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

Setlow, B., Korza, G., Blatt, K.M.S., Fey, J.P., Setlow, P.
Journal of applied microbiology 2016 v.120 no.1 pp. 57-69
Bacillus subtilis, DNA, DNA damage, carbon dioxide, energy metabolism, germination, membrane permeability, mutants, mutation, peracetic acid, plasma membrane, proteins, spores, temperature, viability
AIMS: Determine how supercritical CO₂ (scCO₂) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO₂‐PAA, and if spores inactivated by scCO₂‐PAA are truly dead. METHODS AND RESULTS: Spores of wild‐type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO₂‐PAA in liquid or dry at 35°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°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 scCO₂‐PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO₂‐PAA sensitive. CONCLUSIONS: These findings suggest that scCO₂‐PAA inactivates spores by damaging spores’ inner membrane. The spore coat provided scCO₂‐PAA resistance for both wet and dry spores. DNA protective proteins provided scCO₂‐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 scCO₂‐PAA, an agent with increasing use in sterilization applications.