A mastite bovina é considerada a doença de maior impacto nos rebanhos leiteiros, exercendo efeito econômico negativo sobre a produtividade e perdas significativas à indústria de laticínios. Tendo em vista os impactos na sanidade animal e os prejuízos econômicos acarretados, o objetivo deste estudo foi caracterizar de forma mais abrangente a comunidade microbiana presente em rebanhos leiteiros com mastite subclínica utilizado o sequenciamento parcial do gene 16S ribossomal RNA (rRNA). Especificamente, foram caracterizadas as comunidades bacterianas presentes em amostras de leite vindas de três fazendas comerciais, sendo que cada fazenda contribuiu com amostras com alta contagem de células somáticas (CCS > 200.000 cel./mL) e com baixa contagem (CCS < 200.000 cel./mL) perfazendo um total de 57 animais. O DNA total foi extraído e amplificado com os oligonucleotídeos iniciadores da região V3 e V4 do gene 16S rRNA. O sequenciamento foi realizado utilizando a tecnologia de sequenciamento de nova geração através do equipamento MiSeq (Illumina - San Diego, EUA). Para efeito de comparação, alíquotas de todas as amostras foram destinadas ao cultivo microbiológico para identificação de bactérias causadoras da mastite. Os fragmentos amplicons de todas as amostras foram submetidos a uma série de análises computacionais utilizando o programa QIIME. Após a avaliação adicional das sequências em nível de espécie, verificou-se que em geral as bactérias diagnosticadas por cultura geralmente não corresponderam com as sequências mais abundantes detectadas pelo sequenciamento. A análise da composição da microbiota de amostras de leite provenientes de animais saudáveis revelou a presença de uma grande diversidade de espécies bacterianas, mesmo que nenhuma bactéria tenha sido detectada por técnica de cultura. A espécie bacteriana mais abundante em todas as amostras foi Staphylococcus chromogenes. Staphylococcus aureus também foi detectada na grande maioria das amostras As diferenças na composição microbiana foram observadas entre as amostras quando a comparação foi feita de forma individual. Estas diferenças foram notórias em composição taxonômica e foram refletidas por intermédio das estimativas de alfa e beta diversidade. Quando a comparação foi realizada por separação de grupos com alta e baixa CCS, essa diferença não foi tão evidente. Com este estudo será possível compreender a diversidade dos microrganismos presentes na glândula mamária de animais saudáveis e com mastite subclínica. Essas informações podem ser úteis podendo contribuir no planejamento de medidas terapêuticas e preventivas mais eficazes da doença. / Bovine mastitis is considered the most impact disease in dairy herds, exerting negative economic effect on productivity and significant losses to the dairy industry. In view of the impact on animal health and carted economic losses, the objective of this study was to characterize more comprehensively the microbial community present in dairy herds with subclinical mastitis using the partial sequencing of 16S ribosomal RNA gene (rRNA). Specifically, the bacterial communities present in samples of milk coming from three commercial farms were identified, and each farm contributed samples with high somatic cell count (SCC> 200,000 cel./mL) and low count (SCC <200,000 cel./ml) for a total of 57 animals. Total DNA was extracted and amplified with primers of the V3 and V4 region of the 16S rRNA gene. Sequencing was performed using the new generation of sequencing technology through MiSeq equipment (Illumina - San Diego, USA). For comparison, aliquots of all samples were intended for microbiological culture for identification of bacteria which cause mastitis. The amplicon fragments of all samples were subjected to a series of computer analyzes using the QIIME program. After further evaluation of the sequences at the species level, it was found that in general the bacteria do not generally diagnosed by culture corresponded to the most abundant sequences identified by sequencing. The analysis of milk samples from microbial composition from healthy animals revealed the presence of a diversity of bacterial species, even though no bacteria have been detected by culture technique. The most abundant bacterial species in all samples was Staphylococcus chromogenes. Staphylococcus aureus was also detected in most samples differences in microbial composition were found between the samples when a comparison was made individually. These differences were noticeable in taxonomic composition and were reflected by means of the estimates of alpha and beta diversity. When comparison was performed by separation of high and low groups with CCS, this difference was not so evident. This study will be possible to understand the diversity of microorganisms present in the mammary gland of healthy animals and with subclinical mastitis. This information can be useful and can contribute in the planning of more effective therapeutic and preventive measures of the disease.

