A microbiota normal exerce função protetora na superfície ocular, competindo por nutrientes e secretando substâncias com propriedades bactericidas, o que dificulta a invasão de patógenos. Animais diabéticos apresentam diferentes alterações ceratoconjuntivais, tais como maior concentração de glicose no filme lacrimal, menor quantidade e qualidade da lágrima e diminuição da sensibilidade corneal, diferenças clinicamente relevantes que podem alterar essa microbiota. Traumas oculares também podem apresentar tal alteração por promoverem a quebra da barreira de proteção da superfície ocular. A correta identificação do agente etiológico, além de proporcionar um tratamento eficiente em alterações oculares, direciona a antibioticoterapia profilática tópica nos casos cirúrgicos e evita o uso abusivo de antibióticos, o que pode levar a destruição da microbiota normal da superfície ocular, facilitando assim o crescimento de bactérias e fungos patogênicos. Com o intuito de minimizar as complicações pós-operatórias das cirurgias intra-oculares, como as facectomias, freqüentemente realizadas nos cães diabéticos por serem predispostos a apresentarem catarata, este estudo visou avaliar a prevalência dos principais microrganismos da microbiota conjuntival de cães diabéticos. Foram avaliadas as microbiotas conjuntivais de 29 cães diabéticos, atendidos no Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, totalizando 58 olhos. Estes, não estavam utilizando qualquer medicação tópica e não apresentavam alterações oculares ou doença sistêmica infecciosa que pudesse interferir nos resultados. As amostras foram colhidas do saco conjuntival com auxilio de zaragatoas, cuidando para que não houvesse contaminação pelo contato com pálpebras e cílios. As amostras foram inoculadas em meio enriquecedor Brain Heart Infusion (BHI) e caldo Sabouraud-dextrose e semeados em ágar sangue, MacConkey e Sabouraud-dextrose com cloranfenicol. Os microrganismos foram identificados por suas características morfológicas na bacterioscopia, pela coloração de gram e pelas características macro e microscópicas das colônias, além de testes bioquímicos. Houve maior prevalência de Staphylococcus (32,79%), principalmente coagulase negativos, que são os mais freqüentemente isolados nas infecções oculares: S. klosii, S. simulans, S. capitis, S. pasteuri, S. saprophyticus, S. haemolyticus. Também foram isolados Bacillus sp. (11,48%), S. intermedius (6,56%), Streptococcus sp (1,64%) e alguns fungos, como Cladosporium sp e Candida parapsilosis. Não houve crescimento de microrganismos gram negativos, anaeróbios estritos ou facultativos. Sugere-se que os resultados encontrados se relacionam diretamente às alterações ceratoconjuntivais dos animais analisados, local do exame, clima e diferentes raças. Sabendo-se que cães diabéticos são prováveis candidatos à facectomia, intervenção que promove quebra da barreira de proteção da superfície ocular e que existem microrganismos potencialmente patogênicos na conjuntiva destes animais, sugere-se análise da microbiota ocular antes da intervenção cirúrgica, para uso profilático de agentes antimicrobianos. / Normal ocular flora avoids infection depriving investor microorganisms of nutrients and by the secretion of bactericidal substances. Diabetic animals usually develop keratoconjunctival abnormalities, such as the higher concentration of glucose in the tear film; qualitative and quantitative tear deficiency and a decrease of the cornea sensitivity, what probably changes their conjunctival flora either. The same alteration may happen after ocular traumas, when the ocular barrier of protection is discontinued. The identification of agent etiology allows the efficient treatment of ocular infections, the prophylactic prescription of antibiotics before surgery and avoids destruction of normal ocular flora or multiplication of pathogens. Aiming to minimize complications on intraocular surgeries, such as facectomies, frequently realized in DM dogs for its predisposition to develop cataract, this study evaluated the prevalence of microorganisms in their conjunctival flora. The conjunctival flora of 29 diabetic dogs (58 eyes), attended at the Ophthalmology Service, of the Veterinary Hospital of the Veterinary College University of São Paulo were evaluated. These dogs werent using any kind of eyedrops, didnt present any ocular or systemic disease other than diabetes mellitus, what could change the results. Samples were taken with sterile swabs from the conjunctival sac and inoculated on Brain Heart Infusion (BHI) and Sabouraud-dextrose broth, then sown in blood agar, MacConkey and Sabouraud-dextrose-cloranfenicol. Contamination was avoided during procedure not touching the swab on eyelid or eyelashes. Microorganisms were identified by their morphological aspects at bacterioscopy, gram staining, macro and microscopy characteristics of the colonies and by biochemical tests. Staphylococcus presented the higher prevalence (32,79%), mostly the coagulase negative ones (S. klosii, S. simulans, S. capitis, S. pasteuri, S. saprophyticus, S. haemolyticus), which are frequently isolated from ocular infections. Either isolated, were Bacillus sp. (11,48%), S. intermedius (6,56%), Streptococcus sp (1,64%) and some fungus, such as Cladosporium sp and Candida parapsilosis. Gram negative bacteria, anaerobic stricts or facultative were not identified during the study. We suppose that these results are strictly correlated to keratoconjunctival alterations in DM dogs, procedure, weather and breed. We conclude that, as DM dogs presents potential pathogens on its conjunctiva and are probable candidates to cataract surgery, which discontinues the ocular barrier of protection, we suggest the identification of the agent etiology before surgery and the proper use of antibiotic before and after any intraocular intervention.

