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Archaea et cavité orale / Archaea and oral cavityHuynh, Thi Thuy Hong 18 September 2015 (has links)
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.
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Effect of dietary fiber on the methanogen community in the hindgut of Lantang giltsCao, Z., Liang, J. B., Liao, X. D., Wright, A. D. G., Wu, Y. B., Yu, B. 07 April 2016 (has links)
The primary objective of this study was to investigate the effect of dietary fiber on methanogenic diversity and community composition in the hindgut of indigenous Chinese Lantang gilts to explain the unexpected findings reported earlier that Lantang gilts fed low-fiber diet (LFD) produced more methane than those fed high-fiber diet (HFD). In total, 12 Lantang gilts (58.7±0.37 kg) were randomly divided into two dietary groups (six replicates (pigs) per group) and fed either LFD (NDF=201.46 g/kg) or HFD (NDF=329.70 g/kg). Wheat bran was the main source of fiber for the LFD, whereas ground rice hull (mixture of rice hull and rice bran) was used for the HFD. Results showed that the methanogens in the hindgut of Lantang gilts belonged to four known species (
Methanobrevibacter ruminantium, Methanobrevibacter wolinii
,
Methanosphaera stadtmanae
and
Methanobrevibacter smithii
), with about 89% of the methanogens belonging to the genus
Methanobrevibacter
. The 16S ribosomal RNA (rRNA) gene copies of
Methanobrevibacter
were more than three times higher (
P
<0.05) for gilts fed LFD (3.31×10
9
copies/g dry matter (DM)) than gilts fed HFD (1.02×10
9
copies/g DM). No difference (
P
>0.05) was observed in 16S rRNA gene copies of
Fibrobacter succinogenes
between the two dietary groups, and 18S rRNA gene copies of anaerobic fungi in gilts fed LFD were lower than (
P
<0.05) those fed HFD. To better explain the effect of different fiber source on the methanogen community, a follow-up
in vitro
fermentation using a factorial design comprised of two inocula (prepared from hindgut content of gilts fed two diets differing in their dietary fiber)×four substrates (LFD, HFD, wheat bran, ground rice hull) was conducted. Results of the
in vitro
fermentation confirmed that the predominant methanogens belonged to the genus of
Methanobrevibacter
, and about 23% methanogens was found to be distantly related (90%) to
Thermogymnomonas acidicola. In vitro
fermentation also seems to suggest that fiber source did change the methanogens community. Although the density of
Methanobrevibacter
species was positively correlated with CH
4
production in both
in vivo
(
P
<0.01,
r
=0.737) and
in vitro
trials (
P
<0.05,
r
=0.854), which could partly explain the higher methane production from gilts fed LFD compared with those in the HFD group. Further investigation is needed to explain how the rice hull affected the methanogens and inhibited CH
4
emission from gilts fed HFD.
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Avaliação da presença de biomarcador molecular de contaminação fecal hospedeiro específico em águas de recreação costeira no Estado do Rio de Janeiro / Evaluation the presence of molecular biomarkers of fecal contamination in specific host coastal recreation waters of the State of Rio de JaneiroOliveira, Samara Sant'Anna de January 2014 (has links)
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Previous issue date: 2014 / Instituto Nacional de Controle de Qualidade em Saúde / Atualmente, indicadores bacteriológicos utilizados para avaliar a balneabilidade das águas de recreação estabelecidos em legislações, apresentam limitações como a correlação com seu hospedeiro. Uma variedade de micro-organismos anaeróbios vem sendo considerados alvos promissores no desenvolvimento de indicadores de poluição fecal hospedeiro-especifico. A espécie M. smithii é a única espécie conhecida que coloniza exclusivamente o trato gastrointestinal humano e é altamente prevalente em esgoto. Sendo assim, este projeto tem como objetivo avaliar a presença de M. smithii como bioindicador de contaminação fecal hospedeiro-especifico em águas de recreação costeira no estado do Rio de Janeiro. Para isso foram coletadas amostras de águas superficiais de13 praias (Ilha do governador (n=4), Copacabana (n=1), Leblon (n=2), Prainha (n=2), Ramos (1), Araruama (n=2), Niterói (n=1)) em seguida os parâmetros físico-químicos e as concentrações de coliformes termotolerantes foram determinados. Em seguida foi realizada a caracterização das comunidades de Methano brevibacterspp. através da construção de bibliotecas do gene rrsdo 16S rRNAea detecção e quantificação do M. smithii pela qPCR do gene nifH. Os parâmetros físico-químicos, apresentaram alterações significativas em relação a salinidade e condutividade. Quando analisados em relação a balneabilidade, somente as praias Seca, Pontinha e Ramos apresentaram-se como próprias para atividade recreativa. As análises das bibliotecas demonstraram a prevalência de M. smithiie de organismos não cultivados, muito provavelmente novas espécies do gênero Methano brevibacter ainda não descritas. A PCR convencional detectou M. smithii em 11 amostras, enquanto a qPCR detectou e quantificou cópias do gene nifHem12 amostras. A partir dos dados obtidos podemos concluir que as aplicações de novos indicadores na determinação da origem da contaminação das águas de recreação contribuem para uma avaliação mais precisa da qualidade das águas e consequentemente da saúde da pública. / Currently, bacteriological indicators used to assess bathing water recreation established in laws, have limitations such as the correlation with its host. A variety of anaerobic micro-organisms has been considered promising targets in the development of host-specific indicators of fecal pollution. The specie M. smithiiis the only known species that exclusively colonizes the human gastrointestinal tract and is highly prevalent in sewage. Thus, this project aims to assess the presence of M. smithii as bioindicator of host-specific fecal pollution in coastal recreation waters of Rio de Janeiro. For this surface water samples from 13 beaches (Ilha do Governador (n = 4), Copacabana (n = 1), Leblon (n = 2), Prainha (n = 2), Ramos (1), Araruama (n = 2), Niteroi (n = 1)) then the physico-chemical parameters and concentrations of fecal coliforms were determined. Then the characterization of communities Methanobrevibacterspp was performed. by constructing libraries rrs 16S rRNAe detection and quantification by qPCR of M. smithiinifH. The physico-chemical parameter settings showed significant changes in relation to salinity and conductivity. When analyzed for bathing, only Seca, Pontinha Ramos beaches and presented themselves as fitfor recreational activity. Analyses of libraries demonstrated the prevalence of M. smithii and uncultivated organisms, most likely new species of the genus Methanobrevibacter not yet described. Conventional PCR detected in M. smithii 11 samples, while qPCR detected and quantified copies of the nifH 12 samples. From the data obtained we can conclude that applications of new indicators in determining the source of contamination of water recreation contribute to a more accurate assessment of water quality and consequently the health of the public.