L'établissement d'un répertoire exhaustif ainsi que son élargissement constituent les deux objectifs principaux de ce travail. Nous avons d'abord établi la toute première liste de bactéries identifiées par culture des voies respiratoires au travers de la littérature scientifique. Nous répertorions ici 756 espèces, ce qui représente 27,23% de l'ensemble des bactéries isolées chez l'homme lorsque comparé au répertoire établi récemment par Bilen et al. Parmi ces bactéries, 514 avaient déjà été isolées au moins une fois dans les poumons. Plus de la moitié (i.e., 65,5%) des bactéries isolées pour la première fois dans des échantillons de poumons, ont été identifiées après les années 2000, soulignant la nécessité de poursuivre les efforts pour cultiver des microbes à partir des échantillons de voies respiratoires. Nous pensons que la combinaison de méthodes de culture à grande échelle telles que la culturomique et la métagénomique aidera à mieux décrire le microbiote pulmonaire. Des études antérieures sur le microbiote digestif le démontre. Nous avons ensuite utilisé des approches culturomiques et métagénomiques pour explorer le microbiote respiratoire d'individus sains. Nous avons isolé 193 bactéries par culturomics. Parmi ceux-ci, nous avons ajouté 84 au répertoire du microbiote respiratoire, dont 14 nouvelles espèces. En utilisant des approches métagénomiques, 139 OTU identifiées au rang de l'espèce dont seulement 49 (17,3%) étaient également retrouvées par culturomique, confortant la complémentarité des deux approches. Enfin, nous avons utilisé la taxonogénomique, une nouvelle approche permettant la description de nouvelles espèces bactériennes, pour décrire 19 bactéries. / The establishment of a comprehensive directory and its expansion through the use of high-speed culture methods are the two main objectives of this thesis work. We first established the first list of bacteria identified by airway culture through the scientific literature. Here we list 756 species, representing 27.23% of all bacteria isolated from humans when compared to the recently established repertoire of Bilen et al. Of these bacteria, 514 had already been isolated at least once in the lungs. Considering bacteria isolated for the first time in lung samples, more than half (ie, 65.5%) were identified after the 2000s, highlighting the need for continued efforts to grow microbes from lane samples respiratory. We believe that the combination of large scale culture methods such as culturomics and metagenomics will help to better describe the pulmonary microbiota. Previous studies on the digestive microbiota have shown the complementarity of these two approaches. We then used culturomic and metagenomic approaches to explore the respiratory microbiota of healthy individuals. We isolated 193 bacteria by culturomics. Of these, we added 84 bacteria to the repertoire of the respiratory microbiota, including 14 new species discovered. Using metagenomic approaches, 139 OTUs identified with the rank of the species of which only 49 (17.3%) were also recovered by culturomics, reinforcing the complementarity of the two approaches. Finally, we used taxonogenomics, a new approach for describing new bacterial species by integrating genomic and proteomic data with those that are classically integrated. Using this approach, 19 bacteria were described as part of this work.

Alors que la Guadeloupe possède la plus grande bordure littorale de mangroves des Petites Antilles, la microfaune et la microflore bactérienne marine associées à cet écosystème sont très mal connues. Pourtant, ces diverses communautés de micro-organismes sont à la base du réseau trophique des sédiments marins de mangrove. En effet, grâce à leurs activités basées sur des processus hétérotrophes, ces micro-organismes vont permettre de dégrader la litière constituée de feuilles et branches de palétuviers tombées à la surface du sédiment. En condition anoxique, la dégradation des substrats végétaux par des bactéries sulfato-réductrices entraine la production de sulfures qui vont soutenir l’activité de bactéries chimiosynthétiques. Les protistes ciliés sont des micro-organismes eucaryotes unicellulaires caractérisés par la présence de cils sur la surface cellulaire et appartenant au micro-zooplancton. Leur mode de nutrition basé sur la phagocytose permet non seulement de favoriser la reminéralisation de la biomasse microbienne, ce qui augmente le transfert de nutriments à d'autres organismes du réseau trophique, mais facilite surtout l’émergence de nombreuses associations symbiotiques. Les résultats obtenus durant cette thèse ont permis de mettre en évidence la présence d’associations symbiotiques entre des bactéries sulfo-oxydantes ou hétérotrophes et des espèces de protistes ciliés faisant parties du périphyton de mangrove. / While Guadeloupe has the largest coastal edge of mangroves in the Lesser Antilles, the microfauna and marine bacterial microflora associated with this ecosystem are very poorly understood. However, these diverse communities of microorganisms are at the base of the marine mangrove sediment food web. Indeed, thanks to their activities based on heterotrophic processes, these micro-organisms will make it possible to degrade the litter composed of mangrove leaves and branches that have fallen to the surface of the sediment. In anoxic conditions, the degradation of plant substrates by sulfate-reducing bacteria leads to the production of sulfides that will support the activity of chemosynthetic bacteria. Ciliates protists are unicellular eukaryotic microorganisms characterized by the presence of cilia on the cell surface and belonging to micro-zooplankton. Their phagocytosis-based nutrition not only promotes the remineralization of microbial biomass, which increases the transfer of nutrients to other organisms in the food web, but also facilitates the emergence of many symbiotic associations. The results obtained during this thesis allowed to highlight the presence of symbiotic associations between sulfur-oxidizing or heterotrophic bacteria and ciliates protist species that are part of the mangrove periphyton.

La prise en compte du microbiote intestinal porcin est une approche de plus en plus considérée dans la qualification des mesures de contrôle des pathogènes alimentaires issus de cette filière de production. Toutefois, ces études, en conditions protégées, sont généralement réalisées in vivo, nécessitent des installations spécialisées, coûteuses en fonctionnement. Une alternative possible à l’utilisation des animaux serait la disponibilité d’un système in vitro mimant le microbiote intestinal cultivé en bioréacteur. Ce projet de recherche vise la mise au point d’un système de culture en bioréacteur pour la production d’un microbiote dérivé du microbiote intestinal porcin, puis la mesure de l’effet de deux huiles essentielles sur le microbiote. Un système, original par son alimentation continue par un digestat d’aliment conventionnel porcin, a été utilisé dans le cadre de ce projet. En effet, ce système est composé de huit réacteurs, dont un réacteur mère permettant la croissance du microbiote qui a été distribué dans les sept réacteurs fils afin de disposer de conditions reproductibles. Les résultats obtenus par séquençage métagénomique 16S par Illumina MiSeq des échantillons démontrent une grande évolution de la composition des échantillons à 48 h par rapport au contenu intestinal utilisé pour l’inoculation. Toutefois, cette variation a diminué considérablement entre 48h et 72h, pour offrir une fenêtre d’analyse de microbiotes complexes d’origine intestinale porcine pour l’évaluation d’additifs alimentaires. Dans le cadre de cette étude, l’introduction de deux huiles essentielles, le Thymol et le Carvacrol, a été testée dans le système à 48h, pour deux concentrations (200 ou 1000 ppm). Les résultats de PCR quantitative ont démontré une augmentation significative des populations de Lactobacilles dans les réacteurs contenant du Thymol à 1000 ppm en 24h (entre l’ajout à 48h et la fin de l’expérience, à 72 h) ou par comparaison avec le réacteur contrôle sans huile à la fin de l’essai, mais aucune différence significative n’a été observée pour le Carvacrol. Toutefois, aucune différence significative n’a été observée au niveau de la composition du microbiote entre les réacteurs comportant des additifs différents. Cette étude a permis le développement d’un système de bioréacteur permettant de maintenir un microbiote dérivé du microbiote intestinal porcin sur 72h, ainsi que sa mise en oeuvre pour mesurer l’effet de l’addition d’une huile essentielle dans un réacteur. / Pig intestinal microbiota is an