Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane

Abstract

Aims: To determine the mechanisms of Bacillus subtilis spore resistance to and killing by a novel sporicide, dimethyldioxirane (DMDO) that was generated in situ from acetone and potassium peroxymonosulfate at neutral pH. Methods and Results: Spores of B. subtilis were effectively killed by DMDO. Rates of killing by DMDO of spores lacking most DNA protective α/β‐type small, acid‐soluble spore proteins (α−β− spores) or the major DNA repair protein, RecA, were very similar to that of wild‐type spore killing. Survivors of wild‐type and α−β− spores treated with DMDO also exhibited no increase in mutations. Spores lacking much coat protein due either to mutation or chemical decoating were much more sensitive to DMDO than were wild‐type spores, but were more resistant than growing cells. Wild‐type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with DMDO were sensitized to wet heat. The DMDO‐killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by very high pressures and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after their germination, and none of these treatments were able to revive the DMDO‐killed spores. Conclusions: DMDO is an effective reagent for killing B. subtilis spores. The spore coat is a major factor in spore resistance to DMDO, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent appears to kill spores by damaging the spore's inner membrane in some fashion. Significance and Impact of the Study: This work demonstrates that DMDO is an effective decontaminant for spores of Bacillus species that can work under mild conditions, and the killed spores cannot be revived. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent.

Comparison of the properties of Bacillus subtilis spores made in liquid or on agar plates

“…Spore resistance to 30% Microcyn TM (and almost certainly the full strength product) is attributed largely to the spore coat that is also important in spore resistance to many other oxidizing agents (Loshon et al . 2001; Young and Setlow 2003; Paul et al . 2006).…”

“…However, while the loss of spore coats reduces spore resistance to many oxidizing agents (including 30% Microcyn TM ) 10‐ to 100‐fold, decoated spores are only two‐ to threefold less resistant to nitrous acid than are intact spores (Loshon et al . 2001; Young and Setlow 2003; Cortezzo and Setlow 2005; Paul et al . 2006).…”

Effects of inactivation methods on the analysis of Bacillus atrophaeus endospores using real‐time PCR and MALDI‐TOF‐MS

“…Previous studies have shown how effective different methods of inactivation on endospores can be [31][32][33][34]. Further examination determined in a rather straightforward way, how each treatment affected the characteristic MALDI-TOF-MS spectra of the endospores.…”

“…Further examination determined in a rather straightforward way, how each treatment affected the characteristic MALDI-TOF-MS spectra of the endospores. Previous studies have shown how effective different methods of inactivation on endospores can be [31][32][33][34]. They indicate that each bacterial species has its own optimal inactivation parameters (incubation time, temperature, reagent concentration, etc.).…”

Chemistry and Applications of Iron–TAMLCatalysts in Green Oxidation Processes Based on Hydrogen Peroxide

“…The efficiency of Fe-TAML-TBHP with CTAB for decontaminant of bacterial spores has been separately validated by Setlows group in deactivating Bacillus subtilis spores [65]. A Gram-negative bacterium E. coli strain DH5a was also effectively deactivated on treatment with Fe-TAML-peroxide system.…”