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

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Evidence indicates that 3 gas breath testing (BT) correlates with stool microbial populations. Breath methane (CH 4 ) levels correlate with stool Methanobrevibacter smithii levels and constipation, while hydrogen sulfide (H 2 S) levels correlate with stool H 2 S producers and diarrhea. However, their relationships to small bowel microbes are unknown. To assess relationships between small bowel microbes and breath gases. REIMAGINE study subjects completed a fasting single-sample BT for CH 4 and H 2 S. During esophagogastroduodenoscopy without colon preparation, duodenal aspirates were obtained using double-lumen sterile aspiration catheters. Microbial DNAs underwent shotgun sequencing (NovaSeq6000). Duodenal bacterial profiles differed significantly in subjects with breath H 2 S ≥ 1.5 ppm vs. those with < 1.5 ppm, with 2.08-log 2 fold greater prevalence of phylum Thermodesulfobacteriota. Higher breath H 2 S levels correlated with greater duodenal prevalences of H 2 S 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 H 2 S levels ( P = 0.0001). Duodenal archaeal profiles differed significantly in subjects with intestinal methanogen overgrowth (IMO, CH 4 ≥ 10 ppm), with 2.94-log 2 fold greater prevalence of family Methanobacteriaceae vs. non-IMO subjects. Higher breath CH 4 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, CH 4 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). This study demonstrates that duodenal prevalences of H 2 S-producing bacteria and methanogenic archaea contribute to H 2 S and CH 4 levels, respectively, on BT.