Vitis vinifera subsp. vinifera L., les principales espèces de raisins sont cultivées pour la production de fruits et la production de vin dans le monde est un hôte naturel d'une grande variété de micro-organismes procaryotes et eucaryotes qui interagissent avec la vigne, ayant des effets bénéfiques ou phytopathogènes. Ils pourraient également jouer un rôle majeur dans le rendement des fruits, la qualité du raisin, la protection des plantes et, finalement, dans le modèle de la fermentation du raisin et la production de vin. La phyllosphère (constituée des parties aériennes de la plante) est l'un des habitats microbiens les plus répandus sur terre et est un milieu assez négligé, en particulier dans les vignes et de nombreuses questions liées à cet habitat microbien sont toujours sans réponse.Cette thèse est un effort pour répondre à une question fondamentale en écologie microbienne: quels sont les facteurs qui déterminent le microbiome dans la phyllosphère de la vigne? Les communautés microbiennes de la phyllosphère (PMCs) vivent à l'interface plante-climat et sa capacité à s'établir, prospérer et se reproduire sur la surface des feuilles ou des fruits dépend de plusieurs caractéristiques fonctionnelles microbiennes, comme la capacité de se fixer sur la cuticule et d'utiliser la foliaire. nutriments ainsi que les conditions climatiques dominantes comme la température, l'humidité de l'air et la pluie. La chimie des feuilles ou des fruits, la physiologie et la structure morphologique diffèrent selon le génotype et l'espèce puisque tous ces traits ont une base génétique, et cette variation peut mener à une combinaison différente d'assemblage de PMC parmi les génotypes de plantes. Ainsi, le premier objectif de notre travail était d'évaluer les impacts des cultivars de vigne (variétés de Vitis vinifera L) et des espèces de vigne (espèces Vitis entièrement différentes) sur l'assemblage du microbiome dans la phyllosphère à un endroit géographique particulier (pour minimiser les effets environnementaux) . Plus tard, les impacts de certains cultivars et terroirs de vigne commercialement importants (représentés par trois zones climatiques françaises) ont également été évalués et comparés. Les impacts de la saison et des organes extérieurs de la plante (feuilles et baies) sur la structuration des taxons microbiens dans la phyllosphère ont également été évalués et présentés dans ce travail. De plus, des impacts spécifiques à l'espèce sur le microbiome de la phyllosphère ont également été testés et représentés.Dans l'ensemble, notre étude a évalué et comparé les nombreuses facettes des facteurs qui peuvent influencer structure du microbiome dans la phyllosphère avec un accent particulier sur la pression de sélection relative exercée par le génotype de la vigne et son interaction avec différentes conditions climatiques (ou terroir), ce qui peut améliorer nos chances de trouver des gènes contrôlant les PMCs sur la phyllosphère. les gènes sont réellement importants dans des environnements réalistes et probablement ces gènes nous donneraient de nouvelles idées pour la sélection de nouveaux cépages sains présentant de meilleurs caractères sur leur phyllosphère. De plus, considérant que les PMC végétales jouent un rôle crucial dans la santé et la forme des plantes car elles peuvent moduler la susceptibilité foliaire aux infections, cette étude pourrait également être utile pour développer des méthodes de biocontrôle innovantes et naturelles ou phytostimulation contre les pathogènes de la vigne. de variétés résistantes innovantes. / Vitis vinifera subsp. vinifera L., the main grape species are grown for fruit and wine production over the world is a natural host of a wide variety of prokaryotic and eukaryotic microorganisms that interact with grapevine, having either beneficial or phytopathogenic effects. They could also play a major role in fruit yield, grape quality, plant protection and, ultimately, in the pattern of grape fermentation and wine production. Phyllosphere (consists of the aerial parts of the plant) is one of the most prevalent microbial habitats on earth and is quite a neglected milieu, especially in grapevines and many questions related to this microbial habitat, are still unanswered.This thesis is an effort to answer a very fundamental question in microbial ecology- what are the drivers that shape the microbiome in the grapevine's phyllosphere? The phyllosphere microbial communities (PMCs) live at the plant-climate interface and its ability to establish, thrive and reproduce on the leaf or fruit surface depends on several microbial functional traits, such as the ability to attach to the cuticle and to use the foliar nutrients as well as well as to the prevailing climatic conditions like temperature, air humidity and rain. Leaf or fruit chemistry, physiology, and morphological structure differ among plant genotype and species as all these traits have a genetic basis, and this variation may lead to a different combination of PMCs assemblage among plant genotypes. Hence, the first objective of our work was to assess the impacts of grapevine cultivars (varieties of Vitis vinifera L) and grapevine species (entirely different Vitis species) on microbiome assemblage in the phyllosphere at a particular geographic location (to minimize the environmental effects). Later on, impacts of some commercially important grapevine cultivars and terroirs (represented by three French climate zones) were also assessed and compared. Impacts of the season and exterior plant organs (leaf and berries) on microbial taxa structuring