Rhizobium tropici, Bradyrhizobium yuanmingense and two other rhizobia strains not identified. This is the first work that addresses this ecological interaction between microorganisms of the Brazilian semiarid region and reveals the occurrence of new species related to this negative interaction. Thus, the presence of antagonism among these organisms may lead to “in vivo” studies, contributing to future agricultural and/or ecological uses.
A estrutura das comunidades que vivem no solo é direcionada por vários mecanismos e as interações ecológicas estabelecidas entre os organismos. Da mesma forma, os componentes da microbiota apresentam relações harmônicas ou não entre si, como por exemplo, entre actinobactérias e rizóbios. A competição interespecífica entre os micro-organismos acontece quando muitas espécies procuram pelos mesmos recursos, e o efeito depressor que cada uma tem na disponibilidade dos recursos comuns afeta adversamente os outros. Neste contexto, teve-se como objetivo neste estudo quantificar em região semiárida a população microbiana no solo e serrapilheira nas fisionomias vegetais de caatinga e carrasco (Capítulo I) e caracterizar e avaliar o efeito antagônico de actinobactérias sobre rizóbios do semiárido (Capítulo II). O estudo foi realizado na Estação Ecológica de Aiuaba (Aiuaba, Ceará) caracterizada pelas fisionomias de caatinga e carrasco. No capítulo I, avaliou-se a densidade populacional da comunidade microbiana por meio de coleta de amostras do solo e serrapilheira nas fisionomias vegetais de caatinga e carrasco na Estação Ecológica de Aiuaba. Os micro-organismos foram isolados das amostras em solução salina 0,8% e cultivados, após diluições seriadas, em meios de cultura específicos para populações cultiváveis de bactérias totais, actinobactérias, bactérias celulolíticas, bactérias solubilizadoras de fosfato e fungos. Em seguida foi realizada a contagem das populações pela contagem padrão em placas usando a técnica de espalhamento em superfície, e os valores foram expressos em UFC.g-1. As populações microbianas oriundas do solo e serrapilheira diferiram quantitativamente entre elas e nas duas fisionomias vegetais, mas no geral foram maiores na serrapilheira. Dessa forma, pode-se ampliar o conhecimento da estrutura populacional da comunidade microbiana em solos de clima semiárido. No capítulo II, caracterizaram-se as cepas de actinobactérias isoladas das populações obtidas do primeiro estudo e testou-as para efeito inibidor in vitro sobre estirpes de rizóbios também oriundas de solos semiáridos. As actinobactérias foram purificadas no seu respectivo meio de cultura (meio CDA). As cepas foram caracterizadas quanto a atributos cromogênicos e morfológicos, para tolerância a níveis de pH, na produção de melanina e uso de fontes de carbono. O antagonismo das actinobactérias in vitro sobre rizóbios foi avaliado em meio de cultura ágar manitol levedura (YMA) pela formação de halo de inibição em placa de Petri. Em seguida, as actinobactérias e rizóbios que tiveram maior efeito antagônico ou que não formaram halo inibidor foram caracterizadas molecularmente. Foram obtidas 60 cepas identificados em sete gêneros de actinobactérias que tiveram tolerância a variações de pH, baixa produção de melanina e uso generalista de fontes de carbono. Foi observado antagonismo in vitro com formação de halo inibidor de espécies de actinobactérias, com destaque para o gênero Streptomyces, sobre os rizóbios Rhizobium tropici, Bradyrhizobium yuanmingense e outras duas estirpes não identificadas. Este é o primeiro trabalho que aborda essa relação ecológica em micro-organismos do semiárido e revela a ocorrência de novas espécies relacionados com essa interação negativa. Com isso, confirma-se a presença de antagonismo entre esses organismos que pode direcionar estudos de certificação in vivo, contribuindo para futuros usos agrícolas e/ou ecológicos.

Research in our lab has predicted hundreds of bacterial genes that influence nine different traits in the fruit fly, Drosophila melanogaster. As a practical alternative to creating site-directed mutants for each of the predicted genes, we created an arrayed transposon insertion library using a strain of Acetobacter fabarum DsW_054 isolated from fruit flies. Creation of the Acetobacter fabarum DsW_054 gene knock-out library was done through random transposon insertion, combinatorial mapping and Illumina sequencing. Successful mapping of transposon insertion was achieved for 6418 mutants with hits within 63% of annotated genes within Acetobacter fabarum DsW_054. Insertion sites were verified in 40 mutants through arbitrary PCR and sequencing. To test the utility of the library, genes were selected from MGWAS results on host colonization which show LPS pathway enrichment in the significant gene predicctions. Genes upstream of Lipid-A creation show significant differences in host colonization whereas downstream genes show no effect. In addition, genes were selected from MGWAS results on Drosophila starvation resistance which show Methionine/Cysteine synthesis, Cobalamin synthesis, and Biotin synthesis pathway enrichment. Under our experimental conditions we could not verify influence of these pathways on host starvation resistance. However, they do appear to influence host colonization abundance. This transposon insertion mutant library will be useful for ongoing research in our lab as well as any field studying Acetobacter species, such as other insect microbiome and fermentation research.