approach more considered in the foodborne pathogen control qualification in the pig production. However, these studies are commonly conducted in vivo needing specific installation and significant costs. A possible alternative to the use of animals would be the used of an in vitro system to mimic the intestinal microbiota, in a bioreactor. This research project aims to setup a bioreactor system to produce a microbiota derived from the pig intestinal microbiota, and to assess the effect of two essentials oils on this microbiota. The system used in this project is unique by its design, continuously supplied using digested piglet feed as a culture media. This system is composed of eight reactors, including a mother reactor for the growth of the microbiota that was divided between seven daughter reactors to obtain reproducible conditions. Results obtained by 16S metagenomic sequencing using Illumina MiSeq of the samples showed an evolution of the sample composition at 48h compared to intestinal content used for inoculation. However, this variation decreases between 48h and 72h, offering a window for the analysis of food additives effect on the microbiota from piglet origin. In this study, two essentials’ oils, Thymol and Carvacrol, were added to the system at 48h, at a concentration of 200 and 1000 ppm, respectively. Quantitative PCR showed a significant increase of Lactobacilli in reactors containing Thymol at 1000 ppm in 24h (between the addition at 48h and the end of the experiment at 72h) or when comparing with the control reactor without oils at the end of the experiment, but no significative difference was observed for the Carvacrol. However, no significative difference was observed in microbiota composition between the reactors containing the different additives. This study enabled the development of a bioreactor system to maintain a microbiota derived from the pig intestinal microbiota over 72h, and the possibility to assess the effect of essential oils addition in a reactor.

Bacterial water pollution is a significant problem because it is associated with reduction in the ‘quality’ of water systems with a potential impact on human health. Faecal indicator bacteria (FIB) are usually used to monitor the quality of water, and to indicate the presence of pathogens in water bodies. However, enumeration alone does not enable identification of the precise origin of these pathogens. This study aimed to monitor the quality of bathing water and associated fresh water in and out of the ‘bathing season’ in the UK, and to evaluate the use of microbial source tracking (MST) such as the host-specific based polymerase chain reaction (PCR) and quantitative PCR (qPCR) to recognize human and other animal sources of faecal pollution. The culture-dependent EU method of estimating FIB in water and sediment samples was performed on beach in the South Sands, Kingsbridge estuary, Devon, UK- a previously ‘problematic’ site. FIB were present at significant levels in the sediments, especially mud, as well as fresh water from the stream and pond flowing onto South Sands beach. However, the quality of bathing water was deemed to be ‘good’ and met with the EU bathing water directive 2006. Using MST it was possible to successfully classify the nature of the source from which the bacteria came. PCR was applied to detect the Bacteroides species 16S rRNA genetic markers from human sewage and animal faeces. All water and sediment samples displayed positive results with a general Bacteroides marker indicating the presence of Bacteroides species. Host-specific PCR showed the human Bacteroides genetic marker only in the sediment of the stream. However, limitations in the ‘types’ of probes available and in the persistence of these markers were identified. Thus, novel dog-specific Bacteroides conventional PCR and qPCR primer sets were developed to amplify a section of the 16S rRNA gene unique to the Bacteroides genetic marker from domestic dog faeces, and these were successfully used to quantify those markers in water samples at a ‘dog permitted’ and ‘dog banned’ beach (Bigbury-on-Sea, Devon, UK). Generic, human and dog Bacteroides PCR primer sets were also used to evaluate the persistence of Bacteroides genetic markers in controlled microcosms of water and sediment at differing salinities (< 0.5 and 34 psu) and temperature (10 and 17 ºC). The rates of decline were found did not differ significantly over 14 and 16 days for the water and sediment microcosms, respectively. Beach sediments which were studied in this project may act as a reservoir for adhesive FIB, and this was confirmed using fluorescence in situ hybridisation (FISH). The similarity in the persistence of these Bacteroides 16S rRNA genetic markers in environmental water and sediment suggests that viable but non-culturable (VBNC) Bacteroides spp. do not persist in the natural environment for long. Therefore, 16S rRNA genetic markers can be of value as additional faecal indicators of bathing water pollution and in source tracking. Thus, in this study MST methods were successfully used and in future applications, dog-specific primer sets can be added to the suite of host-specific Bacteroides genetic markers available to identify the source(s) of problem bacteria found on failing beaches.

