Mechanism of Bacillus subtilis spore inactivation by and resistance to supercritical CO2 plus peracetic acid

Aims: Determine how supercritical CO2 (scCO(2)) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO(2)-PAA, and if spores inactivated by scCO(2)-PAA are truly dead. Methods and Results: Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO(2)-PAA in liquid or dry at 35 degrees C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2-PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85 degrees C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO(2)-PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO(2)-PAA sensitive. Conclusions: These findings suggest that scCO(2)-PAA inactivates spores by damaging spores' inner membrane. The spore coat provided scCO(2)-PAA resistance for both wet and dry spores. DNA protective proteins provided scCO(2)-PAA resistance only for dry spores. Significance and Impact of the Study: These results provide information on mechanisms of spore inactivation of and resistance to scCO(2)-PAA, an agent with increasing use in sterilization applications.