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O presente estudo teve por objetivo descrever as alterações morfológicas ocorridas nos cecos de pintos de corte, machos e fêmeas, tratados com Microbiota Cecal (MC) antes e após infecção por Salmonella enterica enterica sorovar Enteritidis (SE) durante a primeira semana de vida. No 1º dia de vida, os pintos foram tratados com MC antes e depois da infecção por SE. Durante todo o período experimental as condições clínicas, assim como a taxa de mortalidade foram observadas. No 1º, 3º, 5º e 7º dias pós infecção (dpi) quatro aves de cada sexo/tratamentos foram sacrificadas por meio de deslocamento cervical para obtenção de amostras do ceco para análises morfológicas (histologia e microscopia eletrônica de varredura), morfométricas e contagem do número de células inflamatórias e células caliciformes PAS+ e AB+. Os dados mostraram alguns episódios de diarréia e quadros de depressão nos pintos infectados com SE no 5º e 7º dpi; nesse período não foi observada mortalidade. No tocante às análise morfológicas, os pintos infectados por SE respondem ao processo infeccioso com um processo inflamatório agudo no 1º dpi, e a partir do 3º dpi, o processo se cronifica. A Microbiota Cecal (MC) antes da infecção, evita as alterações morfofuncionais causadas pela bactéria nos cecos dos pintos de corte, além disso, a MC induz a proliferação de células caliciformes PAS+ e AB+ e, conseqüentemente aumenta a produção de muco, sendo um fator de proteção ao epitélio cecal
This study aimed to describe the morphological changes occurred in the caecu, of males and females broiler chickens treated with Cecal Microbiote (CM) before and after the infection with Salmonella enterica enterica serovar Enteritidis (SE) during the first week of life. On the first day of life, chickens were treated with CM before and after the infection with SE. Throughout the experimental period, the clinical conditions as much as the mortality rates were observed. On 1st, 3rd, 5th and 7th days post infection (dpi), four birds of each sex/treatment were slaughtered by cervical dislocation in order to obtain samples from the caecum for morphological analysis (histology and scanning electron microscopy), morphometrical analysis and counting of the number of inflammatory and goblet cells PAS+ and AB+. The data showed some diarrhea episodes and depression symptoms in the chickens infected with SE on 5th and 7th dpi; in this period it was not observed any mortality. Concerning the morphological analysis, the chicks infected with SE respond to infectious process with and acute inflammatory process on 1st dpi and after the 3rd dpi the process gets chronicle. The Cecal Microbiote (CM) prior to infection avoid the morphological and functional alterations caused by the bacteria in broiler chickens caecum, in addition, the CM induces the proliferation of goblet cells PAS+ and AB+ and consequently increases mucus production being a protection factor to the caecum epithelium

O guaraná (Paullinia cupana) é uma planta nativa da América do Sul, e suas sementes tem sido utilizadas por tribos amazônicas desde antes da colonização. O extrato de guaraná é consumido popularmente nos dias de hoje, entretanto pouco se sabe sobre a relação desse extrato com a microbiota intestinal. A microbiota possui um papel central na absorção de nutrientes da dieta e na regulação do metabolismo energético, sendo modificada na obesidade. A microbiota intestinal também é afetada pelo consumo de compostos vegetais, sendo muitas vezes regulada positivamente por dietas ricas em polifenóis. Todavia, o consumo de extratos vegetais pode gerar um desfecho tóxico, assim como alterações negativas na microbiota. O efeito de extratos ricos em compostos secundários deve ser estudado individualmente. Nesse trabalho avaliamos as alterações no ecossistema intestinal causadas pelo extrato comercial de sementes do Guaraná em animais saudáveis e no contexto da obesidade.Induzimos a obesidade em animais através de uma dieta que mimetiza a dieta ocidental, caracterizada por uma maior quantidade de gordura, sal e açúcar refinado, e uma menor quantidade de fibras. Os animais também foram submetidos ao tratamento com um extrato comercial de Guaraná, cafeína ou salina. A disbiose intestinal foi medida no conteúdo cecal através da amplificação e sequenciamento da porção ribossomal 16S. Também foi medida a atividade enzimática no fígado, rins e intestino delgado, assim como a quantidade de citocinas proinflamatórias