Fecal microbiota transplantation (FMT) describes a therapeutic approach in which feces from healthy donors are delivered into the guts of recipient patients through either the upper or lower gastrointestinal tract. FMT is now a standard therapeutic option for patients with recurrent Clostridium difficile infection (rCDI), but FMT has also been applied to several other gut diseases, not least inflammatory bowel disease (IBD), especially ulcerative colitis (UC). Several clinical trials have now shown that FMT is an efficacious and safe treatment not only for patients with mild-to-moderate active UC but also those in clinical remission.
However, FMT has yet to gain widespread acceptance from physicians and patients as a standard UC treatment. One major concern has been that the underlying mechanism by which FMT modifies the clinical course of UC remains unclear, and as yet there is no standardized protocol for donor selection or feces processing. Most studies in the field have used crude rules for donor screening and various feces preparation protocols that mainly consider safety but not therapeutic efficacy. The "optimal formula" for selecting FMT donors and preparing donor feces for UC treatment remains unknown. Here we summarize the current data on donor selection for FMT treatment in UC, explore areas of uncertainty related to donor feces preparation, and discuss the difficulties in formalizing the rules for donor selection and donor feces preparation.
Clues About Donor Selection: Which Donor Fecal Microbiota Parameters Matter?
The "super donor" phenomenon: In one of the earliest randomized controlled trials (RCTs) in this field, 39% of patients receiving fecal transplantation from one specific donor achieved clinical remission compared with only 10% from the other five donors. This was termed the "super donor" phenomenon. Since then, there have been multiple reports of the phenomenon that feces from certain donors is more efficacious than others in UC and also in other diseases such as obesity and metabolic syndrome. Although the specific underlying mechanisms remain unclear, the existence of "super donors" strongly suggests that donor factors play an essential role in FMT efficacy, providing the rationale for detailed studies on the important characteristics of donor feces.
Alpha diversity and absolute richness:Among numerous donor factors that might affect FMT efficacy, the alpha diversity and absolute microbial richness of the donor fecal microbiota probably have the most supporting evidence. Vermeire et al. first observed that donors donating to UC or Crohn's disease (CD) patients who responded to FMT treatment had significantly higher bacterial richness than those related to non-responders. Kump et al. and Paramsothy et al.also reported significantly higher bacterial richness and diversity in the feces of donors associated with better recipient outcomes. A metagenomic analysis of data from 14 FMT trials found that donor alpha diversity was the strongest positive predictor of donor strain engraftment. Not all studies have confirmed this association; for instance, Nishida et al. did not detect an association between donor feces alpha diversity and FMT outcomes in their trial of 41 patients with UC refractory to standard medical therapy treated with single FMT.
Butyrate-producing bacteria and related metabolic pathways: Compared with healthy subjects, IBD patients not only show different microbial characteristics but also altered metabolic profiles. One metabolic signature that appears to be characteristic of UC patient microbiota is a reduction in short-chain fatty acid (SCFA) production, especially butyrate. In terms of FMT efficacy, fatty acid biosynthesis pathway abundance has been reported to be higher in effective donor feces compared with ineffective feces. SCFA levels were higher after FMT in rCDI and UC patients, especially in those who responded to FMT. Thus, it is reasonable to hypothesize that the overall butyrate-producing function of donor fecal microbiota might influence FMT efficacy. However, a recent meta-analysis reported that levels of gut metabolic modules (GMMs) related to SCFAs in donors were not a marker of increased strain colonization in FMT recipients.
Specific microorganisms: Several specific microorganisms have also been reported as possible FMT-response factors in UC, although the data are inconsistent. For example, higher proportions of Lachnospiraceae, Ruminococcus,Bifidobacterium, Actinobacteria, unclassified Ruminococcaceae, an unclassified Ruminococcus sp., Akkermansia muciniphila,Bacteroides fragilis, and Bacteroides finegoldii have all been reported to be associated with better clinical responses in UC patients. However, none has yet been validated in an independent clinical cohort outside the reporting clinical trial. Conversely, Moayyedi et aland Paramsothy et alreported a negative impact for donor Streptococcus on FMT efficacy in UC patients. Similarly, Proteobacteria, Ruminococcus gnavus,Escherichia,Lactobacillales, Clostridium cluster IV and cluster XI,Bacteroides uniformis, Bacteroides coprocola, Clostridium XIVa, and S. wadsworthensisin donor fecal samples have all been associated with FMT failure in individual studies. Clearly more work is required to validate these potential microbial biomarkers of FMT response.
Other factors: Data from UC patients, especially those comparing FMT responders and non-responders, have indicated that several other factors might also affect FMT efficacy. For example, some metabolic pathways have been reported to be positively or negatively related to FMT efficacy. One study considered immunologic compatibility between donors and recipients as a potential factor influencing engraftment efficacy, but this was not confirmed in a subsequent clinical trial. A recent study analyzed microbiota datasets from 14 public CDI and IBD studies and found that differences in microbial profiles between donors and recipients were significantly associated with FMT outcomes. This finding might partially explain why clinical trials using healthy relatives of patients as FMT donors showed lower efficacy than those using unrelated donors.