in the phyllosphere was also assessed and presented in this work. Furthermore, species-specific impacts on phyllosphere microbiome were also tested and represented.Overall our study assessed and compared the many facets of the factors that may influence themicrobiome structure in the phyllosphere with a special focus on relative selection pressure exerted by grapevine genotype and its interaction with different climatic conditions (or terroir), which may improve our chances to find genes that controls PMCs on phyllosphere, and simultaneously increase our confidence that those genes are actually important in realistic environments and probably those genes would give us new insights for breeding new and healthy grape varieties displaying better traits on their phyllosphere. Moreover, considering that the plant PMCs plays a crucial role in plant health and fitness as it can modulate leaf susceptibility to infection, this study could also be helpful to develop innovative and natural biocontrol methods or phytostimulation against grapevine pathogens or rethink breeding schemes for the creation of innovative resistant varieties.

A água doce no Brasil, assim como o seu consumo é extremamente importante para as diversas atividades criadas pelo ser humano. Por esta razão o consumo deste bem é muito grande e consequentemente, provocando o seu impacto. Os mananciais são normalmente usados para abastecimento doméstico, comercial, industrial e outros fins. Os estudos na área de ecologia de micro-organismos em águas (mananciais e esgoto) vêm sendo realizados com mais intensidade nos últimos anos. Nas últimas décadas foi introduzido o conceito de virioplâncton com base na abundância e diversidade de partículas virais presentes no ambiente aquático. O virioplâncton influencia muitos processos ecológicos e biogeoquímicos, como ciclagem de nutriente, taxa de sedimentação de partículas, diversidade e distribuição de espécies de algas e bactérias, controle de florações de fitoplâncton e transferência genética horizontal. Os estudos nesta área da Virologia molecular ainda estão muito restritos no país, bem como muito pouco se conhece sobre a diversidade viral na água no Brasil. / Freshwater in Brazil, as well as its consumption is extremely important for the various activities created by human being. For this reason the consumption of this good is very great and consequently, causing its impact. The sources are usually used for domestic, commercial, industrial and other purposes. Studies on the ecology of microorganisms in waters (freshwater and sewage) have been carried out more intensively in recent years. In recent decades the concept of virioplankton has been introduced based on the abundance and diversity of viral particles present in the aquatic environment. Virioplankton influences many ecological and biogeochemical processes, such as nutrient cycling, particle sedimentation rate, diversity and distribution of algal and bacterial species, control of phytoplankton blooms and horizontal gene transfer. Studies in this area of molecular Virology are still very restricted in the country, and very little is known about viral diversity in water in Brazil.

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.

In the last years, microbial ecologists have dramatically increased their efforts to elucidate the \"black box\" of patterns and processes that modulate the diversity and functionality of soil microorganisms, examining their genetic diversity (e.g. through metagenomic) and measuring their functional characteristics. The aim of this thesis was to evaluate the interaction of the ecological processes of dispersion, diversification, selection and genetic drift on (1) the soil microbial communities, after conversion of forest to grassland or no-till cropping in long-term and (2) on the microbial communities in the rhizosphere of soybean in long-term no-till system. The cultivation of grassland in long-term led to a homogenizing selection of microbial communities, reducing beta-diversity, with consequent changes in the soil functions related to stress. No-till long-term led to minor changes of diversity, maintaining the functions found in the forest. The soybean plant has shown homogenizing power selection, and this increased with time. However, the functions selected in the rhizosphere were maintained, indicating functional resilience. / Nos últimos anos, ecologistas microbianos aumentaram drasticamente seus esforços para elucidar a \"caixa preta\" dos padrões e processos que modulam a diversidade e funcionalidade dos microrganismos do solo, examinando sua diversidade genética (e.g. através de metagenômica) e medindo suas características funcionais. O objetivo dessa tese foi avaliar a interação dos processos ecológicos de dispersão, diversificação, seleção e deriva gênica, sobre (1) as comunidades microbianas do solo, após a conversão da floresta em pastagem ou plantio direto, em longo período e (2) sobre as comunidades microbianas da rizosfera de soja, em sistema de plantio direto, em longo período. O cultivo de pastagens em longo período levou a uma seleção homogeneizante das comunidades microbianas, reduzindo a beta-diversidade, com conseguinte alteração em funções no solo relacionadas ao estresse. O plantio direto em longo período levou a uma menor alteração da diversidade, com manutenção das funções encontradas na floresta. A planta de soja demonstrou poder de seleção homogeneizante, e este aumentou com o tempo. Contudo, as funções selecionadas na rizosfera foram mantidas, indicando resiliência funcional.

