RABELO, E. M. Avaliação do potencial antimicrobiano do extrato etanólico e frações obtidas de guettarda sericea müll. arg.(rubiaceae). 2013. 83 f. Dissertação ( Mestrado em Biotecnologia) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2013. / Submitted by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-28T15:44:12Z No. of bitstreams: 1 2013_dis_emrabelo.pdf: 10038342 bytes, checksum: 035c474be4424e0dddff689c31e484ba (MD5) / Approved for entry into archive by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-29T12:17:36Z (GMT) No. of bitstreams: 1 2013_dis_emrabelo.pdf: 10038342 bytes, checksum: 035c474be4424e0dddff689c31e484ba (MD5) / Made available in DSpace on 2016-06-29T12:17:36Z (GMT). No. of bitstreams: 1 2013_dis_emrabelo.pdf: 10038342 bytes, checksum: 035c474be4424e0dddff689c31e484ba (MD5) Previous issue date: 2013 / The genus Guettarda (Rubiaceae) comprises plants widely distributed in tropical areas. Regarding the Guettarda sericea species, the literature shows that there is a lack of botanical and phytochemicals studies. Thus, the present study aimed to evaluate the antibacterial effect of ethanol extract of leaves of G. sericea (EEFGS) and its fractions on the growth of Streptococcus oralis ATCC 10557 and S. salivarius ATCC 7073 in both the planktonic and biofilms states. Different methods were employed to verify the antimicrobial potential. Among these are the determination of minimum inhibitory concentration (MIC) determination of the death curve and evaluation of minimum bactericidal concentration (MBC). Furthermore, quantification of biomass and the number of viable cells of the biofilm were performed, respectively, by staining with crystal violet and counting colony forming units (CFU). The negative and positive controls used in all assays were respectively 4% DMSO and chlorhexidine gluconate with concentration adjusted according to the data of the MIC of each microorganism. To determine the toxicity of EEFGS, it was used the toxicity test on Artemia nauplii. The data showed that the extract and subfractions 13 to 17 of the chloroform fraction show a remarkable antimicrobial effect, able to inhibit the growth of planktonic and development of biofilms of S. oralis strain until the concentration of 62.5 μg.mL-1. With respect to S. salivarius, only subfraction 12 interfered on bacterial growth. Regarding the toxicity, it was observed that death of Artemia nauplii occurred at higher concentrations than those that exhibited antibacterial effect. From these results it can be concluded that EEFGS and subfractions 13-17 can be used as agents for the control of biofilm formation of S. oralis. In addition, complementary methodologies that seek purification of the active compounds and their cytotoxic effects on eukaryotic cells need to be held, aiming its use as an herbal agent. / O gênero Guettarda (Rubiaceae) compreende plantas extensamente distribuídas em áreas tropicais. Com relação à espécie Guettarda sericea, a literatura demonstra que existe uma ampla carência acerca de estudos botânicos e fitoquímicos. Assim, o presente trabalho buscou avaliar o efeito antibacteriano do extrato etanólico das folhas de G. sericea (EEFGS) e suas frações sobre o crescimento de Streptococcus oralis ATCC 10557 e S. salivarius ATCC 7073, nas formas planctônicas e de biofilmes. Diferentes metodologias foram empregadas para a verificação do potencial antimicrobiano. Dentre estas estão a determinação da concentração inibitória mínima (MIC), a determinação da curva de morte e a avaliação da concentração bactericida mínima (MBC). Além disso, a quantificação da biomassa e do número de células viáveis do biofilme foi realizada, respectivamente, através da coloração pelo cristal violeta e contagem de unidades formadoras de colônia (UFC). Os controles, negativo e positivo utilizados em todos os ensaios foram, respectivamente, DMSO 4% e Gluconato de Clorexidina com concentração ajustada de acordo com os dados da CIM de cada micro-organismo. Para a determinação da toxicidade do EEFGS utilizou-se o ensaio de toxicidade sobre náuplios de Artemia. Os dados mostraram que o extrato e as subfrações 13 a 17 da fração clorofórmio apresentaram um marcante efeito antimicrobiano, sendo capazes de inibir o crescimento planctônico, bem como o desenvolvimento de biofilmes da cepa de S. oralis até a concentração de 62,5 µg.mL-1. Com relação a S. salivarius, apenas a subfração 12 interferiu sobre o crescimento bacteriano. No tocante à toxicidade, foi observado que a morte dos náuplios de Artemia ocorreu em concentrações mais elevadas do que aquelas que apresentaram efeito antibacteriano. A partir de tais resultados pode-se concluir que EEFGS e as subfrações 13 a 17 podem ser utilizados como insumos para o controle da formação de biofilmes de S. oralis. Em adição, metodologias complementares que busquem a purificação dos compostos ativos e seus efeitos citotóxicos sobre células eucarióticas necessitam ser realizadas, visando sua utilização como um agente fitoterápico.

