Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T

S.F. BLOOMFIELD AND M. ARTHUR. 1992. Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortes material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T.

Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortex material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Sporicidal efficacy of pH-adjusted bleach for control of bioburden on production facility surfaces

pH-adjusted bleach was one of the agents used to disinfect contaminated public buildings in the USA following the 2001 bioterrorist attack with Bacillus anthracis spores. A USEPA fact sheet describes the preparation of pH-adjusted bleach by combining diluted sodium hypochlorite (NaOCl) with a controlled amount of 5 % acetic acid. This paper reports a modification of this procedure to qualify the use of pH-adjusted bleach for routine disinfection of cleanroom surfaces in pharmaceutical manufacturing facilities whenever a short contact time is desirable or there is a need for enhanced germicidal or sporicidal activity. Adjustment of pH was obtained reproducibly with either acetic acid or HCl, confirming the feasibility of developing standard procedures for the controlled addition of acid to diluted NaOCl solutions without compromising operator safety and convenience. Efficacy testing using spores from an in-house isolate of Bacillus pumilus confirmed that NaOCl solutions in the pH 5–8 range have much greater sporicidal activity on surfaces than do unadjusted alkaline solutions (pH > 11). With a contact time of 0.5 min, the log₁₀ reduction in spore viable counts was >5.4 for the five representative surfaces tested relative to untreated controls. Solutions of pH-adjusted NaOCl are known to be less stable than unadjusted alkaline solutions. Stability studies were performed by monitoring sporicidal efficacy, level of free available chlorine (FAC), and pH. Testing included several NaOCl concentrations and adjustment to different starting pHs. The efficacy of pH-adjusted solutions persisted in open containers for at least 12 h even though some FAC degradation occurred. In addition, solutions of 0.29 or 0.50 % NaOCl stored at room temperature protected from light retained efficacy for at least 4 weeks, indicating that short-term storage of solutions is possible following pH adjustment. The inorganic chemical degradation of pH-adjusted NaOCl solutions generates chlorate ion, an undesirable by-product. A comparison of chemical stability for 0.12, 0.25, and 0.50 % NaOCl solutions adjusted to different initial pHs indicated that the least chlorate formation occurred with 0.12 % NaOCl.     

Cloning of Ruminococcus albus endo-β-1,4-glucanase and xylanase genes

Buffered solutions of chlorine-releasing agents, sodium hypochlorite (NaOCl), sodium dichloroisocyanurate (NaDCC), and chloramine-T, showed similar activity against vegetative cells of Bacillus subtilis but there was considerable variation in activity against spores, NaOCl showing higher activity than NaDCC, which in turn was more active than chloramine-T. The effect of coat and cortex extraction on sporicidal activity was determined. It was concluded that although spore coats play a role, they do not totally account for chlorine resistance and that the cortex is also involved, probably through its function in maintaining a low water level in the enclosed core. Observed differences in the sporicidal action of NaOCl, NaDCC, and chloramine-T may be related to their differing ability to produce core and cortex degradation.     

The Influence of High Concentrations of Carbon Dioxide on the Germination of Bacterial Spores

The influence of carbon dioxide at 1–55 atm on the germination of Clostridium sporogenes, Clostridium perfringens and Bacillus cereus spores in a complex medium was studied. The germination studies at atmospheric pressure were done in the pH range 5.2–6.7. Controls at the same pH were done in 100% nitrogen. Carbon dioxide at atmospheric pressure (1 atm) inhibited the spore germination of B. cereus spores but strongly enhanced the germination rate of those of the clostridia. Spore germination of Cl. sporogenes and Cl. perfringens was inhibited completely at 10 atm and at 25 atm, respectively. The germination rate in carbon dioxide or nitrogen was generally higher at pH 6.7 than at 5.2–6.0.     

The effects of some halogen-containing compounds on Bacillus subtilis endospores

Sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were more active against Bacillus subtilis 8236 spores in both viability and in germination and outgrowth studies than were polyvinylpyrrolidone-iodine (PVP-I) and Lugol's solution. Of the two chlorine compounds studied NaOCl proved to be the more active. The two iodine-containing compounds gave contrasting results with the Lugol's solution demonstrating increased antibacterial activity with increasing available iodine concentration. The antibacterial behaviour of PVP-I, however, reflected the more complex nature of aqueous iodine-surfactant mixtures. Initially, non-complexed iodine concentration (the active species) increased with increasing total available iodine concentration, resulting in increasing antibacterial activity. However, due to changes in the physical properties of the mixture, a maximum concentration of non-complexed iodine was reached so that a further increase in total available iodine resulted in a decrease in non-complexed iodine concentration and consequently a decrease in the antibacterial activity of the solution was observed. A greater inhibitory effect was observed in subsequent germination and outgrowth studies when spores were pre-treated with respective biocide than when untreated spores were added to germination media containing biocide at t = 0. This may reflect a combination of different contact times plus the neutralizing effect of the germination media on such halogen compounds.     

