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.
Keywords: Amplicon sequencing; Gut microbiota; Methane; Methanobrevibacter; Methanogens; Molecular thresholds; Quantitative PCR; mcrA gene.
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