Aquatic animals, such as fish and shellfish, provide important economic and nutritional benefits for human society. Due to overexploitation of natural fish sources through traditional wild-caught fisheries, aquaculture (generally described as fish farming or culturing) has grown into an economically important industry. A major focus area for the aquaculture field is related to sustainability by ensuring the health and welfare of the aquatic animals. Communities of microorganisms inhabiting the various niches of a given host comprise its microbiome and provide several key health benefits. The microbiome impacts nutrient acquisition, gut homeostasis, protection against pathogens, and immune system modulation. Therefore, much attention has been placed on studying how various culturing conditions and host factors impact the microbiomes of aquatic animals. Here, multiple studies were conducted to elucidate the impacts of various parameters on the microbiomes of rainbow trout, steelhead trout, and Nile tilapia, including dietary supplementation, administration of probiotics and animal age. Though there is a significant correlation between the diet fed to fish and their microbiome communities, small dietary changes such as the inclusion of a dried and lysed yeast product, acting as a protein source alternative to unsustainable fishmeal did not significantly alter the intestinal adherent microbiome of rainbow trout. Moreover, an optimal percentage of yeast replacement that did not negatively impact weight gain for the aquaculture-raised fish was identified, suggesting its efficacy for the industry. Similarly, the intestinal adherent microbiomes of steelhead trout were not significantly altered by diet supplementation with a Bacillus subtilis probiotic. The total microbiome of steelhead trout (mucosa combined with digesta) was instead significantly changed when they were only fed the probiotic additive at an early stage of intestinal development. This change in the microbiome of steelhead trout correlated with a significant increase in weight gain compared to fish only fed the probiotic during later stages of intestinal development. These findings also corroborate previous observations wherein the intestinal microbiome of fish varies during their developmental stages but then stabilizes over time. Determining the core set of bacteria present in fish microbiomes, independent of treatment variables, is another important factor when considering attempts to manipulate the microbiome. To that end, a literature review was conducted in which the phyla Firmicutes, Proteobacteria and, to a lesser extent, Actinobacteria, Bacteroides, and Tenericutes were identified as likely members of the rainbow trout core microbiome. Bacterial families identified as part of the core phyla included Lactobacilliaceae that are commonly used as probiotics and Mycoplasmataceae that lack cell walls. Preventing dysbiosis of the rainbow trout microbiomes will be crucial to ensuring the health of the fish hosts and increasing longevity and profitability of the aquaculture industry. Another important aquaculture-raised species is the Eastern oyster. This animal is critical for the ecological health of the Chesapeake Bay, and it is also an important source of revenue. A significant portion of the revenue flow is the harvest and sale of live oysters for consumption. Unfortunately, consumption of raw or undercooked oysters is the most common route of infection by the human pathogen Vibrio parahaemolyticus (VP) as oysters are a natural reservoir for VP. This bacterium is responsible for a debilitating acute gastroenteritis with potential to cause fatal septicemia. Despite efforts to mitigate infection by this CDC-reportable pathogen, cases continue to increase. The understudied host-microbe relationship between the Eastern oyster and VP has been implicated as a path to research for potential future therapeutics. A novel culturing system for oysters was created using fermentation jars within a BSL-2 ready biosafety cabinet. Using this system, the effect of harvest season was tested against the inoculation efficiency of VP. It was found that higher native Vibrio levels within the oysters were present during the summer compared to the winter. Moreover, addition of the bacteriostatic antibiotic chloramphenicol (Cm) enabled a higher inoculation efficiency by VP during both the summer and winter compared to oysters not exposed to the antibiotic. During the winter, exposure to Cm led to the highest inoculation efficiency (~100%). These findings confirm the importance of the existing microbial communities against exogenous inoculation. Therefore, a year-long study was conducted to investigate the microbiome of oysters during each season. This pan-microbiome study identified a significant impact of harvest season on the microbiome structure. An increased diversity, including higher levels of Cyanobacteriaceae, was observed during the summer. Whereas an increase in Arcobacteriaceae was observed during the winter. Bacteria that persisted throughout the year included Mycoplamataceae and Spirochaeteacae; these families may represent potential members of the Eastern oyster core microbiome. Further work is needed to study the localization patterns of VP within oysters. Such work includes further optimization of immunohistochemistry (IHC) and intracellular colonization assay methods under development here. Collectively, studies of the oyster-microbe interactions will help improve aquaculture methods and identify mitigation targets to reduce VP-related clinical infections. / Doctor of Philosophy / Fish and shellfish provide important economic and nutritional benefits for human society across the globe. Unfortunately, over-fishing of traditional sources of fish and shellfish has led to a reduced supply for world markets, even as the human population increases. Aquaculture, or fish farming, has been around for centuries, but its role in society has significantly increased in the past 50 years. It currently provides about half of fish and other aquatic products on the market today. To better maintain and increase the sustainability and profitability of this industry, more focus is being placed on the health of the fish. The microbiome is the collection of communities of microorganisms, including bacteria, fungi, and archaea, that inhabit various environments including animal hosts. The majority of this dissertation focuses on the impact of factors like diet and age on the microbiomes of aquaculture-raised animals, especially fish. Dietary changes such as the addition of dried yeast-products had a significant impact on fish health but not on the microbiome communities. However, a common probiotic, Bacillus subtilis, did significantly increase not only the growth rate of trout but it also significantly altered the total intestinal microbiome found in the feces and the intestinal mucosal layer. Moreover, it was found that early exposure of the animals to the probiotic had enhanced benefits even though the microbiome appeared to stabilize over time as the fish developed. Maintaining or improving the microbiomes of fish, paying close attention to the microbes that exist as part of a core group of bacteria always present, is vital to ensuring fish health and understanding vertebrate host-microbe relationships. Thus, an analysis of the core microbiome of trout was performed. The final set of projects within this dissertation focused on the relationship between the Eastern oyster, a mollusk native to the Chesapeake Bay, and the bacterial human pathogen Vibrio parahaemolyticus (VP). VP is the leading cause of seafood-borne acute gastroenteritis worldwide, and efforts are needed to mitigate the increasing rate of human infections. Therefore, a simple system using fermentation jars within the laboratory biosafety cabinet was designed to enable safe culture of oysters that were exposed to VP under experimentally controlled conditions. Oysters harvested during the summer naturally harbored higher amounts of native Vibrio organisms in contrast to the winter oysters that harbored much lower levels. A separate microbiome analysis revealed large shifts in the oyster microbiome between summer and winter, although some microbes were continually present. The lower levels of existing Vibrio species detected in winter oysters may have allowed for the higher efficiency of inoculation of winter animals by VP. In fact, these winter animals had Vibrio microbiomes that were completely dominated by the inoculated strain which will enable future work to observe the pattern by which VP localizes, or colonizes, the oysters. Ultimately, these efforts may lead to the development of future disease mitigation strategies against VP.

