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

Abstract

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 Study These results provide information on mechanisms of spore inactivation of and resistance to scCO2-PAA, an agent with increasing use in sterilization applications.

Killing of spores ofBacillusspecies by cetyltrimethylammonium bromide

“…Indeed, this is what is seen when B. subtilis spores are treated with agents such as oxidizing chemicals and supercritical CO 2 (Setlow ; Setlow et al . ; Li et al . ).…”

“…This behaviour of CTAB‐treated dead spores is essentially identical to that of spores killed by a large number of chemical agents, including many oxidizing agents and supercritical CO 2 , which appear to kill spores by damaging the spore IM in some fashion (Setlow ; Setlow et al . ; Li et al . ).…”

Bacillus spore wet heat resistance and evidence for the role of an expanded osmoregulatory spore cortex

“…In addition to retaining DPA, spores treated at 80°C in 4 mol l −1 CaCl 2 exhibited no staining by propidium iodide (data not shown), consistent with these spores not being killed appreciably (Setlow et al . ).…”

“…Spores were also stained with propidium iodide (5 mg l −1 ) and photographed by fluorescence microscopy (Setlow et al . ).…”

Effects of the microbicide ceragenin CSA‐13 on and properties ofBacillus subtilisspores prepared on two very different media

“…Finally, a key unanswered question is how CSA‐13 kills spores once they germinate or even in the process of germination itself. Preliminary work (data not shown) indicates that following CaDPA release due to CSA‐13 treatment of dormant spores there is damage to the spore IM such that this membrane becomes permeable to a nucleic acid stain, propidium iodide, that normally only accumulates in dead dormant spores with a severely damaged IM or in dead germinated spores (Huesca‐Espitia et al ; Setlow et al ; Li et al ). The next step will be to determine definitively if this is indeed how CSA‐13 kills spores, as well as the features of the killed spores—for example, do the killed spores degrade the cortex peptodoglycan?…”