no soro. A dieta obesogênica gerou um aumento no ganho de peso e no acúmulo de gordura dos animais comparada com a dieta Chow. Tanto o Guaraná quanto a cafeína não foram capazes de reverter o ganho de peso e o acúmulo de gordura. Também observamos a diminuição da atividade da enzima CAT no rim nos animais que receberam guaraná, independente da dieta. A dieta obesogênica induziu alterações na microbiota intestinal semelhantes às da obesidade, diminuindo a proporção de Bacteroidetes sobre Firmicutes, aumentando o gênero Mucispirilum e diminuindo os gêneros Lactobacillus e Bifidobacterium. Os tratamentos com guaraná e cafeína na dieta Chow diminuíram a proporção B/F, assim como o gênero Bifidobacterium. Observamos um aumento no filo Proteobacteria nos animais do grupo controle obeso. Esse aumento foi revertido pelo tratamento com guaraná. Analisando os gráficos de clusterização e landscape, observamos uma maior distância entre a microbiota dos animais que receberam dietas diferentes do que a dos animais que receberam tratamentos diferentes. Os tratamentos com guaraná ou cafeína modificaram o ecossistema intestinal, mas sem a capacidade de diminuir o acúmulo de gordura ou o ganho de peso. A maior alteração na microbiota foi devido à dieta obesogênica e não aos tratamentos, mostrando que os efeitos anti-obesogênicos do guaraná observados em outros estudos provavelmente não são via microbiota intestinal.
Guarana (Paullinia cupana) is a plant native of South America, and its seedshave been used by Amazonian tribes since before colonization. Guarana extract is consumed popularly today, however little is known about the interactions of this extract with the intestinal microbiota. The gut microbiota plays a central role in the absorption of nutrients from the diet and in the regulation of energy metabolism, being altered in obesity. The intestinal microbiota is also affected by the consumption of plant metabolites and is often regulated positively by diets rich in polyphenols. However, the consumption of plant extracts can generate a toxic outcome, as well as negative changes in the microbiota. Therefore, the effect of plant extracts should be studied individually. In this work, we evaluated the changes in the intestinal ecosystem caused by the administration of a commercial extract of Guaraná seeds in healthy animals and in the context of obesity. We induced obesity in animals through a diet that mimics the western diet, characterized by a higher amount of fat, salt and refined sugar, and a smaller amount of fiber. The animals were also treated with a commercial extract of Guarana, caffeine or saline. Intestinal dysbiosis was evaluated by cecal content, amplification and sequencing of the 16S ribosomal portion. The antioxidant enzymatic activity of the liver, kidney and small intestine was measured as well as the concentration of pro-inflammatory cytokines in the serum. The obesogenic diet increased the weight gain and fat accumulation of the animals compared to the Chow diet. Both Guarana and caffeine were unable to reverse weight gain and fat accumulation. We observed a lower activity of the Glutathione Peroxidase enzyme in the kidney and small intestine in the animals that received obesogenic diet compared to the Chow diet, regardless of the treatment. We also observed the decrease of Catalase enzyme activity in the kidney of animals that received Guarana, regardless of diet. The obesogenic diet induced changes in the intestinal microbiota similar to other works in literature, reducing the proportion of Bacteroidetes/Firmicutes (B/F), increasing the genus Mucispirilum and decreasing the genera Lactobacillus and Bifidobacterium. Guarana and caffeine treatments in the Chow diet decreased the B/F ratio, thus the Bifidobacterium genus. We observed an increase in the phylum Proteobacteria, in the animals of the Obese Control group. This increase in the phylum Proteobacteria, was reversed by the treatment with Guaraná. Analyzing the phylogenetic proximity charts and landscape, we observed a greater distance between the animals that received different diets than animals that received different treatments. Treatments with guarana or caffeine modified the intestinal ecosystem, but without the ability to decrease fat accumulation or weight gain. The major change in the microbiota was due to the obesogenic diet and not to the treatments, showing that the antiobese effects of guarana observed in other studies probably are not via intestinal microbiota.

