Browsing by Subject "Caco-2 cell line"

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.