Methanogens and Hydrogen Sulfide Producing Bacteria Guide Distinct Gut Microbe Profiles and Irritable Bowel Syndrome Subtypes

Abstract

Introduction: Irritable bowel syndrome (IBS) includes diarrhea-predominant (IBS-D) and constipation-predominant (IBS-C) subtypes. We combined breath testing and stool microbiome sequencing to identify potential microbial drivers of IBS subtypes.

Methods: IBS-C and IBS-D subjects from 2 randomized controlled trials (NCT03763175 and NCT04557215) were included. Baseline breath carbon dioxide, hydrogen (H 2 ), methane (CH 4 ), and hydrogen sulfide (H 2 S) levels were measured by gas chromatography, and baseline stool microbiome composition was analyzed by 16S rRNA sequencing. Microbial metabolic pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes collection databases.

Results: IBS-C subjects had higher breath CH 4 that correlated with higher gut microbial diversity and higher relative abundance (RA) of stool methanogens, predominantly Methanobrevibacter , as well as higher absolute abundance of Methanobrevibacter smithii in stool. IBS-D subjects had higher breath H 2 that correlated with lower microbial diversity and higher breath H 2 S that correlated with higher RA of H 2 S-producing bacteria, including Fusobacterium and Desulfovibrio spp. The predominant H 2 producers were different in these distinct microtypes, with higher RA of Ruminococcaceae and Christensenellaceae in IBS-C/CH 4 + (which correlated with Methanobacteriaceae RA) and higher Enterobacteriaceae RA in IBS-D. Finally, microbial metabolic pathway analysis revealed enrichment of Kyoto Encyclopedia of Genes and Genomes modules associated with methanogenesis and biosynthesis of methanogenesis cofactor F420 in IBS-C/CH 4 + subjects, whereas modules associated with H 2 S production, including sulfate reduction pathways, were enriched in IBS-D.

Discussion: Our findings identify distinct gut microtypes linked to breath gas patterns in IBS-C and IBS-D subjects, driven by methanogens such as M. smithii and H 2 S producers such as Fusobacterium and Desulfovibrio spp, respectively.

Figure 1.

Breath test results in IBS-D and IBS-C subjects. (a) AUC for breath H₂. (b) Rates of positivity for SIBO based on H₂. (c) AUC for H₂S. (d) AUC for CH₄. Data are shown as mean ± SD. Statistical analyses by the Mann-Whitney U test. AUC, area under the curve; IBS, irritable bowel syndrome; IBS-C, constipation-predominant; IBS-D, diarrhea-predominant; SIBO, small intestinal bacterial overgrowth.

Figure 2.

Associations between breath H₂, H₂S and CH₄ in pooled IBS-D and IBS-C/CH₄+ subjects. (a) Association between H₂ and H₂S AUCs. (b) Association between H₂ and CH₄ AUCs. AUC, area under the curve; IBS, irritable bowel syndrome; IBS-C, constipation-predominant; IBS-D, diarrhea-predominant.

Figure 3.

Stool microbial alpha diversity indices in IBS-D and IBS-C/CH₄+ subjects (a) Chao1, (b) Simpson index, and (c) Shannon index. (d) Associations between microbial alpha diversity indices and breath H₂, H₂S, and CH₄ (Spearman R). Blue gradient, positive correlations; red gradient, negative correlations. Colors indicate ranges of correlation coefficients; circle sizes denote coefficients within each range (Spearman R). ***P < 0.001, **P < 0.01, *P < 0.05. IBS, irritable bowel syndrome; IBS-C, constipation-predominant; IBS-D, diarrhea-predominant.

Figure 4.

(a) Top 25 bacterial families correlated with relative abundance of archaeal family Methanobacteriaceae (Spearman test). (b) Associations between microbial alpha diversity (Shannon and Simpson indices); breath H₂, CH₄, and H₂S AUC; and bacterial families associated with Methanobacteriaceae. Blue gradient, positive correlations; red gradient, negative correlations. Colors indicate ranges of correlation coefficients; circle sizes denote correlation coefficients within each range (Spearman R). ***P < 0.001, **P < 0.01, *P < 0.05. (c) Top 25 bacterial families correlated with H₂S-producing bacterial family Fusobacteriaceae (Spearman test). AUC, area under the curve.