Le tecniche di next generation sequencing costituiscono un potente strumento per diverse applicazioni, soprattutto da quando i loro costi sono iniziati a calare e la qualità dei loro dati a migliorare. Una delle applicazioni del sequencing è certamente la metagenomica, ovvero l'analisi di microorganismi entro un dato ambiente, come per esempio quello dell'intestino. In quest'ambito il sequencing ha permesso di campionare specie batteriche a cui non si riusciva ad accedere con le tradizionali tecniche di coltura. Lo studio delle popolazioni batteriche intestinali è molto importante in quanto queste risultano alterate come effetto ma anche causa di numerose malattie, come quelle metaboliche (obesità, diabete di tipo 2, etc.). In questo lavoro siamo partiti da dati di next generation sequencing del microbiota intestinale di 5 animali (16S rRNA sequencing) [Jeraldo et al.]. Abbiamo applicato algoritmi ottimizzati (UCLUST) per clusterizzare le sequenze generate in OTU (Operational Taxonomic Units), che corrispondono a cluster di specie batteriche ad un determinato livello tassonomico. Abbiamo poi applicato la teoria ecologica a master equation sviluppata da [Volkov et al.] per descrivere la distribuzione dell'abbondanza relativa delle specie (RSA) per i nostri campioni. La RSA è uno strumento ormai validato per lo studio della biodiversità dei sistemi ecologici e mostra una transizione da un andamento a logserie ad uno a lognormale passando da piccole comunità locali isolate a più grandi metacomunità costituite da più comunità locali che possono in qualche modo interagire. Abbiamo mostrato come le OTU di popolazioni batteriche intestinali costituiscono un sistema ecologico che segue queste stesse regole se ottenuto usando diverse soglie di similarità nella procedura di clustering. Ci aspettiamo quindi che questo risultato possa essere sfruttato per la comprensione della dinamica delle popolazioni batteriche e quindi di come queste variano in presenza di particolari malattie.

O Brasil é um dos países com a maior biodiversidade do mundo, embora existam ainda poucos estudos sobre a biodiversidade microbiana dulcícola, assim, o objetivo deste trabalho foi avaliar a diversidade e estrutura das comunidades bacterianas presentes no corpo e afluentes do Rio Tietê e relacioná-las às variáveis ambientais. Foram obtidos 385 fragmentos terminais de restrição representando a temporada de estiagem em 2013 e 217 TRFs representando a temporada de cheias em 2014. As análises de Redundância (RDA) apresentaram separação entre as amostras de acordo com o período de coleta seguida da qualidade da água de origem e a temporada 2013 apresentou maior riqueza e diversidade com relação à 2014. Já a técnica de sequenciamento por MiSeq Illumina, apresentou 2.130.122 sequências com boa qualidade e o parâmetro temperatura representou a principal variável ambiental agindo sobre a riqueza das comunidades avaliadas, além disso, a localização geográfica dos rios e suas conexões representaram fatores importantes para a distribuição dos gêneros observados.
Brazil is considered one of the countries with the highest biodiversity; however, there are few studies on microbial diversity in freshwaters. Thereby, the aim of present work was to evaluate the diversity and structure of bacterial community present in Tietê river and its tributaries, for that, 28 points along Tietê River Basin were evaluated by T-RFLP and 14 points were chosen based on these results for partial sequencing of rRNA 16S gene by MiSeq Illumina. 385 Terminal Restriction Fragments were obtained for season 2013 and 217 for 2014 and Redundancy Analysis presented separation between samples according to seasons followed by water quality group separation. Season 2013 presented higher richness and diversity compared to season 2014 and high throughput sequencing presented 2.130.122 sequences with good quality. Temperature parameter represented the main environmental variable acting on the richness of assessed communities, in addition, the geographic location of the rivers and their connections were important factors for the distribution of observed genera.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by persistent sinonasal inflammation and sinus microbiome dysbiosis. The basis of this heterogeneity is poorly understood. We sought to address the hypothesis that a limited number of compositionally distinct pathogenic bacterial microbiota exist in CRS patients and invoke discrete immune responses and clinical phenotypes in CRS patients. Results: Sinus brushings from patients with CRS (n = 59) and healthy individuals (n = 10) collected during endoscopic sinus surgery were analyzed using 16S rRNA gene sequencing, predicted metagenomics, and RNA profiling of the mucosal immune response. We show that CRS patients cluster into distinct sub-groups (DSI-III), each defined by specific pattern of bacterial co-colonization (permutational multivariate analysis of variance (PERMANOVA); p = 0.001, r(2) = 0.318). Each sub-group was typically dominated by a pathogenic family: Streptococcaceae (DSI), Pseudomonadaceae (DSII), Corynebacteriaceae [DSIII(a)], or Staphylococcaceae [DSIII(b)]. Each pathogenic microbiota was predicted to be functionally distinct (PERMANOVA; p = 0.005, r(2) = 0.217) and encode uniquely enriched gene pathways including ansamycin biosynthesis (DSI), tryptophan metabolism (DSII), two-component response [DSIII(b)], and the PPAR-gamma signaling pathway [DSIII(a)]. Each is also associated with significantly distinct host immune responses; DSI, II, and III(b) invoked a variety of pro-inflammatory, T(H)1 responses, while DSIII(a), which exhibited significantly increased incidence of nasal polyps (Fisher's exact; p = 0.034, relative risk = 2.16), primarily induced IL-5 expression (Kruskal Wallis; q = 0.045). Conclusions: A large proportion of CRS patient heterogeneity may be explained by the composition of their sinus bacterial microbiota and related host immune response-features which may inform strategies for tailored therapy in this patient population.