Background: Enteric methane from rumen methanogens is responsible for 25.9 % of total methane emissions in the United States. Rumen methanogens also contribute to decreased animal feed efficiency. For methane mitigation strategies to be successful, it is important to establish which factors influence the rumen methanogen community and rumen volatile fatty acids (VFA). In the present study, we used next-generation sequencing to determine if dairy breed and/or days in milk (DIM) (high-fiber periparturient versus high-starch postpartum diets) affect the rumen environment and methanogen community of primiparous Holstein, Jersey, and Holstein-Jersey crossbreeds. Results: When the 16S rRNA gene sequences were processed and assigned to operational taxonomic units (OTU), a core methanogen community was identified, consisting of Methanobrevibacter (Mbr.) smithii, Mbr. thaueri, Mbr. ruminantium, and Mbr. millerae. The 16S rRNA gene sequence reads clustered at 3 DIM, but not by breed. At 3 DIM, the mean % abundance of Mbr. thaueri was lower in Jerseys (26.9 %) and higher in Holsteins (30.7 %) and Holstein-Jersey crossbreeds (30.3 %) (P < 0.001). The molar concentrations of total VFA were higher at 3 DIM than at 93, 183, and 273 DIM, whereas the molar proportions of propionate were increased at 3 and 93 DIM, relative to 183 and 273 DIM. Rumen methanogen densities, distributions of the Mbr. species, and VFA molar proportions did not differ by breed. Conclusions: The data from the present study suggest that a core methanogen community is present among dairy breeds, through out a lactation. Furthermore, the methanogen communities were more influenced by DIM and the breed by DIM interactions than breed differences.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / The larval stages of Culicoides sonorensis Wirth and Jones, a confirmed vector of bluetongue and epizootic hemorrhagic disease viruses affecting ruminants in North America, have been observed to occur typically in animal waste enhanced muds. In this dissertation, I studied the larval development (first instar to adult stage) and oviposition (four-choice assays) of C. sonorensis on sterilized mud (autoclaved) enriched with manure of different farm animal species (dairy cattle, beef cattle, sheep, goats, pigs, horses, white-tailed deer, and chicken). In addition, to determine why only some manure-polluted sites are colonized by C. sonorensis even when they are in close proximity to each other, I examined the moisture levels and microbial concentrations (mud) and physicochemical characteristics (standing water) of a manure-overflow pond site producing C. sonorensis and compared them to nearby cattle stock pond site(s) that produced different Culicoides species. Finally, as the first step in examining the role of microbiome in various physiological functions of C. sonorensis and other suspected/potential vector Culicoides species, I assessed the bacterial communities in field-collected adult females of C. sonorensis, C. crepuscularis, C. haematopotus, and C. stellifer (Illumina sequencing of 16S rRNA gene). In larval development experiments, the proportion of adults emerged and development time to adult stage varied with manure type and its concentration present in the substrate. Mud supplemented with chicken manure did not support C. sonorensis development, mud enriched with white-tailed deer manure poorly supported midge development, while C. sonorensis development in mud enhanced with manure of sheep, goats, beef cattle, dairy cattle, pigs, and horses varied. In oviposition experiments, colonized females preferred to deposit eggs on substrates without animal manure over substrates with animal manure. In subsequent studies, the manure-overflow pond site that produced mainly C. sonorensis contained significantly higher total aerobic culturable bacteria, pH, salinity, total dissolved