L’identification, chez divers organismes, d’enzymes de dégradation des N-acyl homosérineslactones (NAHLs) impliquées dans la signalisation QS pose la question de leurs rôles dans lesinteractions bactéries-eucaryotes. Dans une première partie, une synthèse bibliographique analyse lesconnaissances acquises sur ces enzymes dégradant les NAHLs. Dans une seconde partie, la croissancedes bactéries dégradant les signaux NAHLs a été stimulée par l’application de g-caprolactone (GCL)dans la rhizosphère de plants de pommes de terre à des fins de phytoprotection. L’effet de cetraitement sur la diversité des communautés bactériennes rhizosphériques a été évalué en combinantdifférentes approches d’écologie microbienne moléculaire comme la DGGE, le pyroséquençaged’amplicons rrs, et la métagénomique fonctionnelle. Cette dernière approche appliquée à une banquede 30 000 clones environ a conduit à l’identification d’un gène qsdB codant la dégradation des signauxNAHL. Ce travail révèle ainsi l’existence d’une nouvelle classe d’enzymes de dégradation des NAHLsappartenant à la famille des enzymes possédant une signature amidase (AS) dont des membres sontpar ailleurs impliqués dans la dégradation de composés xénobiotiques. Dans une troisième partie, unsystème expérimental a été développé afin de mesurer le transfert conjugatif du plasmide de virulenceTi (tumor inducing) chez des dérivés du pathogène Agrobacterium tumefaciens, appelés «tricheurs»,incapables de produire des signaux NAHLs mais utilisateurs de ceux produits par les autres bactéries.Ce modèle a permis de montrer l’effet modérateur de lactonases dégradant les NAHLs exprimées chezdes agrobactéries produisant les NAHLs, chez des bactéries réceptrices du plasmide Ti, ou des planteshôtes des agrobactéries sur le transfert conjugatif initié par les tricheurs. L’ensemble de ce travailrévèle à la fois une nouvelle famille d’enzymes impliquées dans la dégradation des NAHLs, ainsiqu’un nouveau rôle de ces enzymes dans la modulation des flux de gènes entre bactériesphytopathogènes en interaction avec une plante hôte. / Identification of bacterial and eukaryotic enzymes that degrade N-acyl homoserine lactones(NAHLs) involved in QS signaling raises the question of their roles in bacteria-eucaryotesinteractions. In a first part of this study, a bibliographic report analyzes the current data on thoseNAHL-degrading enzymes. In a second part, the growth of NAHL-degrading bacteria was stimulatedby g-caprolactone (GCL) amendment in potato rhizosphere to protect this plant against the soft-rotpathogen Pectobacterium. The effect of the GCL treatment on rhizospheric bacterial communities wasevaluated by a combination of different molecular microbial ecology techniques such as DGGE,pyrosequencing and functional metagenomic. This last approach was applied to generate ametagenomic library of ca. 30,000 clones and lead to the identification of the qsdB gene that encodesNAHL degradation, This work revealed the occurrence of a novel class of NAHL-degrading enzymesthat belong to the amidase signature (AS) family, some members of which being involved inxenobiotic compound degradation. In a third part, an experimental system was developed to measurethe conjugative transfer of Ti plasmid in various strains of the pathogen Agrobacterium tumefaciens,including “cheaters”, i.e. bacteria unable to produce NAHL signals but capable to use signals producedby other bacteria. Using this model; variations of the plasmid transfer of cheaters were measured whenNAHL-degrading lactonases were expressed in agrobacteria that produce NAHL signals, in recipientbacteria of Ti plasmid, or in agrobacterial host plant. Taken together, thesis experiments revealed anovel class of enzymes involved in NAHL-degradation and a new role for thoses enzymes in themodulation of gene transfer between pathogenic bacteria interacting with host plants.

