Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. METHODS AND RESULTS: Bacillus anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to > or =1000 ppm hydrogen peroxide gas for 20 min. Hydrogen peroxide exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials except G. stearothermophilus on industrial carpet. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with both surrogates. The effectiveness of gaseous hydrogen peroxide on the growth of biological indicators and spore strips was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days postexposure, decontaminated biological indicators and spore strips exhibited no growth, while the nondecontaminated samples displayed growth. CONCLUSIONS: Significant differences in decontamination efficacy of hydrogen peroxide gas on porous and nonporous surfaces were observed when comparing the mean log reduction in B. anthracis spores with B. subtilis and G. stearothermophilus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using hydrogen peroxide gas.     

Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials

Aims: We evaluated whether lowering pH (with acetic acid) and raising free available chlorine (FAC) levels in bleach solutions would improve efficacy in inactivating Bacillus spores on different materials. We also determined how varying pH and FAC levels affected bleach stability. Methods and Results: Acidified bleach solutions with pH levels of 4·5, 6 and 7·5 and FAC levels between 5000 and 10 000 ppm were evaluated for decontamination efficacy against Bacillus subtilis spores inoculated onto test coupons made from wood, ceramic and galvanized steel. Lowering the pH or increasing the FAC level improved efficacy in some of the tests, but depended on the material, which significantly affected decontamination efficacy. The acidified bleach at pH of 7·5 was significantly less effective than bleach at a pH of 4·5 or 6. The FAC levels in the bleach were the most stable at pH 4·5, and stability at pH 4·5 was not significantly affected by the initial FAC level. Conclusions: It may be advisable to use bleach solutions with lower pH (rather than high FAC levels) in light of both the decontamination efficacy and bleach stability results. For wood materials, use of sporicides other than acidified bleach may be warranted. Significance and Impact of the Study: These results may be useful in preparing acidified bleach solutions for decontamination of materials contaminated with spores such as Bacillus anthracis.     

A preliminary assessment of Bacillus anthracis spore inactivation using an electrochemically activated solution (ECASOL)

AIM: To evaluate the efficacy of electrochemically activated solution (ECASOL) in decontaminating Bacillus anthracis Ames and Vollum 1B spores, with and without changing the source water hardness and final ECASOL pH. METHODS AND RESULTS: Five different ECASOL formulations were generated, in which the source water hardness and final ECASOL pH were varied, resulting in cases where significant changes in free available chlorine (FAC) and oxidative-reduction potential (ORP) were observed. B. anthracis Ames and Vollum 1B spores were suspended in the various ECASOL formulations for 30 min, and decontamination efficacy was determined; calcium hypochlorite [5% high-test hypochlorite (HTH)] was used as a positive control. The five different ECASOL formulations yielded mean FAC levels ranging from 305 to 464 ppm, and mean ORP levels ranging from +826 to +1000 mV. Exposure to all the ECASOL formulations and 5% HTH resulted in >or=7.0 log reductions in both B. anthracis Ames and Vollum 1B spores. CONCLUSIONS: The present testing demonstrated that ECASOL with a minimum of c. 300-ppm FAC levels and +800-mV ORP inactivated the B. anthracis spores in suspension, similar to 5% HTH. Significance and Impact of the Study: These results provide information for decontaminating B. anthracis Ames and Vollum 1B spores in suspension using ECASOL.     

Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores

Aims:  This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants.
Methods and Results:  The first phase determining the availability of the liquid decontaminant within the SAP showed that the SAP gel containing pH-adjusted sodium hypochlorite (NaOCl) inhibited B. anthracis growth while the water control SAP gel had no affect on growth. For testing surface decontamination, B. anthracis spores were dried onto steel coupons painted with chemical agent resistant coating and exposed to SAP containing either pH-adjusted NaOCl, chlorine dioxide (ClO₂) or hydrogen peroxide/peracetic acid (H₂O₂/PA) for 5 and 30 min. At contact times of both 5 and 30 min, all of the SAP gels containing pH-adjusted NaOCl, ClO₂ or H₂O₂/PA inactivated B. anthracis spores at levels ranging from 2·2 to ≥7·6 log reductions.
Conclusions:  Incorporation of three commercially available decontaminant technologies into a SAP gel promotes inactivation of B. anthracis spores without observable physical damage to the test surface.
Significance and Impact of the Study:  This work provides preliminary data for the feasibility of using SAP in inactivating B. anthracis spores on a nonporous surface, supporting the potential use of SAP in surface decontamination.     

Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to the general biological decontamination agents, Decon and Oxone. METHODS AND RESULTS: Spores of B. subtilis treated with Decon or Oxone did not accumulate DNA damage and were not mutagenized. Spore killing by these agents was increased if spores were decoated. Spores prepared at higher temperatures were more resistant to these agents, consistent with a major role for spore coats in this resistance. Neither Decon nor Oxone released the spore core's depot of dipicolinic acid (DPA), but Decon- and Oxone-treated spores more readily released DPA upon a subsequent normally sublethal heat treatment. Decon- and Oxone-killed spores initiated germination with dodecylamine more rapidly than untreated spores, but could not complete germination triggered by nutrients or Ca(2+)-DPA and did not degrade their peptidoglycan cortex. However, lysozyme treatment did not recover these spores. CONCLUSIONS: Decon and Oxone do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents is the spore coat. Spore killing by both agents renders spores defective in germination, possibly because of damage to the inner membrane of spore. SIGNIFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of the killing of bacterial spores by Decon and Oxone.     

Inhibitory effects of single-walled carbon nanotubes on biofilm formation from Bacillus anthracis spores

This study reports the inhibitory effect of single walled carbon nanotubes (SWCNTs) on biofilm formation from Bacillus anthracis spores. Although the presence of 50 to 100 μg ml^?1 of SWCNTs in the suspension increased spore attachment in the wells of 96-well plates, the presence of 200 μg ml^?1 of SWCNTs in the germination solution decreased the germination percentage of the attached spores by 93.14%, completely inhibiting subsequent biofilm formation. The inhibition kinetics of 50 μg ml^?1 SWCNTs on biofilm formation showed that this concentration inhibited biofilm formation by 81.2% after incubation for 48 h. SWCNT treatment in the earlier stages of biofilm formation was more effective compared to treatment at later stages. Mature biofilms were highly resistant to SWCNT treatment.     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.     

Validation of a polynomial regression model: the thermal inactivation of Bacillus subtilis spores in milk

AIMS: The predicted survival of Bacillus subtilis 168 spores from a polynomial regression equation was validated in milk. METHODS AND RESULTS: Bias factor suggested as an index of model performance was used to validate the polynomial model predictions in ultrahigh temperature (UHT) treated and sterilized whole and skim milk. Model predictions were fail safe, predicting higher D-values (decimal reduction times) in buffer than actually noted in milk. CONCLUSIONS: The D-values for spores were lower in milk as compared with those predicted in potassium phosphate buffer contrary to the popular expectation of better spore survival in complex food systems. The Bias factor, a quantitative measure of the model performance, indicated that on average the model predictions exceed the observations by 40% in the case of whole milk and by 60% in the case of skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is an attempt to ascertain the extent of reliability that one can safely place in polynomial model predictions, without compromising on the safety or palatability of foods where it is eventually intended to be applied. The work has also highlighted the differences in the thermal inactivation pattern of spores in buffer and in milk with a possible influence of the various constituents of milk. The work will assist the dairy industry to better design thermal processes to ensure longer shelf life of dairy foods.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

CEMOVIS on a pathogen: analysis of Bacillus anthracis spores

Background information. Under conditions of starvation, bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions, the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the aetiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analysing the organization of the spore and the changes during germination at the electron microscopy level were only previously performed with techniques relying on fixation with aldehydes and osmium, and subsequent dehydration, which can produce artefacts. Results and conclusions. In the present study, we describe the morphology of dormant spores using CEMOVIS (Cryo‐Electron Microscopy of Vitreous Sections). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore, we chose to analyse spores of the closely related non‐pathogen Bacillus cereus T. For none of the investigated structures could we find a difference in morphology induced by aldehyde fixation compared with the native preparations for CEMOVIS. This result legitimizes work with aldehyde‐fixed spores from B. anthracis. Using CEMOVIS, we describe two new structures present in the spore: a rectangular structure, which connects the BclA filaments with the basal layer of the exosporium, and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated, and the layered organization of the coat, as well as the repetitive structure within it, remain unchanged.