Made available in DSpace on 2014-06-12T18:27:29Z (GMT). No. of bitstreams: 2 arquivo1004_1.pdf: 1251472 bytes, checksum: 1f6e464692ea89b8cd077af78f9fa70b (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A injeção intravítrea representa um dos maiores desafios na terapêutica oftalmológica atual para o tratamento da degeneração macular relacionada à idade, na forma exsudativa, embora as injeções intravítreas possam levar a endoftalmite póscirurgica, onde a fonte pricipal é a microbiota conjuntival. Objetivos: descrever a microbiota conjuntival bacteriana e fúngica de portadores de degeneração macular relacionada à idade no peri-operatório com uso de injeção intravítrea de antiangiogênico em um esquema terapêutico trimestral. Métodos: foi realizado um estudo de série de casos com 35 pacientes submetidos a injeção intravítrea de antiangiogênico para o tratamento de DMRI e 30 pacientes controles sadios, na Fundação Altino Ventura-PE. O material da conjuntiva foi coletado em 4 momentos: (t0) antes da primeira aplicação de antiangiogênico; (t1) após uso tópico de 3 dias de antibiótico; (t2) antes da terceira aplicação de antiangiogênico e (t3) no retorno de 30 dias após término, para o grupo DMRI. Com relação ao grupo controle, foi feita apenas uma coleta para o islomento bacteriano e fúngico, correspondente a t0. A coleta foi através de swab no fundo de saco conjuntival inferior, e colocado em meio BHI.O processo de investigação microbiológica seguia as seguintes etapas:bacterioscopia, cultura e antibiograma. Resultados: para o grupo DMRI, o houve crescimento positivo das culturas para os momentos t0, t1, t2 e t3, cujos percentuais foram respectivamente de 91,4%; 28,6%; 74,3% e 94,3%. Para o grupo controle, foi realizada a cultura da microbiota apenas no momento t0, evidenciando-se um percentual de 93,3% de culturas positivas. Para ambos os grupos houve maior freqüência de bactérias Gram-positivas em todos momentos, prevalecendo às espécies Staphyloccocus aureus e Staphyloccocus coagulase negativo. Os fungos mais frequentemente isolados Aspergillus sp e Penicillium sp para ambos grupos.Conclusão: não houve diferença na distribuição dos micro-organismos isolados na conjuntiva dos portadores de DMRI, comparada aos pacientes controles

Made available in DSpace on 2014-06-12T23:00:51Z (GMT). No. of bitstreams: 2 arquivo8537_1.pdf: 5921633 bytes, checksum: 36de0e6e5c490e680c08c8691edd422e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo deste estudo foi observar a resposta bacteriológica e histológica nos processos de alveolite em ratos Wistar machos tratados com Mercurius solubilis 12 CH. Os animais foram anestesiados com quetamina (10 mg/kg de peso) + xilazina (0,5 mg/kg de peso), submetidos à extração do incisivo superior direito e subseqüente alveolite, perfazendo, inicialmente, 3 grupos: I água (controle), II - Mercurius solubilis 12 CH 0/20 mL (placebo) e III - Mercurius solubilis 12 CH. Posteriormente, estes grupos foram divididos em 3 subgrupos: A, B e C, correspondendo ao 6o, 15o e 21o dia de alveolite, respectivamente. A microbiota oral foi coletada através de swab em BHI para semeio e bacterioscopia. Após o semeio, as placas de Petri foram incubadas a 37°C por 48 horas. Para o exame histológico, os espécimens foram fixados em formol a 10% por 24 horas, descalcificados e processados para microscopia de luz. Observaram-se alterações quantitativas e qualitativas da microbiota oral ao compararem-se os grupos após extração e alveolite. Os grupos I e II apresentaram maior surgimento de bactérias patogênicas, enquanto o grupo III conservou a microbiota com as características mais próximas da normal. O Mercurius solubilis 12 CH não apresentou atividade bactericida, porém conservou a microbiota dentro dos parâmetros de normalidade. A cicatrização alveolar procedeu-se mesmo diante da alveolite, ocorrendo discretamente no grupo I, mais desenvolvida no grupo II e comparável aos achados normais no grupo III. Tanto do ponto de vista bacteriológico quanto histológico, os melhores resultados foram obtidos após 21 dias de tratamento com o Mercurius solubilis 12 CH

