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.     

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.     

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.     

Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.     

The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration.

Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB. Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance. The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores. These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration. This defect in spore core dehydration was exaggerated by the additional loss of dacB expression. However, loss of dacB alone did not alter the spore core water content. Spores produced by spmA and spmB mutants germinated faster than did those of the wild type. Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth. Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant. Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants. These results are consistent with spore core water content being the major determinant of spore heat resistance. The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.     

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.     

Amino acids in the Bacillus subtilis morphogenetic protein SpoIVA with roles in spore coat and cortex formation

Bacterial spores are protected from the environment by a proteinaceous coat and a layer of specialized peptidoglycan called the cortex. In Bacillus subtilis, the attachment of the coat to the spore surface and the synthesis of the cortex both depend on the spore protein SpoIVA. To identify functionally important amino acids of SpoIVA, we generated and characterized strains bearing random point mutations of spoIVA that result in defects in coat and cortex formation. One mutant resembles the null mutant, as sporulating cells of this strain lack the cortex and the coat forms a swirl in the surrounding cytoplasm instead of a shell around the spore. We identified a second class of six mutants with a partial defect in spore assembly. In sporulating cells of these strains, we frequently observed swirls of mislocalized coat in addition to a coat surrounding the spore, in the same cell. Using immunofluorescence microscopy, we found that in two of these mutants, SpoIVA fails to localize to the spore, whereas in the remaining strains, localization is largely normal. These mutations identify amino acids involved in targeting of SpoIVA to the spore and in attachment of the coat. We also isolated a large set of mutants producing spores that are unable to maintain the dehydrated state. Analysis of one mutant in this class suggests that spores of this strain accumulate reduced levels of peptidoglycan with an altered structure.     

An Electrophoretic Study of Peptidases in Starter Streptococci and in Cheddar Cheese

Disinfection capacity determinations using solutions prepared from effervescent tablets of sodium dichloroisocyanurate (NaDCC) and from solutions of sodium hypochlorite (NaOCI) indicated high activity against a range of organisms, which represent adequate safety margins for disinfection. NaDCC showed significantly higher activity than NaOCl solution against all bacterial species tested but activity against Candida albicans was similar. Results indicate that differences in activity between NaDCC and NaOC1 formulations are not entirely due to pH effects and that fundamental differences exist between properties and mode of action of the 2 hypochlorite systems.     

Destruction of Bacillus licheniformis spores by microwave irradiation

Aims:  To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores.
Methods and Results:  We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0·5 kW) was modified to output power at 2·0 kW, which allowed a shorter sterilization cycle. A 2·0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0·5 kW microwave oven. In contrast to boiling and 0·5 kW microwave treatments, the 2·0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2·0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane.
Conclusions:  These results suggest that 2·0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study:  This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.     

Influence of cations on lysozyme-induced germination of coatless spores of Clostridium perfringens 8-6

Bacterial endospore germination is powerfully influenced by inorganic salts, cations having especially important effects. Spores of Clostridium perfringens 8-6 are unusual in lacking a spore coat; these spores germinate only in the presence of lysozyme, which readily digests the exposed cortex. Lysozyme-induced germination showed the same response to ionic strength and valence of cations as does lysozyme hydrolysis of peptidoglycan, and close parallels are evident in the influence of inorganic cations on germination of normal spores. La3+ and transition element cations inhibited lysozyme-induced germination at low concentration, again demonstrating parallels with their action on lysozyme digestion of peptidoglycan and on the germination of normal spores. The poly-cations poly(L-lysine) and Ruthenium Red inhibited at extremely low concentrations. Mn2+ and Co2+, at appropriately low concentrations, stimulated lysozyme germination of 8-6 spores and also lysis of Micrococcus lysodeikticus.     

Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde

G orman , S.P. S cott , E.M. H utchinson , E.P. 1984. Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde. Journal of Applied Bacteriology 56 , 95–102.
Bacillus subtilis spores with altered ionic content were tested for their susceptibility to lysis with lysozyme or sodium nitrite following treatment with glutaraldehyde. The Ca-form was more sensitive to glutaraldehyde (pH 4.0.and pH 7.9) than the untreated or H-form. Removal of spore coat dramatically increased sensitivity of the spore to glutaraldehyde. Pretreatment of spores, the coats of which had been extensively removed, with glutaraldehyde (pH 7.9) reduced the rate of lysis by lysozyme and by sodium nitrite, whereas glutaraldehyde at pH 4.0.had little effect. Glutaraldehyde pretreatment (pH 4.0 and pH 7.9) reduced the amount of hexosamine released by lysozyme but not by nitrite from isolated cortical fragments. Spore protoplasts were more susceptible to 0.01% (w/v) glutaraldehyde at pH 4.0 and isolated spore coats adsorbed alkaline glutaraldehyde more rapidly. These results are discussed in terms of a possible mode of action of glutaraldehyde on the bacterial spore.     