A pele dos anfíbios, assim como de outros animais, atua como primeira proteção contra agentes patogênicos. A comunidade microbiológica ali residente é composta de algumas espécies de bactérias, e estas, possuem ação antifúngica contra patógenos conhecidos, inclusive Batrachochytrium dendrobatidis (Bd), o suposto agente principal de declínios de populações de anfíbios em diversas partes do mundo. Uma vez que as variáveis químicas e físicas de um ecossistema influenciam o crescimento, sobrevivência e atividade metabólica dos microorganismos, a microbiota cutânea que atua como barreira de proteção nos anfíbios contra agente infecciosos, provavelmente é afetada quando determinados parâmetros ecofisiológicos são alterados em ambientes florestais fragmentados, modulando assim a vulnerabilidade das populações de anfíbios aos agentes patogênicos. Nossa pesquisa esteve focada na caracterização das comunidades microbianas residentes da pele dos anfíbios em dois contextos de paisagem: fragmento e área contínua. Os parâmetros utilizados para essas análises foram a densidade microbiana e a riqueza de morfotipos de colônias bacterianas. O potencial inibitório do crescimento de patógenos também foi testado em ensaios do tipo cross-strak. As diferenças de densidade e riqueza microbiana entre as paisagens e a presença de táxons típicos de ambiente, apontam para o ambiente como um componente importante na determinação dos perfis das comunidades microbianas dos anfíbios estudados. Essas mudanças são muito provavelmente conseqüências, mas para o entendimento da extensão e natureza de tais conseqüências são necessários estudos adicionais. / The skin of amphibians, as well as that of other animals, acts as a first protection barrier against pathogens. The microbial community resident in the amphibian skin is composed of some species of bacteria that may have antibacterial or antifungal action against known pathogens, including Batrachochytrium dendrobatidis, the alleged principal agent Tleading to declines of amphibian populations around the world. Because the chemical and physical variables of the landscape influence the growth, survival and metabolic activity of microorganisms, the function of skin as a protective barrier against infectious agents in amphibians, is likely affected by parameters that are altered in fragmented forest habitats. Thus, it is important to understand how environmental conditions affect the skin microbiota of amphibians, and the possible induced changes on vulnerability of amphibians to pathogens. Our research aimed to characterize the microbial communities living skin of amphibians in two contexts of landscape: fragment and continuous area. The parameters used for this analysis were density and richness of microbial morphotypes of bacterial colonies. The potential inhibition of pathogen growth was also evaluated using a cross-streak test, and some taxa in these communities were identified using international protocols. The observed differences in microbial density and richness across landscapes, and the presence of bacterial taxa typical of given environments, point out to the role of environmental change as an important component determining the profiles of microbial communities living on the skin of amphibians. These changes are very likely consequential, but understanding the scope and nature of consequences require additional study.

