T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype

Introduction: Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. Methods: Forty-four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real-time quantitative polymerase chain reactions. Results: Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non-synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl-coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. Conclusion: Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.