The effects of high-power microwaves on the ultrastructure of Bacillus subtilis

Aims:  To investigate the microbicidal mechanisms of high-power microwave (2·0 kW) irradiation on Bacillus subtilis and to determine the effect of this procedure on the ultrastructure of the cell wall.
Methods and Results:  We performed viability test, examined cells using transmission electron microscopy (TEM), and measured the release of intracellular proteins and nucleic acids. The inactivation rate of B. subtilis by 2·0-kW microwave irradiation was higher than that of a domestic microwave (0·5 kW). Few proteins were released from either microwaved or boiled cells. However, the leakage of nucleic acids from 2·0-kW-microwaved cells was significantly higher than that of 0·5-kW-microwaved or boiled cells. Therefore, we examined ultrastructural alterations of microwaved or boiled cells to analyse the pattern of release of cytoplasmic contents. Although boiled cells did not show any ultrastructural changes on TEM, 2·0-kW-microwaved cells showed disruption of the cell wall.
Conclusion:  The microbicidal mechanisms of 2·0-kW microwave irradiation include damage to the microbial cell wall, breakage of the genomic DNA, and thermal coagulation of cytoplasmic proteins.
Significance and Impact of the Study:  TEM images showed that the cytoplasmic protein aggregation and cell envelope damage by microwave irradiation were different from the ultrastructural changes observed after boiling.     

Analysis of dye binding by and membrane potential in spores of Bacillus species

Aims:  To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.
Methods and Results:  Staining by four dyes and autofluorescence of B. subtilis spores that lack some ( cotE , gerE ) or most ( cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC₆(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC₆(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC₆(3) bound well to all germinated spores.
Conclusions:  The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.
Significance and Impact of the Study:  The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.     

Bovicin HC5 reduces thermal resistance of Alicyclobacillus acidoterrestris in acidic mango pulp

Aims:  To test the effect of bovicin HC5 against vegetative cells and endospores of Alicyclobacillus acidoterrestris DSMZ 2498 in synthetic media and in acidic mango pulp.
Methods and Results:  Alicyclobacillus acidoterrestris was grown in synthetic medium at 40°C and pH 4·0. The effect on vegetative cells was assayed by adding bovicin HC5 to synthetic medium (40–160 AU ml⁻¹) or to mango pulp (100 AU ml⁻¹) at various pH values and determining the effect on growth (OD_600nm) and viable cell number, respectively. The effect of bovicin HC5 on spore germination and thermal sensitivity of A. acidoterrestris was tested in mango pulp (pH 4·0) containing 80 AU ml⁻¹ of bovicin HC5. Bovicin HC5 was bactericidal against vegetative cells of A. acidoterrestris at different pH values and showed sporicidal activity against endospores of this bacterium. When spores of A. acidoterrestris were heat treated in the presence of bovicin HC5, D -values decreased 77% to 95% compared to untreated controls at temperatures ranging from 80 to 95°C.
Conclusion:  Bovicin HC5 was bactericidal and sporicidal against A. acidoterrestrsi DSMZ 2498.
Significance and Impact of the Study:  These results indicated that bovicin HC5 has potential to prevent spoilage of acidic fruit juices by thermocidophilic spore-forming bacteria.     

The formation of spores in biofilms of Anoxybacillus flavithermus

Aims:  To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable – temperature – on spore formation.
Methods and Results:  A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8·5 h period at three different temperatures of 48, 55 and 60°C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6–8 h. Spores formed at 55 and 60°C and amounted to approx. 10–50% of the biofilm. No spores formed at 48°C.
Conclusions:  The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores.
Significance and Impact of the Study:  This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus .     

Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H₂O₂) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H₂O₂, as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Clostridia spore formation during aerobic deterioration of maize and sorghum silages as influenced by Lactobacillus buchneri and Lactobacillus plantarum inoculants

Aims:  The effect of the inoculation of maize and sorghum silages with Lactobacillus plantarum (LP) and Lactobacillus buchneri (LB) on the clostridia spore formation during aerobic deterioration has been studied.
Methods and results:  The crops were ensiled in 30 l jars, without a lactic acid bacteria inoculant (C), and with an LP or LB inocula (theoretical rate of 1 × 10⁶). After 90 days of conservation, the silages were analysed for the chemical and microbiological characteristics and subjected to an aerobic stability test, during which pH, temperature, nitrate, yeast, mould and clostridia spores were measured. Compared to the C and LP silages, yeasts were reduced in the LB silages, resulting in an increased aerobic stability. Clostridia spores, determined by most probable number (MPN) procedure, increased to 6 log₁₀ MPN g⁻¹ in the C and LP maize silages, whereas they reached 3 log₁₀ MPN g⁻¹ in C and LP sorghum silages.
Conclusions:  Clostridia spore count only slightly increased in the LB maize silages after 342 h (2·59 log₁₀ MPN g⁻¹), whereas it did not show any increase in the LB sorghum silages for the whole period of air exposure.
Significance and impact of the study:  The data indicated that clostridia spore outgrowth can take place during silo feedout in aerobic-deteriorated silages and that LB inoculation reduces the risk of clostridia outgrowth after silage opening by increasing the aerobic stability.     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .     

Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone. METHODS AND RESULTS: Killing of B. subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity. Spores (termed alpha-beta-) lacking the two major DNA protective alpha/beta-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and alpha-beta- and alpha-beta-recA spores exhibited identical ozone sensitivity. Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat. Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores. However, ozone-killed spores did not germinate with either nutrients or Ca(2+)-DPA and could not be recovered by lysozyme treatment. CONCLUSIONS: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat. Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of spore killing by and resistance to ozone.     

A combined immunomagnetic separation and lateral flow method for a sensitive on-site detection of Bacillus anthracis spores – assessment in water and dairy products

Aim:  Combination of immunomagnetic separation (IMS) and lateral flow device (LFD) assays for the development of a sensitive, rapid, on-site methodology that enables concentration and detection of Bacillus anthracis spores in complex samples.
Methods and Results:  The data presents the development of an optimized, 30 min, IMS assay, with about 95% capture of B. anthracis spores from different dairy products ( n  = 38). No cross reactivity was detected with typical milk flora and some closely related Bacilli. To enable direct application of the IMS captured spores on the LFD, spores were eluted from the bead–spore complex utilizing 95% (v/v) formamide-10 mmol l⁻¹ EDTA for 30 s in a microwave oven. Detached spores were analysed on LFD enabling detection within 10 min. The combined IMS–LFD methodology (40 min) demonstrates a 60-fold improvement in sensitivity, relative to samples that were applied directly on the LFD without the IMS concentrating step.
Conclusions:  The IMS–LFD method is a powerful platform, combining rapidity, specificity and efficiency for concentrating and detecting B. anthracis from water and milk contaminated samples.
Significant and Impact of the Study:  The combination of IMS and LFD enhances the sensitivity and flexibility of B. anthracis spore detection from complex samples. This method can potentially be extended to other toxins and micro-organisms in a variety of matrices.     

Analysis of damage due to moist heat treatment of spores of Bacillus subtilis

Aims:  To determine conditions for generation and recovery of Bacillus subtilis spore populations heavily damaged by moist heat treatment.
Methods and Results:  Bacillus subtilis spores were treated with moist heat and spore viability was assessed on different media. A rich medium and several minimal media gave similar spore recoveries after moist heat treatment, but lack of glucose in minimal media greatly decreased spore recovery. High NaCl levels also greatly decreased the recovery of moist heat-treated spores on minimal media, and addition of good osmoprotectants reversed this effect. Moist heat treatment did not decrease spore recovery on minimal media with high salt through DNA damage or by eliminating spore germination, but by affecting spore outgrowth.
Conclusions:  Conditions for generating B. subtilis spore populations with high levels of conditional moist heat damage have been determined. The major conditional damage appears to be in spore outgrowth, perhaps because of damage to one or more important metabolic enzymes.
Significance and Impact of the Study:  This work has provided new insight into the mechanism of B. subtilis spore killing by moist heat.     