The bronchiectatic lung is a diseased state in which the airways are chronically damaged and dilated. This state is found in the clinical entities of cystic fibrosis and non-cystic fibrosis bronchiectasis. These are two highly relevant chronic suppurative lung diseases in which an understanding of the microbiology of these patients is considered key to appropriate management. This has traditionally been via the use of traditional culture techniques. However, with the development of molecular methodologies, the previously perceived wisdom is being challenged. In both cystic fibrosis and non-cystic fibrosis bronchiectasis, Pseudomonas aeruginosa is considered the most significant pathogen. In CF there has been considerable concern surrounding the risk of transmission of Pseudomonas aeruginosa between patients on the basis of a significant quantity of research into this matter. In contrast, there has been very little research performed into the equivalent risk in non-cystic fibrosis bronchiectasis. In this thesis we describe an extensive single-centre epidemiological review of Pseudomonas aeruginosa spanning both these diseases. Via this we have shown evidence of cross-infection within a non-cystic fibrosis bronchiectasis cohort. This epidemiological review has included multiple genotyping methods including multilocus sequence typing and whole genome sequencing, As an extension of the epidemiological review, we have performed an in silico prediction of hypermutator status from the whole genome sequencing data to provide greater understanding of the likelihood of cross-infection, and have also demonstrated a culture-independent adaption of multilocus sequence typing for potential screening for cross-infection. In addition to Pseudomonas aeruginosa, we have also looked at the wider bacterial community in the lungs of patients with these two conditions via culture-independent techniques. We have shown that whilst Pseudomonas aeruginosa is often an important component, these are clearly complex communities. We have primarily investigated the cohort with non-cystic fibrosis bronchiectasis, but we have demonstrated associations between clinically-relevant markers and complexity of the bacterial communities within the lungs of both these cohorts of patients. Whilst we have used the gold-standard technique of 16S rRNA sequencing, we have also shown the validity of a simple and potentially more feasible profiling technique for standard clinical care. In summary, through the application of culture-dependent and independent molecular techniques, this research has shed light on the epidemiology of Pseudomonas aeruginosa within our respiratory cohorts, and the complexity and clinical relevance of the wider microbial communities within these patients. Such studies are essential if we are to advance our understanding of the bronchiectatic lung and optimise strategies for patient management.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-08T17:37:16Z No. of bitstreams: 1 RenataHenriqueSantanaTeseParcial2017.pdf: 1215291 bytes, checksum: cc35f4b9a014e3e823968429c1849485 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-08T17:37:28Z (GMT) No. of bitstreams: 1 RenataHenriqueSantanaTeseParcial2017.pdf: 1215291 bytes, checksum: cc35f4b9a014e3e823968429c1849485 (MD5) / Made available in DSpace on 2017-09-08T17:37:28Z (GMT). No. of bitstreams: 1 RenataHenriqueSantanaTeseParcial2017.pdf: 1215291 bytes, checksum: cc35f4b9a014e3e823968429c1849485 (MD5) Previous issue date: 2017-03-31 / Termites are well known for their ability to cause agricultural losses and building damage through their ability to digest lignocellulose; however, termites provide important ecosystem services, such as soil bioturbation and nutrient cycling. Microorganisms in the digestive tract of termites are responsible for several of these functions. Syntermes wheeleri (Syntermitinae) is a litter-feeding termite species of the Termitidae family that is abundant in the Brazilian Cerrado. In this work, we characterized the gut microbiota of S. wheeleri, aiming to better understand the ecosystem function of termite-microbe symbiosis and its evolutionary history. First, the gut microbiota composition of worker termites was described by pyrosequencing phylogenetic markers (amplicons), specifically 16S rRNA (archaea and bacteria) and 18S rRNA (fungi). The resulting bacterial taxonomic profile was then compared with the gut microbiota of several other termite species and feeding guilds. To identify the functional groups and activity of the microbiota in the whole gut and the largest proctodeal segments (P1 and P3), the metagenome and metatranscriptome were sequenced by next-generation sequencing (NGS). Amplicon sequences revealed a high abundance of Firmicutes in S. wheeleri, which was not observed in the other termite species. Most archaea belonged to the methanogen orders, and the most abundant fungal classes were Dothideomycetes, Sordariomycetes, and Eurotiomycetes, which have saprophytic species. Ordination analysis of the relative abundance of bacterial taxa showed that the S. wheeleri gut microbiota did not cluster