Il est maintenant bien établi que le microbiote du tractus digestif humain joue un rôle important dans la santé humaine. Pourtant, nous commençons à peine à comprendre les mécanismes moléculaires par lesquels les bactéries agissent sur les cellules hôtes, des connaissances qui pourraient fournir des nouvelles orientations dans le traitement et la prévention de maladies. Cette dernière décennie a vu un développement rapide des études du microbiote intestinal, et à présent des quantités importantes de données métagénomiques ainsi que des centaines de séquences génomiques de bactéries commensales sont disponibles. Ensemble, ces données fournissent une plateforme pour des approches in silico pour l'identification de molécules bactériennes impliquées dans la communication moléculaire avec l'hôte. Le défi consiste à développer des stratégies efficaces d'exploration de données et de validation, permettant de passer de corrélations et prédictions à des interactions bactérie - hôte fonctionnelles, validées expérimentalement. Le travail présenté dans cette thèse vise à démontrer l'importance d'analyses in silico afin d'élargir nos connaissances sur les interactions bactéries - hôtes. Il montre également comment cette information peut être appliquée dans des études fonctionnelles visant à identifier des molécules effectrices bactériennes fonctionnelles. Les principaux résultats peuvent être divisés en trois parties. La première partie traite de l'élaboration et de la validation d'un système hôte - vecteur pour des études de (méta)génomique fonctionnelle. La deuxième partie décrit une étude fonctionnelle où un certain nombre d'effecteurs candidats ont été identifiés parmi les protéines sécrétées et de surface de bactéries à Gram positif par une approche d'exploration in silico. Il décrit également l'application du nouveau système hôte - vecteur pour l'évaluation du rôle de ces candidats dans l'immuno-modulation. Enfin, dans la troisième partie, nous présentons une étude in silico qui a permis l'identification de fonctions bactériennes sur- ou sous-représentées dans une sélection de bactéries à Gram positif du tractus digestif humain. / It is now well established that the human gastrointestinal tract microbiota plays an intricate role in human health. However, we are only beginning to understand the molecular mechanisms by which bacteria act on the host cells, knowledge that could provide new directions in treating and preventing disease. The last decade has seen a rapid development of the gut microbiota field, and presently abundant metagenome data and hundreds of genome sequences of individual commensal bacteria are available. Together, these data provide a platform for in silico mining approaches to identify bacterial molecules involved in communication with the host. The challenge is to develop efficient mining and validation strategies, in order to move from correlations and predictions to experimentally validated functional bacteria – host relationships. The work presented in this thesis aims to demonstrate the importance of in silico analyses to broaden our knowledge on bacteria - host interactions. It also shows how this information can be applied in functional studies aiming to identify functional bacterial effector molecules. The main results can be divided in three parts. The first part deals with the development and validation of a host - vector system for functional (meta)genomics studies. The second part describes a functional study where a number of candidate effectors were identified among secreted and surface-exposed proteins from Gram-positive bacteria using an in silico mining approach. It also describes the application of the newly developed host - vector system to evaluate the role of these candidates in immune modulation. Finally, in the third part we present an in silico study that identified new bacterial functions over- or under-represented in a selection of Gram-positive human gut bacteria.