Decontamination of a hard surface contaminated with Bacillus anthracisΔSterne and B. anthracis Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO2)

Aims: To evaluate the inactivation of Bacillus anthracisΔSterne and Ames spores using electrochemically generated liquid‐phase chlorine dioxide (eClO₂) and compare two sporulation and decontamination methods with regard to cost, safety and technical constraints. Methods and Results: Spores were prepared via agar and broth methods and subsequently inoculated and dried onto clean, autoclave‐sterilized glass coupons. Bacillus anthracis spore inactivation efficacy was evaluated using the modified three‐step method (AOAC 2008.05) and a single‐tube extraction method. Spores (7·0 ± 0·5 logs) were inactivated within 1 min at room temperature using freshly prepared eClO₂. Bacillus anthracisΔSterne spores decreased in size after eClO₂ treatment as measured using a Beckman Coulter Multisizer. Conclusions: eClO₂ saturation of a hard surface was an effective B. anthracis sporicide. Broth sporulation and the single‐tube extraction method required less time and fewer steps, yielded a higher percentage of phase‐bright spores and showed higher spore recovery efficiency compared with AOAC 2008.05, making it more amenable to biosafety level 3 (BSL3) testing of virulent spores. Significance and Impact of the Study: Two test methods demonstrated the sporicidal efficacy of eClO₂. A new single‐tube extraction test protocol for decontaminants was introduced.     

Mechanisms of Bacillus subtilis spore killing by and resistance to an acidic Fe-EDTA-iodide-ethanol formulation

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to an acidic solution containing Fe(3+), EDTA, KI and ethanol termed the KMT reagent. METHODS AND RESULTS: Wild-type B. subtilis spores were not mutagenized by the KMT reagent but the wild-type and recA spores were killed at the same rate. Spores (alpha(-)beta(-)) lacking most DNA-protective alpha/beta-type small, acid-soluble spore proteins were less resistant to the KMT reagent than wild-type spores but were also not mutagenized, and alpha(-)beta(-) and alpha(-)beta(-)recA spores exhibited nearly identical resistance. Spore resistance to the KMT reagent was greatly decreased if spores had defective coats. However, the level of unsaturated fatty acids in the inner membrane did not determine spore sensitivity to the KMT reagent. Survivors in spore populations killed by the KMT reagent were sensitized to killing by wet heat or nitrous acid and to high salt in plating medium. KMT reagent-killed spores had not released their dipicolinic acid (DPA), although these killed spores released their DPA more readily when germinated with dodecylamine than did untreated spores. However, KMT reagent-killed spores did not germinate with nutrients or Ca(2+)-DPA and were recovered only poorly by lysozyme treatment in a hypertonic medium. CONCLUSIONS: The KMT reagent does not kill spores by DNA damage and a major factor in spore resistance to this reagent is the spore coat. KMT reagent treatment damages the spore's ability to germinate, perhaps by damaging the spore's inner membrane. However, this damage is not oxidation of unsaturated fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanism of spore resistance to and killing by the KMT reagent developed for killing Bacillus spores.     

Mechanism of microwave sterilization in the dry state.

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Mechanism of microwave sterilization in the dry state

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.
Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca²⁺ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination.
Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores.
Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.     

Interaction of iodine with Bacillus subtilis spores and spore forms

Buffered solutions of iodine (pH 7.0) were effective against Bacillus subtilis spores, but concentrations and contact times for effective sporicidal action were relatively high. Concentrations of 500 to 1000 ppm available iodine with a contact time of 30–45 min were required to produce a 3–5 log reduction. Treatment of spores with agents which caused progressive extraction of coat protein and cortex hexosamine was associated with increased sensitivity to iodine. Treatment of spores with iodine produced extraction of spore coat protein which was potentiated in the presence of NaOH, but there was no evidence of breakdown of cortex hexosamines or release of dipicolinic acid, either from intact spores or spore protoplasts. Sporicidal concentrations of iodine stimulated the uptake of (³²P) phosphate over an initial period of 30–40 min, but phosphate then leaked from the cells; 1000 ppm available iodine produced total loss within 60 min. Results of this investigation are consistent with previous findings which suggest that the resistance of spores to biocides is related to the barrier properties of the spore outer layers and that the sporicidal action of halogen-releasing agents is related to their ability to cause coat and cortex degradation, leading to rehydration of the spore protoplast and allowing diffusion to their site of action on the underlying protoplast.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane

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 alpha/beta-type small, acid-soluble spore proteins (alpha- beta- spores) or the major DNA repair protein, RecA, were very similar to that of wild-type spore killing. Survivors of wild-type and alpha- beta- 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.