VERAS, I. N. Análise do perfil morfológico de candida tropicalis durante a formação do biofilme sob influência de metabólitos extracelulares de bactérias do gênero Streptococcus. 2014. 77 f. Dissertação ( Mestrado em Biotecnologia) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2014. / Submitted by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-29T14:58:00Z No. of bitstreams: 1 2014_dis_insveras.pdf: 2042523 bytes, checksum: adf18ca2b4b9df61f42a9d6cff04ce95 (MD5) / Approved for entry into archive by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-29T14:58:25Z (GMT) No. of bitstreams: 1 2014_dis_insveras.pdf: 2042523 bytes, checksum: adf18ca2b4b9df61f42a9d6cff04ce95 (MD5) / Made available in DSpace on 2016-06-29T14:58:25Z (GMT). No. of bitstreams: 1 2014_dis_insveras.pdf: 2042523 bytes, checksum: adf18ca2b4b9df61f42a9d6cff04ce95 (MD5) Previous issue date: 2014 / Bacteria and fungi are found together in a multitude of environments, and particularly in the form of biofilm adherent species which interact through various signaling mechanisms. In the oral cavity, Candida species coexist with numerous bacterial species, and evidence suggests that bacteria can modulate biofilm formation as well as induce the formation of hyphae. Thus, to characterize such interactions are essential to the understanding of pathogenesis of diseases and possibly the discovery of new therapeutic strategies. In this sense, the main objective of this study was to evaluate, in vitro, the mechanisms involved in the interaction between bacteria Streptococcus oralis, Streptococcus sanguinis, and Streptococcus parasanguinis yeast Candida tropicalis. In the study the effect of extracellular metabolites present in the supernatant of C. tropicalis (SCT) on the pre-formed biofilm (6H) S. oralis, S. sanguinis and S. parasanguinis was analyzed. The effect of extracellular metabolites of S. oralis (SSO), S. sanguinis (SSS) and S. parasanguinis (SSP) on planktonic growth and biofilm / Bactérias e fungos são encontrados juntos em uma infinidade de ambientes e, particularmente, na forma de biofilme, onde as espécies aderentes interagem através de diversos mecanismos de sinalização. Na cavidade oral, espécies de Candida coexistem com inúmeras espécies bacterianas, e evidências sugerem que bactérias podem modular a formação de biofilme, bem como induzir a formação de hifas e pseudo-hifas. Assim, caracterizar tais interações é essencial para o entendimento da patogênese das doenças e, possivelmente, a descoberta de novas estratégias terapêuticas. Nesse sentido, o objetivo principal deste trabalho foi avaliar, in vitro, os mecanismos envolvidos na interação entre as bactérias Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis e a levedura Candida tropicalis. No estudo foi analisado o efeito dos metabólitos extracelulares presentes no sobrenadante de C. tropicalis (SCT) sobre o biofilme pré-formado (6h) de S. oralis, S. sanguinis e S. parasanguinis. Também foi avaliado o efeito dos metabólitos extracelulares de S. oralis (SSO), S. sanguinis (SSS) e S. parasanguinis (SSP) sobre o crescimento planctônico, formação de biofilme e capacidade de filamentação de C. tropicalis, utilizando apenas o sobrenadante da cultura de cada um dos estreptococos em diferentes concentrações (100, 50 e 25%). Além disso, foi analisado o efeito dos metabólitos extracelulares dos estreptococos sobre o biofilme pré-formado (6h) de C. tropicalis. Para verificar o efeito dos metabólitos extracelulares foram utilizados dois métodos: o método turbidimétrico que se baseia na leitura da densidade óptica (OD) das suspensões celulares e a coloração cristal violeta (CV) que permite a quantificação indireta da formação de biofilme através da coloração com cristal violeta. Em seguida, foram examinadas as características dos biofilmes, formados por 24 horas, através da análise por microscopia óptica comum. Os resultados referentes ao biofilme foram submetidos ao ANOVA com pós-teste Bonferroni, com diferença estatística de p<0,01. Nossos resultados sugerem que substâncias solúveis produzidas por S. oralis, S. sanguinis e S. parasanguinis induzem a formação de hifas de C. tropicalis sem interferir no crescimento planctônico. Além de diminuir drasticamente o desenvolvimento do biofilme dessa levedura quando em contato com SSS e SSP. Tal fato reforça a ideia de que existe grande heterogeneidade dentro de biofilmes polimicrobianos, especialmente entre leveduras e bactérias. E que o resultado dessa interação depende das condições as quais estes micro-organismos serão submetidos.