The effect of different C. difficile MLST strains on viability and activity of macrophages.

Objectives: Clostridioides difficile is the most common infectious agent of nosocomial diarrhea. C. difficile infection (CDI) pathogenesis and disease severity depend on its toxins (toxins A, B and binary) and on the host's immune response, especially the innate immune system. The current study examined the efficacy of macrophage activity, macrophages viability and cytokine secretion levelsin response to different sequence type (ST) strains of C. difficile. Methods: RAW 264.7 macrophages were exposed to six different strains of C. difficile as well as to both toxins A and B and macrophage viability was measured. The levels of four secreted cytokines were determined by RT-PCR and ELISA. Morphological changes to the macrophages were investigated by fluorescent microscopy. Results: Strains ST37 and ST42 affected macrophages' vitality the most. Toxins A and B led to a significant reduction in macrophages' vitality at most time points. In addition, starting at 30-min post-exposure to 5 ng/µl of both toxins led to significant differences in macrophage viability versus at lower concentrations. Furthermore, cytokine secretion levels, including IL-12, IL-6 and TNF-α, increased dramatically when macrophages were exposed to strains ST42 or ST104. Finally, gene expression surveys point to increases in IL-12 gene expression in response to both ST42 and ST104. Conclusions: C. difficile strains with higher toxins levels induced an increased activation of the innate immune system and may activate macrophages more profoundly resulting in secretion of higher levels of pro-inflammatory cytokines. However, higher toxin levels may also damage macrophages' normal skeletal structure, reducing macrophage viability.

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.