C. difficile Spores and Macrophage Interactions Figure 8. Factors affecting survival of C. difficile spores inside Raw 264.7 cells. A) Monolayers of Raw 264.7 cells were infected at an MOI of 10 with dormant C. difficile spores; and with spores incubated for 30 min with 1% sodium taurocholate (ST) in DMEM and 1% sodium taurocholate- 5 mM L-glycine (STG) in DMEM. After 30 min of infection, unbound spores were washed, and Raw 264.7 cells were incubated in DMEM with no FBS under: Aer, aerobic conditions-5% CO2 for 24 h; Ana, anaerobic conditions for 24 h. Loss of spore viability was determined as described in Methods section. B) Monolayers of Raw 264.7 cells were infected with non-heat activated C. difficile spores treated for 30 min with: C, DMEM; 0.1-STG, 0.1% sodium taurocholate-5 mM L-glycine in DMEM; HS, 50% human serum in DMEM; HS 0.1-STG, 0.1% sodium taurocholate-5 mM L-glycine-50% human serum in DMEM; HA HS 0.1-STG, with heat activated C. difficile spores treated for 30 min with 0.1% sodium taurocholate-5 mM L-glycine-50% human serum in DMEM. Infected monolayers of Raw 264.7 cells were incubated for 24 h and viable spores were determined by plating aliquots of lysed Raw 264.7 cells onto BHIS agar plates as described in Methods section. Treatment C corresponds to data from Fig. 7A and is shown for comparative purposes. C,D) Fluorescence micrographs of Raw 264.7 cells infected with 1.0%-STG C. difficile spores. Infected Raw 264.7 cells were incubated for 24 h under aerobic (panel C) and anaerobic (panel D) conditions, fixed and DNA material of either Raw 264.7 cells and C. difficile vegetative cells were stained with DAPI as described in Methods section. White arrows denote growing C. difficile vegetative cells. Results are the average of at least three independent experiments and error bars are standard error of the mean. doi:10.1371/journal.pone.0043635.g008 interesting to note that C. difficile spores are well suited to survive inside Raw 264.7 murine-like macrophages for up to 72 h under aerobic conditions, which is due in part to their ability to remain dormant. It can be argued that survival of C. difficile spores during infection of Raw 264.7 cells could be due to macrophage cell death. While this was observed at an MOI of 10, where 49% of Raw 264.7 cells were dead after 48 h of infection, however, at an MOI of 1, only 12% of Raw 264.7 cells were dead, indicating that C. difficile spores’ survival ability is due to their intrinsic properties. In fact, no alterations in the spore ultrastructure was observed after 24 h of infection, indicating that C. difficile spores are able to resist successive attacks from the phagolysosome’s antimicrobial ma- chinery. The ability of C. difficile spores to survive inside the macrophage environment is most likely to the lack of germination, since the macrophage environment lacks the co-germinants ST and L-glycine. Indeed, a recent study demonstrated that B. anthracis spores with mutations in the main gerA receptors were able to survive inside Raw 264.7 macrophages primarily due to their ability to remain dormant during infection of Raw 264.7 cells [46]. Germination-dependent survival has also been observed in C. perfringens, with spores of isolates that are able to germinate during macrophage infection being more easily killed by macrophages than with spores of isolates that are less able to germinate during infection [13]. Similar spore killing became evident when C. difficile PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e43635