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O gÃnero Guettarda (Rubiaceae) compreende plantas extensamente distribuÃdas em Ãreas tropicais. Com relaÃÃo à espÃcie Guettarda sericea, a literatura demonstra que existe uma ampla carÃncia acerca de estudos botÃnicos e fitoquÃmicos. Assim, o presente trabalho buscou avaliar o efeito antibacteriano do extrato etanÃlico das folhas de G. sericea (EEFGS) e suas fraÃÃes sobre o crescimento de Streptococcus oralis ATCC 10557 e S. salivarius ATCC 7073, nas formas planctÃnicas e de biofilmes. Diferentes metodologias foram empregadas para a verificaÃÃo do potencial antimicrobiano. Dentre estas estÃo a determinaÃÃo da concentraÃÃo inibitÃria mÃnima (MIC), a determinaÃÃo da curva de morte e a avaliaÃÃo da concentraÃÃo bactericida mÃnima (MBC). AlÃm disso, a quantificaÃÃo da biomassa e do nÃmero de cÃlulas viÃveis do biofilme foi realizada, respectivamente, atravÃs da coloraÃÃo pelo cristal violeta e contagem de unidades formadoras de colÃnia (UFC). Os controles, negativo e positivo utilizados em todos os ensaios foram, respectivamente, DMSO 4% e Gluconato de Clorexidina com concentraÃÃo ajustada de acordo com os dados da CIM de cada micro-organismo. Para a determinaÃÃo da toxicidade do EEFGS utilizou-se o ensaio de toxicidade sobre nÃuplios de Artemia. Os dados mostraram que o extrato e as subfraÃÃes 13 a 17 da fraÃÃo clorofÃrmio apresentaram um marcante efeito antimicrobiano, sendo capazes de inibir o crescimento planctÃnico, bem como o desenvolvimento de biofilmes da cepa de S. oralis atà a concentraÃÃo de 62,5 Âg.mL-1. Com relaÃÃo a S. salivarius, apenas a subfraÃÃo 12 interferiu sobre o crescimento bacteriano. No tocante à toxicidade, foi observado que a morte dos nÃuplios de Artemia ocorreu em concentraÃÃes mais elevadas do que aquelas que apresentaram efeito antibacteriano. A partir de tais resultados pode-se concluir que EEFGS e as subfraÃÃes 13 a 17 podem ser utilizados como insumos para o controle da formaÃÃo de biofilmes de S. oralis. Em adiÃÃo, metodologias complementares que busquem a purificaÃÃo dos compostos ativos e seus efeitos citotÃxicos sobre cÃlulas eucariÃticas necessitam ser realizadas, visando sua utilizaÃÃo como um agente fitoterÃpico. / The genus Guettarda (Rubiaceae) comprises plants widely distributed in tropical areas. Regarding the Guettarda sericea species, the literature shows that there is a lack of botanical and phytochemicals studies. Thus, the present study aimed to evaluate the antibacterial effect of ethanol extract of leaves of G. sericea (EEFGS) and its fractions on the growth of Streptococcus oralis ATCC 10557 and S. salivarius ATCC 7073 in both the planktonic and biofilms states. Different methods were employed to verify the antimicrobial potential. Among these are the determination of minimum inhibitory concentration (MIC) determination of the death curve and evaluation of minimum bactericidal concentration (MBC). Furthermore, quantification of biomass and the number of viable cells of the biofilm were performed, respectively, by staining with crystal violet and counting colony forming units (CFU). The negative and positive controls used in all assays were respectively 4% DMSO and chlorhexidine gluconate with concentration adjusted according to the data of the MIC of each microorganism. To determine the toxicity of EEFGS, it was used the toxicity test on Artemia nauplii. The data showed that the extract and subfractions 13 to 17 of the chloroform fraction show a remarkable antimicrobial effect, able to inhibit the growth of planktonic and development of biofilms of S. oralis strain until the concentration of 62.5 &#956;g.mL-1. With respect to S. salivarius, only subfraction 12 interfered on bacterial growth. Regarding the toxicity, it was observed that death of Artemia nauplii occurred at higher concentrations than those that exhibited antibacterial effect. From these results it can be concluded that EEFGS and subfractions 13-17 can be used as agents for the control of biofilm formation of S. oralis. In addition, complementary methodologies that seek purification of the active compounds and their cytotoxic effects on eukaryotic cells need to be held, aiming its use as an herbal agent.