C. difficile Spores and Macrophage Interactions Figure 9. C. difficile spores affect viability of Raw 264.7 cells. Monolayers of Raw 264.7 cells were infected for 24 (grey bars) and 48 h (black bars) with C. difficile spores at MOIs of 1 and 10 and effects on Raw 264.7 cells was measured by quantification of: A) dead Raw 264.7 cells with ethidium homodimer-1; or B) live Raw 264.7 cells with calcein AM. Results were also confirmed with: C) cell viability assay with the membrane impermeable trypan blue as described in Methods section. Dark cells indicate disruption of the plasma membrane. Double asterisk indicate statistical significant difference (p,0.05) between treatments highlighted by brackets. 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.g009 spores were pre-incubated with STG or HS prior to infecting Raw 264.7 cells: HS increased the killing of C. difficile spores by Raw 264.7 cells. Serum lysozyme has been previously shown to trigger germination of C. perfringens spores through spore’s PG cortex hydrolysis [36], suggesting that similarly, HS lysozyme might be sensitizing C. difficile spores to macrophage killing, most likely by degrading the spore’s PG cortex. Since the environment inside the phagosome is cholate free, untreated C. difficile spores remain PLOS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e43635