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

Dissertação de Mestrado Integrado em Medicina Veterinária
Investigations of the relationships between the gut microbiota and gastrointestinal parasitic nematodes are attracting growing interest by the scientific community. These studies have however been carried out mainly in humans and experimental animals, while knowledge of the make-up of the gut commensal microbiota in presence or absence of infection by parasitic nematodes in domestic animals is limited. In this study, we investigate the qualitative and quantitative impact that infections by a widespread parasite of cats (i.e. Toxocara cati) exert on the gut microbiota of feline hosts. The faecal microbiota of cats with patent infection by T. cati (= Tc+), as well as that of negative controls (= Tc-) was examined via high-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data. A total of 2,325,366 useable high-quality sequences were generated from the faecal samples analysed in this study and subjected to further bioinformatics analyses, which led to the identification of 128 OTUs and nine bacterial phyla, respectively. The phylum Firmicutes was predominant in all samples analysed (mean of 53.0%), followed by the phyla Proteobacteria (13.8%), Actinobacteria (13.7%) and Bacteroidetes (10.1%). Among others, bacteria of the order Lactobacillales, the family Enterococcaceae and genera Enterococcus and Dorea showed a trend towards increased abundance in Tc+ compared with Tc- samples, while no significant differences in OTU richness and diversity were recorded between Tc+ and Tcsamples (P = 0.485 and P = 0.581, respectively). However, Canonical Correlation and Redundancy Analyses were able to separate samples by infection status (P = 0.030 and P = 0.015, respectively), which suggests a correlation between the latter and the composition of the feline faecal microbiota.
RESUMO - Alterações na microbiota intestinal felina associadas a infecções por Toxocara cati - A investigação das interações entre a microbiota intestinal e os nematodes intestinais tem vindo a atrair o interesse da comunidade cientifica. No entanto, a maioria destes estudos tem sido desenvolvida em humanos e animais de laboratório, e deste modo o conhecimento da composição da microbiota comensal do intestino na presença de nematodes intestinais é reduzido. Neste estudo, foi investigado o impacto qualitativo e quantitativo da infeção pelo parasita comum dos gatos (i.e. Toxocara cati) na microbiota intestinal do hospedeiro felino. A microbiota fecal de gatos com infeção patente por T. cati (Tc+), bem como controlos negativos (Tc-) foi avaliada através de sequenciação de alto débito da região hiper-variável V3-V4 do gene 16S, seguido de análise bioinformática e bioestatística. Das amostras fecais incluídas no estudo foram obtidas um total de 2 325 366 sequências de alta qualidade e sujeitas a analise bioinformática, o que levou à identificação de 128 OTUs e nove filos bacterianos. O filo Firmicutes foi encontrado em predominância em todas as amostras (média de 53,0%), seguido do filo Proteobacteria (13,8%), Actinobacteria (13,7%) e por fim Bacteroidetes (10,1%). A abundância de determinados grupos de bactérias tendeu a aumentar nas amostras Tc+ quando comparadas com as amostras Tc-, tais como a ordem Lactobacillales, a família Enterococaceae e o género Enterococcus e Dorea. No entanto, a riqueza e diversidade das OTUs não apresentou diferenças significativas entre as amostras Tc+ e Tc- (P=0,485 e P=0,581, respetivamente). Todavia, a análise canónica de redundância demonstrou uma separação das amostras de acordo com o estado de infeção (P=0,030 e P=0,015, respetivamente), o que sugere uma correlação entre este e a composição da microbiota fecal felina.
