Moist-heat resistance, spore aging, and superdormancy in Clostridium difficile

Abstract

Clostridium difficile spores 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 killing C. difficile, we quantified (D values) the effect of moist heat at 85°C (145°F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63°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°C was also reproducible (P < 0.001), but heating at 96°C reduced 6 log(10) within 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 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°C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores.

Fig. 1.

Effect of moist heat in phosphate-buffered saline at 63 and 85°C on viability of fresh (1-week-old) and aged (20-week-old) spores of C. difficile. Open symbols represent isolate means; gray squares with interval bars represent 95% CI of the means.

Fig. 2.

Effect of storage on cultivability of aged C. difficile spores during 18 days of refrigeration at 4°C. (A) Results for nonheated spores illustrate reduced cultivability over time (12 strains; triangles and interval bars represent means and 95% CI). (B) Results for spores heated at 85°C indicate reproducible estimation and cultivability (PCR ribotype 078 strain; regression fits shown for 0, 3, 14, and 18 days).

Fig. 3.

Germination and outgrowth studies of C. difficile to determine if 15 min of heat shock impairs spore germination or the subsequent cell division. (A) Germination study. Optical density (OD₆₀₀) reductions of heavily inoculated BHI spore suspensions, incubated at 37°C for 7 h, are similar for heated and nonheated spores, indicating normal germination of heated spores. Only PCR ribotype 078 and the ATCC strain are shown; results are means ± standard deviations. (B) Outgrowth study. Enumeration of ethanol-resistant cells from aliquots in Fig. 3A after 7 h of incubation. Countable colonies on blood agar indicate adequate cell division. Reduced counts after heat shock with 85°C (and normal germination shown in Fig. 3A) indicate impaired cell division. Dots represent replica averages; results are means (solid bars) ± 95% CI.

Fig. 4.

Effect of heat shock on immediate cultivability and subsequent germination of C. difficile spores determined by enumeration of ethanol-resistant cells before and after 7 h of incubation in BHI. (A) Line plots represent paired enumeration of heat-shocked and nonheated individual spore replicas. Linear equations and extended dashed lines represent average effects (slopes). The highest and steepest dashed line (63°C) indicates immediate enhanced cultivability and subsequently increased spore germination. The panel for 85°C shows immediate inhibition and subsequent increased cultivability. (B) Spore germination relative to baseline. Normalized paired differences (before and after BHI incubation). Heat shock with 63 and 71°C enhanced germination in spores that otherwise would have remained dormant after an additional 7 h of incubation in BHI and 72 h in blood agar at 37°C (here referred to as superdormant spores). Dots represent average difference per replica; means ± 95% CI (see also Table S1 in the supplemental material).

Fig. 5.

Minutes of heat needed to inhibit a mixture of aged C. difficile spores heated in three food matrices. Dots indicate individual D value estimates; results are means ± 95% CI. Identical superscripts connect variables with nonsignificant differences (i.e., GLM; P > 0.05), whereas different superscripts indicate significance. See also Table S2 in the supplemental material for coefficient estimates.