A simbiose desenvolvida entre seres vivos e microrganismos desempenha um importante papel na relação saúde-doença do hospedeiro. Neste sentido, o corpo humano abriga uma grande e diversa comunidade de microrganismos, sendo as mucosas vaginal, intestinal e oral as principais superfícies mucosas do corpo feminino que abrigam as comunidades bacterianas de fundamental importância para a mulher. Estes microrganismos atuam no desenvolvimento e modulação do sistema imune, na manutenção e otimização de vias metabólicas e competem por sítios de colonização, prevenindo que microrganismos patogênicos estabeleçam colonização. A composição da microbiota feminina varia com a idade, pH, secreção hormonal, ciclo menstrual, uso de anticoncepcional e atividade sexual. O presente estudo buscou caracterizar a composição da microbiota do corpo feminino durante o período gestacional, comparando os achados entre gestantes e não gestantes saudáveis, através de técnicas de biologia molecular. Foram selecionadas 60 mulheres saudáveis para o estudo e coletadas amostras de secreção vaginal, fezes e swab oral de cada participante. O DNA das amostras foi extraído e submetido à sequenciamento do gene 16S rRNA e quantificado através da técnica de PCR em tempo real. Das participantes selecionadas, 42 eram gestantes e 18 eram mulheres não gestantes em idade reprodutiva. Observamos que a quantificação total de bactérias na vagina não apresentou diferenças entre gestantes e não gestantes. Houve aumento na abundância de Lactobacillus no sítio vaginal, bactérias produtoras de butirato na microbiota intestinal e Streptococcus na microbiota oral de mulheres grávidas quando comparadas com mulheres não gestantes. Além disso, observamos que a composição e a disposição dos gêneros encontrados sofrem uma modificação, tal como aumento de gêneros relacionados com a manutenção da homeostase no grupo de mulheres gestantes. O período gestacional influencia positivamente na composição da microbiota, garantindo assim a prevalência de gêneros bacterianos responsáveis pela manutenção das condições ideais para o desenvolvimento da gestação saudável. / The symbiosis developed between living organisms and microorganisms plays an important role in the health-disease relationship of the host. In this sense, the human body harbor a large and diverse community of microorganisms, the vaginal, intestinal and oral mucosa are the main mucosal surfaces of the female body that harbor bacterial communities of fundamental importance for women. These microorganisms act in the development and modulation of the immune system, in the maintenance and optimization of metabolic pathways and compete for colonization sites, preventing pathogenic microorganisms from establishing colonization. The composition of the female microbiota varies with age, pH, hormonal secretion, menstrual cycle, contraceptive use and sexual activity. The present study aimed to characterize the microbiota composition of the female body during the gestational period, comparing the findings between healthy and non - pregnant women through molecular biology techniques. Sixty healthy women were selected for the study and samples of vaginal secretion, stool and oral swab from each participant were collected. The DNA of the samples was extracted and submitted to the 16S rRNA gene sequencing and quantified by the real-time PCR technique. Were select, 42 were pregnant and 18 were non-pregnant women of reproductive age. We observed that the total quantification of bacteria in the vaginal samples did not present differences between pregnant and non-pregnant women. There was an increase in the abundance of Lactobacillus in the vaginal site, butyrate producing bacteria in the intestinal microbiota and Streptococcus in the oral microbiota of pregnant women when compared to nonpregnant women. In addition, we observed that the composition and arrangement of the genera found undergo a modification, such as an increase in genera related to the maintenance of homeostasis in the group of pregnant women. The pregnancy influences the composition of the microbiota, thus ensuring the prevalence of bacterial genera responsible for the maintenance of the ideal conditions for the development of healthy pregnancy.