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Se han evidenciado cambios en la composición de la microbiota fecal en pacientes con CU en actividad frente a sanos, pero no queda claramente establecido si los cambios disbióticos son secundarios a la enfermedad o por el contrario pueden jugar cierto papel en la fisiopatología de la inflamación intestinal. La literatura es controvertida con respecto a si estos cambios se mantienen en períodos de remisión de la enfermedad. La primera parte de esta tesis trata de establecer si los cambios disbióticos desaparecen durante los periodos de remisión y la estabilidad de la microbiota se restituye completamente. Para ello se estudia una población de sujetos incluidos en el estudio METAHIT, compuesto por pacientes con colitis ulcerosa en remisión con alta tasa de brotes, baja tasa de brotes, familiares emparentados y voluntarios sanos. Bajo la hipótesis de que en la colitis ulcerosa hay una disbiosis intestinal permanente que puede demostrarse durante los periodos de remisión de la enfermedad, comparamos la composición de la microbiota fecal de pacientes con CU en remisión con la de personas sanas empleando técnicas de secuenciación de alto rendimiento. Apreciamos que existe una gradación en cuanto a riqueza genética entre pacientes con CU en remisión, familiares emparentados y voluntarios sanos. La segunda parte de esta tesis, trata de establecer si los cambios que tienen lugar en la microbiota de pacientes con colitis ulcerosa, se deben a un mayor tránsito colónico (efecto de lavado por aceleración del tránsito intestinal). Para ello se analiza si la preparación de limpieza colónica con PEG produce cambios permanentes en la composición de la microbiota intestinal de individuos sanos y pacientes con colitis ulcerosa en remisión mediante la aplicación de técnicas de secuenciación del gen 16S rRNA. Los resultados indican que tras la administración del PEG se produce una pérdida en la diversidad de la microbiota que tiende a recuperarse a los 2 meses.
There have been described changes in fecal microbiota composition in patients with UC with inflammatory activity versus healthy individual, but it is not clear whether the dysbiotic changes are secondary to the disease itself or, on the contrary, may play a role in the pathophysiology of intestinal inflammation. The literature is controversial as to whether these changes are maintained during remission periods. The first part of this thesis tries to establish whether the dysbiotic changes disappear during the remission periods and the stability of the microbiota is completely restored. For this purpose, a population of subjects included in the METAHIT study, composed with patients with ulcerative colitis in remission with high outbreak rate, low outbreak rate, related relatives and healthy volunteers are studied. Under the hypothesis that ulcerative colitis has permanent intestinal dysbiosis changes that can be demonstrated during periods of remission, we compare the fecal microbiota composition of UC patients in remission with that of healthy individuals using high throughput sequencing techniques. We appreciate that there is a gradation in bacterial gene count among patients with CU in remission, related relatives and healthy volunteers. The second part of this thesis, tries to establish if the fecal dysbiosis are due to a greater colonic transit. To this purpose, it is analyzed whether the colonic cleansing with PEG induces permanent changes in the composition of the intestinal microbiota of healthy individuals and patients with ulcerative colitis in remission, through the application of 16S rRNA gene sequencing techniques. The results indicate that after the administration of the PEG there is a loss in the diversity of the microbiota that tends to recover at 2 months.

L’exploration du microbiote humain a connu un tournant ces dernières années avec l’apport significatif de l'approche « microbial culturomics », qui a permis d’augmenter de façon considérable le répertoire de microbes humain cultivables. Mais cette approche n’a jamais été appliquée à l’étude d’écosystèmes anciens. Dans cette thèse, nous avons associé culturomics et métagénomique pour explorer la composition bactérienne d’un échantillon environnemental ancien vieux de 2,7 millions d’années : le permafrost sibérien. Nous avons identifié 28 espèces bactériennes que nous avons séquencées puis comparer à leurs homologues contemporaines en analysant leurs profils de résistances et en étudiant leurs évolutions génomiques principalement par détermination de la composition en SNPs dans les génomes. Les souches du permafrost hébergeaient de 2-51 gènes de résistances appartenant à 20 classes d’antibiotiques différentes et étaient phénotypiquement résistantes à 2-8 classes d’antibiotiques différentes. L’étude génomique montre que la contenance génomique des souches contemporaines et anciennes étaient similaires mettant ainsi en évidence une évolution moléculaire réduite pendant 2,7 millions d’années.Dans un second travail, nous avons évalué l’impact de « la désinfection à l’éthanol » sur la composition du microbiote intestinal pouvant servir de bactériothérapie. L’application de 22 conditions de culture sur 11 selles de donneurs de greffes fécales désinfectées à l’éthanol a identifié 254 espèces bactériennes majoritairement anaérobies. Parmi elles, 242 n’avaient jamais été signalées en essais de bactériothérapie et représentent des candidats potentiels pour des études futures