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / BactÃrias e fungos sÃo encontrados juntos em uma infinidade de ambientes e, particularmente, na forma de biofilme, onde as espÃcies aderentes interagem atravÃs de diversos mecanismos de sinalizaÃÃo. Na cavidade oral, espÃcies de Candida coexistem com inÃmeras espÃcies bacterianas, e evidÃncias sugerem que bactÃrias podem modular a formaÃÃo de biofilme, bem como induzir a formaÃÃo de hifas e pseudo-hifas. Assim, caracterizar tais interaÃÃes à essencial para o entendimento da patogÃnese das doenÃas e, possivelmente, a descoberta de novas estratÃgias terapÃuticas. Nesse sentido, o objetivo principal deste trabalho foi avaliar, in vitro, os mecanismos envolvidos na interaÃÃo entre as bactÃrias Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis e a levedura Candida tropicalis. No estudo foi analisado o efeito dos metabÃlitos extracelulares presentes no sobrenadante de C. tropicalis (SCT) sobre o biofilme prÃ-formado (6h) de S. oralis, S. sanguinis e S. parasanguinis. TambÃm foi avaliado o efeito dos metabÃlitos extracelulares de S. oralis (SSO), S. sanguinis (SSS) e S. parasanguinis (SSP) sobre o crescimento planctÃnico, formaÃÃo de biofilme e capacidade de filamentaÃÃo de C. tropicalis, utilizando apenas o sobrenadante da cultura de cada um dos estreptococos em diferentes concentraÃÃes (100, 50 e 25%). AlÃm disso, foi analisado o efeito dos metabÃlitos extracelulares dos estreptococos sobre o biofilme prÃ-formado (6h) de C. tropicalis. Para verificar o efeito dos metabÃlitos extracelulares foram utilizados dois mÃtodos: o mÃtodo turbidimÃtrico que se baseia na leitura da densidade Ãptica (OD) das suspensÃes celulares e a coloraÃÃo cristal violeta (CV) que permite a quantificaÃÃo indireta da formaÃÃo de biofilme atravÃs da coloraÃÃo com cristal violeta. Em seguida, foram examinadas as caracterÃsticas dos biofilmes, formados por 24 horas, atravÃs da anÃlise por microscopia Ãptica comum. Os resultados referentes ao biofilme foram submetidos ao ANOVA com pÃs-teste Bonferroni, com diferenÃa estatÃstica de p<0,01. Nossos resultados sugerem que substÃncias solÃveis produzidas por S. oralis, S. sanguinis e S. parasanguinis induzem a formaÃÃo de hifas de C. tropicalis sem interferir no crescimento planctÃnico. AlÃm de diminuir drasticamente o desenvolvimento do biofilme dessa levedura quando em contato com SSS e SSP. Tal fato reforÃa a ideia de que existe grande heterogeneidade dentro de biofilmes polimicrobianos, especialmente entre leveduras e bactÃrias. E que o resultado dessa interaÃÃo depende das condiÃÃes as quais estes micro-organismos serÃo submetidos. / Bacteria and fungi are found together in a multitude of environments, and particularly in the form of biofilm adherent species which interact through various signaling mechanisms. In the oral cavity, Candida species coexist with numerous bacterial species, and evidence suggests that bacteria can modulate biofilm formation as well as induce the formation of hyphae. Thus, to characterize such interactions are essential to the understanding of pathogenesis of diseases and possibly the discovery of new therapeutic strategies. In this sense, the main objective of this study was to evaluate, in vitro, the mechanisms involved in the interaction between bacteria Streptococcus oralis, Streptococcus sanguinis, and Streptococcus parasanguinis yeast Candida tropicalis. In the study the effect of extracellular metabolites present in the supernatant of C. tropicalis (SCT) on the pre-formed biofilm (6H) S. oralis, S. sanguinis and S. parasanguinis was analyzed. The effect of extracellular metabolites of S. oralis (SSO), S. sanguinis (SSS) and S. parasanguinis (SSP) on planktonic growth and biofilm formation capacity filamentation of C. tropicalis was also evaluated using only the supernatant culturing each of streptococci in different concentrations (100, 50 and 25%). Furthermore, the effect of extracellular metabolites of streptococci on the pre-formed biofilm (6h) of C. tropicalis were analyzed. To verify the effect of extracellular metabolites