with the microbiota of termites of different clades and feeding guilds. This finding suggests that both variables are important in microbial assembly in termites. The taxonomic profile generated by NGS analysis of the metagenome was similar to the results of amplicon analysis, with higher Firmicutes abundance in P1 and higher Spirochaetes abundance in P3. Additionally, alignment of ribosomal protein S3 identified possible compartment-specific bacterial lineages of Firmicutes, Spirochaetes, Actinobacteria, Bacteroidetes, and Tenericutes. The results of our study revealed a vast metabolic repertoire of termite gut microbes, which supplies nutrients for the host. Differences identified in the P1 and P3 metabolic pathways are likely caused by differences in O2 concentration. As for biomass conversion, the most abundant glycosyl hydrolases (necessary for lignocellulose digestion) detected in the S. wheeleri gut microbiota are a mixture of those that are most abundant in litter-feeding species (fungus growers), as well as wood-, soil-, and dung-feeding species. In addition, putative bacterial laccases (involved in lignin degradation) were detected. Besides the known function of the termite gut microbiota in carbon and nitrogen cycling, we found evidence of its possible involvement in arsenic cycling. This study adds information about microbial establishment in the termite gut, with functional groups selected based on feeding substrate, instead of specific microbial lineages. In addition, our results show that the metagenome can be used to identify molecules with potential industrial applications, such as enzymes useful in biofuel production. / Os cupins s??o conhecidos por causar perdas agr??colas e danos a edifica????es devido a sua capacidade de digerir celulose. No entanto, os cupins tamb??m fornecem importantes servi??os ecossist??micos como a modifica????o do solo e a ciclagem de nutrientes. Os micro-organismos do trato digestivo dos cupins s??o respons??veis por muitas dessas fun????es. Syntermes wheeleri (Syntermitinae) ?? uma esp??cie de cupim da fam??lia Termitidae, comedora de serapilheira e muito abundante no Cerrado. Neste trabalho caracterizamos a microbiota intestinal de Syntermes wheeleri, buscando esclarecer a fun????o ecossist??mica da simbiose entre cupins e micro-organismos e sua hist??ria evolutiva. Inicialmente, a composi????o da microbiota dos oper??rios foi descrita por meio de pirosequenciamento dos marcadores filogen??ticos (amplicons) 16S rRNA (arqueia e bact??ria) e 18S rRNA (fungo). Em seguida, o perfil taxon??mico de bact??rias foi comparado com o apresentado por diferentes esp??cies de cupins e h??bitos alimentares. Finalmente, para identificar os grupos funcionais presentes e ativos da microbiota no intestino inteiro e em seus compartimentos mais volumosos (primeiro - P1 e terceiro - P3 proctodeo) o metagenoma e metatranscriptoma foram submetidos a sequenciamento de alto rendimento (NGS). As sequ??ncias de amplicons revelaram alta abund??ncia de Firmicutes, n??o observada em outras esp??cies de cupins. A maioria das arqueias pertence ??s ordens de metanog??nicas e as classes de fungos mais abundantes foram Dothideomycetes, Sordariomycetes, e Eurotiomycetes, que possuem membros saprof??ticos. Uma an??lise de ordena????o da abund??ncia relativa dos t??xons de bact??ria mostrou que a microbiota de S. wheeleri n??o agrupou com nenhum clado de cupins da fam??lia Termitidae nem com outras guildas alimentares. Esse resultado indica que ambas as vari??veis afetam a composi????o da microbiota. An??lises do metagenoma apontaram perfil taxon??mico semelhante ao obtido pelas an??lises das sequ??ncias de DNA dos marcadores filogen??ticos, com abund??ncia de Firmicutes maior em P1 e de Spirochaetes maior em P3. Adicionalmente, o alinhamento das prote??nas ribossomais S3, mostrou poss??veis linhagens de bact??rias compartimento-espec??ficas de Firmicutes, Spirochaetes, Actinobacteria, Bacteroidetes e Tenericutes. Os resultados deste estudo revelaram vasto repert??rio metab??lico da microbiota, que fornece nutrientes ao hospedeiro. As diferen??as identificadas nas vias metab??licas de P1 e P3 s??o provavelmente causadas por diferen??as na concentra????o de O2. Em rela????o ?? convers??o de biomassa, as fam??lias de glicosil hidrolases (necess??rias para digest??o da lignocelulose) mais abundantes detectadas na microbiota de S. wheeleri s??o uma mistura daquelas mais abundantes na microbiota de cupins comedores de madeira, de esterco e de cultivadores de fungos. Tamb??m foram detectadas lacases putativas (envolvidas na degrada????o de lignina) de origem bacteriana. Al??m da conhecida fun????o da microbiota de cupins no ciclo do carbono e do nitrog??nio, encontramos evid??ncias de sua poss??vel participa????o no ciclo do ars??nio. Este estudo adiciona informa????o sobre o estabelecimento da microbiota no intestino de cupins, com sele????o de grupos funcionais baseados no alimento, ao inv??s de linhagens microbianas espec??ficas. Adicionalmente, os dados aqui gerados podem ser explorados na produ????o de biocombust??veis e em outros processos biotecnol??gicos.

