DIVERSITY OF METHANOGENIC ARCHAEA IN HUMAN MICROBIOTA. Thesis co-director Pr Raymond RUIMY, Nice

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1 DIVERSITY OF METHANOGENIC ARCHAEA IN HUMAN MICROBIOTA GRINE GHILES 1st YEAR THESIS Thesis director Pr Michel DRANCOURT, Marseille Thesis co-director Pr Raymond RUIMY, Nice

2 Bacteria Eukaryotes - From greek archaios «ancient» formerly called «Archaebacteria» -Initially founded in extreme environments, 2.7 billion years ago - Initially classified in Bacteria domain Archaea Virus Balch et al. (1977), De lang et al. (2001), Boyer M et al. (2010)

3 DIVERSITY OF ARCHAEA Archaea known Archaea unknown (Brochier et al., 2004); (Eme et al., 2013)

4 Euryarchaeota DIVERSITY OF ARCHAEA Halophils Archaea known Methanogens Archaea unknown Nanoarchaeota Traumarchaeota Crenarchaeota Thermophils (Brochier et al., 2004); (Eme et al., 2013) Konarchaeota

5 Methanogens Strict anaerobes Producing methane gas (CH4) as a metabolic byproduct in presence of H 2 and substrates Groups depending on substrates use for methanogenesis Hydrogenotrophs Carbon dioxide : CO 2 + 4H 2 CH 4 + 2H 2 O Order concerned: Methanobacteriales, Methanomicrobiales, Methanopyrales, Methanococcales, Methanocellales Methylotrophs Methylamine: 4CH 3 NH 2 + 2H 2 O 3CH 4 + CO 2 + 4NH 3 Methanol: 4CH 3 OH 3CH 4 + CO 2 + 2H 2 O Order concerned: Methanomassiliicoccales Acetogenotrophs Acetate: CH 3 COOH CH 4 + CO 2 Order concerned: Methanosarcinales Tabatabaei, (2010); Thauer, (2006); Gribaldo S, (2006); Dridi, (2012); Hyunh, (2016)

6 Oral microbiota Methanogens (Euryarchaeota) Methanobrevibacter oralis (48%) Methanobreviacter smithii Methanobrevibacter massiliense Methanosarcinia mazei Methanobacterium congolense Cutaneous microbiota - Methanogens (Euryarchaeota) Methanosarcinia sp - Methanosaeta concilii Traumarchaoata - Traumachaeota sp Vaginal microbiota Methanogens (Euryarchaeota) Methanogens as members of human microbiota Methanogens Intestinal microbiota Methanobrevibacter smithii (95.7%) Methanosphaera stadtmanae (27%) Methanomassilicoccus luminyensis (4%) Methanobrevibacter arboriphilicus Methanobrevibacter oralis Methanobrevibacter millerae Methanocellus chikugoensis Candidatus Methanomassilococcus intestinalis Candidatus Methanomethylophilus alvus Methanobrevibacter smithii Stark and Lee, (1982); Palmer,(2007); Maczulak, (1989); Koenig, (2011); Mihajlovski, (2010)

7 Methanogenics archaea diversity years 70 years 3-15 years 4 days-10 months M. smithii Methanobrevibacter spp Methanobacteriales Methanomassiliicocales sp M. smithii Methanobrevibacter spp Methanobacteriales Methanomassiliicocales sp Palmer,(2007); Mihajlovski; (2010) Koenig, (2011); ottman, (2012) M. smithii Methanobrevibacter oralis Methanobrevibacter arboriphilus Methanobrevibacter millerae Methanosphaera stadtmanae Methanosalsum zhilinae Methanomassiliicoccus luminyensis Candidatus Methanomassiliicoccus intestinalis Candidatus Methanomethylophylus alvus M. smithii Methanosphaera stadtmanae Methanomassiliicoccus luminyensis Candidatus Methanomassiliicoccus intestinalis

8 Methanogenic archaea diversity years 70 years 3-15 years 4 days-10 months M. smithii Methanobrevibacter spp Methanobacteriales Methanomassiliicocales sp M. smithii Methanobrevibacter spp Methanobacteriales Methanomassiliicocales sp Palmer,(2007); Mihajlovski; (2010) Koenig, (2011); ottman, (2012) M. smithii Methanobrevibacter oralis Methanobrevibacter arboriphilus Methanobrevibacter millerae Methanosphaera stadtmanae Methanosalsum zhilinae Methanomassiliicoccus luminyensis Candidatus Methanomassiliicoccus intestinalis Candidatus Methanomethylophylus alvus M. smithii Methanosphaera stadtmanae Methanomassiliicoccus luminyensis Candidatus Methanomassiliicoccus intestinalis

9 Potential implications of methanogens archaea in human diseases M. oralis was correlated with the severity of periodontitis in patient A strong association between the presence of M. smithii and chronic constipation in human was shown Association between high numbers of M. smithii were observed in obese individuals than in normal-weight or post-gastric-bypass individuals Association between the absence of M. smithii and severity of diarrhea/ Malnutrition was also shown Bringuier. (2013) ; Zhang, (2009); Koenig. (2011); Million et al. (2016)

10 Potential implications of methanogens archaea in human diseases M. oralis was correlated with the severity of periodontitis in patient A strong association between the presence of M. smithii and chronic constipation in human was shown Association between high numbers of M. smithii were observed in obese individuals than in normal-weight or post-gastric-bypass individuals Association between the absence of M. smithii and severity of diarrhea/ Malnutrition was also shown The repertoire and the prevalence of human methanogens and their variations still underestimated : Fastidious culture, standard protocols for DNA extraction have not been optimized for such microorganisms Bringuier. (2013) ; Zhang, (2009); Koenig. (2011); Million et al. (2016)

11 Project 1 Defining the sources of methanogens archaea Project 2 Increase the repertory of methanogens archaea Project 3 Improve methods of culture and detection of archaea

12 Molecular approach Echantillon à analyser Selles, G.N.N. 250µl of suspension + lysis buffer g glass powder mecanical lysis DNA extraction 16S rrna and mcra gene amplification Seqquencing, analysis and identification Culture approach Echantillon à analyser Selles, G.N.N. Hungate Tube (SAB-medium for cultivating methanogens) Incubation at 37 C With 150rpm CH 4

13 culture of methanogens archaea with H2-producing bacteria (Khelaifia et al. 2016)

14 Gastric of newborn Number of samples 50 PCR 1OO% (+) Séquençage M.smithii Culture 10 (+)

15 Gastric of newborn Number of samples 50 PCR 1OO% (+) Séquençage M.smithii Culture 10 (+)

16 Gastric of newborn Number of samples 50 PCR 1OO% (+) Séquençage M.smithii Culture 10 (+)

17 Gastric of newborn Newborn Stool Necrotizing Enterocolitis of stool newborn Number of samples 50 Number of samples 35 Number of samples 48 vaginal samples Number of samples 30 PCR 1OO% (+) PCR (-) PCR (-) PCR (-) Séquençage M.smithii Témoin externe (tiss) (+) Témoin externe (tiss) (+) Témoin interne (actine) 18 (+) 12 (-) Culture 10 (+) Témoin externe (tiss) +

18 Stool samples Number of samples 100 PCR 98% (+) Séquençage M.smithii Culture (en cours)

19 ON GOING Multilocus sequence typing Archaea culture medium with rumen 30% de rumen 70% de SAB Lyophilisation as Method for the conservation of archaea strains

20 THANKS FOR YOUR ATTENTION