The human microbiome - defined as the microbial communities that live in and on our bodies - is emerging as a key factor in human diseases. The expanding research field that investigates the role of the microbiome on human cancer development, termed oncobiome, has led to important discoveries such as the role of Fusobacterium nucleatum in colorectal cancer carcinogenesis and tumor progression. Motivated by these discoveries, this thesis studied the oncobiome from different perspectives, investigating whether alterations to microbial profiles were associated with disease status or an adverse response to treatment. We used both biopsy tissue samples and 16S rRNA amplicon sequencing (N = 36), as well as privately and publicly available fecal whole metagenomes (N = 764) to investigate microbiome-colorectal cancer (CRC) associations. We observed significant increases in species richness in CRC, regardless of sample type or methodology, which was partially due to expansions of species typically from the oral cavity, as well as an overabundance of specific taxa such as Bacteroides fragilis, Fusobacterium, Desulfovibrio and Bilophila in CRC. Functional potential analysis of CRC metagenomes revealed that the choline trimethylamine-lyase (cutC) gene was over-abundant in CRC, with the strength of association dependent on four identified sequence variants, pointing at a novel potential mechanism of CRC carcinogenesis. Predictive microbiome signatures trained on the combination of multiple datasets showed very high and consistent performances on distinct cohorts (average AUC 0.83, minimum 0.81). To investigate the microbiomes role in response to treatment, we profiled microbial communities of gastric wash samples in gastric cancer patients (N = 36) before and after neoadjuvant chemotherapy through 16S rRNA amplicon sequencing. Gastric wash microbial communities presented remarkably high inter-individual variation, with significant decreases in richness and phylogenetic diversity after treatment and associations with pH, pathological response and sample collection. The most abundant genera found in patients before or after chemotherapy treatment included Streptococcus, Prevotella, Rothia and Veillonella. Despite limitations inherent to differing experimental choices, this thesis provides microbiome signatures that can be the basis for clinical prognostic tests and hypothesis-driven mechanistic studies, as well as supporting the role of the human oral microbiome in whole-body diseases. / O microbioma humano - definido como as comunidades microbianas que vivem sobre e dentro do corpo humano - está se tornando um fator cada vez mais importante em doenças humanas. O campo de estudo que investiga o papel do microbioma no desenvolvimento do câncer humano, denominado oncobioma, está crescendo e já levou a importantes descobertas como o papel da espécie Fusobacterium nucleatum na carcinogênese e progressão tumoral de tumores colorretais. Motivado por estas descobertas, esta tese de doutorado analisou o oncobioma por diferentes perspectivas, investigando se alterações nos perfis microbianos estavam associados à presença da doença ou a uma resposta adversa ao tratamento. Usamos tanto amostras de tecidos de biópsias e o sequenciamento do gene 16S rRNA (N = 36), quanto metagenomas fecais públicos e privados (N = 764), para investigar associações entre o microbioma e o câncer colorretal (CCR). Observamos um aumento significativo da riqueza microbiana no CCR, independentemente do tipo da amostra ou metodologia, que era em parte, devido ao aumento de espécies tipicamente presentes na cavidade oral. Observamos também um aumento da abundância de táxons específicos no CCR, que incluíam Bacteroides fragilis, Fusobacterium, Desulfovibrio e Bilophila. Analisando o potencial funcional dos metagenomas, encontramos um aumento significativo da enzima liase colina trimetilamina (cutC) no CCR, cuja associação era dependente de 4 variantes de sequência, demonstrando ser um possível novo mecanismo de carcinogênese no CCR. Assinaturas preditivas do microbioma treinadas na combinação dos estudos demonstraram ser altamente preditivas e consistentes nos diferentes estudos (média de AUC 0.83, mínimo de 0.81). Para investigar o possível papel do microbioma na resposta ao tratamento, analisamos os perfis microbianos do suco gástrico de pacientes com câncer gástrico (N = 36) antes e depois do tratamento quimioterápico neoadjuvante. As comunidades microbianas apresentaram uma variabilidade inter-individual notavelmente grande, com diminuições significativas na riqueza e diversidade filogenética pós tratamento, além de estarem associadas principalmente ao pH, mas também à resposta patológica e ao tempo da coleta. Os gêneros mais abundantes encontrados nos pacientes antes ou depois da quimioterapia incluíam Streptococcus, Prevotella, Rothia e Veillonella. Apesar das limitações inerentes às escolhas experimentais, esta tese proporciona assinaturas do microbioma que podem servir de base para testes clínicos prognósticos e estudos mecanísticos, além de dar mais suporte ao papel do microbioma oral em doenças humanas.

The human microbiome - defined as the microbial communities that live in and on our bodies - is emerging as a key factor in human diseases. The expanding research field that investigates the role of the microbiome on human cancer development, termed oncobiome, has led to important discoveries such as the role of Fusobacterium nucleatum in colorectal cancer carcinogenesis and tumor progression. Motivated by these discoveries, this thesis studied the oncobiome from different perspectives, investigating whether alterations to microbial profiles were associated with disease status or an adverse response to treatment. We used both biopsy tissue samples and 16S rRNA amplicon sequencing (N = 36), as well as privately and publicly available fecal whole metagenomes (N = 764) to investigate microbiome-colorectal cancer (CRC) associations. We observed significant increases in species richness in CRC, regardless of sample type or methodology, which was partially due to expansions of species typically from the oral cavity, as well as an overabundance of specific taxa such as Bacteroides fragilis, Fusobacterium, Desulfovibrio and