two methods were used: The turbidimetric method based on the reading of the optical density (OD) of cell suspension and coloring crystal violet (CV) which permits indirect quantification of the biofilm formation by staining with crystal violet. Then were examined characteristics of biofilms formed by 24 hours, through the analysis simple optical microscope. The results for the biofilm were subjected to ANOVA with Bonferroni post-test, with a statistical difference of p<0.01.Our results suggest that soluble substances produced by S. oralis, S. sanguinis and S. parasanguinis induce the formation of hyphae of C. tropicalis without interfering with planktonic growth.In addition to dramatically decrease biofilm development of oral yeast when in contact with SSP and SSS. This reinforces the idea that there is great heterogeneity within polymicrobial biofilms, especially between yeasts and bacteria. And the result of this interaction depends on the conditions which these micro-organisms will be subjected.

Les Archaea methanogènes sont des organismes environnementaux ayant été également détectés dans certaines flores associées aux muqueuses des mammifères. Chez l’homme ces microorganismes ont été associés avec les muqueuses intestinale, vaginale et orale. Ces organismes sont des procaryotes anaérobies stricts et leurs conditions de culture restent fastidieuses et très mal connues. En effet, uniquement trois Archaea methanogènes ont été cultivées à partir de prélèvements humains, Methanobrevibater smithii et Methanosphaera stadtmanae à partir des selles puis Methanobrevibater oralis à partir de la plaque dentaire. Récemment l’ADN d’autres Archaea methanogènes et d’Archaea non-methanogènes a été détecté dans des selles humaines, y compris des séquences indiquant la présence d’espèces appartenant à un nouvel ordre de méthanogènes n’ayant aucun représentant cultivé. La connaissance actuelle sur la diversité de ces methanogènes chez l’homme et sur leurs effets potentiels sur la santé humaine est en grande partie basée sur les techniques de détection de l’ADN par PCR et métagénomique. Ces techniques fondées sur la détection de l’ADN ribosomal 16S et du gène mcrA codant la sous-unité alpha du methyl-coenzyme M reductase, une enzyme clé dans le processus de méthanogenèse, ont montré dans un premier temps que M. smithii était détecté chez moins de 50% des individus et M. stadtmanae chez 0-20 % seulement. Ces résultats étaient contradictoires avec le rôle de la méthanogenèse dans l’élimination des acides et d’autres produits du processus digestion, et nous avons émis l’hypothèse que ces résultats pouvaient ne pas refléter la quantité réelle des méthanogènes dans le tube digestif humain, suggérant la mise au point de nouvelles méthodes de détection moléculaire et de culture adaptées aux caractéristiques de ces organismes fastidieux. Dans ce travail, nous nous sommes fixés comme premier objectif de mettre au point une méthode moléculaire permettant de détecter M. smithii chez tous les individus testés et nous avons mis au point un protocole d’extraction et de détection d’ADN d’Archaea à partir des selles en se basant sur les génomes séquencés de M. smithii et M. stadtmanae. Ce protocole nous a permis de détecter M. smithii chez 95,5% des individus et M. stadtmanae chez 29,4% des individus. En ce basant sur ce protocole et moyennant une approche moléculaire basée sur une PCR universelle de l’ADN ribosomal 16S des méthanogènes, le séquençage et le clonage, nous avons également détecté chez 4% de la population, une séquence correspondant à un phylotype (FJ823135) ayant déjà été rapporté comme représentant un nouvel ordre de méthanogènes. A partir de là, nous avons choisi un prélèvement de selle susceptible de contenir le plus fort ratio de FJ823135/ M. smithii et nous avons réussi à isoler et à cultiver une nouvelle Archaea que nous avons nommé Methanomassiliicoccus luminyensis, premier représentant cultivé d’un nouvel ordre de méthanogènes et la quatrième Archaea cultivée chez l’homme. M. luminyensis et M. stadtmanae présentent des métabolismes similaires en réduisant le méthanol en méthane en utilisant l’hydrogène comme donneur d’électrons, cette observation nous a incité à tester l’addition de tungstate de sélénium, requis pour la croissance de