Mechanism of killing of spores ofBacillus cereusandBacillus megateriumby wet heat

Abstract

Aims: To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. Methods and Results: Bacillus cereus and B. megaterium spores wet heat‐killed 82–99% gave two bands on equilibrium density gradient centrifugation. The lighter band was absent from spores that were not heat‐treated and increased in intensity upon increased heating times. These spores lacked dipicolinic acid (DPA) were not viable, germinated minimally and had much denatured protein. The spores in the denser band had viabilities as low as 2% of starting spores but retained normal DPA levels and most germinated, albeit slowly. However, these largely dead spores outgrew poorly if at all and synthesized little or no ATP following germination. Conclusions: Wet heat treatment appears to kill spores of B. cereus and B. megaterium by denaturing one or more key proteins, as has been suggested for wet heat killing of Bacillus subtilis spores. Significance and Impact of the Study: This work provides further information on the mechanisms of killing of spores of Bacillus species by wet heat, the most common method for spore inactivation.

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

“…Indeed, there is one report that spores incubated at lower temperatures for longer periods germinate more slowly than untreated spores (12). In addition, spores heat treated at even higher temperatures (ϳ90°C) that retain DPA and at least some viability germinated more slowly than untreated spores (19,20,29). However, the surprising result obtained with purified wild-type B. megaterium spores that had been incubated at 75°C was that they germinated more rapidly than untreated spores (Fig.…”

“…These spores were also not treated as severely as the heat-treated B. subtilis spores that were significantly slower in outgrowth than untreated spores. Slower outgrowth of even more severely heat-treated B. subtilis spores has been seen previously and has been suggested to be due to partial inactivation of spore core proteins essential for metabolism essential for spore outgrowth, with more complete inactivation of such proteins leading to spore death (19,20). Notably, heat-treated spores of a number of species can often exhibit conditional survival on poor or stressful medium, even medium with small amounts of added salt (35)(36)(37).…”

“…However, to ensure that all of the spores that were analyzed for rRNA were neither germinated nor dead, spores incubated at these temperatures were layered on a high-density solution and centrifuged. The density of the solution was chosen such that spores that retain DPA pellet because of their high core wet density while spores that have lost DPA either through germination or heat-induced release float because their wet density has gone down significantly (11,19,20). Previous work has shown that spores treated in water at even higher temperatures that have lost all DPA are dead, while spores that have retained DPA retain significant viability (19,20).…”

“…The density of the solution was chosen such that spores that retain DPA pellet because of their high core wet density while spores that have lost DPA either through germination or heat-induced release float because their wet density has gone down significantly (11,19,20). Previous work has shown that spores treated in water at even higher temperatures that have lost all DPA are dead, while spores that have retained DPA retain significant viability (19,20). We felt that this purification step after heat treatment would be essential for ultimate analysis of rRNA only in spores that are still dormant and not dead.…”

“…Following all incubations, spore preparations were centrifuged and washed with water and final pellets were suspended in 0.2 ml of 20% HistoDenz (Sigma Chemical Co., St. Louis, MO), applied to the top of 1.9 ml of 50 or 55% HistoDenz in water for B. subtilis and B. megaterium spores, respectively, and centrifuged at 4°C at ϳ8,000 ϫ g for 10 min. Under these centrifugation conditions, dormant spores that retain their DPA pellet while spores that have lost DPA and are germinated, dead, or both float (11,19,20). Spore pellets from these centrifugations were isolated, washed several times with 1.5 ml of water to remove HistoDenz, suspended in water at an OD 600 of ϳ5, and stored at 4°C prior to analyses.…”