Bilophila in CRC. Functional potential analysis of CRC metagenomes revealed that the choline trimethylamine-lyase (cutC) gene was over-abundant in CRC, with the strength of association dependent on four identified sequence variants, pointing at a novel potential mechanism of CRC carcinogenesis. Predictive microbiome signatures trained on the combination of multiple datasets showed very high and consistent performances on distinct cohorts (average AUC 0.83, minimum 0.81). To investigate the microbiomes role in response to treatment, we profiled microbial communities of gastric wash samples in gastric cancer patients (N = 36) before and after neoadjuvant chemotherapy through 16S rRNA amplicon sequencing. Gastric wash microbial communities presented remarkably high inter-individual variation, with significant decreases in richness and phylogenetic diversity after treatment and associations with pH, pathological response and sample collection. The most abundant genera found in patients before or after chemotherapy treatment included Streptococcus, Prevotella, Rothia and Veillonella. Despite limitations inherent to differing experimental choices, this thesis provides microbiome signatures that can be the basis for clinical prognostic tests and hypothesis-driven mechanistic studies, as well as supporting the role of the human oral microbiome in whole-body diseases. / O microbioma humano - definido como as comunidades microbianas que vivem sobre e dentro do corpo humano - está se tornando um fator cada vez mais importante em doenças humanas. O campo de estudo que investiga o papel do microbioma no desenvolvimento do câncer humano, denominado oncobioma, está crescendo e já levou a importantes descobertas como o papel da espécie Fusobacterium nucleatum na carcinogênese e progressão tumoral de tumores colorretais. Motivado por estas descobertas, esta tese de doutorado analisou o oncobioma por diferentes perspectivas, investigando se alterações nos perfis microbianos estavam associados à presença da doença ou a uma resposta adversa ao tratamento. Usamos tanto amostras de tecidos de biópsias e o sequenciamento do gene 16S rRNA (N = 36), quanto metagenomas fecais públicos e privados (N = 764), para investigar associações entre o microbioma e o câncer colorretal (CCR). Observamos um aumento significativo da riqueza microbiana no CCR, independentemente do tipo da amostra ou metodologia, que era em parte, devido ao aumento de espécies tipicamente presentes na cavidade oral. Observamos também um aumento da abundância de táxons específicos no CCR, que incluíam Bacteroides fragilis, Fusobacterium, Desulfovibrio e Bilophila. Analisando o potencial funcional dos metagenomas, encontramos um aumento significativo da enzima liase colina trimetilamina (cutC) no CCR, cuja associação era dependente de 4 variantes de sequência, demonstrando ser um possível novo mecanismo de carcinogênese no CCR. Assinaturas preditivas do microbioma treinadas na combinação dos estudos demonstraram ser altamente preditivas e consistentes nos diferentes estudos (média de AUC 0.83, mínimo de 0.81). Para investigar o possível papel do microbioma na resposta ao tratamento, analisamos os perfis microbianos do suco gástrico de pacientes com câncer gástrico (N = 36) antes e depois do tratamento quimioterápico neoadjuvante. As comunidades microbianas apresentaram uma variabilidade inter-individual notavelmente grande, com diminuições significativas na riqueza e diversidade filogenética pós tratamento, além de estarem associadas principalmente ao pH, mas também à resposta patológica e ao tempo da coleta. Os gêneros mais abundantes encontrados nos pacientes antes ou depois da quimioterapia incluíam Streptococcus, Prevotella, Rothia e Veillonella. Apesar das limitações inerentes às escolhas experimentais, esta tese proporciona assinaturas do microbioma que podem servir de base para testes clínicos prognósticos e estudos mecanísticos, além de dar mais suporte ao papel do microbioma oral em doenças humanas.

Orientador: Ida Maria Foschiani Dias Batista / Resumo: As bactérias Mycobacterium leprae e mais recentemente Mycobacterium lepromatosis são os agentes etiológicos da hanseníase que causam sérios danos neuromotores e podem evoluir para incapacidades irreversíveis. A incidência de casos novos de hanseníase em todo o mundo foi de 2.77/100 mil habitantes. No Brasil, em 2016, foram 2.665 casos somente do Estado de Mato Grosso na Amazônia Meridional, esses valores representam 88,9/100 mil habitantes no índice geral de detecção para a hanseníase. Foram capturados animais silvestres naturalmente infectados por Mycobacterium leprae e Mycobacterium lepromatosis das Ordens Cingulata, Didelphimorphia, e Rodentia, todos estavam em fragmentos florestais próximos a grupos humanos. Um total 327 amostras de biópsias foram avaliados, dos quais recuperou-se 254, sendo 187 amostras de orelhas, 77 baço, e 63 fígado de 187 animais silvestres das Ordens Cingulata, Rodentia e Didelphimorphia. Após extraídos DNA e RNA de baço, fígado, e orelha, foram avaliados por qPCR para os genes RLEP (enumerador) e 16S rRNA (viabilidade). Três gêneros apresentaram positividade nas orelhas para ambos os genes RLEP e 16S rRNA, sendo 18% para Dasypus (Cingulata), 60% para Proechimys (Rodentia) e 64% para Marmosa (Didelphimorphia). Enquanto que nos testes utilizando PCR multiplex obteve-se 12 amostras positivas para o gene henM referente a Mycobacterium lepromatosis, dos 13 gêneros avaliados apenas Proechimys e Marmosa apresentaram presença para o bacilo. A presença freq... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bacteria Mycobacterium leprae and more recently Mycobacterium lepromatosis are the etiological agents of leprosy that cause serious neuromotor damage and can progress to irreversible impairments. The incidence of new cases of leprosy worldwide was 2.77 / 100 thousand inhabitants. In Brazil, in 2016, there were 2,665 cases of the State of Mato Grosso alone in the Southern Amazon, which represent 88.9 / 100 thousand inhabitants in the general detection index for leprosy. Wild animals naturally infected with Mycobacterium leprae and Mycobacterium lepromatosis from the Cingulata, Didelphimorphia, and Rodentia orders were all captured in forest fragments close to human groups. A total of 327 biopsy specimens were evaluated, of which 254 were recovered, being 187 samples of ears, 77 spleen, and 63 liver of 187 wild animals of the Orders Cingulata, Rodentia and Didelphimorphia. After extracting DNA and RNA from spleen, liver, and ear, they were assessed by qPCR for the RLEP (enumerator) and 16S rRNA (viability) genes. Three genera presented positivity in the ears for both RLEP and 16S rRNA genes, 18% for Dasypus (Cingulata), 60% for Proechimys (Rodentia) and 64% for Marmosa (Didelphimorphia). While in the tests using multiplex PCR, 12 samples were positive for the henM gene for Mycobacterium lepromatosis, of the 13 genera evaluated only Proechimys and Marmosa showed presence for the bacillus. The frequent presence of man in the forest fragments where M. leprae or Mycobacterium lepro... (Complete abstract click electronic access below) / Doutor