M. luminyensis, dans une culture M. stadtmanae, et nous avons observé une accélération de la vitesse de croissance de M. stadtmanae par un facteur 3. Nous avons ensuite étudié la sensibilité des méthanogènes isolés chez l’homme aux antibiotiques et établi qu’ils sont seulement sensibles à des molécules efficaces contre les bactéries et les eucaryotes, ceci étant en accord avec leur position phylogénétique en tant qu’un des quatre domaines de la vie. [...] / Methanogenic Archaea are environmental organisms which have also been associated to mammals mucosa. In humans these microorganisms have been detected in the vaginal, intestinal and oral mucosa. These organisms are strict anaerobes and their culture conditions remains fastidious and poorly known. In fact only three methanogens have been isolated from human samples, both Methanobrevibater smithii and Methanosphaera stadtmanae from stool and Methanobrevibater oralis from dental plaque. Current knowledge on the diversity of methanogens in humans and their potential effects on human health were largely based on DNA detection methods as PCR and metagenomics. These techniques based on 16S rDNA and mcrA gene (encoding the alpha subunit of methyl coenzyme-M-reductase, a key enzyme in methanogenesis process) detection, showed that M. smithii was the most present in man and that the presence of M. stadtmanae was transient. Recently, the DNA of other methanogenic and non- methanogenic Archaea, has been detected in human feces, including sequences indicating the presence of non-cultured species belonging to potential new order of methanogens with no cultured representative. However, these studies detected M. smithii with variable prevalence in less than half of the tested individuals and no M. stadtmanae; such results does not confirm the paramount role of methanogenesis in preventing the accumulation of acids and other reaction end products during the digestion process, and can not reflect the actual amount of these two methanogens in the human digestive tract because of their specific association with the intestinal mucosa. Therefore, these studies pointed that the diversity of methanogens in humans has been underestimated suggesting the development of new molecular detection methods and cultural approaches adapted these fastidious organisms. In this work, we preset as first criteria, the detection of M. smithii in all tested individuals, therefore we developed an improved protocol for archaeal DNA extraction and detection from stool based on sequenced genomes of M. smithii and M. stadtmanae, this protocol allowed us to detect the first one DNA in 95.5% tested individuals and the second in a prevalence of 29.4%. Based on this protocol and through molecular approach based on universal amplification of methanogenic 16S rDNA, sequencing and cloning, we detected in 4% of the tested population, a sequence corresponding to a new phylotype (FJ823135) that has been previously reported and proposed as a representative of a new order of methanogens. From there, we chose one stool specimen susceptible to contain the highest amount of FJ823135 and successfully isolated Methanomassiliicoccus luminyensis B10T clone, the first cultured representative of a new order of methanogens and the fourth Archaea cultured in humans.This archaeon exhibited a similar type of metabolism to that of M. stadtmanae by oxidizing H2 and reducing methanol to methane but require tungstate-selenite, an element essential for its growth, this fact prompted us testing tungstate-selenite addition on M. stadtmanae growth and establishing that it was strongly stimulatory with a growth rate three times faster. We have thereafter studied the sensitivity of methanogens isolated from humans to antibiotics and established that they are susceptible only to molecules also effective against both Bacteria and Eucarya, in agreement with their phylogenetic location as a unique domain of life. The aim of the latter part of this work was to test the effectiveness of MALDI-TOF mass spectrometry identification of environmental and host-associated Archaea. The obtained data indicated that that MALDI-TOF-MS protein profiling is an efficient first-line step for the rapid phenotypic identification of cultured Archaea organisms including host-associated ones. [...]