solids, and conductivity levels than cattle stock pond sites that produced different Culicoides species. Finally, bacterial composition of field-collected C. sonorensis adult females comprised mainly of the phyla Proteobacteria and Firmicutes, while the majority of bacterial taxa identified from C. crepuscularis, C. haematopotus, and C. stellifer belonged to Proteobacteria. An unidentified bacterial genus (related to Tumebacillus), Propionibacterium, and Curvibacter were detected commonly across all four midge species. These results suggest that manure of several farm animal species can contribute to C. sonorensis development in the field. However, oviposition preferences remain uncertain, as colonized females appeared to show aversion to animal manure, which is in contradiction to the typical presence of C. sonorensis larvae in animal waste enhanced muds. Nonetheless, variations in microbial and/or physicochemical conditions in the larval habitats likely play a role in the differential emergence of C. sonorensis from various manure-polluted sites. Moreover, some bacterial taxa are associated commonly with C. sonorensis and other suspected/potential vector Culicoides species. Future studies are needed to examine oviposition preferences of field-collected females, life history traits of adults emerging from various manure-enriched substrates, developmental requirements of larvae, and the role of microbiome in various physiological functions of the host including vector competence for orbiviruses.

This dissertation examines the microbial and functional diversity in Kartchner Caverns, a limestone cave in Arizona, USA. Kartchner is highly oligotrophic due to the lack of photosynthesis and the limited inputs of organic material from the surface. This characteristic poses a challenge for microbial life in the cave. The first objective of this work was to evaluate the bacterial richness, diversity and taxonomic composition of speleothems surfaces within Kartchner Caverns in order to gain insight into the distribution patterns associated with these communities. Secondly, the metabolic strategies used by cave communities to survive harsh cave conditions were investigated based on phylogenetic associations and metagenomics. Both objectives were directed toward answering the questions "who are there?" and "what are they doing?". The 454-pyrotag analysis of the V6 region of the 16S rRNA gene revealed an unexpectedly high bacterial diversity with each speleothem supporting a unique bacterial community profile. A focused study on one room of the cave revealed three community types: Type 1 was dominated by the phylum Proteobacteria; Type 2 by Actinobacteria; and Type 3 by Acidobacteria. Phylogenetic associations of the sequences generated by the 454 sequencing and by a Sanger clone library suggested cave microbial communities are supported by chemoautotrophic activities such as nitrite and iron oxidation. Results from the phylogenetic associations guided the metagenomic analysis which supports the presence of chemoautotrophic activities in the cave. Genes for two complete CO2 fixation mechanisms, the Calvin-Benson-Bashan and the rTCA cycles were identified in the cave metagenome, as well as genes for ammonia and nitrite oxidation. These genes are associated with both Bacteria and Archaea suggesting members of both domains are acting as primary producers in the cave ecosystem. Comparative analysis of cave samples to other environments suggests an overabundance of DNA repair mechanisms which could be potentially used by cave communities to overcome the toxicity due to high concentrations of calcium on the speleothem surfaces. This work provides the first comprehensive analysis of the microbial diversity and potential strategies used by microbial communities to survive under the extreme conditions found in a semi-arid limestone cave environment.