A microbiota intestinal é um ecossistema complexo que geralmente vive em harmonia com seu hospedeiro. É essencial para o desenvolvimento e funcionamento adequado do sistema imunológico da mucosa durante o início da vida, um processo que atualmente é conhecido por ser importante para a imunidade de adultos. A microbiota intestinal compreende aproximadamente 1000 espécies, das quais 80% não são cultiváveis. Tendo em vista a importância de se conhecer a microbiota humana e a utilização da ferramenta da construção da biblioteca genômica RNAr 16S ser relativamente recente, esse estudo tem como objetivo analisar diferentes protocolos para avaliar o uso desta ferramenta para o estudo da microbiota intestinal humana. Para a realização dos ensaios experimentais, o DNA extraído de fezes de seis crianças, em diferentes faixas etárias, foi utilizado para a criação de um pool, o qual foi utilizado nos ensaios de PCR. As bibliotecas RNAr 16S foram construídas utilizando 2 pares de iniciadores bactéria-específicos 27F-1492R e 63F-1387R, variando o tempo de desnaturação inicial de cada reação de amplificação do gene RNAr 16S entre 5 e 10 minutos, e 1 par de iniciadores 341F-518R. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em banco de dados do gene RNAr 16S. A diversidade da microbiota foi menor quando os iniciadores 63F-1387R foram utilizados, em comparação aos resultados dos iniciadores 27F-1492R, no entanto, apenas o par de iniciadores 63F-1387R identificou Bifidobacterium sp., gênero importante para o desenvolvimento da microbiota intestinal humana. Não houve diferenças significativas na diversidade quando o tempo de desnaturação inicial da reação de PCR foi estendido para 10 minutos. Com o uso do par de iniciadores 341F-518R mostrou uma diversidade satisfatória, uma maior riqueza, quando comparada com os outros pares de iniciadores e detectou a presença de Bifidobacterium sp. Os dados obtidos sugerem que mais de um par de iniciadores deve ser empregado para o estudo da microbiota fecal quando se utilizar a biblioteca de RNAr 16S como ferramenta. / The intestinal microbiota is a complex ecosystem that usually lives in harmony with its host. It is essential for the development and proper functioning of the mucosal immune system during beginning of the life, a process that is currently known to be important for overall immunity of adults. The intestinal microbiota comprises about 1000 species, 80% of which are not cultivable. Given the importance of understanding the human microbiota and that the use of the tool library construction genomic 16S rRNA is relatively recent, this study aims to analyze different protocols to evaluate the use of this tool to study of the human intestinal microbiota. To perform the experimental tests, the DNA extracted from feces of six children, in different age groups, was used for the creation of a pool, which was used in the PCR assays. The 16S rRNA libraries were constructed using 2 pairs of primers specific-bacterium 27F-1492R and 63F-1387R, varying the denaturation time of each initial amplification reaction between 5 and 10 minutes, and the pair of primers 341F - 518R.The clones were randomly selected, partially sequenced and analyzed based on database 16S rRNA gene. The diversity of the microbiota was lower when the primers 63F - 1387R were used when compared with the results of the primers 27F - 1492R. However, only the pair 63F - 1387R was able to identified Bifidobacterium spp., important genus for the development of the microbiota from human gut. No significant differences in diversity were observed when the time of initial denaturation of the PCR reaction was extended to 10 minutes. By using the pair of primers 341F - 518R a satisfactory diversity, with detection of Bifidobacterium spp., and greater richness were observed, compared with the other pairs of primer. The data suggests that more than one pair of primers should be used for the study of fecal microbiota when using the library of 16S rRNA as a tool.

Forest biomes cover approximately 38 million km2 worldwide, from which one third represent tropical and subtropical forests. Among these biomes, the Amazon forest is one of the most important for its roles in global climate regulation and dueling high levels of plant, animal and microbial diversity. The Amazon forest represents 60% of Brazilian territory and has been constantly threatened by the expansion of agricultural and animal husbandry areas. The reduction of the biodiversity levels in the Amazon may result in unforeseen impacts on the stability of the biome. The role of the microorganisms in this process is unknown. In general, the knowledge about the microbial diversity and community structure in the Amazon forest, as well the drivers of these community are poorly understood. It has been observed in the Brazilian Atlantic forest that the bacterial communities associated to the phyllosphere, dermosphere and rhizosphere of several tree species are unique and depend on the plant taxon. In order to unravel the drivers of the bacterial communities associated to plants of the Amazon forest in specific microenvironments, we evaluated the bacterial communities associated with the phyllosphere, litter and rhizospheric soil of nine tree species at three time points in a pristine Amazon forest in Brazil, using high-throughput sequencing of 16S rRNA genes. Our