L’analyse du microbiote oral et de son évolution séculaire se fait principalement à partir de l’analyse du tartre dentaire ancien des populations passées et du biofilm dentaire des populations modernes. Nous avons dans un premier temps fait le point des connaissances sur la paléomicrobiologie des bactéries et des archaea contenues dans le tartre dentaire et montré que les archaea faisaient partie du microbiote oral chez l'homme. Dans la deuxième partie, nous avons mis en évidence le répertoire des archaea méthanogènes vivant dans la cavité orale par la culture (une nouvelle espèce Methanobrevibacter massiliense, Methanobrevibacter smithii et Methanobrevibacter oralis). La prévalence de ces archaea était significativement plus élevée chez les patients atteints de parodontite que chez les personnes contrôles. Ensuite, nous avons développé une méthode de génotypage Multispacer Sequence Typing pour typer M. oralis et M. smithii et révélé différents génotypes. Enfin, nous avons analysé le répertoire des archaea méthanogènes dans des échantillons de tartre dentaire ancien datant du 14ème au 19ème siècle. La prévalence et la diversité des archaea méthanogènes dans la cavité orale ont diminué significativement au cours des sept derniers siècles. Des archaea méthanogènes ont été retrouvées dans 75% des prélèvements de tartre dentaire (Candidatus M. massiliense à 44,6%, M. oralis à 19,6%, Methanomassiliicoccus luminyensis-like à 12,5%, Candidatus Nitrososphaera evergladensis-like dans un prélèvement et Methanoculleus bourgensis dans un autre prélèvement). Un prélèvement de tartre positif pour Candidatus M. massiliense a été documenté par hybridation in situ en fluorescence. / The analyses of oral microbiome and its secular evolution mainly use dental calculus in past populations and dental plaque in modern populations. In our thesis, we initially reviewed the knowledge actual about bacteria and archaea paleomicrobiology of the dental calculus. The review disclosed that archaea taked part in the secular core-microbiota in past and modern populations. In the second work, we demonstrated the repertoire of methanogenic archaea currently living in the oral cavity using culture-based approach and succeeded in isolating for the first time a new species named Methanobrevibacter massiliense in addition to Methanobrevibacter smithii and Methanobrevibacter oralis from dental plaque in periodontitis patients. This work showed that the prevalence of methanogens was significantly higher in periodontitis patients than in controls. Some methanogenic archaea were involved in periodontitis. Then, we developed Multispacer Sequence Typing to evaluate M. oralis and M. smithii and revealed different genetic variants in these archaea. Finally, we examined the repertory of methanogenic archaea in ancient dental calculus dating from the 14th to the 19th century. The prevalence and diversity of methanogenic archaea in the oral cavity decreased significantly during the last seven centuries. Methanogenic archaea were found in 75% of dental calculis (Candidatus M. massiliense, 44.6%; M. oralis, 19.6%; Methanomassiliicoccus luminyensis-like, 12.5%; Candidatus Nitrososphaera evergladensis-like in one and Methanoculleus bourgensis in one specimen). One Candidatus M. massiliense dental calculus was further documented by fluorescent in situ hybridization.