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.

Orientador: Jayme Augusto de Souza Neto / Resumo: Aedes aegypti (Ae. aegypti) é o principal vetor de dengue e é também responsável por transmitir outras arboviroses de importância em saúde pública, como as febres zika e chikungunya. Devido a falhas no controle da transmissão destas arboviroses, que tem como base a eliminação do mosquito vetor, o Brasil é um país endêmico para a dengue e a cada ano nos deparamos com epidemias cada vez mais graves. Ao se alimentar de sangue humano infectado o primeiro local de interação do vírus com o organismo do mosquito é o intestino. Além das respostas imunológicas antivirais para conter a infecção, neste ambiente também está presente a microbiota intestinal do mosquito, um importante modulador na infecção de patógenos. Compreender como a microbiota intestinal de mosquitos é definida e se modifica em uma determinada população é de grande interesse uma vez que isso pode elucidar a relação entre mosquitos e seus organismos simbiontes, e consequentemente auxiliar em processos de paratransgênese. A principal forma de aquisição das bactérias intestinais é através do contato com o ambiente, mas outros mecanismos como transmissão transestadial e vertical também devem influenciar no estabelecimento da microbiota intestinal. Neste estudo, nós analisamos a composição da microbiota intestinal de mosquitos Ae. aegypti de uma população de campo, coletados na cidade de Botucatu, SP, através do sequenciamento em larga escala da região hipervariavel V4 do gene 16S rRNA, e acompanhamos como esta composição... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Orientador: Daniela Cardoso Umbelini Cavallini / Orientador no exterior: Bruce A. Vallance / Coorientador: Caetano Martha Antunes / Banca: Bruce Vallance / Banca: Carla Fontana / Banca: Xiaonan Lu / Banca: Ligia Sassaki / Resumo: O trato gastrointestinal abriga a maior população de microrganismos no corpo humano, onde eles desempenham um papel importante na promoção da saúde do hospedeiro. A alteração na composição da microbiota pode levar à disbiose intestinal, que consequentemente desencadeia ou agrava doenças intestinais e extra-intestinais. Microrganismos benéficos também conhecidos como probióticos são constantemente investigados como uma terapia complementar nas doenças relacionadas à disbiose. No entanto, sua eficácia no tratamento de condições severas, como por exemplos as doenças inflamatórias intestinais (DII), é bastante variável e apresenta resultados controversos. Para abordar a importância de uma abordagem probiótica personalizada para tratar a inflamação intestinal, primeiro examinamos o efeito de bactérias personalizadas usando um modelo de colite induzida por produtos químicos. Os animais que receberam comensais isolados de suas próprias fezes foram mais protegidos contra a inflamação, pois mostraram sinais reduzidos de colite, menor dano histológico e menores níveis de marcadores inflamatórios, quando comparados aos ratos que receberam uma cepa probiótica comercial. Em seguida, o papel da mucina intestinal Muc2 e da enzima Core-1 que glicosilam foram explorados usando o modelo de colite infecciosa Citrobacter rodentium. A camada de muco intestinal é a primeira linha de defesa no intestino e é composta em grande parte pela mucina Muc2. Uma vez que quase todas as bactérias entéricas de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The intestinal tract harbours the largest population of microbes in the human body where they play an important role in promoting the health of their host. If the composition of these microbes is altered, this may lead to dysbiosis that triggers or exacerbates intestinal and extra-intestinal diseases. Probiotics have been investigated as a complementary therapy in dysbiosis-related diseases. However, their effectiveness in treating severe conditions such as Inflammatory Bowel Disease (IBD) is quite variable and have shown controversial results. To address the importance of a personalized probiotic approach to treat intestinal inflammation, we first examined the effect of personalized bacteria using a model of chemical induced colitis. The animals that received commensals isolated from their own feces were more protected against inflammation as they showed reduced signs of colitis, less histological damage and lower levels of inflammatory markers as compared to mice given a commercial probiotic strain. Next, the role of the intestinal mucin Muc2 and the Core-1 enzyme that glycosylates it were explored using the Citrobacter rodentium model of infectious colitis. The intestinal mucus layer is the first line of defense in the intestine and is largely composed of the secreted mucin Muc2. Since almost all enteric bacteria must cross the overlying mucus layer to infect the host, the mucus-enteric bacterial interactions provide fundamental knowledge about infectious diseases as well as inflammatory conditions linked to dysbiosis... (Complete abstract click electronic access below) / Doutor

