Clostridium difficile infection (CDI) is a microbiota-related disease. Typically, antibiotic-induced perturbation of gut microbiota precedes the infection, while a healthy gut microbiota provides protection, i.e. colonization resistance, against it. Furthermore, in the case of recurrent CDI that is not resolved by antibiotics, restoring the gut microbiota with a fecal microbiota transplantation (FMT) is among the few treatment options that really work. Although FMT is effective in the treatment of CDI, the factors behind treatment success remain unclear. Both, the key species and the functions that are necessary to restore the healthy microbiota and eradicate C. difficile, are a matter of speculation. This study was based on the hypothesis that the adherence of some commensal bacteria to the gut epithelial cells could play a role in eradicating C. difficile by competing for epithelial binding with it. Furthermore, the isolation of those bacteria from the donor feces would enable more detailed mechanistic studies and development of a bacterial product for the treatment of CDI in the future. As a pre-selection step, bacterial adhesion to Caco-2 cells was utilized to isolate and cultivate epithelium-adherent bacteria from the donor feces. Microbiota composition of fecal sample, and the adhered and cultured sub-populations thereof, was determined by partial 16S rDNA amplicon sequencing using MiSeq method. The pre-selection approach was successful, since the obtained populations were different, both after the adhesion and cultivation, as compared to the original fecal sample. In addition, most obtained pure isolates adhered well to enterocytes. The ability of fecal bacteria to compete with C. difficile for binding to gut epithelial cells in vitro was also studied. Isolated bacteria from Caco-2-adhered populations were applied in competition and exclusion assays with C. difficile as purified or multi-species cultures, and reduction in C. difficile binding was observed due to the certain bacteria or bacterial populations. These assays still need developing and the results must be confirmed with more repetitions. However, the results are promising and a useful ground for future work in developing bacteriotherapeutic formulations for the treatment of CDI.

Abstract Background Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (rCDI). It restores the disrupted intestinal microbiota and subsequently suppresses C. difficile. The long-term stability of the intestinal microbiota and the recovery of mucosal microbiota, both of which have not been previously studied, are assessed herein. Further, the specific bacteria behind the treatment efficacy are also investigated. Methods We performed a high-throughput microbiota profiling using a phylogenetic microarray analysis of 131 faecal and mucosal samples from 14 rCDI patients pre- and post-FMT during a 1-year follow-up and 23 samples from the three universal donors over the same period. Results The FMT treatment was successful in all patients. FMT reverted the patients’ bacterial community to become dominated by Clostridium clusters IV and XIVa, the major anaerobic bacterial groups of the healthy gut. In the mucosa, the amount of facultative anaerobes decreased, whereas Bacteroidetes increased. Post-FMT, the patients’ microbiota profiles were more similar to their own donors than what is generally observed for unrelated subjects and this striking similarity was retained throughout the 1-year follow-up. Furthermore, the universal donor approach allowed us to identify bacteria commonly established in all CDI patients and revealed a commonly acquired core microbiota consisting of 24 bacterial taxa. Conclusions FMT induces profound microbiota changes, therefore explaining the high clinical efficacy for rCDI. The identification of commonly acquired bacteria could lead to effective bacteriotherapeutic formulations. FMT can affect microbiota in the long-term and offers a means to modify it relatively permanently for the treatment of microbiota-associated diseases.