Comparison of coats and surface-dependent properties of Bacillus cereus T prepared in two sporulation environments

The surface or coat-associated properties of Bacillus cereus T spores produced from modified G medium (MGM) and fortified nutrient agar (FNA) were compared. The two populations appeared structurally similar by transmission electron microscopy. Spores prepared on FNA were more susceptible to ozone inactivation than MGM-prepared spores. When activated by heating for 15 min at 70–85°C, FNA-prepared spores were optimally activated at 85°C and did not become hydrophilic on heat activation while MGM spores were optimally activated at 70°C and became hydrophilic on activation. Susceptibility to removal of coat and outer membrane by chemical and enzymatic extraction treatments was measured by monitoring reduced ability to germinate in nutrients and acquired ability to germinate in the presence of lysozyme. Bacillus cereus T MGM-prepared spores germinated in lysozyme upon<1 h exposure to sodium dodecyl sulphate-dithiothreitol. FNA-prepared spores were lysozyme sensitive after > 2 h treatment. Thus, B. cereus T FNA spore coats and outer membranes were more resistant to these denaturing agents. Transmission electron micrographs revealed no change in appearance of extracted spores. Sporulation environment must be considered when laboratory-prepared spores are used to assess or predict the effect of control procedures on spores present in nature.     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Effects of sporulation pH on the heat resistance and the sporulation of Bacillus cereus

Spores of Bacillus cereus ATCC 7004, 4342 and 9818 were obtained in nutrient agar at several pH from 5·9 to 8·3. The optimum pH for sporulation was around 7, but good production of spores was obtained in the range 6·5–8·3. With all three strains, D -values clearly dropped with sporulation pH, decreasing by about 65% per pH unit. z -Values were not significantly modified ( P > 0·05) by this factor. Mean z -values of 7·13 °C ± 0·16 for strain 7004, 7·67 °C ± 0·04 for 4342 and 8·80 °C ± 0·64 for 9818 were obtained.     

Inactivation of Bacillus anthracis spores by liquid biocides in the presence of food residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H(2)O(2)) for 10 min at 10, 20, or 30 degrees C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H(2)O(2) concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20 degrees C, the minimum concentrations of peroxyacetic acid, H(2)O(2), and NaOCl (as total available chlorine) predicted to inactivate 6 log(10) CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10 degrees C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log(10) CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H(2)O(2) sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log(10) CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.     

The effects of some halogen-containing compounds on Bacillus subtilis endospores

Sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were more active against Bacillus subtilis 8236 spores in both viability and in germination and outgrowth studies than were polyvinylpyrrolidone-iodine (PVP-I) and Lugol's solution. Of the two chlorine compounds studied NaOCl proved to be the more active. The two iodine-containing compounds gave contrasting results with the Lugol's solution demonstrating increased antibacterial activity with increasing available iodine concentration. The antibacterial behaviour of PVP-I, however, reflected the more complex nature of aqueous iodine—surfactant mixtures. Initially, non-complexed iodine concentration (the active species) increased with increasing total available iodine concentration, resulting in increasing antibacterial activity. However, due to changes in the physical properties of the mixture, a maximum concentration of non-complexed iodine was reached so that a further increase in total available iodine resulted in a decrease in non-complexed iodine concentration and consequently a decrease in the antibacterial activity of the solution was observed.
A greater inhibitory effect was observed in subsequent germination and outgrowth studies when spores were pre-treated with respective biocide than when untreated spores were added to germination media containing biocide at t = 0. This may reflect a combination of different contact times plus the neutralizing effect of the germination media on such halogen compounds.     

Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis

Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.     

Hypochlorite effects on spores and spore forms of Bacillus subtilis and on a spore lytic enzyme

G orman , S.P., S cott , E.M. & H utchinson , E.P. 1984. Hypochlorite effects on spores and spore forms of- Bacillus subtilis and on a spore lytic enzyme. Journal of Applied Bacteriology 56 , 295–303.
Spores of Bacillus subtilis NCTC 10073 were converted to ion-exchange (Ca, H) forms and coat-defective (urea-mercaptoethanol, urea-dithiothreitol-sodium lauryl sulphate) forms. The resistance of these to sodium hypochlorite (1000 parts/10⁶ free chlorine) was compared and related to uptake from which the assumed monolayer capacities were calculated. Hypochlorite effects on spore protoplasts and cortical fragments were also examined in relation to DPA and hexosamine release. A spore lytic enzyme was extracted and examined in respect of hypochlorite activity. The results are discussed in terms of the mechanism and site of action of hypochlorite on the bacterial spore.     

