Aims: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them. Methods and Results: Spores of B. subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective α/β‐type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway. Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins. Spores prepared at a higher temperature were also much more resistant to these agents. Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite‐ and chlorine dioxide‐treated spores much more readily released DPA upon a subsequent normally sub‐lethal heat treatment than did untreated spores. Hypochlorite‐killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+‐DPA, and these spores could not be recovered by lysozyme treatment. Chlorine dioxide‐treated spores also did not germinate with Ca2+‐DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients. However, while germinated chlorine dioxide‐killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties. Conclusions: Hypochlorite and chlorine dioxide do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat. Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane. While chlorine dioxide‐killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage. Significance and Impact of the Study: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide.

Aims: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone. Methods and Results: Killing of B. subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity. Spores (termed a ) b ) ) lacking the two major DNA protective a ⁄ b-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and a ) b ) and a ) b ) recA spores exhibited identical ozone sensitivity. Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat. Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores. However, ozone-killed spores did not germinate with either nutrients or Ca 2+ -DPA and could not be recovered by lysozyme treatment. Conclusions: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat. Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane. Significance and Impact of the Study: These results provide information on the mechanisms of spore killing by and resistance to ozone.

Aims: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to the general biological decontamination agents, Decon and Oxone TM . Methods and Results: Spores of B. subtilis treated with Decon or Oxone TM did not accumulate DNA damage and were not mutagenized. Spore killing by these agents was increased if spores were decoated. Spores prepared at higher temperatures were more resistant to these agents, consistent with a major role for spore coats in this resistance. Neither Decon nor Oxone TM released the spore core's depot of dipicolinic acid (DPA), but Decon-and Oxone TM -treated spores more readily released DPA upon a subsequent normally sublethal heat treatment. Deconand Oxone TM -killed spores initiated germination with dodecylamine more rapidly than untreated spores, but could not complete germination triggered by nutrients or Ca 2+ -DPA and did not degrade their peptidoglycan cortex. However, lysozyme treatment did not recover these spores. Conclusions: Decon and Oxone TM do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents is the spore coat. Spore killing by both agents renders spores defective in germination, possibly because of damage to the inner membrane of spore. Significance and impact of study: These results provide information on the mechanisms of the killing of bacterial spores by Decon and Oxone TM .