Made available in DSpace on 2014-06-12T23:13:15Z (GMT). No. of bitstreams: 2 arquivo3053_1.pdf: 2529352 bytes, checksum: ea122352da45d79a2b72d5634d4999af (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco / O zoantídeo Palythoa caribaeorum é um cnidário colonial cujo os pólipos são conectados por um espesso tecido que possui partículas minerais incorporadas e habita extensas áreas dos recifes costeiros no Brasil. Este zoantídeo é popularmente conhecido por "baba-de-boi", por secretar um muco muito viscoso. O muco produzido por corais, animais que são filogeneticamente próximos ao zoantídeos, consiste de um conjunto complexo de Eukarya Archaea e Bacteria. O objetivo deste estudo foi realizar uma caracterização taxonômica da microbiota presente no muco de colônias saudáveis e branqueadas de P. caribaeorum. O muco foi coletado nos recifes costeiros de Porto de Galinhas e de Suape, litoral sul de Pernambuco, em 2010. O isolamento das bactérias foi realizada utilizando o meio Ágar Marinho. O PCR dos segmentos 16S rDNA foi realizado utilizando os primers universais 27F e 1492R. O Sequênciamento dos fragmentos foi realizado no sequênciador ABI 3100 e a qualidade das sequências foram verificadas usando o programa Sequencing analysis 3.4 e, então, comparadas com as sequencias depositadas no GenBank usando o algoritmo Blastn. Um total de 50 amostras de bactérias isoladas de colônias saudáveis e branqueadas foram analisadas. O grupo dominante foi -Proteobacteria com 36 isolados (72%), seguido por - Proteobacteria e Actinobacteria com seis isolados (12%) cada, e Firmicutes com dois isolados (4%). O gênero Vibrio foi o mais comum (50%), corroborando trabalhos anteriores em que este gênero foi o mais comum associado aos cnidários. Sequências de alguns isolados foram relacionados ao nível de espécie (97%) aos já depositados no GenBank, entre elas, espécies com potencial biotecnológico interessante, como bactérias do gênero Alcanivorax, que usa hidrocarbonetos derivados do petróleo como fonte de carbono e energia; e bactérias dos gêneros Vibrio, Photobacterium, Pseudomonas, Micrococcus e Pseudoalteromonas, grupos conhecidos por produzir compostos antimicrobianos e potentes toxinas marinhas. Algumas das sequências dos isolados foram relacionados com patógenos de seres humanos como V. alginolyticus e V. proteolyticus, e outras relacionadas a espécies do gênero Pseudoalteromonas, que podem ter participação no fenômeno do branqueamento. Outros isolados podem representar novas espécies uma vez que suas seqüências mostrou uma baixa similaridade com os taxa já conhecidos e muitos deles foram registrados pela primeira vez no muco de P. caribaeorum. É importante intensificar os estudos de diversidade microbiana neste zoantídeo para entender melhor o papel dessas bactérias nas propriedades farmacológicas do muco

The dense and diverse community of microorganisms inhabiting the gastrointestinal tract of ruminant animals plays critical roles in the metabolism and absorption of nutrients, and gut associated immune function. Understanding microbial colonization in the small intestine of new born ruminants is a vital first step toward manipulating gut function through interventions during early life to produce long-term positive effects on host productivity and health. Yet the knowledge of microbiota colonization and its induced metabolites of small intestine during early life is still limited. In the present study, we examined the microbiota and metabolome in the jejunum and ileum of neonatal sika deer (Cervus nippon) from birth to weaning at days 1, 42, and 70. The microbial data showed that diversity and richness were increased with age, but a highly individual variation was observed at day 1. Principal coordinate analysis revealed significant differences in microbial community composition across three time points in the jejunum and ileum. The abundance of Halomonas spp., Lactobacillus spp., Escherichia-Shigella, and Bacteroides spp. tended to be decreased, while the proportion of Intestinibacter spp., Cellulosilyticum spp., Turicibacter spp., Clostridium sensu stricto 1 and Romboutsia spp. was significantly increased with age. For metabolome, metabolites separated from each other across the three time points in both jejunum and ileum. Moreover, the amounts of methionine, threonine, and putrescine were increased, while the amounts of myristic acid and pentadecanoic acid were decreased with age, respectively. The present study demonstrated that microbiota colonization and the metabolome becomes more developed in the small intestine with age. This may shed new light on the microbiota-metabolome-immune interaction during development.

Master of Science / Department of Animal Sciences and Industry / Joann M. Kouba / The objectives of these studies were to (1) characterize mare milk and fecal bacteria, and foal fecal microbiota from birth to 4 mo and (2) determine the effect of weaning and weaning method on foal fecal bacterial composition. Next generation sequencing of the V4 region of the bacterial 16S rRNA gene was performed using the Illumina Miseq according to Earth Microbiome Project protocols and sequencing data was analyzed using QIIME. In experiment 1, mare milk, mare fecal, and foal fecal samples were collected from 9 mare and foal pairs at birth (d 0), d 2, 7, and 1, 2, 3 and 4 mo. In experiment 2, 9 foals were separated into 2 treatments: abrupt (n = 5) and gradual (n = 4) weaning methods. Fecal samples were collected the day before weaning (d-1), the day of weaning (d 0) and post-weaning on d 1, 2, 3, 4, and 7. Blood was collected for analysis of cortisol concentration at 0800 h on d -1, 1, 2, and at 0800 h and 1100 h on d 0 and 4. Heart rate was recorded in 10 min intervals on the day of weaning starting 1 h before weaning to 2 h post-weaning, and again for 1 h starting 24 h after weaning. Results from experiment 1 showed newborn foal meconium and mare milk were similar in species diversity and composition; however, large shifts in composition and increases in foal fecal bacterial diversity occurred within the first week. By 1 mo, foal fecal bacterial composition did not differ in composition from mare feces at the phylum level (P = 1.0). Firmicutes, Bacteriodetes, Verrucomicrobia, and Spirochaetes were the dominant phyla found in feces of foals 1 mo and older and adult mare feces. For experiment 2, there were no differences in species diversity (P > 0.05) or separations in bacterial community structure between weaning methods or before and after weaning. There were minor shifts in relative abundance of specific phyla and genera in response to weaning. Foals in the abrupt treatment group had increased cortisol concentrations on d 1 (P < 0.05) and increased heart rate for 50 min after weaning on d 0 (P < 0.05). The foal is born with fecal microbial communities similar to milk that rapidly change during the first week of life, reaching the same composition of its dam by the first month. The foal fecal microbiota matures prior to weaning, therefore weaning did not cause drastic changes in bacterial composition. Although acute stress was increased in abruptly weaned foals, stress associated with different weaning methods did not influence the fecal microbiota within the first week post-weaning.