Orientador: André Rodrigues / Coorientador: Fernando Carlos Pagnocca / Banca: Allen Norton Hagler / Banca: Maurício Bacci Junior / Resumo: As formigas cortadeiras (Hymenoptera: Formicidae: tribo Attini) mantêm um mutualismo com o fungo Leucoagaricus gongylophorus, utilizado como alimento por esses insetos. Nos jardins de fungo, as formigas abrigam o fungo mutualista, pupas, larvas, além de uma microbiota composta por bactérias, leveduras e outros fungos. Uma grande quantidade de machos (bitus) e fêmeas aladas (içás) é produzida anualmente em ninhos adultos de formigas do gênero Atta. Esses indivíduos deixam os ninhos para acasalarem durante o voo nupcial (revoada), onde provavelmente ocorre a transferência de micro-organismos entre os indivíduos. Neste trabalho, foi avaliado a diversidade de leveduras no integumento de 200 bitus provenientes de dois ninhos de Atta sexdens rubropilosa, nas revoadas de 2010 e 2011. Foram isoladas também leveduras do solo dos túneis e do solo adjacente aos ninhos na tentativa de descobrir a origem das leveduras no integumento dos bitus. Buscando entender o papel das leveduras nos ninhos, foi estudada a capacidade de degradação de polímeros vegetais presentes no jardim de fungo e no consumo de açúcares gerados da quebra desses substratos. Um total de 242 estirpes distribuídas em 29 espécies foi recuperado do integumento dos insetos. Bitus do ninho #1 (Rio Claro, SP) apresentaram maior riqueza e diversidade de espécies de leveduras do que bitus do ninho #2 (Botucatu, SP). Embora a riqueza e diversidade tenham variado entre as diferentes amostragens, a estrutura da comunidade foi semelhante, a qual não foi correlacionada com o local de coleta ou o ano em que ocorreram as revoadas. O isolamento de leveduras do solo dos túneis e do solo próximo aos ninhos revelou poucas espécies em comum com aquelas encontradas no integumento dos bitus; o que sugere que a maioria das leveduras presentes nos bitus... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leafcutter ants (Hymenoptera: Formicidae: tribe Attini) maintain an obligate mutualism with Leucoagaricus gongylophorus, a basidiomycetous fungus cultivated for food. Attine fungus gardens harbor the mutualistic fungus, pupae, larvae and several additional microorganisms such as bacteria, yeasts and filamentous fungi. Annually, several alates are produced in Atta nests and leave their nests for the matting flight, when the transmition of microorganisms probably occur between them. Here, we evaluated the yeast diversity found on the integument of 200 males of Atta sexdens rubropilosa sampled from two different nests in the mating flights of 2010 and 2011. Yeasts were also isolated from the soil of tunnels and soil adjacent to nests in order to study if yeasts found on males were originated from this environment. In an attempt to understand the role of yeasts in the fungus garden, we study the degratation of plants polymers and the assimilation of sugars generated after hydrolysis of these substrates. A total of 242 strains distributed in 29 species were recovered on the integument of these insects. Males from nest #1 (Rio Claro, SP) showed the highest yeast species richness and diversity in comparison to males from nest #2 (Botucatu, SP). Although species richness and diversity varied between samples, the community structure was similar between nests. Thus, the community structure was not correlated with collection sites or the year when the mating flights occurred. Yeast isolation from soil samples suggested that few species were shared between this type of sample and the yeasts found on the integument of males; this result shows that most yeast species on the integument of males were derived from the fungus garden. Cryptococcus haglerorum and Trichosporon chiarellii isolated from males of nest #1 (in 2011) indicate a putative... (Complete abstract click electronic access below) / Mestre