results showed that bacterial alpha diversity in the rhizosphere is higher than in the phyllosphere. However, the phyllosphere showed higher levels of heterogeneity (i.e. higher beta diversity). We also observed that an extreme drought during the ENSO 2015-2016 affected mainly the phyllosphere bacterial communities, inducing decreases in alpha diversity and increases in beta diversity. Our results also showed that plant species and plant functional traits are important drivers of the bacterial communities in the Amazon forest. In general, our data indicate that even though plant species is an important determinant of phyllosphere, litter and rhizospheric soil bacterial community structures, extreme climatic events (such as drought) may induce significant changes in bacterial diversity and community structure of the Amazon forest trees, with possible changes in functionality. / Biomas flroestais cobrem aproximadamente 38 milhões de km2 do globo terrestre, dos quais um terço é representado por florestas tropicais e subtropicais. Dentre esses biomas, a floresta Amazônica é uma das mais importantes, uma vez que possui papéis chave na regulação climética e na manutenção da diversidade vegetal, animal e microbiana. A floresta Amazônica representa 60 % do território brasileiro e tem sido constantemente ameaçada pela expanção da agricultura e pecuária. A redução dos níveis de biodiversidade na floresta Amazônica podem resultar em impactos graves e desconhecidos na estabilidade do bioma, uma vez que es papéis desempenhados por microganismos são desconhecidos. Em geral, a diversidade e estrutura da comunidade microbiana na floresta Amazônica, bem como os fatores que moldam essas comunidade são pouco estudados. Tem sido observado na Mata Atlântica que comunidades bacterianas associadas a filosfera, dermosfera e rizosfera de diversas espécies vegetais são únicas e dependentes da espécie vegetal. Com o intuito de revelar quais são os fatores moduladores das comunidades bacterianas associadas a espécies vegetais em micro-ambientes específicos da floresta Amazônica, nós avaliamos a comunidade bacteriana associada a filosfera, serapilheira e solo rizosférico de nove espécies vegetais em três épocas ao longo de um ano em uma parcela natural de floresta Amazônica no Brasil, utilizando plataforma de sequenciamento de alto rendimento do gene 16S rRNA. Nossos resultados destacam que alfa diversidade bateriana na rizosfera é maior que na filosfera. Contudo, a filosfera apresentou alto níveis de heterogeneidade, (altos valores de beta diversidade). Nós também observamos que a extreme seca ocasionada durante o evento climático ENSO 2015-2016 afetou principalmente as comunidade bacterianas na filosfera, induzindo a diminuição da alfa diversidade e o aumento da beta diversidade. Nossos resultados também mostraram que a espécie vegetal e parâmetros funcionais relaciados a espécie vegetal foram importantes moduladores das comunidades bacterianas na floresta Amazônica. Em geral, nossos dados indicam que a embora a espécie vegetal seja um importante determinante das comunidades bacterianas associadas a filosfera, serapilhiera e solo rizosférico, eventos climáticos extremos (tal como secas severas) podem induzir significantes mudanças na diversidade e estrutura das comunidades bacterianas das espécies vegetais da floresta Amazônica, com possíveis mudanças na funcionalidade.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-03-04T16:25:51Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Roger Dissertação Mestrado 2014_ versão Final biblioteca_ Entregue_.pdf: 1359994 bytes, checksum: ce3bbcf3906e6cb72bdfea9a7b7e7d06 (MD5) / Made available in DSpace on 2016-03-04T16:25:51Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Roger Dissertação Mestrado 2014_ versão Final biblioteca_ Entregue_.pdf: 1359994 bytes, checksum: ce3bbcf3906e6cb72bdfea9a7b7e7d06 (MD5) Previous issue date: 2014-02-20 / CAPES / CNPq / FINEP / O gênero Shewanella é um representante da classe das Gammaproteobactérias, família Shewanellaceae, compondo um grupo de bactérias gram-negativas, móveis, baciliforme, oxidase positiva, comumente encontrada em ambiente marinho e isolada do trato digestivo de animais aquáticos. Devido a suas características vem sendo amplamente testado como probiótico na carcinicultura. Estes têm sido utilizados na aquicultura para o controle biológico, aumento da taxa de conversão alimentar e sistema imune dos camarões. Este estudo teve por objetivo identificar potenciais bactérias probióticas retiradas do hepatopâncreas e estômago do camarão cultivado em sistema heterotrófico, avaliar as relações filogenéticas das cepas com o gênero Shewanella e caracteriza-las através de análisesmorfológicas, bioquímicas, produção de biofilme e antibiograma. A partir do cultivo heterotrófico de Litopenaeus vannamei, foram selecionadas as cepasIPA-S.51, IPA-S.111 e IPA-S.252para identificação através do sequenciamento parcial do gene 16S rRNA e comparados ao GenBank – NCBI e RDP - Seqmatch. As cepas foram alinhadas a 45 espécies do gênero Shewanella e avaliadas filogeneticamente utilizando os métodos Neighbor-Joining, Máxima Verossimilhança e Inferência Bayesiana. Quanto àsanálises morfológicas foram avaliadas parâmetros de acordo com a similaridade a padrões estabelecidos. Os testes bioquímicos foram realizados com o auxílio dos kits BACTRAY I, BACTRAY II e BACTRAY III (Labroclin®), totalizando 30 testes bioquímicos. A avaliação da capacidade de formação de biofilme foi realizada segundo Christensen e colaboradores. No antibiograma as cepas bacterianas foram submetidas a 13 antibióticos distintos segundo à técnica de Kirby e Bauer, com três repetições. O sequenciamento das cepas revelou alta similaridade a espécie Shewanella algae, utilizando o GenBank e o RDP-seqmath. A Inferência Bayesiana apresentou maior aporte estatístico e fidelidade dentre os métodos analisados. A Shewanella upenei apresentou alta similaridade as cepas estudadas, assim como a S. algae. As análises filogenéticas não descartam a hipótese de novas espécies para IPA-S.51, IPA-S.111 e IPAS. 252. As cepas estudadas foram sensíveis aos antibióticos Ampicilina/Subactan, Ofloxacina e Tetraciclina. A caracterização fenotípica fortalece a hipótese de especiação