Polyphasic molecular approach to the characterization of methanogens in the saliva of Tunisian adults

Objectives: Methanogenic archaea are a minor component of human oral microbiota. Due to their relatively low abundance, the detection of these neglected microorganisms is challenging. This study concerns the presence of methanogens in salivary samples collected from Tunisian adults to evaluate their prevalence and burden using a polyphasic molecular approach. Methods: A total of 43 saliva samples were included. Metagenomic and standard 16S rRNA sequencing were performed as an initial screening to detect the presence of methanogens in the oral microbiota of Tunisian adults. Further investigations were performed using specific quantitative real-time PCR targeting Methanobrevibacter oralis and Methanobrevibacter smithii. Results: Methanobrevibacter was detected in 5/43 (11.62 %) saliva samples after metagenomic 16S rRNA data analysis. The presence of M. oralis was confirmed in 6/43 samples by standard 16S rRNA sequencing. Using realtime PCR, methanogens were detected in 35/43 (81.39 %) samples, including 62.79 % positive for M. oralis and 76.74 % positive for M. smithii. These findings reflect the high prevalence of both methanogens, revealed by the high sensitivity of the real-time PCR approach. Interestingly, we also noted a significant statistical association between the detection of M. smithii and poor adherence to a Mediterranean diet, indicating the impact of diet on M. smithii prevalence. Conclusion: Our study showed the presence of methanogens in the oral microbiota of Tunisian adults with an unprecedented relatively high prevalence. Choice of methodology is also central to picturing the real prevalence and diversity of such minor taxa in the oral microbiota.