As doenças associadas ao circovírus suíno (DACS) causam impacto econômico negativo nos sistemas de criação de suínos no mundo todo. As perdas incluem tratamento da doença, mortalidade, bem como diminuição no desempenho dos animais. Uma das manifestações mais relevantes das DACS é a síndrome multissistêmica do definhamento dos suínos (SMDS). O principal agente patogênico na causa da SMDS é o circovírus suíno tipo 2 (PCV2), no entanto, estudos observacionais e experimentais demonstraram que outros agentes estão envolvidos na patogênese e manifestação dos sinais clínicos. O sequenciamento de alto desempenho aliado a metagenômica são ferramentas que tornam possível a identificação da microbiota total de uma determinada amostra, independentemente de cultivo dos microrganismos. Visando contribuir para o conhecimento dos possíveis vírus envolvidos na SMDS, o presente trabalho realizou o sequenciamento genético de alto desempenho de soros de suínos e posterior análise do metagenoma resultante. Foram utilizadas amostras de soro coletadas em 2008, de 16 suínos com sinais clínicos da SMDS, entre 80 e 100 dias de idade, em uma granja no interior do Rio Grande do Sul. Os dados revelaram sequências virais de PCV2, parvovírus suíno tipo 1 a 6 (PPV1-6), torque teno vírus de suínos (TTSuV) tipo 1b, k2a e k2b e vírus de DNA circular de fita simples codificador de proteína associada a replicação (CRESS). A ocorrência de PCV2, PPV1-5 e TTSuV já foi descrita em suínos com SMDS, portanto este estudo reforça resultados anteriores. O PPV6 foi recentemente descrito na China, Europa e Estados Unidos, e os estudos não relacionaram o vírus com nenhuma doença específica de suínos. Os CRESS já foram identificados em todos os continentes, em vários tipos de amostras, incluindo fezes de suínos, mas sem nenhuma conexão com doenças de animais. Portanto, este é o primeiro relato de PPV6 e CRESS em suínos apresentando sinais de SMDS. Porém estudos posteriores são necessários para poder atribuir relação entre PPV6 e CRESS no desenvolvimento da SMDS. / Porcine circovirus associated disease (PCVAD) is one of the causes of negative economic impact on pig farming systems described worldwide. Losses include expenditures with treatment, increased mortality rates, and decreased productivity. One of the most relevant manifestations of PCVAD is the post-weaning multisystemic wasting syndrome (PMWS). The main pathogen present in PMWS is porcine circovirus type 2 (PCV2). However, observational and experimental studies have shown that other agents may be involved in the pathogenesis and clinical manifestation. High-throughput sequencing combined with metagenomics analyses make it possible to identify the total microbiota in a given sample, regardless of microorganism culture. In order to contribute to the knowledge of the viruses involved in PMWS, the present study carried out the high-throughput sequencing of swine sera and subsequent analysis of the resulting metagenome. Sixteen serum samples collected in 2008 on a farm in Rio Grande do Sul, from 80 and 100 days old pigs with clinical signs of PMWS, were examined. Data revealed viral sequences of PCV2, porcine parvovirus type 1 through 6 (PPV1-6), torque teno sus virus (TTSuV) types 1b, k2a and k2b and circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses. The occurrences of PCV2, PPV1-5 and TTSuV have already been described in pigs with PMWS, so this study reinforces previous results. PPV6 was recently described in China, Europe and the United States, and the studies did not correlate the virus to any specific disease. CRESS DNA viruses have been identified on all continents in various types of samples, including swine feces, but without any connection to animal diseases. The present study is the first report of PPV6 and CRESS in pigs presenting PMWS signals. However, further studies are necessary to be able to attribute the relationship between PPV6 and CRESS in the development of SMDS.

La flore microbienne est un ensemble de micro-organismes comme les bactéries, archées, eucaryotes inférieurs et virus, jouant un rôle important dans l’équilibre d’un écosystème. Cette flore reste encore mal définie car en 2012, seules ~30% des micro-organismes de la flore intestinale humaine étaient caractérisés, et moins de 50% des micro-organismes de la flore fromagère traditionnelle étaient caractérisés au niveau fonctionnel. Depuis 2006, les séquenceurs à ADN de seconde génération permettent d’analyser directement le contenu génique d’un prélèvement de flore sans contrainte d’isolement ou de culture. Toutefois, les séquences d’ADN générées ne sont pas structurées en génome et sont hautement fragmentées, freinant considérablement l’analyse et l’exploitation de ces données. Dans ce travail, nous avons développé de nouvelles méthodes dites de métagénomiques quantitatives, car elles permettent de regrouper les courtes séquences d’ADN ayant la même abondance au sein de plusieurs échantillons métagénomiques, pouvant provenir d’une même espèce microbienne.Au niveau du microbiote intestinal humain, nous avons structuré un catalogue de 3,9 millions de gènes de la flore intestinale humaine en 741 unités ou « clusters » correspondant à des génomes de bactéries, d’archées et d’eucaryotes, que nous appelons espèces métagénomiques (MGS) et 6640 unités correspondant principalement à des génomes de virus, plasmides et divers ilots génomiques comme des CRISPR, que nous appelons unités métagénomiques (MGU). Cette méthode de structuration a ensuite été utilisée pour faciliter des analyses d’associations de la composition de la flore intestinale avec des maladies humaines comme la maladie de Crohn, l’obésité ou le diabète de type 2. Enfin, au niveau des flores alimentaires, nos méthodes ont été utilisées pour constituer un catalogue de 134 génomes d’espèces bactériennes fromagères et caractériser la flore de surface de fromages traditionnels. Ceci nous a permis de détecter la présence de nouvelles bactéries alimentaires, pouvant enrichir la liste des bactéries à possible intérêt technologique dans les produits laitiers fermentés. / The microbial flora is a micro-organism complex containing for example bacteria, archaea, lower eukaryotes and viruses, which play an important role in ecosystem equilibrium. This flora remains poorly defined as in 2012, only ~30% of the intestinal flora micro-organisms have been characterized, and less than 50% of traditional cheese floras were characterized at the functional level. Since 2006, the second generation of DNA sequencers have allowed the direct analysis of the genetic content from a microbial flora sample without isolation or culture limitation. However, the DNA reads generated are not structured with respect to genomes and also are highly fragmented, slowing down dramatically the exploitation and analysis of these data.In this work, a new methodology based on quantitative metagenomic are described., This allows the clustering of short DNA sequences with the same abundance in multiple metagenomic samples, which should originate from the same microbial species. A 3.9 million gene catalog has been built from the human intestinal tract microbiota and divided into 741 units or clusters corresponding to bacteria, archaea and eukaryote genomes. These have been defined as metagenomic species (MGS) and 6640 units of them corresponds mainly to viral genomes, plasmids and genetic islands like CRISPR, with the sub-name of metagenomic units (MGU). This methodology was then used to facilitate the association analysis of the intestinal flora composition with human diseases such as Crohn’s disease, obesity or type 2 diabetes. Within, the alimentary flora, our methods have also been used to constitute a 134 genomic catalog of cheese bacteria and characterize them from the surface of traditional cheeses. This allowed the detection of new alimentary bacteria, that will enriched the list of bacteria with potential interest for the commercial exploitation of fermented products.

A recent study using human fecal samples in conjunction with a culture-independent approach revealed immense diversity of antibiotic resistance (AR) genes in the human gut microflora. We hypothesize that food animal gut microflora also contain diverse and novel AR genes which could contribute to the emergence and transmission of AR in pathogens important in animal and human health. To test this, we examined AR reservoir in chicken gut microflora using a metagenomic, functional cloning method. Total genomic DNA was extracted from individual cecal contents of two free range chickens and two conventionally raised chickens. The DNAs were physically sheered into 1 to 3 kb fragments, cloned into expression vector pZE21-MCS, and transformed into E. coli TOP10 host strain, resulting in four metagenomic libraries of a total size of 108 base pairs per library. The AR transformants from the libraries were selected on plates containing the specific antibiotic of interest; six antibiotics including ampicillin, tetracycline, chloramphenicol, spectinomycin, ciprofloxacin and norfloxacin were used for screening. Plasmids from selected transformants were extracted and subjected to sequence analysis of inserted fragments. Identified AR genes were annotated and aligned with homologs that have been deposited in GenBank. A total of 12 AR genes and 3 AR genes were identified from the microbiome in conventionally raised chickens and free-range chickens, respectively. Of the identified 15 AR genes, 8 genes that confer resistance to ampicillin, spectinomycin or chloramphenicol shared low sequence similarity (58% - 76% at amino acid level) with the corresponding AR genes previously identified using culture-dependent approaches. Notably, among the 8 novel AR genes identified in this study, 4 genes also shared low sequence similarities (59%-76% at amino acid level) with recently identified AR genes in human gut. An E. coli-Campylobacter shuttle vector bearing the flaA sigma 28 promoter was constructed. Two novel genes conferring resistance to ampicillin (FRAmp1.1) and spectinomycin (FRSpe1.1) were cloned into this new expression vector, respectively. The derived vectors have conferred increased AR in C. jejuni, a leading zoonotic bacterial pathogen causing human gastroenteritidis in many industrialized countries. Together, findings from this study showed the effectiveness of the metagenomic approach for examination of AR reservoir in food animals, revealed novel AR resistance genes in chicken gut microflora, and demonstrated the functionality of such AR genes in foodborne human pathogens.