Our study addressed the microbiological mechanisms underlying the FMT treatment for rCDI. We showed, for the first time, that FMT has long-term effects on the microbiota and offers a means to modify it relatively permanently. The rapid changes induced by FMT explain the prompt and high clinical efficacy – it drastically altered the patients’ intestinal microbiota by restoring the anaerobic community. Patients’ faecal microbiota prior to FMT was dominated with facultative anaerobic bacteria such as Bacilli and Proteobacteria, which are known for their proinflammatory properties [37]. Post-FMT, their microbiota composition resembled that of the donors as early as 3 days following transplantation, containing bacteria typical for a healthy microbiota such as strict anaerobes from the Clostridium clusters IV and XIVa. These observed changes confirmed previous findings [14, 38] and, importantly, we were able to show that these modifications persisted long-term. We also addressed the effects induced by FMT on the rectal mucosa, which have not been studied previously. Furthermore, our universal donor approach allowed the identification of commonly acquired bacterial taxa, potentially underlying the treatment efficacy.

Antibiotics suppress anaerobic commensals and induce profound changes to microbiota, resulting in loss of colonisation resistance [39, 40]. We observed a similar effect in patient P3, who mistakenly took vancomycin after the first FMT. The transplanted microbiota was unable to engraft and there was no change in the microbial composition before the second FMT treatment. We also showed that the patients’ microbiota composition pre-FMT represents the effects of multiple antibiotic treatments, including low diversity and depletion of anaerobes. The FMT-treatment restored these levels very rapidly.

The novel mucosal microbiota findings showed that similar to the faecal microbiota, FMT restored the anaerobic bacterial community due to the increase of Bacteroidetes. The faecal and mucosal tissues are distinct communities and have specific microbial compositions [41, 42]. Therefore, it was not surprising that a sub-population of the transplanted microbiota was selected to the mucosal compartment. Further, our in vitro experiment showed that the epithelium-adherent fraction of faecal microbiota was enriched in Bacteroidetes. This group is abundant in the healthy intestinal mucosa and is known to enforce epithelial integrity [43] and maintains immunological homeostasis [44, 45]. Thus, it can be hypothesised that the increase of Bacteroidetes in the mucosa was part of the efficacy of the FMT treatment.

One of the main findings of this study was the high similarity in the microbiota profiles between patients and their own donors that lasted throughout the 1-year follow-up. This was not altered even by antimicrobial treatments taken by some patients during the follow-up period. The microbial stability was effected by the antibiotics, but it recovered to its original composition, in line with recent observations with healthy subjects [40]. Regardless of the antibiotics, we were able to identify specific donor-derived bacterial signatures, which persisted throughout the follow-up. This surprisingly high similarity between the donor-patient pair led us to speculate that there is no major selection pressure from the host to alter the transplanted microbial composition. The hypothesis could be that the transplant provides a functional microbial ecosystem, which outweighs the individual-based bacterial selection.

Previously, three FMT-trials have addressed the engraftment of donors’ microbiota in patients, with shorter 4- to 6-month follow-up periods and less detailed microbial analysis [17, 38]. Our comprehensive investigation extends the previous preliminary observations on the establishment of donors’ microbiota post-FMT; both the high patient–donor similarity and the donor-specific bacterial signatures in patients indicate a long-term establishment of the donors’ microbiota. This is in line with a recent metagenomics study that revealed colonisation of donor bacteria at strain level persisting for 3 months after FMT treatment [46]. Since one of the characteristics of a healthy microbiota is its resilience to change [35], it was unexpected that the donors’ microbiota was so strongly established and maintained. Our hypothesis is that the depletion of the microbiota with broad-spectrum antibiotics and bowel cleansing creates an open ecological niche for the transplanted microbiota. This novel finding on the long-term stability is promising when considering other indications where changing the intestinal microbiota composition could be used as a potential treatment.

One of our main aims was to determine a group of bacteria necessary for the resolution rCDI. This was addressed by the universal study setup, where faecal preparations from three donors were used to treat several patients, allowing better evaluation of the commonly acquired bacteria, which were transferred to all patients. We identified 24 bacterial taxa that were absent in patients before the treatment and present afterwards. Thus, it would be plausible to hypothesise that such a specific subpopulation within the complex faecal microbiota could underlie the treatment efficacy of FMT for rCDI. This commonly acquired core identified in our study was taxonomically diverse and included bacterial genera from four major phyla. The therapeutic core determined in our study showed considerable overlap with health-associated microbial cores determined in other studies [47], highlighting its potential in restoring health.

The impact of these 24 taxa to intestinal health potentially lies in their ecological functions and nutrient utilisation networks as well as immunomodulatory capacity. One of these genera, Bacteroides spp. has been previously found to increase significantly after FMT for rCDI and to have a key role in restoring the intestinal ecosystem [14]. Our findings on the increase of Bacteroides spp. in the mucosa also underline their importance in maintaining intestinal homeostasis. There is evidence that the human commensal B. fragilis fortifies epithelial integrity [43] and, more recently, the bacterium was shown to interact with intestinal mucosa to suppress inflammation [48]. Furthermore, mice studies have shown that the Bacteroidetes taxa are required in successful colonisation of a health-associated Faecalibacterium prauznitzii [49].

The majority (22/24) of the commonly transplanted bacterial taxa belonged to three Clostridium clusters (Firmicutes). The Clostridium taxa of the therapeutic core have been shown to play key roles in the nutrient utilisation networks and, therefore, can be considered to be essential for the general restoration of the complex ecosystem [50–52]. For example, the therapeutic core bacteria Eubacterium, Coprococcus, Anaerostipes and Ruminococcus spp. are known to participate in bacterial cross-feeding pathways that are responsible for the production of short-chain fatty acids (SCFA) – the major microbial metabolites from carbohydrate fermentation [50]. Concurrently, with the appearance of the therapeutic core taxa, we also observed a more than 20-fold increase in Ruminococcus obeum and Subdoligranulum variable, both of which are major SCFA-producing bacteria in the gut [50]. SCFAs promote intestinal homeostasis by both strengthening epithelial cell layer integrity and stimulating regulatory T cells [53]. Recently, Atarashi et al. [54] treated inflammatory colitis in a mouse model with a combination of 17 clostridial strains, which affected SCFA and regulatory T cell levels.

In summary, the therapeutic core seems to consist of intestinal bacteria that are able to regenerate key interaction-networks within the microbiota and consequently restore the complex intestinal ecosystem that carries out essential functions for the host and provides colonisation resistance against pathogens, especially C. difficile. Therefore, isolation and characterisation of these commensal bacteria would be of high importance when developing microbiota-based therapies for rCDI. We consider that there are multiple alternatives to combine intestinal bacterial strains as an effective bacteriotherapy mixture.