. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the nonpathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreakassociated strains ribotype 017, 027, and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-B phosphorylation, and increased proinflammatory cytokine TNF⣠protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-B phosphorylation, and TNF⣠protein expression caused by infection with all strains. Moreover, S.b-conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b-conditioned medium also inhibited toxin A-and B-mediated actin cytoskeleton disruption. S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins. (46). C. difficile is an anaerobic bacterium that produces two toxins -toxin A and toxin B -that mediate diarrhea, inflammation, and apoptosis of the mucosal epithelium in animals and humans (32). The effects of the toxin in target intestinal cells involve inactivation of the Rho family of GTPase, leading to cytoskeletal disorganization, epithelial cell apoptosis, and ultimately cell death (42, 54). C. difficile toxins also stimulate transcription of several proinflammatory genes, including tumor necrosis factor-⣠(TNFâŁ) (19) and activate transcription factors and mitogen-activated protein kinases involved in their proinflammatory effects (18,25). The mainstream CDI regimen includes the use of metronidazole, vancomycin, and fidaxomicin (55). However, many C. difficileinfected patients may also suffer from recurrent infections (13), while the recent epidemics that also involve new epidemic outbreak-associated strains (29) pose a major medical problem and epidemiological concern. These challenges have been met with a broad range of preventive approaches against CDI, including the use of probiotics, most notably Saccharomyces boulardii (S.b) alone or in combination with established antibiotic treatment (23,24).S.b is a nonpathogenic yeast that represents one of the well-studied probiotics against CDI in both experimental and clinical settings (24,40). Randomized double-blind placebocontrolled clinical trials have shown that S.b CNCM I-745 is an effective probiotic in the prophylaxis of antibi...