Abstract Background Fecal microbiota transplantation (FMT), which aims to transfer viable donor-derived gut microbiota, has demonstrated efficacy in ulcerative colitis (UC). However, manufacturing parameters and quality assessment for fecal microbiota suspensions (FMS) remain insufficiently standardized. This study quantified the impact of manufacturing, storage, and thawing conditions on viable bacterial counts and adenosine triphosphate (ATP) concentrations in FMS and explored their association with clinical efficacy of antibiotic FMT(A-FMT). Methods In FMS derived from healthy subjects, colony-forming units (CFU) and ATP were measured, and microbiota were analyzed by 16S rRNA amplicon sequencing with propidium monoazide (PMA) treatment to assess viable communities. Furthermore, a prospective multicenter clinical trial of A-FMT for active UC (jRCTs031220542) was conducted between January 2023 and August 2025. The protocol comprised a 2-week course of oral amoxicillin, fosfomycin, and metronidazole, followed by FMS administration with a single 200mL colonoscopic infusion and two additional 100mL enemas. In 23 patients who received FMS from the same donor, clinical efficacy was evaluated using the change in Modified Mayo Disease Activity Index (MMDAI) from baseline to 8 weeks. Results Both CFU and ATP tended to be lower in FMS stored at − 20 °C than in those stored at − 80 °C for 12 months. FMS were frozen at − 80 °C for 1 day and then stored at − 20 °C for 6weeks of storage, PMA-treated samples, representing viable bacteria only, formed clusters that were positioned close to the corresponding non–PMA-treated, representing all bacteria including dead bacteria, clusters on PCoA (weighted UniFrac distances). In contrast, FMS were stored at − 20 °C for 12 weeks, PMA-treated samples formed clusters that were clearly separated from the corresponding non–PMA-treated clusters in all subjects, suggesting that FMS stored at − 20 °C for 12 weeks induces compositional changes in the viable microbiota. A subset of bacterial taxa commonly affected by freezing and temperature-shift conditions was identified. In the prospective clinical cohort, FMS stored for ≥180 days and thawed at 37 °C for 90 min had significantly higher ATP than those stored for 180 days and thawed for 30 min (p 0.001). Under these conditions, ATP concentrations in FMS were positively correlated with ΔMMDAI (R² = 0.47, p 0.05), suggesting that extended thawing may enhance bacterial activity in long-stored preparations and improve clinical outcomes. Conclusion Freezing, storage, and thawing conditions of FMS influence viable bacterial counts and ATP levels and may affect therapeutic efficacy of A-FMT in UC, supporting development of standardized quality control criteria for FMS used in FMT for UC. References: 1. Ishikawa D, Sasaki T, Osada T, et al. Changes in intestinal microbiota following combination therapy with fecal microbial transplantation and antibiotics for ulcerative colitis. Inflamm Bowel Dis. 2017;23(1):116-125. doi:10.1097/MIB.0000000000000975 2. Ishikawa D, Watanabe H, Nomura K, et al. Patient–donor similarity and donor-derived species contribute to the outcome of fecal microbiota transplantation for ulcerative colitis. J Crohns Colitis. 2025;19(4):jjaf054. doi:10.1093/ecco-jcc/jjaf054 3. Cammarota G, Ianiro G, Kelly CR, et al. International consensus conference on stool banking for faecal microbiota transplantation in clinical practice. Gut. 2019;68(12):2111-2121. doi:10.1136/gutjnl-2019-319548 4. Bottino P, Vay D, Leli C, et al. Evaluation of bacterial viability for fecal microbiota transplantation: impact of thawing temperature and storage time. Microorganisms. 2024;12(7):1294. doi:10.3390/microorganisms12071294 Conflict of interest: Dr. Koma, Masao: No conflict of interest Katsumata, Ryo: No conflict of interest Nomura, Kei: No conflict of interest Mizutani, Sayaka: No conflict of interest Nakato, Gaku: No conflict of interest Kogure, Shota: No conflict of interest Odakura, Rina: No conflict of interest Omori, Masashi: No conflict of interest Maruyama, Takafumi: No conflict of interest Zhang, Xiaochen: No conflict of interest Yamada, Takuji: No conflict of interest Fukuda, Shinji: No conflict of interest Shibuya, Tomoyoshi: No conflict of interest Nagahara, Akihito: No conflict of interest Ishikawa, Dai: Dr. Ishikawa is a co-founder of Metagen Therapeutics and has received salary support as well as research funding from the company.
Koma et al. (Thu,) studied this question.