The exploration of the human microbiota has been a turning point in recent years with the significant contribution of the « microbial culturomics » a high throughput culture technique that has significantly increased the repertoire of cultivable human microbes. But this approach has never been applied to the study of ancient ecosystems. To start, we have combined culturomics approach with metagenomics to explore the bacterial composition of an ancient environmental sample dated to 2.7 million years old: Siberian permafrost. We identified 28 bacterial species that we have sequenced and then compared to their contemporary counterparts by analyzing their resistance patterns and studying their genomic evolution mainly by determining the single nucleotide polymorphism (SNPs) composition in the genomes. We found that permafrost strains harbored two to fifty-one antibiotic resistance genes belonging to twenty different antibiotic class and were phenotypically resistant to 2-8 different antibiotic class. Genomics studies show that contemporary genomes and ancient strains were quite similar, thus revealing a reduced molecular evolution for 2.7 million years.Secondly, we evaluated the impact of "ethanol disinfection" on the intestinal microbiota's composition that can be used as bacteriotherapy, particularly to treat Clostridium difficile infections. The application of twenty-two culture conditions on 11 stools of faecal graft donors previously disinfected with ethanol allowed the isolation of 254 predominantly anaerobic bacterial species. Among these, 242 had never been reported in bacteriotherapy trials and represent potential candidates for future studies

The purpose of this project was to determine the interaction between dietary protein and the gut microbiome in the production of genotoxic metabolites, with a particular focus on the poorly characterised metabolite 4-cresol. The thesis describes, in the first instance, data from a large human observation study (n=205 healthy Omani adults). In which dietary records and urinary nitrogen excretion were used to estimate protein consumption in relation to urinary 4-cresol excretion. The study observed positive correlations between excreted 4 cresol and protein intake and then sought to explain the inter-individual variance in this by evaluating the influence of the colonic microbiota. Then the study focused on predicting 4-cresol exposures in the colon using in vitro gut fermentation models. The microbiota composition and metabolic profiles from these models are evaluated against different substrates, including comparisons of animal and plant proteins. We show that the total production of 4-cresol is dependent both on the host microbiota and also upon the dietary nitrogen source. The metabolite profiles of these fermentations may be used to predict DNA damage, with 4-cresol emerging as the greatest correlate of fermentation supernatant mediated genotoxicity. Finally, the study explored whether specific tumour isolates of F. nucleatum produce 4-cresol, or other genotoxins, that could drive intestinal carcinogenesis. At this stage the study is unable to conclude whether or not these isolates are passengers or drivers of intestinal disease. This work suggests the need for better models of the effects of the tumour environment on microbial growth. The most significant aspect of this thesis is that it evidences both the potential genotoxic contribution of 4-cresol in the colonic milieu, but also that urinary 4-cresol sulfate may be used as a biomarker of genotoxic colonic fermentation and thus, may be of use as a cancer risk endpoint in future dietary intervention study.

To the colonising bacterium, the human body represents a number of ecological niches, some of which it could be argued are as hostile to the coloniser as even the most extreme of ex vivo environments; by the activities of the immune system, these living host niches are actively dedicated to the prevention of their colonisation. Paradoxically, the normal human microbiota of each human extends in estimated total number to approximately 1014 per human. This thesis is a compilation of published work that focuses on two anaerobic bacteria of the normal human microbiota: Bacteroides fragilis predominantly found in the large intestine; and Propionibacterium acnes predominantly found in the skin microbiota. When given the opportunity these bacteria can cross the divide between commensal and pathogen and cause infection. The papers included in this thesis address aspects of the characteristics of these bacteria that may relate to virulence and the association of these bacteria with clinical infection.

