Hypothesis

In individuals aged >70 years, luminal hydrogen sulfide (H2S) produced by sulfate-reducing bacteria (SRB) accumulates to concentrations that directly inhibit the butyrate synthesis enzymes butyrate kinase (buk) and butyryl-CoA dehydrogenase (but) in Firmicutes, particularly butyrate‑producing taxa such as Roseburia and Faecalibacterium. This enzymatic suppression reduces colonic butyrate despite stable or even increased Firmicutes abundance, leading to a functional loss of butyrate producers. Because Bacteroidetes are relatively resistant to H2S-mediated inhibition of their metabolic pathways, their relative abundance rises, causing the observed decline in the Firmicutes/Bacteroidetes (F/B) ratio after the seventh decade. The hypothesis predicts that (1) fecal H2S levels will positively correlate with the inverse F/B ratio in centenarians, (2) metatranscriptomic buk/but expression will be downregulated in high-H2S samples irrespective of Firmicutes 16S abundance, and (3) pharmacological scavenging of luminal H2S (e.g., bismuth subsalicylate) in aged mice will restore buk/but transcription, elevate colonic butyrate, and prevent the age-associated F/B ratio reversal.

Predictions

• Positive correlation: Higher fecal H2S (>= µmol/g) predicts lower F/B ratio (Spearman rho < -0.4, p < 0.01) in human cohorts aged 70-90.
• Enzyme suppression: In samples with high H2S, buk/but transcript counts (TPM) will be reduced by >= 50 % compared with low-H2S matched samples, even after adjusting for Firmicutes 16S copy number.
• Intervention rescue: Oral bismuth subsalicylate (200 mg/kg/day) administered to 24-month-old mice for 8 weeks will decrease fecal H2S by >= 60 %, increase colonic butyrate concentration by >= 30 %, and maintain the F/B ratio at youthful levels (>= 1.2) relative to vehicle-treated controls.

Experimental Design

1. Human observational cohort – Collect stool, blood, and dietary records from 150 participants stratified by age (60-69, 70-79, 80+). Measure fecal H2S (gas chromatography), perform shotgun metagenomics for taxa abundance, and metatranscriptomics for buk/but. Model F/B ratio as outcome with H2S, age, sex, and fiber intake as covariates.
2. Mouse intervention – Use C57BL/6J mice aged 24 months. Randomize to vehicle or bismuth subsalicylate (oral gavage) for 8 weeks. At endpoint, quantify luminal H2S (lead acetate assay), colonic butyrate (GC-MS), F/B ratio (16S qPCR), and barrier integrity (FITC-dextran permeability).
3. Mechanistic validation – Incubate isolated Faecalibacterium prausnitzii cultures with physiologic H2S concentrations (0-5 mM) and quantify buk/but expression via RT-qPCR; test whether addition of the H2S scavenger zinc acetate restores expression.

Potential Confounds and Controls

• Dietary sulfur intake (e.g., cruciferous vegetables, meat) will be recorded and included as a covariate.
• Antibiotic or PPI use in the last 3 months will be exclusion criteria.
• Sex-specific analyses will be performed because prior work shows males exhibit more severe mucus thinning.

Falsifiability

If fecal H2S shows no correlation with F/B ratio, buk/but expression remains unchanged across H2S gradients, or H2S scavenging fails to restore butyrate producers and the F/B ratio in aged mice, the hypothesis would be refuted.