Forces involved in adhesion of Bacillus cereus spores to solid surfaces under different environmental conditions

H usmark , U. & R önner , U. 1990. Forces involved in adhesion of Bacillus cereus spores to solid surfaces under different environmental conditions. Journal of Applied Bacteriology 69 , 557–562.
The adhesion of Bacillus cereus spores (NCTC 2599) to hydrophobic and hydro-philic glass surfaces was studied when environmental conditions were varied. The spores were exposed in media of different polarities as well as different pH and ionic concentrations. With increasing ethanol concentrations, the polarity of the medium was decreased and the predominant force of attraction was found to be hydrophobic. The spore surface was uncharged at a pH around 3, at which value the spore was most adhesive to both hydrophobic and hydrophilic glass. This could be attributable to the absence of electrostatic repulsion. An increased ionic concentration of the bulk increased the degree of adhesion especially to the hydrophilic surfaces. This indicates the suppression of a solvation barrier at high ionic concentrations, when the polymers of the spore surface become dehydrated.     

Growth of Bacillus cereus during rice tapé fermentation

Growth of Bacillus cereus NCIB 8579 was studied on four varieties of rice with and without tapé fermentation. Fermented and unfermented rice supported growth of B. cereus to 10⁷–10⁹cfu/g. With fermentation the pH fell and numbers of B. cereus remained high ( ca 10⁸cfu/g) except on black glutinous rice where numbers declined. Cells added at different fermentation times survived less well as fermentation progressed and the pH fell. Once growth on rice is established, B. cereus is able to survive fermentation, probably as spores.     

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium. Methods and RESULTS: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h). CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.     

Revival of Bacillus subtilis spores from biocide-induced injury in the germination process

Spores of Bacillus subtilis NCTC 8236 were treated with glutaraldehyde, Lugol's iodine, polyvinylpyrrolidone-iodine (PVP-I), sodium hypochlorite or sodium dichloroisocyanurate (NaDCC). After exposure survivors were enumerated on nutrient agar containing potential revival agents (subtilisin, lysozyme, calcium dipicolinate, calcium lactate). Of these, only calcium lactate had any significant enhancing effect and then only with iodine-treated spores. Calcium lactate (9 mmol 1⁻¹) in nutrient broth enhanced the rate and extent of germination of iodine-treated spores but not of spores previously subjected to glutaraldehyde, hypochlorite or NaDCC.     

An effective sporicidal reagent against Bacillus subtilis spores

We developed a reagent which showed significant sporicidal activity against Bacillus subtilis spores. This reagent was composed of ethylenediaminetetraacetic acid, disodium salt (EDTA-2Na), ferric chloride hexahydrate (FeCl3 x 6H2O) and ethanol (tentatively designated as the ethanol reagent). The ethanol reagent showed pH- and temperature-dependent sporicidal activity. At pH 0.3, its activity was almost the same as that of 0.05% sodium hypochlorite at 20 C and was higher at 37 C than at 20 C. The activity of the ethanol reagent was similar both with and without 10% serum. The ethanol reagent might be applicable for disinfecting Bacillus spores.     

Inhibition of bacterial growth, enterotoxin production, and spore outgrowth in strains of Bacillus cereus by bacteriocin AS-48

Bacteriocin AS-48 showed high bactericidal activity for mesophilic and psychrotrophic strains of Bacillus cereus over a broad pH range. AS-48 inhibition of the enterotoxin-producing strain LWL1 was enhanced by sodium nitrite, sodium lactate, and sodium chloride. The latter also enhanced AS-48 activity against strain CECT 131. Bacterial growth and enterotoxin production by strain LWL1 were completely inhibited at bacteriocin concentrations of 7.5 microg/ml. At subinhibitory bacteriocin concentrations, enterotoxin production decreased markedly and sporulation was delayed. Intact spores were resistant to AS-48 but became gradually sensitive to AS-48 during the course of germination.     

Inactivation of Bacillus anthracis Spores by Liquid Biocides in the Presence of Food Residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H₂O₂) for 10 min at 10, 20, or 30°C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H₂O₂ concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20°C, the minimum concentrations of peroxyacetic acid, H₂O₂, and NaOCl (as total available chlorine) predicted to inactivate 6 log₁₀ CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10°C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log₁₀ CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H₂O₂ sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log₁₀ CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.