Viable gut bacterial metrics associated with intestinal eubiosis and dysbiosis

Gut microbial turnover in humans remains poorly understood. We evaluated gut bacterial viability by quantifying viable cells and relating them to indicators of gut environmental status. Fecal microbial loads (microbial cells per gram feces) were quantified by flow cytometry using the DNA-intercalating dye SytoBC, and viable bacteria were identified by fluorescence in situ hybridization with the 16S rRNA-targeted Eub338 probe. Bacteria retaining sufficient rRNA were defined as viable, and the living bacterial rate was calculated as the proportion of Eub338-positive cells among SytoBC-positive cells. These metrics were applied to starvation‑cultured gut bacterial strains and fecal samples from patients with ulcerative colitis (UC), older adults, and healthy individuals. Starvation culture showed time‑dependent decreases in living bacterial rate and viable cell counts, consistent with colony-forming unit measurements. Patients with active UC exhibited reduced fecal microbial loads and viable microbial loads. Living bacterial rates negatively correlated with fecal calprotectin and Mayo score. In older adults, reduced living bacterial rates and viable Bifidobacteriaceae were associated with higher fecal calprotectin. In healthy individuals, lower living bacterial rates and viable Lachnospiraceae were associated with harder stools and reduced defecation frequency. Quantitative profiling of viable fecal bacteria may aid in characterizing intestinal homeostasis and dysbiosis‑associated conditions.