Immune-mediated inflammatory diseases (IMID) represent a group of ostensibly unrelated, chronic and highly disabling diseases that preferentially affect different organ systems. IMID are assumed to manifest as a result of the accumulation of genetic, environmental and immunological factors. A fundamental commonality between IMID is the idiopathic nature of disease, and moreover, substantial similarities are apparent in disease etiopathogenesis. The complex assemblage of microbes and their genes that exists within and on the human body, collectively known as the microbiome has emerged as a critical factor in human health and, altered microbial populations within the gastrointestinal tract lumen and mucosa have been linked to several IMID. Accordingly, we conducted several studies investigating the association of the gut microbiome with IMID. Our main study investigated differences in the microbial profile and functional potential of multiple IMID utilizing 16S rDNA amplicon sequencing and analysis of stool. We also investigated the mucosal-associated microbiome in IBD to characterize the microbial populations and their functions residing in distinct gastrointestinal compartments from inflamed and noninflamed mucosa. We also explored a potential environmental factor; specifically assessing whether microbes present in drinking water in low or high incidence areas of IBD might contribute to disease etiology. The findings of these studies are manifold. First, we show important differences of the stool microbial profile in IMID. In doing so, we were able to identify distinct states of gut dysbiosis and have revealed numerous microbes that are consistently or uniquely disproportionate between IMID. Second, we have shown the microbial profile associated with inflamed and noninflamed mucosa and have reported that a localized dysbiosis is not observed in the presence of inflammation. Third, we have revealed that distinct gastrointestinal compartments are comprised of similar microbial communities. Lastly, we have reported the drinking water microbiome to differ between low and high incidence areas of IBD, thus suggesting a potential role in IBD etiology. Understanding the role of the gut microbiome in human disease will enable the development and application of more appropriate therapeutic strategies that specifically target microbes within the gut. / May 2017

Os micro-organismos desempenham importante papel na reciclagem dos elementos em ecossistemas de manguezais, uma vez que como produtores primários podem controlar reações químicas. O grupo particular das cianobactérias atua promovendo a entrada de carbono e nitrogênio por meio da sua capacidade de realizar fotossíntese oxigênica e fixação de nitrogênio atmosférico. No Brasil, as florestas de manguezais ocupam uma área de aproximadamente 25.000 km2 e no Estado de São Paulo, 240 km2 da área total está coberta por este ecossistema. O objetivo deste trabalho foi avaliar a diversidade de cianobactérias que colonizam as folhas de Avicennia schaueriana, Rhizophora mangle e Laguncularia racemosa do manguezal da Ilha do Cardoso, um ambiente pristino, bem como acessar e comparar a população de cianobactérias dos solos dos manguezais da Ilha do Cardoso e de Bertioga, este último contaminado com óleo bruto. Para este propósito, as técnicas de DGGE e biblioteca de clone do gene RNAr 16S, ARISA e TRFLP do gene nifH foram utilizadas. Os resultados da filosfera evidenciaram uma sutil influência do gênero de árvore na colonização das cianobactérias, entretanto, um forte efeito da localização destas dentro do manguezal foi observado. As folhas das árvores do meio da área de manguezal apresentaram uma maior diversidade de gêneros de cianobactérias. No geral, foram identificados 19 gêneros e várias sequências de cianobactérias não cultiváveis. Uma predominância de sequências com alta similaridade com representantes das ordens Nostocales e Oscillatoriales foi observada. Sequências com identidade com o gênero Symphyonemopsis (ordem Stigonematales) foram recuperadas em maiores quantidade. Com relação à diversidade no solo, os resultados de DGGE e ARISA demonstraram que a população de cianobactérias é distinta entre os manguezais estudados, porém os perfis eletroforéticos das amostras coletadas próximo ao mar se agruparam, sugerindo que a colonização é influenciada pelas condições de inundação. O perfil mais diferente foi obtido no ponto próximo à floresta no manguezal de Bertioga, local mais afetado pela contaminação de óleo. As bibliotecas de clones claramente