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Les Archaea méthanogènes comme pathogènes opportunistes / Methanogenic Archaea as human opportunistic pathogensNkamga, Vanessa Demonfort 16 September 2016 (has links)
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.
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Etude de microbiote digestif africain par culturomics et nouvelle technique d'isolement et de culture de Methanobrevibacter smithii / African digestive microbiote studies by culturomics and a new studies culturomics and a new technique of isolation and culture of Methanobrevibacter smithiiTraore, Sory Ibrahima 23 November 2018 (has links)
L’étude du microbiote digestif a connu un regain d’intérêt au début des années 2000, avec l’avènement des techniques moléculaires. La culturomics a démontré sa complémentarité depuis 2010 en réduisant une partie des biais des méthodes moléculaires. Une revue sur les techniques d’étude du microbiote digestif et l’analyse du microbiote des sujets africains. Les études de métagénomique en Afrique ont révélé une augmentation de la biodiversité, en particulier des Spirochaetes et des Prevotella chez les africains par rapport aux occidentaux. Sur les 1162 bactéries isolées par culturomics, 476 n'étaient pas africaines, 445 étaient communes et 241 étaient d’origine africaine dont 68 nouvelles espèces. Pour ma participation au travail de culturomics, 102750 colonies testées par MALDI-TOF,ont permis d'identifier 377 espèces incluant 40 nouvelles espèces,17 nouveaux genres et 2 nouvelles familles.Ces nouvelles espèces ont été décrites par taxonogenomics ou new species announcement.Les archaea méthanogènes ont une prévalence de 97,4% pour M. smithii et associés à des pathologies comme l’abcès du cerveau,les parodontites etc. La culture est fastidieuse et nécessitait une source extérieure d’hydrogène. Sous enceinte anaérobie, nous avons cultivé avec succès M. smithii à partir d’un milieu de culture liquide inoculé d’échantillon de selle. L’isolement en culture pure a été un succès sur milieu gélosé en réalisant une coculture avec Bacteroides thetaiotaomicron. Nous avons aussi testé avec succès la coculture de M. smithii avec d’autres bactéries productrices d’hydrogène connues. Les tests de chromatographie en phase gazeuse montraient que ces souches produisaient de l’hydrogène. / The study of the digestive microbiota was a renewed interest in the early 2000s, with the advent of molecular techniques. The culturomics has demonstrated its complementarity since 2010 by reducing some of the biases of molecular methods. A review on the techniques of studying the digestive microbiota and the analysis of the microbiota of African subjects. Metagenomic studies in Africa have revealed an increase in biodiversity, especially Spirochaetes and Prevotella among Africans compared to Westerners. Of the 1162 bacteria isolated by culturomics, 476 were non-African, 445 were common, and 241 were of African origin, including 68 new species. For my participation in the work of culturomics, 102750 colonies tested by MALDI-TOF, identified 377 species including 40 new species, 17 new genera and 2 new families. These new species have been described by taxonogenomics or new species announcement.Methanogenic archaea have a prevalence of 97.4% for M. smithii and associated with pathologies such as brain abscess, periodontitis and so on. The cultivation is tedious and required an external source of hydrogen. Under anaerobic enclosure, we successfully cultivated M. smithii from a liquid culture medium inoculated with a stool sample. The isolation in pure culture was a success on agar medium by performing a coculture with Bacteroides thetaiotaomicron. We have also successfully tested the co-culture of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen.
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Détection et culture des archaea associées aux muqueuses intestinale et orale humainesKhelaifia, Saber 07 June 2013 (has links)
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.
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Partial purification and characterization of F₄₂₀-dependent NADP reductase from Methanobrevibacter smithii strain DE1Sheridan, Scott D. 01 January 1985 (has links)
The F420-dependent NADP reductase of Methanobrevibacter smithii has been partially purified employing a combination of affinity chromatography with Blue Sepharose (Cl-6B) and molecular sieve chromatography with Sephacryl S-200, The enzyme, which requires reduced F420 as an electron donor, has been purified over 145 fold with a recovery of 6%. A molecular weight of 120,00 for the native enzyme was determined by Sephacryl S-200 chromatography. A subunit molecular weight of 28,200 was determined by SDS-PAGE, indicating that the native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 45°C, with a pH optimum of 7.5. The NADP reductase had an apparent Km of 42 uM for reduced F420, and an apparent Km of 4l uM for NADP. The enzyme was stable in 0.05 M sodium phosphate buffer (plus 10 mM cysteine) at pH 7.0, when gassed with nitrogen or hydrogen and stored at 4°C.
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