Mechanisms of killing Bacillus subtilis spores by hypochlorite, chlorine dioxide, Decon, Oxone(TM) and aqueous ozone

Date of Completion

January 2004


Biology, Microbiology




In this thesis the mechanisms of killing spores by hypochlorite, chlorine dioxide, Decon, Oxone™ and aqueous ozone are studied. Using Bacillus subtilis strains deficient in the small acid soluble spore proteins that protect spore DNA or in the RecA DNA repair pathway or both, as well as mutagenesis studies on spores that survived treatment with these agents, it was determined that damage to the spore's DNA was not the mechanism of killing. As spores are invariably more resistant to decontaminants than growing cells, some spore component must offer protection from them. Chemically decoated spores and spores with a coat mutation were killed much more rapidly by these agents, indicating that the spore coat is a major protective factor. Initial treatment with hypochlorite, chlorine dioxide, Decon, Oxone™ and aqueous ozone did not cause the loss of DPA from the spore's core, but subsequent sublethal heat treatment of these treated spores did cause DPA loss, indicating possible damage to the spore's inner membrane permeability barrier. Rapid loss of DPA from Decon-, Oxone™- and ozone-treated spores after germination with dodecylamine also indicated alteration of the inner membrane. Treatment with hypochlorite, Decon, Oxone™ and aqueous ozone, but not chlorine dioxide, abolished spore germination. These spores did not swell, become phase dark, release DPA or hexosamine fragments or initiate metabolism when incubated with germinants. Damage to the spore's germination apparatus, while possible, could not be the sole mechanism of killing as spores were not rescued by alternative germination pathways that bypass the germinant receptors or the cortex lytic enzymes. The mechanism of killing by these four agents appears to be damage to the spore's inner membrane. Chlorine dioxide-treated spores entered germination but did not initiate metabolism. A bacterial viability stain based on the permeability of dead cells to propidium iodide indicated that many cells were dead, probably resulting from some type of inner membrane damage. ^