The ethics committee at Keio University School of Medicine approved the protocol (#20130383), and all participants provided written informed consent. The study was registered at the University hospital Medical Information Network (UMIN) Center (UMIN 000012814).

In this single-center, open-label, non-randomized study, the safety and efficacy of FMT was evaluated in patients with moderate-to-severe active UC. Clinical follow-up was performed 12 weeks post-FMT. The primary endpoint of the study was at week 12.

As we previously reported,²¹ the donors were instructed to collect a fecal sample in an AneroPack™ (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) and bring the pack to the hospital at 4℃ on the day of the scheduled FMT. Approximately 50 to 300 g of feces was collected from donors, dissolved in 50 to 100 mL of saline, and filtered through a metal strainer to make a liquid slurry. Fecal materials were administered to the patient within 6 hours after collection by the donor via colonoscopy following standard bowel preparation (2 L polyethylene glycol solution).

The primary endpoint of the study was the presence or absence of serious adverse events related to FMT. The secondary endpoint was a change in the partial Mayo (pMayo) score 12 weeks after FMT. Scores ≤2 were considered a clinical response

Fecal samples were longitudinally collected from patients at weeks 0, 1, 2, 4, 8, and 12 post-FMT and from donors on the day of the FMT. In total, 58 fecal samples were collected from 10 patients, and the collected fresh feces were stored under anaerobic conditions in an AneroPack™ (Mitsubishi Gas Chemical Co., Inc.) at 4℃. Within 24 hours after sampling, the feces were frozen in 20% glycerol (Wako Pure Chemical Industries Ltd., Osaka, Japan)/phosphate-buffered saline solution (Life Technologies, Tokyo, Japan) by liquid nitrogen and stored at −80℃ until use.

Bacterial DNA were isolated as described previously.¹⁹ Briefly, bacterial DNA was isolated by the enzymatic lysis method using lysozyme (Sigma-Aldrich Co., LCC., Tokyo, Japan) and achromopeptidase (Wako Pure Chemical Industries Ltd.). DNA samples were then purified by treatment with ribonuclease A (Wako Pure Chemical Industries Ltd.), followed by precipitation with 20% polyethylene glycol solution (PEG6000 in 2.5 M sodium chloride). The DNA was then pelleted by centrifugation, rinsed with 75% ethanol, and dissolved in tris-EDTA buffer.

The fecal DNA samples were sequenced using the 454 GS FLX Titanium and FLX₊ (Roche, Basel, Switzerland) sequencing system. The detailed protocols were described previously.¹⁹ Briefly, the 16S rRNA gene V1 to V2 region was amplified by PCR by using the primers 27Fmod and 338R containing 454 primer sequences A and B and a unique 10-bp barcode sequence in 27Fmod. The PCR amplicons were sequenced to obtain reads, and the reads with an average quality value <25, mismatches to both universal primers, and possible chimeric reads were removed. Among the high-quality reads, 3,000 reads per sample were randomly selected and grouped into operational taxonomic units (OTUs) by clustering using the UCLUST algorithm with a 96% identity threshold. Taxonomic assignments for each OTU were made by similarity search against the public 16S and National Center for Biotechnology Information (NCBI) genome databases using the GLSEARCH program. For assignment at the phylum, family, genus, and species levels, sequence similarity thresholds of 70%, 90%, 94%, and 96%, respectively, were employed. All of the high-quality 16S V1 to V2 sequences analyzed in this study were deposited into the DNA Data-Bank of Japan (DDBJ)/GenBank/ European Molecular Biology Laboratory (EMBL) database under accession number DRA004886. UniFrac distance and principal coordinate analysis were used to assess the similarity of the microbiota structure of each pair of samples.²²

Results are expressed as mean±SEM. Groups of data were compared using Student t-test. For multiple comparisons, the statistical analysis was performed using the Kruskal-Wallis one-way ANOVA test and the Tukey-Kramer test. Differences were considered statistically significant when the P-value was <0.05. All of the analyses were conducted using GraphPad Prism Software version 6 (GraphPad Software, San Diego, CA, USA).

We recruited 10 patients from March 2014 through October 2015, including three with mild disease and seven with moderate disease. Patients' baseline and clinical characteristics are shown in Table 2. All patients had a history of immunosuppressive treatment, while six were naïve to biologics treatment.

Screening of 12 healthy subjects for stool pathogens and serology resulted in 10 eligible donors; two were excluded because of positive screening results for infectious agents in their feces. Donors comprised two spouses, four parents, and four sisters. All donors donated feces that were used for patient infusion. The donors' mean age was 45 years (range, 31-66 years), of which four were male.

Previous studies reported serious adverse events following FMT, including small bowel perforation, cytomegalovirus infection, and carcinoma. In our study, two patients required additional therapy during the 12 weeks following FMT; however, no severe FMT-associated adverse event was observed except for exacerbation of the UC itself. Six patients showed exacerbation of colitis and three showed amelioration of colitis. Only one patient (“J” in Fig. 1A) showed a clinical response (pMayo score pre-FMT, 4; post-FMT, 1). Two other patients (“D” and “E” in Fig. 1A) did not satisfy the definition of clinical response. Overall, no significant difference was found between the pre-FMT and post-FMT pMayo scores (Fig. 1). These results suggested that our single FMT protocol was safe; however, it had limited effectiveness for active UC.

Intestinal microbial profiling was conducted by extracting genomic DNA from the patient and donor fecal samples using the protocol described above. Fifty-nine samples were collected from the 10 patients and 10 samples were collected from the 10 donors.

Comparison of the donor and patient samples at baseline revealed that the diversity index of the fecal microbiota in the healthy donors showed higher diversity than that of the patients, although there was no significant difference (P=0.11). The diversity of the microbiota in the patients' samples increased slightly at the 12-week post-FMT evaluation; however, the findings were not significant (P=0.29) (Fig. 2A).

The taxonomic profiles showed that the phylum Firmicutes was dominant in almost all of the donors and patients with no significant difference among them. The taxonomic profiles of the patient samples showed no significant change pre-FMT versus post-FMT (Fig. 2B, Supplementary Fig. 1). Interestingly, the abundance of Bifidobacterium longum was restored in patients with active UC following FMT; however, there was no significant difference pre-FMT versus post-FMT (P=0.08) (Fig. 2C, Supplementary Fig. 2). Redundancy analysis showed that the microbiota composition of donors overlapped with that of patients at baseline and at 12 weeks post-FMT (Supplementary Fig. 3). These results indicate that the single FMT via colonoscopy for patients with UC was conducted safe; however, it had limited clinical effectiveness and potential to change the intestinal microbiota.