An effective iodide formulation for killing Bacillus and Geobacillus spores over a wide temperature range

AIMS: To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures. METHODS AND RESULTS: Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N. Kida, Y. Mochizuki and F. Taguchi, Microbiology and Immunology 2003; 47: 279-283). The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3. The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature. The KMT reagent had many practical advantages, i.e. activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures. The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen. CONCLUSIONS: The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores.     

Sporicidal Properties of Some Halogens

S ummary . Sodium hypochlorite, sodium dichloro iso cyanurate, dichlorodimethyl hydantoin, dibromodimethyl hydantoin and an iodophor have been examined for disinfectant activity against spores of Bacillus cereus. Under the test conditions used sodium hypochlorite was the most effective compound and dibromodimethyl hydantoin was least affected by increase in pH from 6.5 to 8; the activity of the iodophor was unaffected in the pH range 2.3–6.5. Bacillus subtilis spores were much more resistant to the disinfectants than were B. cereus spores. The addition of KBr to solutions of sodium dichloro iso yanurate enhanced its activity at pH 9 but not at pH 7 or 8.
Mixtures of 1.5–4% of NaOH with NaOCl (200 p of available chlorine/m) were much more rapidly sporicidal than either NaOH or NaOCl (pH 9 and above) alone.     

Mechanism of resistance of Bacillus subtilis spores to chlorhexidine

Chlorhexidine diacetate (CHA) was rather more sporicidal at 20C to ureadithreitol-sodium lauryl sulphate (UDS)-treated spores of Bacillus subtilis NCTC 8236 than to urea-dithiothreitol (UDT)-treated or normal (untreated) spores. UDS spores adsorbed more CHA from solution than did the other two forms. No differences in hydrophobicity, as determined by hydrophobic interaction chromatography (HIC) or bacterial adherence to hydrocarbon (BATH), could be detected between the three spore types. Germinating spores took up much less CHA than did outgrowing spores. Germinating cells were considerably more hydrophobic, as measured by the BATH technique, than outgrowing cells or normal spores. Chlorhexidine diacetate increased the apparent hydrophobicity of the two latter forms, but this effect could be partially reversed by subsequent exposure to a non-ionic surfactant.     

Mechanism of resistance of Bacillus subtilis spores to chlorhexidine

Chlorhexidine diacetate (CHA) was rather more sporicidal at 20 degrees C to urea-dithreitol-sodium lauryl sulphate (UDS)-treated spores of Bacillus subtilis NCTC 8236 than to urea-dithiothreitol (UDT)-treated or normal (untreated) spores. UDS spores adsorbed more CHA from solution than did the other two forms. No differences in hydrophobicity, as determined by hydrophobic interaction chromatography (HIC) or bacterial adherence to hydrocarbon (BATH), could be detected between the three spore types. Germinating spores took up much less CHA than did outgrowing spores. Germinating cells were considerably more hydrophobic, as measured by the BATH technique, than outgrowing cells or normal spores. Chlorhexidine diacetate increased the apparent hydrophobicity of the two latter forms, but this effect could be partially reversed by subsequent exposure to a non-ionic surfactant.     

PCR detection of potentially pathogenic aeromonads in raw and cold-smoked freshwater fish

AIMS: To determine the mechanism of killing of spores of Bacillus subtilis by ortho-phthalaldehyde (OPA), an aromatic dialdehyde currently in use as an antimicrobial agent. METHODS AND RESULTS: OPA is sporicidal, although spores are much more OPA resistant than are vegetative cells. Bacillus subtilis mutants deficient in DNA repair, spore DNA protection and spore coat assembly have been used to show that (i) the coat appears to be a major component of spore OPA resistance, which is acquired late in sporulation of B. subtilis at the time of spore coat maturation, and (ii) B. subtilis spores are not killed by OPA through DNA damage but by elimination of spore germination. Furthermore, OPA-treated spores that cannot germinate are not recovered by artificial germinants or by treatment with NaOH or lysozyme. CONCLUSIONS: OPA appears to kill spores by blocking the spore germination process. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to, and spore killing by, the disinfectant, OPA.     

Cortex peptidoglycan lytic activity in germinating Bacillus anthracis spores

Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that found in other species. During germination, B. anthracis released peptidoglycan fragments into the surrounding medium more quickly than some other species. A major lytic enzymatic activity was a glucosaminidase, probably YaaH, that cleaved between N-acetylglucosamine and muramic-delta-lactam. An epimerase activity previously proposed to function on spore peptidoglycan was not detected, and it is proposed that glucosaminidase products were previously misidentified as epimerase products. Spore cortex lytic enzymes and their regulators are attractive targets for development of germination inhibitors to kill spores and for development of activators to cause loss of resistance properties for decontamination of spore release sites.     

Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures

AIMS: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition. METHODS AND RESULTS: Bacillus subtilis spores prepared at temperatures from 22 to 48 degrees C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures. As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures. Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox. However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures. In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased. However, this latter change was not due to a reduction in cotA gene expression at higher temperatures. CONCLUSIONS: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The precise conditions for the formation of B. subtilis spores have a large effect on many spore properties.     

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.