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The gut microbiota has been implicated in onset and progression of ulcerative colitis (UC). Here, we assess potential causal involvement of the microbiota and -associated fecal water (FW) metabolome in altering key functional parameters of the colonic epithelium. Fecal samples were collected from N = 51 healthy controls (HC), N = 36 patients with active UC (UC-A), and N = 41 subjects in remission N = 41 (UC-R). Using in vitro incubation experiments, the FW metabolome’s impact on butyrate oxidation rates/gene expression and cell death (cytotoxicity) of HT-29 cells, cytokine production by PBMC, and barrier integrity of Caco2 monolayers was evaluated. The FW metabolome from patients and individuals hosting the Bacteroides 2 (Bact2) enterotype (69% of UC-A, 31% of UC-R, 3% of HC), characterized by lower levels of median- and short-chain fatty acids and furan compounds, left butyrate oxidation rates unaltered but affected associated gene expression profiles. UC patients/Bact2-carriers’ FW lowered PBMC IL-8 production and increased IL-1β production. Patients’ FW increased cytotoxicity, associated with sulfide compound levels. Bact2 carriers’ FW, displaying higher levels of bile acids, lowered barrier function upon incubation of monolayers. The FW metabolome of patients and individuals hosting a dysbiotic microbiota could contribute to the disruption of functional processes of the colonic epithelium as observed in UC.

ABSTRACT Advances in gut microbiota research have triggered interest in developing colon butyrate producers as niche-specific next-generation probiotics, targeted at increasing colon butyrate production and countering disease-associated microbiota alterations. Crucial steps in the development of next-generation probiotics are the design of formulations with a reasonable shelf life as well as the safety demonstration of an intervention in healthy volunteers. One such potential next-generation butyrate-producing probiotic is Butyricicoccus pullicaecorum 25-3T, with demonstrated safety in in vitro as well as animal models. Here, we examined the strain’s safety, tolerability, and impact on microbiota composition and metabolic activity in healthy volunteers in a randomized, double-blind, placebo-controlled crossover study in 30 healthy volunteers. The study design consisted of two 4-week intervention periods (108 CFU B. pullicaecorum [treatment] or maltodextrin [placebo] per day) with a 3-week washout in between. We assessed adverse events, blood parameters (primary endpoints), and fecal microbiota composition and metabolite profiles (secondary endpoints). The number of reported adverse events during the B. pullicaecorum treatment was similar to that of placebo intervention, as were observed changes in blood chemistry parameters, bowel habits, and fecal calprotectin concentrations. Administration of the strain did not induce any disruptive effect in microbiota composition or metabolic activity. In this first human intervention trial with a butyrate-producing Clostridium cluster IV isolate, we demonstrated B. pullicaecorum 25-3T administration to be both safe and well tolerated by healthy participants. This safety study paves the way for the further development of the strain as a next-generation probiotic. IMPORTANCE This study is the first to determine the safety and tolerance in humans of a butyrate-producing Clostridium cluster IV next-generation probiotic. Advances in gut microbiota research have triggered interest in developing colon butyrate producers as next-generation probiotics. Butyricicoccus pullicaecorum 25-3T is one such potential probiotic, with demonstrated safety in vitro as well as in animal models. Here, we produced an encapsulated B. pullicaecorum formulation that largely preserved its viability over an 8-month storage period at 4°C. Administration of this formulation to healthy volunteers allowed us to establish the intervention as safe and well tolerated. The probiotic intervention did not cause disruptive alterations in the composition or metabolic activity of health-associated microbiota. The results presented pave the way for the exploration of the impact of the strain on microbiota alterations in a clinical setting.