para as cepas testadas. Portanto, a capacidade de formação de biofilme, adicionada ao alto potencial enzimático e antagonismo a patógenos, característico do gênero Shewanella, tornam estas cepas potenciais probióticos para carcinicultura. / The genus Shewanella is one representant of Gammoproteobacteria class, family Shewanellaceae, being part of gram-negative bacteria group, mobile, bacilliform, oxidasepositive, commonly found on marine environments and isolated from the digestive tract of aquatic animals. Due to its characteristics, some tests as probiotics are being carried out in carciniculture. They are being used on aquaculture for biological control, augmentations on feed conversion rates and immune system of shrimps. This study has as objective identify potentials probiotics bacteria found in the hepatopancreas and stomach of shrimps cultivated on heterotrophic based system, evaluating the strain phylogenetic relationships with Shewanella genus and characterizing them through morphological and biochemical analysis, biofilm production and antibiogram. The strains IPA-S.51, IPA-S.111 e IPA-S.252 were selected from the heterotrophic cultivation of Litopenaeus vannamei, identified through partial 16S rRNA sequencing and compared to GenBank – NCBI and RDP - Seqmatch.The strains were aligned to 45 species of Shewanella genus and phylogenetically evaluated using Neighbor-Joining,Maximum-Likelihood estimation and Bayesian Inference.Regarding the morphological analysis parameters were evaluated according with established standard similarities. The biochemical tests were conducted with the assistance of BACTRAY I, BACTRAY II and BACTRAY III (Labroclin®) kits, totalizing 30 biochemical tests.The Biofilm capacity evaluation was made according Christensen et al. Regarding the antibiogram, the bacterial strains had undergone 13 distinct antibiotics according Kirby and Bauer, with three repetitions. The strains sequencing showed high similarity to Shewanella algae species, using GenBank and RDP-seqmath.The Bayesian inference displayed higher statistic contribution e fidelity among the other methods.The Shewanella upenei showed high similarity to the strains in study also as Shewanella algae.The phylogenetic analysis does not exclude the hypothesis of IPA-S.51, IPA-S.111 e IPA-S.252 being new species.The strains studied were sensitives to the antibiotics Ampicillin/Sulbactam, Ofloxacin, Tetracyclin. The phenotypic characterization of the strains supports the hypothesis of speciation. Thus, the capacity of biofilm formation plus the high enzymatic potential and antagonism interactions to pathogens, characteristics found in the Shewanella genus, make these strains potential probiotics to carciniculture.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-03-16T15:01:11Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CAROLINA KROPNICZKI GOUVEIA - DISSERTAÇÃO.pdf: 1168935 bytes, checksum: ec9fc0cb4665cb4641bd7162f5449c56 (MD5) / Made available in DSpace on 2016-03-16T15:01:11Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CAROLINA KROPNICZKI GOUVEIA - DISSERTAÇÃO.pdf: 1168935 bytes, checksum: ec9fc0cb4665cb4641bd7162f5449c56 (MD5) Previous issue date: 2012 / A produção de camarões marinhos na região Nordeste do Brasil vem crescendo principalmente pela introdução da espécie Litopenaeus vannamei. Adicionadas à dieta ou diretamente na água, as bactérias probióticas têm sido utilizadas para controle biológico e aumento da digestibilidade alimentar e do sistema imune dos animais, elevando a lucratividade dos empreendimentos dedicados à carcinicultura. A influência de enzimas exógenas de bactérias na digestão dos camarões não está bem elucidada e ainda existe uma grande lacuna no que diz respeito a aspectos nutricionais tanto dos microrganismos, quanto dos animais cultivados. Dessa forma, objetivou-se avaliar as atividades proteolítica e amilolítica de cepas bacterianas isoladas do hepatopâncreas e estômago do L. vannamei e identificar as bactérias produtoras destas enzimas pelo sequenciamento do gene 16S rRNA. Para estudo do ambiente em tanques heterotróficos experimentais utilizando dois probióticos comerciais (HP1 e HP2) e em tanques controles heterotrófico (Het) e autotrófico (Aut), amostras de água foram tomadas ao final do experimento para contagem de bactérias heterotróficas, autotróficas e víbrios por plaqueamento em meios específicos. As médias de população bacteriana foram submetidas à análise de variância (ANOVA) complementada pelo teste de Tukey (p<0,05). Dos órgãos das amostras de camarão (n=3) foram retirados os materiais internos para isolamento das cepas bacterianas e seleção in vitro pela capacidade de produção de enzimas digestivas. Uma cepa de Bacillus subtilis (ATCC 6633) foi utilizada como testemunha na identificação utilizando os inicializadores universais para o domínio bactéria fD1 (Forward) e rD1 (Reverse). A população de bactérias heterotróficas foi mais representativa quando comparada às de autotróficas e víbrios, e o experimento Het atingiu a maior média (1,91x107 UFC/mL). As menores cargas de víbrios foram encontradas nos tanques experimentais HP1 e HP2 com médias de 2,46x104 e 2,00x104 UFC/mL, respectivamente. De 64 cepas isoladas, 11 possuíram índices enzimáticos satisfatórios (IE ≥ 2,0) para a produção de protease e amilase. Os amplicons após sequenciamento do DNA apresentaram tamanho maior que 1,4Kb e homologia com o GenBank e RDP, identificando bactérias como Bacillus subtilis e Shewanella algae. O incremento da população heterotrófica está relacionado à adição de melaço como fonte de carbono no sistema de cultivo e nos experimentos com os probióticos comerciais, a carga vibrionácea foi reduzida. A técnica de sequenciamento do gene 16S rRNA utilizando os primers fD1 e rD1 foi eficiente na caracterização bacteriana a nível de espécie e até subespécie, revelando a presença de bactérias com potencial para utilização como probiótico. / The marine shrimps cash crop in the Northeast region of Brazil is raising due to the introduction of Litopenaus vannamei. The probiotics bacteria added in the diet or directly in the water have been used for biological control and augmentations in the alimental digestibility and in the immune system, increasing the profits