indicaram diferenças das sequências do gene RNAr 16S entre os pontos amostrados. Um total de 99 UTOs foi obtido, com 61 "singletons". Na localidade próxima ao mar os gêneros Procholorococcus e Synechococcus foram dominantes em ambos os manguezais. A maioria das sequências de RNAr 16S encontradas nos outros pontos foram relacionadas com cianobactérias não cultiváveis. A diversidade alfa sugeriu que o local com menor diversidade foi o meio do manguezal de Bertioga, e o maior foi em Bertioga próximo à floresta, os demais pontos tiveram valores similares. Os maiores índices de riqueza foram encontrados nos pontos próximos à floresta, enquanto menores valores foram observados nos pontos próximos ao mar. A maioria das sequências de RNAr 16S obtidas em Bertioga no meio do manguezal e próximo à floresta tiveram identidades menores do que 90% com as disponíveis no GenBank. Estas sequências podem representar novos táxons ou cianobactérias conhecidas, porém ainda não sequenciadas. O TRFLP do gene nifH indicou que os locais próximos ao mar e meio do manguezal na Ilha do Cardoso abrigaram populações de diazotróficos semelhantes, enquanto que em Bertioga estes pontos apresentaram diferenças nos perfis de TRFLP. As maiores diferenças estavam nos locais próximos à floresta em ambos os manguezais. / Microorganisms play important role in the recycling of elements in mangrove ecosystems, since as primary producers they can control chemical reactions. The particular cyanobacteria group act promoting the input of carbon and nitrogen through their ability to realize oxygenic photosynthesis and fixing atmospheric nitrogen. In Brazil, the mangrove forests occupy an area of approximately 25.000 km2, and in the total area of São Paulo State, 240 km2 are covered by this ecosystem. The aim of this work was to evaluate the cyanobacterial diversity that colonize Avicennia schaueriana, Rhizophora mangle and Laguncularia racemosa leaves from Cardoso Island mangrove, a pristine site, as well as to assess and compare the soil cyanobacterial population from both Cardoso Island and Bertioga mangroves, this last one contaminated with crude oil. For this purpose, the techniques of DGGE and clone library of 16S rRNA gene, ARISA, and TRFLP of nifH gene were used. The phyllosphere results evidenced a subtle difference of the genus of tree on the colonization of cyanobacteria, however a strong effect from tree’s location within the mangrove was observed. The tree leaves from the middle of mangrove area showed a greater diversity of cyanobacterial genera. In geral, 19 genera and several uncultivated cyanobacteria were identified. A predominance of sequences with high similarities to representatives of the order Nostocales and Oscillatoriales were observed. Sequences with similarities to the genus Symphyonemopsis (order Stigonematales) were recovered in higher quantity. Regarding to the soil diversity, DGGE and ARISA results showed that the cyanobacterial population is distinct among both mangroves studied, however the electrophoretic profiles from samples collected near to the sea grouped together, suggesting that colonization is influenced by flood conditions. The most different profile was obtained in the site near to the forest in Bertioga mangrove, location more affected by the oil contamination. Clone libraries clearly showed 16S rRNA sequences differences among sites sampled. A total of 99 OTUs were obtained, with 61 singletons. In the site near to the sea the Procholorococcus and Synechococcus genera were dominant in both mangroves. The majority of 16S rRNA sequences found in the other sites were related to uncultured cyanobacteria. Alpha diversity suggested that the site with lowest diversity was middle of the Bertioga mangrove, and the highest was Bertioga near to the forest, the remainder sites had similar values. The highest richness indices were found in the sites near to the forest, while lower values were observed in the sites near to the sea. The majority of the 16S rRNA sequences obtained from the middle of the mangrove and near to the forest in Bertioga showed identities lower than 90% with that available in the GenBank. These sequences may represent novel cyanobacterial taxa or known cyanobacteria not yet sequenced. The TRFLP of nifH gene indicated that the sites near to the sea and middle of the Cardoso Island mangrove harbored similar diazotrophic populations, while in Bertioga these sites presented differences in TRFLP profiles. The greatest differences were in the sites near to the forest in both mangroves.