The toxic effects of C. difficile culture supernatants on Vero cells (a kind gift of Eric Snijder [39] ) were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium (Dulbecco modified Eagle medium (Lonza) supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum), before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin (Techlab) was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB (Techlab) was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects (cell rounding) were observed.