Barrett's oesophagus (BO) arises from chronic gastro-oesophageal reflux disease(GORD). Patients have an increased risk of adenocarcinoma (ADC), which is the sixth most common cause of cancer mortality in the UK. All ADC develop from BO, and over the last twenty years there has been a marked increase in both conditions. The reasons for this are not known, however, as with some forms of gastric cancer, it is possible that there may be a bacterial aetiology. This study employed both culturebased and molecular techniques to characterise microbial communities colonising the distal oesophageal mucosae in individuals with GORD, BO and ADC, together with healthy controls. Furthermore, in vitro models were designed to create an oral microbiota, from which an oesophageal community could develop. Microbial analysis identified a shift in oesophageal population composition with disease progression, with an incremental increase in total eubacterial scores related to the metaplasia-dysplasia sequence. Additionally, an increased proportion of Gram negative species and potentially pathogenic organisms, such as Peptostreptococcus were identified. Campylobacter spp. were isolated from 75%, 50% and 60% of GORD, BO and ADC patients, respectively, compared with 20% of controls. Helicobacter pylori, which has been proposed to be protective in oesophageal disease, was significantly reduced in disease, especially in ADC patients. In vitro models were successful, with a simple oral microbiota leading to the development of unique, varied oesophageal populations representative of those found in vivo. Additionally, after exposure of this community to bile acid, population dynamics were altered, with an increase in Gram negative species, associated with a rise in haemolytic and mucinolytic activities. Exposure of oesophageal cell lines to these stressed biofilms resulted in increased cell death, and in some cases, amplified expression of p53 and COX-2. In conclusion, this research proved an association between bacterial composition and oesophageal disease. With progression to adenocarcinoma, the community becomes increasingly diversified and Gram negative in character, and therefore, is proposed to be more pathogenic. Further research is required to investigate causal relationships, through which mechanisms for disease initiation and/or maintenance can be understood.

Metagenomics, or genomic sequence of the community of microbiota (bacteria, fungi, virus), enables an investigation of the full complement of genetic material, including virulence, antibiotic resistance, and strain differentiating markers. The granularity to distinguish between closely related strains is important as within one species, these strains possess distinct functions and relationships to a host. To analyze metagenomic samples, I developed a reference-based approach that utilizes both single nucleotide variants and genetic content to assign species and strain-level designations. After refining this approach with complex simulated communities, I utilized it to analyze the microbial communities present in skin samples from healthy and diseased individuals. First, to investigate strain-level heterogeneity in healthy adults, I focused on the common skin commensals Propionibacterium acnes and Staphylococcus epidermidis with well-documented sequence variation. Results indicated that an individual’s strains of P. acnes are shared across multiple sites of his or her body, and that those strains are more similar within than between individuals. For S. epidermidis, in addition to individual site similarities, there were also site-specific strains. Overall these results emphasize that both individuality and site specificity shape our bodies’ microbial communities. Based on longitudinal data, an individual’s strain signatures remain stable for up to a year despite external, environmental perturbations. I then used metagenomic data to explore microbial temporal dynamics in atopic dermatitis (AD; eczema), an inflammatory skin disease commonly associated with Staphylococcal species. Species-level investigation of AD flares demonstrated a microbial dichotomy in which S. aureus predominated on more severely affected patients while S. epidermidis predominated on less severely affected patients. Strain-level analysis determined that S. aureus-predominant patients were monocolonized with distinct S. aureus strains, while all patients had heterogeneous S. epidermidis strain communities. To assess the host immunologic effects of these species, I topically applied patient-derived strains to mice. AD strains of S. aureus were sufficient to elicit a skin immune response, characteristic of AD patients. This suggests a model whereby staphylococcal strains contribute to AD progression through activation of the host immune system. Overall, this strain-level analysis of healthy and disease communities provides previously unexplored resolution of human skin microbiome. / 2018-03-24T00:00:00Z