Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

Abstract

Aims: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. Methods and Results: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone TM , ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. Conclusions: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. 2004, 97, 838-852 doi:10.1111/j.1365-2672.2004.02370.x Significance and Impact of the Study: The more rapid heat killing and germination with dodecylamine, the g...

Gas plasma sterilization of microorganisms and mechanisms of action

“…Figure 12. Representation of the interior and exterior structure of a bacterial spore (21). the inner membrane and dna, SaSp and dpa, ca in the core are essential targets and indicators of effective sterilization.…”

Disinfection efficiency of chlorine dioxide gas in student cafeterias in Taiwan

“…Following the 9-11 attacks in the USA, ClO 2 gas was used to treat the Bacillus anthracis spores detected in certain government buildings and on the exterior of mail packages addressed to a small number of government institutions (EPA, 2006;Canter et al, 2005). Finally, various studies have demonstrated the efficiency of ClO 2 in deactivating Bacillus endospores (surrogates for B. anthracis spores) (Buttner et al, 2001;Cortezzo et al, 2004;Young and Setlow, 2003).…”

Sensing and inactivation of Bacillus anthracis Sterne by polymer–bromine complexes

“…In a previous study, the damage of the spore inner membrane by oxidative species was reported (Cortezzo et al 2004). Spores exposed to non-lethal concentrations of chlorine dioxide and sodium hypochlorite, among others, were more susceptible to heat killing, and membrane permeability had been compromised (Cortezzo et al 2004). Perhaps a similar mechanism occurs with our system, and the spore surface and spore integrity have been compromised by our brominated polymers, allowing hypobromous acid to act against the spore.…”

“…While the mechanism of action of halogens against spores is not well understood, researchers have suggested that halogens may produce coat and cortex degradation in spores, which would allow halogens to enter the spore and oxidize proteins located on the inner membrane of the spore (Cousins and Allan 1967). In a previous study, the damage of the spore inner membrane by oxidative species was reported (Cortezzo et al 2004). Spores exposed to non-lethal concentrations of chlorine dioxide and sodium hypochlorite, among others, were more susceptible to heat killing, and membrane permeability had been compromised (Cortezzo et al 2004).…”