Hydrogen Sulfide and Methane on Breath Test Correlate with Human Small Intestinal Hydrogen Sulfide Producers and Methanogens

Abstract

Background: Evidence indicates that 3 gas breath testing (BT) correlates with stool microbial populations. Breath methane (CH₄) levels correlate with stool Methanobrevibacter smithii levels and constipation, while hydrogen sulfide (H₂S) levels correlate with stool H₂S producers and diarrhea. However, their relationships to small bowel microbes are unknown.

Aims: To assess relationships between small bowel microbes and breath gases.

Methods: REIMAGINE study subjects completed a fasting single-sample BT for CH₄ and H₂S. During esophagogastroduodenoscopy without colon preparation, duodenal aspirates were obtained using double-lumen sterile aspiration catheters. Microbial DNAs underwent shotgun sequencing (NovaSeq6000).

Results: Duodenal bacterial profiles differed significantly in subjects with breath H₂S ≥ 1.5 ppm vs. those with < 1.5 ppm, with 2.08-log₂fold greater prevalence of phylum Thermodesulfobacteriota. Higher breath H₂S levels correlated with greater duodenal prevalences of H₂S producers, including Proteus mirabilis (P = 0.002), Desulfosarcina widdelii (P = 0.027), and Desulfobulbus oligotrophicus (P = 0.041); co-occurrence of all 3 species correlated with ~ 50% higher breath H₂S levels (P = 0.0001). Duodenal archaeal profiles differed significantly in subjects with intestinal methanogen overgrowth (IMO, CH₄ ≥ 10 ppm), with 2.94-log₂fold greater prevalence of family Methanobacteriaceae vs. non-IMO subjects. Higher breath CH₄ correlated with greater prevalences of methanogens including M. smithii (P = 0.02), Halarchaeum sp. CBA1220 (P = 0.003), Desulfurococcus mucosus (P = 0.046), and Halobaculum rubrum (P = 0.049). IMO was more common in subjects with co-occurrence of all 4 species (P = 0.04). In IMO-positive subjects, CH₄ levels correlated with greater constipation severity (P = 0.019); P. mirabilis (P = 0.021) and D. oligotrophicus (P = 0.003) correlated with looser stool in IMO-negative subjects. M. smithii prevalence correlated with known hydrogen-producing syntrophs, e.g., Christensenella minuta (P < 0.001).

Conclusion: This study demonstrates that duodenal prevalences of H₂S-producing bacteria and methanogenic archaea contribute to H₂S and CH₄ levels, respectively, on BT.

Keywords: Methanobrevibacter smithii; Proteus mirabilis; Breath testing; Duodenum; Hydrogen sulfide; Methane; Microbiome.