of enterprises related to carciniculture. The influence of bacterial exogenic enzymes on shrimp digestion is not clear and still has a gap concerning the nutrition aspects of shrimps and microorganisms. Therefore, this study had as objective the evaluations of proteolytic and amylolytic activities of bacterial strains isolated from hepatopancreas and stomach of L. vannamei and identify the bacteria responsible for enzymes production by sequencing the 16S rRNA gene. For environmental study in heterotrophic tanks using two commercial probiotics (HP1 e HP2) and in heterotrophic (Het) and autotrophic (Aut) control tanks, water samples were collected at the end of the experiment in order to count the autotrophic and heterotrophic bacteria and vibrio by plating in specific media. The means of bacterial population were submitted to variance analysis (ANOVA) and complemented by Tukey’s test (p<0.05). Internal materials were extracted from shrimp organs samples (n=3) in order to isolate the bacterial strains and “in vitro” selection due to its capacity to produce digestive enzymes. One strain of Bacillus subtilis (ATCC 6633) was used as witness on the identification using the universal primers fD1 (Forward) e rD1 (Reverse). The population of heterotrophic bacteria was more representative compared with autotrophic and vibrio population and the Het experiment presented the greatest mean (1,91x107 CFU.mL-1). The minor loads of vibrio were found in the experimental tanks HP1 and HP2 with means 2,46x104 e 2,00x104 CFU.mL-1 respectively. From sixty four strains, only eleven showed satisfactory enzymatic index (EI ≥ 2.0) to protease and amylase production. The amplicons after the DNA sequencing showed sizes bigger than 1,4Kb and homology with GenBank e RDP, identifying bacteria as Bacillus subtilis and Shewanella algae. The increment of heterotrophic population is related with the addition of molasses as carbon source in the crop system and the vibrio load were reduced in the experiments with commercial probiotics. The sequencing technique of 16S rRNA gene using fD1 and rD1 were efficient in the characterization of bacteria at the level of species and even at the level of subspecies, revealing the presence of bacteria with potential use as probiotic.

A microbiota intestinal é um ecossistema complexo que geralmente vive em harmonia com seu hospedeiro. É essencial para o desenvolvimento e funcionamento adequado do sistema imunológico da mucosa durante o início da vida, um processo que atualmente é conhecido por ser importante para a imunidade de adultos. A microbiota intestinal compreende aproximadamente 1000 espécies, das quais 80% não são cultiváveis. Tendo em vista a importância de se conhecer a microbiota humana e a utilização da ferramenta da construção da biblioteca genômica RNAr 16S ser relativamente recente, esse estudo tem como objetivo analisar diferentes protocolos para avaliar o uso desta ferramenta para o estudo da microbiota intestinal humana. Para a realização dos ensaios experimentais, o DNA extraído de fezes de seis crianças, em diferentes faixas etárias, foi utilizado para a criação de um pool, o qual foi utilizado nos ensaios de PCR. As bibliotecas RNAr 16S foram construídas utilizando 2 pares de iniciadores bactéria-específicos 27F-1492R e 63F-1387R, variando o tempo de desnaturação inicial de cada reação de amplificação do gene RNAr 16S entre 5 e 10 minutos, e 1 par de iniciadores 341F-518R. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em banco de dados do gene RNAr 16S. A diversidade da microbiota foi menor quando os iniciadores 63F-1387R foram utilizados, em comparação aos resultados dos iniciadores 27F-1492R, no entanto, apenas o par de iniciadores 63F-1387R identificou Bifidobacterium sp., gênero importante para o desenvolvimento da microbiota intestinal humana. Não houve diferenças significativas na diversidade quando o tempo de desnaturação inicial da reação de PCR foi estendido para 10 minutos. Com o uso do par de iniciadores 341F-518R mostrou uma diversidade satisfatória, uma maior riqueza, quando comparada com os outros pares de iniciadores e detectou a presença de Bifidobacterium sp. Os dados obtidos sugerem que mais de um par de iniciadores deve ser empregado para o estudo da microbiota fecal quando se utilizar a biblioteca de RNAr 16S como ferramenta. / The intestinal microbiota is a complex ecosystem that usually lives in harmony with its host. It is essential for the development and proper functioning of the mucosal immune system during beginning of the life, a process that is currently known to be important for overall immunity of adults. The intestinal microbiota comprises about 1000 species, 80% of which are not cultivable. Given the importance of understanding the human microbiota and that the use of the tool library construction genomic 16S rRNA is relatively recent, this study aims to analyze different protocols to evaluate the use of this tool to study of the human intestinal microbiota. To perform the experimental tests, the DNA extracted from feces of six children, in different age groups, was used for the creation of a pool, which was used in the PCR assays. The 16S rRNA libraries were constructed using 2 pairs of primers specific-bacterium 27F-1492R and 63F-1387R, varying the denaturation time of each initial amplification reaction between 5 and 10 minutes, and the pair of primers 341F - 518R.The clones were randomly selected, partially sequenced and analyzed based on database 16S rRNA gene. The diversity of the microbiota was lower when the primers 63F - 1387R were used when compared with the results of the primers 27F - 1492R. However, only the pair 63F - 1387R was able to identified Bifidobacterium spp., important genus for the development of the microbiota from human gut. No significant differences in diversity were observed when the time of initial denaturation of the PCR reaction was extended to 10 minutes. By using the pair of primers 341F - 518R a satisfactory diversity, with detection of Bifidobacterium spp., and greater richness were observed, compared with the other pairs of primer. The data suggests that more than one pair of primers should be used for the study of fecal microbiota when using the library of 16S rRNA as a tool.