C. difficile toxin cytotoxicity assay

C. difficile toxin specific cytotoxicity was determined using a Green African monkey kidney epithelial (Vero) cell-rounding cytotoxicity assay^18,31. Briefly, fresh fecal samples were normalized to weight, homogenized in sterile PBS, and pelleted at 13,000 x g. Tenfold serial dilutions of supernatants were added to monolayers of Vero cells and cell-rounding was assessed for each dilution. The presence of C. difficile toxin A and toxin B were confirmed by neutralizing cell-rounding activity with a combined antitoxin (toxin A and toxin B) antisera (Techlab, cat# T5000). Cytotoxicity data are expressed as the log-ten reciprocal value of the highest dilution that rounded 100% of the Vero cells.