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

Abstract

ABSTRACTClostridium difficilespores can survive extended heating at 71°C (160°F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killingC. difficile, we quantified (Dvalues) the effect of moist heat at 85°C (145°F, for 0 to 30 min) onC. difficilespores and compared it to the effects at 71 and 63°C. Fresh (1-week-old) and aged (≥20-week-old)C. difficilespores from food and food animals were tested in multiple experiments. Heating at 85°C markedly reduced spore recovery in all experiments (5 to 6 log10within 15 min of heating;P< 0.001), regardless of spore age. In ground beef, the inhibitory effect of 85°C was also reproducible (P< 0.001), but heating at 96°C reduced 6 log10within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85°C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63°C reduced counts for fresh spores (1 log10, 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 inBacillusspp., and it is likely to occur inC. difficileas 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°C would be a simple and important intervention to reduce the risk of inadvertent ingestion ofC. difficilespores.