Les méthanogènes sont des Archaea anaérobies stricts, connus pour être les seuls êtres vivants capables de produire du méthane comme sous-produit de leur métabolisme. Notre revue de littérature a montré qu’outre les espèces environnementales, l’ordre des Methanomassiliicoccales ne comptait à ce jour qu’une seule espèce Methanomassiliicoccus luminyensis isolée et cultivée dans notre laboratoire. Aucours de notre thèse, nous avons développé une nouvelle méthode permettant la culture et l’isolement des méthanogènes en absence de source externe de dihydrogène (H2) d’une part et d’autre part une méthode de génotypage Multispacer Sequence Typing (MST) basée sur le séquençage d’espaces intergéniques pour typer Methanobrevibacter smithii et M. oralis. Par la suite, nous avons mis en évidence la présence de méthanogènes en situation pathologique chez l’homme. Nous avons détecté et isolé pour la première fois au sein de flores anaérobies, M. oralis à partir d’échantillons d’abcès cérébraux, et de sinusite d’une part, et d’autre part M. smithii à partir d’un échantillon de patient souffrant d’abcès para-vertébral. Enfin, dans la cinquième partie de notre thèse, nous avons testé in vitro la sensibilité de cinq méthanogènes associés aux flores humaines à la lovastatine qui est une pro-drogue utilisée pour abaisser la concentration de cholestérol chez l’homme dans le cadre de certaines pathologies. Les cinq méthanogènes se sont avérées sensibles à une concentration minimale inhibitrice de 1µg/mL, après activation par hydrolyse de la lovastatine par des bactéries anaérobies du microbiote digestif, via une l’inhibition de la croissance et de la production du méthane. / Methanogens are strict anaerobic Archaea, known to the only being able to producing methane gas as a byproduct. Methanogens which are not detected in clinical microbiology laboratories were present in oral, digestive, vaginal and cutaneous microbiota of human. Only five species on the thirteen known in human have been cultivated before the beginning of our thesis. In our thesis, we initially reviewed the state of knowledge about methanogens in human microbiota, particularly the new order Methanomassiliicoccales of methylotrophic methanogens, the as member of human microbiota. In the second part of this work, we performing new method for methanogens culture and isolation without any dihydrogen atmosphere, by co-cultured in tubes methanogens with Bacteroides thetaiotaomicron, which produces hydrogen. We also developed Multispacer Sequence Typing (MST), a genotyping method based on intergenic spacers sequencing, to genotype M. oralis and Methanobrevibacter. Smithii. We demonstrated that methanogens could be part of polymicrobial infection in the case of brain and sinusal abscesses, and also in skeletal muscle abscess, by isolating for the first time M. oralis and M. smithii in these pathologies, using culture-based and molecular-based approaches, and suggested that methanogens could be considered as human opportunistic or emerging pathogens. Finally, we tested the in vitro susceptibility of lovastatin which is a prodrug used as a powerful serum cholesterol-lowering drug in some human diseases and showed that it’s inhibits growth and methane production in human-associated methanogens without affecting intestinal bacteria.

Les archaea constituent l'un des quatre domaines connus du vivant. Contrairement à ce que leur nom laisse supposer, elles ont colonisé tous les écosystèmes et les microbiotes de certains hôtes dont l'Homme. Chez l'homme, certaines espèces d'archaea méthanogènes ont été associées aux muqueuses orale, intestinale et vaginale. Ces archaea méthanogènes sont des procaryotes anaérobies stricts et leurs conditions de culture restent fastidieuses et très mal connues. Quatre archaea methanogènes seulement ont été isolées à partir de prélèvements humains y compris dans le microbiote digestif Methanobrevibacter smithii détectée dans 95,7% des individus, Methanosphaera stadtmanae retrouvée chez environ un tiers des individus et plus récemment dans notre laboratoire Methanomassilicoccus luminyensis détectée en moyenne chez 4% des individus avec une prévalence liée à l'âge ; et dans le microbiote orale Methanobrevibacter oralis isolée à partir de la plaque dentaire. / Archaea is one of four known domains of life. Unlike what their name suggests, they some species of methanogenic archaea have been associated with oral, vaginal and intestinal mucosa. These methanogenic archaea are obligate anaerobic prokaryotes and their culture conditions are fastidious and very poorly known. Only four methanogenic archaea have been isolated from human samples including the digestive microbiota; Methanobrevibacter smithii detected in 95.7% of individuals Methanosphaera stadtmanae found in approximately one third of individuals and more recently in our laboratory Methanomassilicoccus luminyensis detected on average in 4% of individuals with a prevalence of age-related, and in the oral microbiota Methanobrevibacter oralis isolated from dental plaque.