The investigation of extreme environments has accelerated in the last 3 decades with the advent of molecular techniques, allowing a greater insight into the diversity of microbial communities in these regions. These have allowed the discovery of new bacteria and a greater understanding into the interactions which occur between bacteria, fungi and archaea which are found in these environments. This study attempted to use a number of techniques common in microbial ecology in the analysis of samples taken from the Qedem region of the Dead Sea. The areas sampled showed high salinity, decreased pH and low redox potential. Forty strains were isolated from various environmental samples with optimisation of growth media for in vitro studies of these isolates. Phenotypic analysis showed a clear optimum salt requirement in the majority of the strains isolated. Using chemical based MALDI-TOF-MS technology the forty strains were analysed and compared according to their protein profiles. Principal Component analysis was used to show that the majority isolates were relatively tightly clustered depending on their salt requirements. Four cyanobacterial samples were analysed using both MALDI-TOF MS and 2D gel electrophoresis and a large number of eubacteria were also found to be present, showing the existence of communities. Two of the cyanobacterial samples, KM8 from a shoreline spring and KM39 from a submarine spring, were analysed using ION Torrent next generation sequencing technology and the multiple databases of the MG-RAST pipeline. This showed a diverse species range present in the samples based on the ribosomal databases used. When the samples were analysed using protein databases, archaea including methanogens were found in the samples. A number of springs of the Qedem region were analysed using 454 pyrosequencing and showed a high level of variation in the microbial communities. Overall this study has shown there are various rich and diverse microbial communities within the Qedem area of the Dead Sea.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
O microbiota intestinal é o conjunto dos microrganismos que existem no intestino humano. O microbioma intestinal diz respeito ao genoma desses microrganismos. Estes microrganismos estabelecem com o hospedeiro uma relação de mutualismo, em que ambos contribuem e beneficiam. O microbiota intestinal é bastante diversificado e numeroso. Com o progresso de técnicas de genética, o avanço no estudo do microbioma foi conseguido, permitindo a classificação do microbiota. Estudos de metagenómica, metatranscriptómica, metaproteómica e metametabolómica, permitiram descrever a diversidade de espécies microbianas existentes no intestino. O microbiota intestinal caracteriza-se pelo seu constante dinamismo, sendo que este pode ser afetado por inúmeros fatores ambientais como dieta, estilo de vida, consumo de antibióticos e idade. O desenvolvimento do microbiota ocorre logo após o nascimento e vai ter influência na fisiologia do hospedeiro, nomeadamente no desenvolvimento e morfogénese de órgãos e na manutenção do equilíbrio de tecidos e órgãos. Irá também contribuir para o desempenho de funções metabólicas, principalmente na obtenção de energia a partir da dieta e no desenvolvimento do sistema imunológico. O desenvolvimento adequado tanto do GALT (isto é, do tecido linfóide associado à mucosa intestinal, que constitui o sistema imunológico do trato gastrointestinal), como da tolerância imunológica, são de extrema importância para o hospedeiro pois permitem que este seja menos suscetível a desenvolver patologias. Alterações no microbiota estão associadas a diversas doenças como diabetes tipo 1, obesidade, do foro cardiovascular, entre outras. Por sua vez, estas patologias podem causar uma modificação considerável do microbiota e suas funções, afetando a relação de simbiose com o hospedeiro. Se ocorrer uma alteração extensa do microbiota, poderá ser necessário recorrer ao uso de probióticos, prebióticos e, em último recurso, transplante fecal para se poder restabelecer ou modificar a microbiota intestinal, minimizando os danos causados. O estudo do microbiota humano e, em particular, do microbiota intestinal está em franco desenvolvimento, tendo vindo a surgir novas evidências relativamente à sua associação a diferentes patologias e ao seu papel na fisiologia humana. The gut microbiota is composed by the microorganisms that exist in the human intestine. The gut microbiome refers to the genome of these organisms. The gut microorganisms live in mutualism with the host, both contributing to and benefiting of each other. The gut microbiota is quite diverse and numerous. With the progress of genetic techniques, advances in the study of the microbiome has been achieved, allowing classification of the microbiota. Metagenomic studies, metatranscriptomics, metaproteomics and metametabolomics allowed to describe the diversity of microbial species in the intestine. The intestinal microbiota is characterized by its constant dynamics, and can be affected by many environmental factors such as diet, lifestyle, age and antibiotic consumption. The development of the microbiota occurs soon after birth and will influence the physiology of the host, including the development and organ morphogenesis and in maintaining the homeostasis of tissues and organs. It will also contribute to the performance of the metabolic functions, particularly in the production of energy from the diet and development of the immune system. The proper development of both GALT (i.e. the lymphoid tissue associated with the intestinal mucosa, which is the immune system of the gastrointestinal tract), and of immune tolerance, are extremely important to the host because they contribute to a lower susceptibility to disease development. Changes in the microbiota are associated with various diseases such as type 1 diabetes, obesity, cardiovascular diseases, among others. On the other hand, these diseases may also cause considerable modification of the microbiota and its functions, affecting the symbiotic relationship with the host. If an extensive alteration of the microbiota occurs, the use of probiotics, prebiotics or even fecal transplantation may be necessary to restore or modify the intestinal microbiota, minimizing damage. The study of the human microbiota and, in particular, of the intestinal microbiota is rapidly developing, with new evidences regarding their association with different pathologies and their role in human physiology.