D2.1 Report on results from toxicity assays (classical toxicity tests and High Content Analysis)

This task was launched on M4. Among the 21 B. cereus strains and 40 C. perfringens strains selected in WP1, a subset was selected for cytotoxicity test. The growth conditions of the strains as well as the cytotoxicity assays were defined and optimized. Protocols for the production of culture supernatants of B. cereus and C. perfringens (vegetative and sporulating) have been implemented and optimized by ANSES, INRAE and INSTITUT PASTEUR.
Following obtention of the strain supernatant of B. cereus and C. perfringens, their cytotoxicity was tested following 2h and 24h treatments of intestinal Caco2 cells using classical toxicity tests including the MTT assay. Potential effects of bacterial toxins on the pro-inflammatory response in Caco-2 cells (IL-8 secretion) was also investigated. Cellular imaging-based High Content Analysis approaches was developed and used to characterize the mechanisms of toxicity of a subset of specific strains in detail. Various markers of cellular toxicity were evaluated. These makers include apoptosis, genotoxicity (γH2AX, phospho-ATM), the pro-inflammatory response (nuclear translocation of NF-kB), mitochondrial membrane potential (TMRE) and cell cycle (Nuclear DAPI staining). The cytotoxic profiles of bacterial strains were then reported.