Methanogen Abundance Thresholds Capable of Differentiating In Vitro Methane Production in Human Stool Samples
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ORIGINAL ARTICLE
Methanogen Abundance Thresholds Capable of Differentiating In Vitro Methane Production in Human Stool Samples Levi Teigen1 · Prince P. Mathai2 · Michael Matson1 · Sharon Lopez1 · Daria Kozysa1 · Amanda J. Kabage1 · Matthew J. Hamilton2 · Byron P. Vaughn1 · Michael J. Sadowsky2,3,4 · Alexander Khoruts1,2,5 Received: 3 September 2020 / Accepted: 15 November 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract 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 C H4 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 C H4 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 C H4+ 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 Gut microbiota · Methanogens · Methane · Methanobrevibacter · mcrA gene · Quantitative PCR · Amplicon sequencing · Molecular thresholds
Introduction
Prince P. Mathai contributed equally to this manuscript. Michael J. Sadowsky, Levi Teigen and Alexander Khoruts share equal senior authorship. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10620-020-06721-5) contains supplementary material, which is available to authorized users. * Alexander Khoruts [email protected] Extended author information available on the last page of the article
Intestinal methane ( CH4) gas production has been associated with a number of clinical conditions such as obesity, constipation-dominant irritable bowel syndrome (IBS-C) and diverticulosis [1]. In individuals with obesity and prediabetes, eradication of CH4 can result in positive metabolic changes [2]. Physiologically, C H4 slows intestinal transit and increases gut contr
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