dormant and therefore are likely to persist even after macrophage death. It is worth noting that C. difficile vegetative cells were efficiently inactivated by Raw 264.7 cells under aerobic conditions, environment typically found in the sub-mucosal intestinal layers. However, although under anaerobic conditions C. difficile vegeta- tive cells were first efficiently killed by a fixed number of Raw 264.7 cells, C. difficile cell counts increased over 24 h infection. It can be speculated that this might be the case in the mucosal environment and towards the colonic lumen; however, the constant infiltration of macrophages might produce a more host favorable outcome than the in vitro results shown in this work. Regardless of the fate of C. difficile vegetative cells, C. difficile spores were particularly resistant to macrophage killing under aerobic conditions. Collectively, these results provide support to the hypothesis that C. difficile spore survival inside macrophages is primarily due to their ability to remain dormant. The extent of inactivation of C. difficile spores by Raw 264.7 cells after 72 h of incubation (i.e., ,0.6 decimal reduction), from a host perspective, is not effective in terms of pathogen-clearance by the innate immune system, and in the case of CDI, it might hold implications in persistence and high relapse episodes of CDI. Indeed, it might be possible that C. difficile isolates with high sproulation efficiencies persist in the host’s colonic tract due to the virtue of the C. difficile spore’s elevated resistance to the hosts’ immune system. Recent developments of animal models suited to study the role of C. difficile spores in the initiation and persistence of CDI [47] will facilitate our future investigation in this direction. Another contribution of this work is that C. difficile spores seem to directly interact with the phagosome’s membrane of Raw 264.7 cells and presumably induce membrane disruption and cell death. The precise nature of this interaction is unclear but is likely to involve interactions between the spore surface proteins and the phagosome’s membrane and/or membrane embedded proteins. It is also unclear if these interactions might have any implication in References 1. Deneve C, Janoir C, Poilane I, Fantinato C, Collignon A (2009) New trends in Clostridium difficile virulence and pathogenesis. Int J Antimicrob Agents 33 Suppl 1: S24–28. 2. Rupnik M, Wilcox MH, Gerding DN (2009) Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7: 526–536. 3. Goulding D, Thompson H, Emerson J, Fairweather NF, Dougan G, et al. (2009) Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1. 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In conclusion, our work provides evidence that C. difficile spores are recognized by phagocytic cells but remain impermeable to attacks by their antimicrobial components and are able to exert cytotoxic effects to Raw 264.7 cells. The implications of these findings clearly suggest that C. difficile spores are able to subvert attacks of host’s immune system avoiding spore clearance and persist in the colonic tract. Acknowledgments The authors wish to thank Drs Ling Jin and Lia Danelishivili of the Department of Biomedical Sciences at Oregon State University for critical discussions. Authors thank Dr. Luiz Bermudez of the Department of Biomedical Sciences of Oregon State University and Dr. Claudia Saavedra of the Department of Biological Sciences of Universidad Andre´s Bello for providing us with Raw 264.7 macrophages. Authors also wish to thank Dr. Peter Setlow of University of Connecticut Health Center for providing strain PS832. 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