Controversies in Donor Material Preparation Protocols: What is the Best Way to Process Feces Samples?
Despite intense clinical research activity in FMT treatment for UC patients, there is still no agreed standard procedure for donor feces preparation. Interestingly, even seemingly conflicting protocols can result in similar clinical outcomes.
Washed microbiota transplantation (WMT) vs. fecal filtrate transplantation (FFT): Zhang et al. developed the washed microbiota transplantation (WMT) protocol, a technique that preserves intact bacteria and washes off small molecules, and applied it in both CD and UC clinical trials. Compared with traditional FMT with a manual feces processing step, the WMT technique resulted in similar efficacy but improved safety. On the contrary, some groups chose another technique named fecal filtrate transplantation (FFT), which only retains bacterial debris, proteins, metabolites, and oligonucleotides but not intact bacteria. FFT has been successfully piloted in rCDI and necrotizing enterocolitis. A study of FFT in UC patients is also ongoing (ClinicalTrials.gov: NCT03843385). Therefore, these two entirely different FMT methods have both been successful, making it difficult to conclude which fecal component is most important mechanistically. One study compared the efficacy of fecal supernatant and fecal sediment in rCDI, and the former was more effective. One potential explanation is that products like metabolites and enzymes in the filtrate are sufficient to induce the therapeutic effect of FMT but, when small molecules are washed off, intact bacteria can still function by re-synthesizing these active constituents in the gastrointestinal tracts of recipients.
Frozen vs. fresh feces: Fecal samples can be stored at -80°C for up to two years. It remains uncertain whether the efficacy of FMT using frozen or fresh feces differs. Some studies have shown that freezing may increase the Firmicutes/Bacteroidetes ratio in feces, which might be beneficial for UC patients. In some clinical trials, frozen feces seemed to have greater efficacy than fresh feces. In early RCTs investigating FMT efficacy in UC patients, most of the trials reporting positive results used frozen feces,[1,2] while the only negative trial used fresh feces. However, a meta-analysis of FMT treatment for UC did not detect a significant difference between frozen and fresh feces. On the contrary, a meta-analysis of FMT treatment for CD indicated that patients receiving fresh feces had a higher rate of clinical remission than those receiving frozen feces. Therefore, how freezing changes the microbial profiles of donor feces and whether the change benefits UC treatment remain unclear.
Why it is Difficult to Find an "Optimal Formula" for Donor Selection and Feces Processing?
Up to now, only a few groups have tried to establish definitive rules for FMT donor selection, with related pilot clinical trials in rCDI and hepatic encephalopathy showing some promise. While these studies encourage the design of standards for selecting FMT donors for UC treatment, some challenges remain.
First, it is still unclear how the gastrointestinal microbiota participates in UC pathogenesis and progression, with a lot of conflicting findings reported. A meta-analysis found that lower Bacteroides levels were related to higher UC or CD activity scores. However, some studies have indicated that some Bacteroidetes species might promote IBD development. IBD patients frequently exhibit an enterotype dominated by Bacteroides, and donors with this enterotype also tend to produce poor FMT results. These sometimes contradictory results remind us of the complexity of the gut microbiota and its interaction with the host.
Second, it is important to remember that FMT does not simply describe the replacement of a deficiency in the patients' gastrointestinal tracts. We cannot directly infer which materials are suitable for transplantation solely from the differences observed between the microbial profiles of UC patients and healthy donors. For example, the abundances of Actinobacteria and Proteobacteria were increased in IBD patients, but Bifidobacterium, an Actinobacteria genus, was positively associated with FMT efficacy in UC patients.Clostridium clusters IV and XIVa contain butyrate-producing species, and these species may be beneficial factors since they tend to be more abundant in responders than non-responders.[8,15] However, they have also been reported to be significantly more abundant in donor feces associated with failed FMT.[5,7]
Finally, we still have little idea about the relative importance of different donor factors. A recent bioinformatics study demonstrated that factors related to recipients, not donors, are more predictive of FMT success. Zou et al. constructed a predictive model of gut microbiota composition in post‐FMT patients, in which no donor-related factor contributed to the predictive accuracy.
In conclusion, there remain difficulties in standardizing donor selection and feces preparation for FMT in UC patients. More clinical trials focusing on donor selection are needed to overcome these difficulties. Randomized clinical trials comparing different donor feces samples are of course essential, but bioinformatics analyses and animal experiments are also useful for supporting protocol development. Only by gaining further insights into the mechanisms governing the efficacy of this novel therapeutic method can we establish valid rules for donor selection and feces preparation.
This work is supported by the grants from the Beijing Municipal Natural Science Foundation (No. 7212078), the National key clinical specialty construction project (No. ZK108000), and the National High Level Hospital Clinical Research Funding (No. 2022-PUMCH-B-022).
Conflicts of interest
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