Multifactorial resistance of Bacillus subtilis spores to low-pressure plasma sterilization

ABSTRACT Common sterilization techniques for labile and sensitive materials have far-reaching applications in medical, pharmaceutical, and industrial fields. Heat inactivation, chemical treatment, and radiation are established methods to inactivate microorganisms, but pose a threat to humans and the environment and can damage susceptible materials or products. Recent studies have demonstrated that cold low-pressure plasma (LPP) treatment is an efficient alternative to common sterilization methods, as LPP’s levels of radicals, ions, (V)UV-radiation, and exposure to an electromagnetic field can be modulated using different process gases, such as oxygen, nitrogen, argon, or synthetic (ambient) air. To further investigate the effects of LPP, spores of the Gram-positive model organism Bacillus subtilis were tested for their LPP susceptibility including wild-type spores and isogenic spores lacking DNA-repair mechanisms such as non-homologous end-joining (NHEJ) or abasic endonucleases, and protective proteins like α/β-type small acid-soluble spore proteins (SASP), coat proteins, and catalase. These studies aimed to learn how spores resist LPP damage by examining the roles of key spore proteins and DNA-repair mechanisms. As expected, LPP treatment decreased spore survival, and survival after potential DNA damage generated by LPP involved efficient DNA repair following spore germination, spore DNA protection by α/β-type SASP, and catalase breakdown of hydrogen peroxide that can generate oxygen radicals. Depending on the LPP composition and treatment time, LPP treatment offers another method to efficiently inactivate spore-forming bacteria. IMPORTANCE Surface-associated contamination by endospore-forming bacteria poses a major challenge in sterilization, since the omnipresence of these highly resistant spores throughout nature makes contamination unavoidable, especially in unprocessed foods. Common bactericidal agents such as heat, UV and γ radiation, and toxic chemicals such as strong oxidizers: (i) are often not sufficient to completely inactivate spores; (ii) can pose risks to the applicant; or (iii) can cause unintended damage to the materials to be sterilized. Cold low-pressure plasma (LPP) has been proposed as an additional method for spore eradication. However, efficient use of LPP in decontamination requires understanding of spores’ mechanisms of resistance to and protection against LPP. Surface-associated contamination by endospore-forming bacteria poses a major challenge in sterilization, since the omnipresence of these highly resistant spores throughout nature makes contamination unavoidable, especially in unprocessed foods. Common bactericidal agents such as heat, UV and γ radiation, and toxic chemicals such as strong oxidizers: (i) are often not sufficient to completely inactivate spores; (ii) can pose risks to the applicant; or (iii) can cause unintended damage to the materials to be sterilized. Cold low-pressure plasma (LPP) has been proposed as an additional method for spore eradication. However, efficient use of LPP in decontamination requires understanding of spores’ mechanisms of resistance to and protection against LPP.