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

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 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.

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Acknowledgment

University of Minnesota Center for Mass Spectrometry and Proteomics for conducting the short chain fatty acid analysis. We also thank the University of Minnesota Supercomputing Institute for providing resources.

Funding

Healthy Foods Healthy Lives (A.Kh); Achieving Cures Together (A.Kh); the University of Minnesota MnDRIVE Initiative (MJS); Allen Foundation (MJS).

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Correspondence to Alexander Khoruts.

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Teigen, L., Mathai, P.P., Matson, M. et al. Methanogen Abundance Thresholds Capable of Differentiating In Vitro Methane Production in Human Stool Samples. Dig Dis Sci (2020). https://doi.org/10.1007/s10620-020-06721-5

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Keywords

  • Gut microbiota
  • Methanogens
  • Methane
  • Methanobrevibacter
  • mcrA gene
  • Quantitative PCR
  • Amplicon sequencing
  • Molecular thresholds