A monitoring effort that spanned across one and a half years was conducted to examine three types of produce-associated microbiota. Produce type was determined to be the predominant factor affecting the microbial communities. Other significant factors that resulted in differences in the microbial populations were the origin and sampling date. Specifically, produce-associated microbiota among lettuce and tomatoes clustered based on the sampling period. Through molecular and cultivation-based approaches, sporadic presence of extended spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae and Acinetobacter baumannii was detected on lettuce and cucumbers during certain periods of sampling. Quantitative microbial risk assessment denoted varying levels of ingestion risks associated with different types of produce. In particular, the risks arising from ESBL-positive K. pneumoniae in the lettuce were higher than the acceptable annual risk of 10-4. Commonly used approaches to clean and wash the produce were insufficient in removing majority of the produce-associated microbiota. More invasive cleaning approaches or thorough cooking of the produce would be required to mitigate the associated risks. Most of the current reports of ESBL-positive bacterial isolates were identified in nosocomial environment. However, the carriage of such drug-resistant bacteria in food that is consumed daily

Methylmercury (MeHg) is a global pollutant that can bioaccumulate and biomagnify along the aquatic food chain, causing adverse outcomes in humans and wildlife. Effective biomonitoring programs are needed to identify high exposure populations and to develop proper mitigation strategies. However, biomonitoring results showed high inter-individual variability in the relationship between MeHg exposure and body burden. Moreover, the gut microbiota can potentially play a role in MeHg transformations, and it is widely believed that the gut microbiota may be the underlying reason for the variability between and within a population. However, the microbially-mediated mechanisms of Hg transformation in the gastrointestinal environment is poorly understood. The overarching goal of my thesis is to investigate the role of gut microbiota in MeHg transformation in human, and the relationship between environmental pollutants and the gut microbiota of sentinel species such as river otters (Lontra canadensis) and seabirds (Arctic Tern [Sterna paradisaea], Black Guillemot [Cepphus grille], Common Eider [Somateria mollissima], Double-crested Cormorant [Phalacrocorax auratus], and Leach’s Storm Petrel [Oceanodroma leucorhoa]). My thesis consists of four research papers. In the first paper, I discovered that the gut microbiota`s ability to demethylate MeHg is significantly enhanced by altering the diet. In my second paper, I discovered a novel MeHg degradation pathway. In the third and fourth papers, I explored the effect of Hg and other environmental contaminant exposure on river otters and seabirds gut microbial community structures and found a relationship between prey selection and diet to the gut microbial structure. In conclusion, my thesis explores the relationship between diet, prey selection, environment contaminants and the humans and wildlife gut microbiota and contributes to understanding the gut microbiota’s role in biomonitoring of ecosystem and human health.

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.

There is an increasing interest in how therapeutic drugs could alter the human gut microbiota composition and function. While some knowledge is accumulating on the antimicrobial impact of some psychotropics on isolated strains or the gut microbiota of animal models, information about other classes of psychotropics and representative species from the human gut is poorly investigated. The antimicrobial effect of psychotropic drugs is usually neglected as a confounding factor when investigating gut microbiome biomarkers, knowing that patients are generally put in long-term medication. The purpose of the present study was to investigate (in vitro and ex-vivo) the antimicrobial activity of some oral commonly prescribed psychotropics from different therapeutic classes on colonic microbiota diversity and metabolism and the potential capacity of probiotics to alleviate related dysbiosis. The findings of this study revealed an important in vitro inhibitory activity of psychotropic drugs, which were also expressed as drastic alterations in gut microbiota composition ex-vivo. Indeed, the relative abundances of Firmicutes and Actinobacteria were lowered while the Proteobacteria population was increased. Families of Lachnospiraceae, Lactobacillaceae, and Erysipelotrichaceae were also declined by psychotropics (aripiprazole) treatment. These microbial changes were translated into a decrease of the major SCFA (butyrate, acetate, and propionate) at the metabolic level. The addition of a probiotic combination (Lactobacillus rhamnosus and Bifidobacterium longum) concomitantly with a psychotropic (aripiprazole) had a protective effect by attenuating the decline of microbiota composition and increasing the concentrations of SCFA. These findings provide evidence that psychotropics, through their antimicrobial effect, have the potential to alter the human gut microbiota composition and metabolism, while probiotics can mitigate the related dysbiosis.

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