Different bile acids have versatile effects on sporulation, toxin levels and biofilm formation of different Clostridioides difficile strains

Clostridioides difficile infection develops following ingestion of virulent stains by a susceptible host. Once germinated, toxins TcdA and TcdB, and in some of the strains binary toxin, are secreted, eliciting disease. Bile acids play a significant role in the process of spore germination and outgrowth, with cholate and its derivative enhancing colony formation, while chenodeoxycholate inhibit germination and outgrowth. This work investigated bile acids' impact on spore germination, toxin levels and biofilm formation in various strain types (STs). Thirty C. difficile isolates (A⁺ B⁺ CDT^-\+) of different STs were exposed to increasing concentrations of the bile acids, cholic acid (CA), taurocholic acid (TCA) and chenodeoxycholic acid (CDCA). Following treatments, spore germination was determined. Toxin concentrations were semi-quantified using the C. Diff Tox A/B II™ kit. Biofilm formation was detected by the microplate assay with crystal violet. SYTO® 9 and propidium iodide staining were used for live and dead cell detection, respectively, inside the biofilm. Toxins levels were increased by 1.5–28-fold in response to CA and by 1.5–20-fold in response to TCA, and decreased by 1–37-fold due to CDCA exposure. CA had a concentration-dependent effect on biofilm formation, with the low concentration (0.1%) inducing- and the higher concentrations inhibiting biofilm formation, while CDCA significantly reduced biofilm production at all concentrations. There were no differences in the bile acids effects on different STs. Further investigation might identify a specific bile acids' combination with inhibitory effects on C. difficile toxin and biofilm production, which could modulate toxin formation to reduce the likelihood of developing CDI.