Methanogen Abundance Thresholds Capable of Differentiating In Vitro Methane Production in Human Stool Samples

Background: Intestinal methane (CH₄) gas production has been associated with a number of clinical conditions and may have important metabolic and physiological effects. Aims: In this study, taxonomic and functional gene analyses and in vitro CH₄ gas measurements were used to determine if molecular markers can potentially serve as clinical tests for colonic CH₄ production. Methods: We performed a cross-sectional study involving full stool samples collected from 33 healthy individuals. In vitro CH₄ gas measurements were obtained after 2-h incubation of stool samples and used to characterize samples as CH₄ positive (CH₄+) and CH₄ negative (CH₄–; n = 10 and 23, respectively). Next, we characterized the fecal microbiota through high-throughput DNA sequencing with a particular emphasis on archaeal phylum Euryarchaeota. Finally, qPCR analyses, targeting the mcrA gene, were done to determine the ability to differentiate CH₄+ versus CH₄− samples and to delineate major methanogen species associated with CH₄ production. Results: Methanobrevibacter was found to be the most abundant methane producer and its relative abundance provides a clear distinction between CH₄+ versus CH₄− samples. Its sequencing-based relative abundance detection threshold for CH₄ production was calculated to be 0.097%. The qPCR-based detection threshold separating CH₄+ versus CH₄− samples, based on mcrA gene copies, was 5.2 × 10⁵ copies/g. Conclusion: Given the decreased time-burden placed on patients, a qPCR-based test on a fecal sample can become a valuable tool in clinical assessment of CH₄ producing status.