Moist-Heat Resistance, Spore Aging, and Superdormancy in Clostridium difficile

Clostridium difficile spores can survive extended heating at 71 degrees C (160 degrees F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killing C. difficile, we quantified (D values) the effect of moist heat at 85 degrees C (145 degrees F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63 degrees C. Fresh (1-week-old) and aged (>= 20-week-old) C. difficile spores from food and food animals were tested in multiple experiments. Heating at 85 C markedly reduced spore recovery in all experiments (5 to 6 log(10) within 15 min of heating; P<0.001), regardless of spore age. In ground beef, the inhibitory effect of 85 degrees C was also reproducible (P < 0.001), but heating at 96 degrees C reduced 6 log(10) within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85 degrees C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63 degrees C reduced counts for fresh spores (1 log(10), 30 min; P < 0.04) but increased counts of 20-week-old spores by 30% (15 min; P < 0.02), indicating that sublethal heat treatment reactivates superdormant spores. Superdormancy is an increasingly recognized characteristic in Bacillus spp., and it is likely to occur in C. difficile as spores age. The potential for reactivation of (super) dormant spores with sublethal temperatures may be a food safety concern, but it also has potential diagnostic value. Ensuring that food is heated to >85 degrees C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores.