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.     

Effect of lipid materials on heat resistance of bacterial spores

The apparent heat resistance of spores of Bacillus megaterium, B. subtilis, B. cereus, B. stearothermophilus, and Clostridium botulinum type E in lipids was investigated and compared with the resistance of the spores in phosphate buffer solution. The most pronounced increase in heat resistance was noted for B. subtilis and C. botulinum type E, the increase varying with the type of lipid used. A high water content of the lipids used as heating menstruum lowered the heat resistance of the spores. Possible explanations for the high heat resistance of spores in lipids are discussed.     

Effect of Temperature on Germination of Spores from Some Bacillus and Clostridium Species

The effect of temperature on germination of spores of Bacillus subyilis, B. megaterium. B. cereus, Clostridium sporogenes, Cl. butyricum and Cl. bifermentans was studied. At lower temperatures (+5°C to +10°C) the three Glostridium species germinated to a less extent than the three Bacillus. species. The optimum temperature for germination of the six species varied between +35°C and +45°C. The Clostridium species were more tolerant to heat than the Bacillus species.     

Effects of heat-, CaCl2- and ethanol-treatments on activation of Bacillus spores

The effects of heat, CaCl2, and ethanol on activation of Bacillus spores were determined by monitoring the absorbance decrease during germination in inosine. Bacillus cereus T, B. subtilis A and B. megaterium QM B1551 spores were activated by heat- and CaCl2-treatments. Ethanol activated B. megaterium and B. subtilis spores yet did not activate B. cereus spores. CaCl2- and ethanol-activations were less effective than heat-activation as judged by optimal germination rates and germination extents. The presence of CaCl2 during heat-treatment inhibited heat-activation of all three Bacillus spores without affecting viability or dipicolinic acid content of the spores. The electrophoretic patterns of coat plus outer membrane proteins extracted from Bacillus spores treated with CaCl2 and heat in the presence of CaCl2 were similar to each other and were distinctively different from the patterns of proteins from unactivated spores or the spores treated with heat and/or ethanol.     

Sensitivity of Three Selected Bacterial Species to Ozone

THE MINIMAL LETHAL CONCENTRATION OF OZONE IN WATER WAS DETERMINED FOR THREE BACTERIAL SPECIES: Escherichia coli, Bacillus cereus, and Bacillus megaterium. A contact period of 5 min was selected. The lethal threshold concentration for the cells of B. cereus was 0.12 mg/liter while that for E. coli and B. megaterium was 0.19 mg/liter. Low concentrations of ozone were ineffective when organic matter was present to interfere with the action of ozone on the bacterial cells. Also determined during the study was the sensitivity of spores of B. cereus and B. megaterium to ozone in water. The threshold concentration required to kill the spores of both species was 2.29 mg/liter. The cells and spores of these organisms exhibited the "all-or-none" die-away phenomenon normally associated with ozone treatment.     

Comparison of various properties of low-molecular-weight proteins from dormant spores of several Bacillus species.

Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared. All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan. The proteins could be subdivided into two groups: group I (B. megaterium A and C proteins, B. cereus A protein, and B. subtilis alpha and beta proteins) and group II (B. cereus and B. megaterium B proteins and B. subtilis gamma protein). Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline. Similarly, proteins in each group were more closely related immunologically. However, antisera against a B. megaterium group I protein cross-reacted more strongly with the B. megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B. megaterium group II protein cross-reacted most strongly with B. megaterium group I proteins. Analysis of the primary sequences at the amino termini and in the regions of the B. cereus and B. subtilis proteins cleaved by the B. megaterium spore protease revealed that the B. cereus A protein was most similar to the B. megaterium A and C proteins, and the B. cereus B protein and the B. subtilis gamma protein were most similar to the B. megaterium B protein. However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.     

Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Perturbation of the heat resistance of bacterial spores by sporulation temperature and ethanol

Spores ofBacillus megaterium, B. subtilis, andB. stearothermophilus, harvested from cultures grown and sporulated at different temperatures or in the presence of ethanol, had different thermal resistance. There was a direct relationship between the sporulation temperature and the spore-killing temperature. The spores were more temperature-sensitive when formed in ethanol-supplemented media. Temperature and ethanol are known to perturb the degree of order within membranes and to alter membrane functions. Thus, alteration of spore membranes is an additional factor in the multifactorial nature of heat resistance. Another interpretation may be that heat shock proteins, known to be induced by heat, are formed during sporulation and may increase the thermostability of the spores.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Photodynamic inactivation of Bacillus spores, mediated by phenothiazinium dyes

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.     

Identification of homologues of a functionally important 50 S ribosomal protein in different bacterial species.

Functional homology between the 50 S ribosomal protein L3 from Bacillus stearothermophilus and a protein from each of three other species of Bacillaceae (Bacillus licheniformis, Bacillus megaterium, and Bacillus subtilis) is demonstrated by substituting each of the proteins for B. stearothermophilus L3 in active reconstituted ribosomes. The structurally related protein from Escherichia coli, L2, cannot replace B, stearothermophilus L3, nor can any other E. coli protein.     

Dielectric properties of native and decoated spores of Bacillus megaterium.

A general model for use in interpreting dielectric data obtained with bacterial endospores is developed and applied to past results for Bacillus cereus spores and new results for Bacillus megaterium spores. The latter were also subjected to a decoating treatment to yield dormant cells with damaged outer membranes that could be germinated with lysozyme. For both spore types, core ions appeared to be completely immobilized, and decoating of B. megaterium spores did not affect this extreme state of electrostasis in the core. The cortex of B. megaterium appeared to contain a high level of mobile ions, in the cortex of B. cereus. The outer membrane-coat complex of B. megaterium acted dielectrically as an insulating layer around the cortex, so that native dormant spores showed a Maxwell-Wagner dispersion over the frequency range from about 1 to 20 MHz. The decoating treatment resulted in a shift in the dispersion to frequencies below the range of observation. Increases in cell conductivity in response to increases in environmental ionic strength indicated that the coats. of B. megaterium could be penetrated by environmental ions and that they had an inherent fixed charge concentration of about 10 to 20 milliequivalents per liter. In contrast, the dispersion for B. cereus spores was very sensitive to changes in environmental ion concentration, and it appeared that some 40% of the spore volume could be penetrated by environmental ions and that these ions traversed a dielectrically effective layer, either the exosporium or the outer membrane. It appears that dormancy is associated with extreme electrostasis of core ions but not necessarily of ions in enveloping structures and that the coat-outer membrane complex is dielectrically effective but not required for maintenance of extreme electrostasis in the core.     

Room temperature sterilization of surfaces and fabrics with a One Atmosphere Uniform Glow Discharge Plasma

We report the results of an interdisciplinary collaboration formed to assess the sterilizing capabilities of the One Atmosphere Uniform Glow Discharge Plasma (OAUGDP). This newly-invented source of glow discharge plasma (the fourth state of matter) is capable of operating at atmospheric pressure in air and other gases, and of providing antimicrobial active species to surfaces and workpieces at room temperature as judged by viable plate counts. OAUGDP exposures have reduced log numbers of bacteria, Staphylococcus aureus and Escherichia coli, and endospores from Bacillus stearothermophilus and Bacillus subtilis on seeded solid surfaces, fabrics, filter paper, and powdered culture media at room temperature. Initial experimental data showed a two-log₁₀ CFU reduction of bacteria when 2 × 10² cells were seeded on filter paper. Results showed ≥3 log₁₀ CFU reduction when polypropylene samples seeded with E. coli (5 × 10⁴) were exposed, while a 30 s exposure time was required for similar killing with S. aureus-seeded polypropylene samples. The exposure times required to effect ≥6 log₁₀ CFU reduction of E. coli and S. aureus on polypropylene samples were no longer than 30 s. Experiments with seeded samples in sealed commercial sterilization bags showed little or no differences in exposure times compared to unwrapped samples. Plasma exposure times of less than 5 min generated ≥5 log₁₀ CFU reduction of commercially prepared Bacillus subtilis spores (1 × 10⁶); 7 min OAUGDP exposures were required to generate a ≥3 log₁₀ CFU reduction for Bacillus stearothermophilus spores. For all microorganisms tested, a biphasic curve was generated when the number of survivors vs time was plotted in dose-response cures. Several proposed mechanisms of killing at room temperature by the OAUGDP are discussed. Received 06 June 1997/ Accepted in revised form 01 November 1997     

Quantification of magnetic susceptibility in several strains of Bacillus spores: implications for separation and detection

Three strains of Bacillus: Bacillus atrophaeus (formally Bacillus globigii), Bacillus thuringiensis, and Bacillus cereus were tested for their intrinsic magnetic susceptibility. All three strains when sporulated demonstrated significant magnetic susceptibility using an instrument referred to as Cell Tracking Velocimetry. Energy dispersive spectroscopy also confirmed the presence of paramagnetic elements, Fe and Mn, in the spore form of the bacteria. It was demonstrated that this magnetic susceptibility is sufficient to separate and deposit these spores on glass slides in a magnetic deposition system. These results indicate the potential to separate spores with intrinsic magnetic susceptibility directly out of water or air samples.     

多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

巨大芽孢杆菌是微生物肥料生产中的常用菌种, 与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌, 传统方法区分两者费时费力, 有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物, 并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增, 以检验其特异性。结果显示, 巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物, 其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105 CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测, 其鉴定结果与常规鉴定结果一致。以上结果表明, 本文建立的多重PCR方法具有较高的特异性和灵敏度, 可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌, 在微生物肥料检测方面有良好的实用前景。     

Cloning and nucleotide sequence of a plasmid-carried gene coding for a minor small, acid-soluble protein from Bacillus megaterium spores.

The gene (termed sspG) coding for a small, acid-soluble protein (SASP) from spores of Bacillus megaterium QMB1551, termed SASP-G, has been cloned, and its nucleotide sequence has been determined. SASP-G is a 42-residue protein containing 2 tryptophan and 11 lysine residues, including a hexalysine sequence, and is not homologous to any previously described SASP. The sspG gene appears to be an additional member of the sigma G regulon. No gene homologous to sspG is present in B. cereus T or B. subtilis 168. The reason for the absence of sspG from other Bacillus species appears to be that in B. megaterium, sspG is present only on a 111-kb plasmid; this plasmid is not present in B. cereus T or B. subtilis 168. The sspG gene is the first forespore-expressed gene found to be on a plasmid.     

Isolation of endospore-forming bacilli toxic to Culiseta longiareolata (Diptera: Culicidae) in Jordan

Ten of 80 endospore-forming bacilli, isolated from various habitats of Jordan, were found to be highly toxic to the 4th instar larvae of Culiseta longiareolata (Macquart). The bacilli were identified into the following species and strains: Bacillus sphaericus (H6), B. sphaericus (H9a, 9b), B. cereus Frankland and Frankland, B. brevis Migula and B. megaterium Bary. Bacillus cereus comprised 50% of the isolates. The toxic concentrations of these isolates against C. longiareolata ranged between 1.2 x 10(7) and 1.1 x 10(9) viable spores ml-1.     

3种细菌芽胞遗传同源性及抗力对比研究

比较细菌芽胞的遗传同源性、结构和抗力差异,为炭疽芽胞的替代试验菌的可靠评价提供依据。采用资料检索、生物信息分析、显微镜观察和微生物学技术分析不同芽胞的遗传同源性、超微结构和抗力差异。炭疽芽胞与腊样芽胞的结构和大小相似,生物遗传同源性最近,对热力、UVC和有效氯的抗力相近。腊样芽胞对炭疽芽胞的代表性最好,可以倾向性选用其替代炭疽芽胞进行试验研究。     

Effect of the internal water activity of bacterial spores on their heat resistance in water

The water contents and effective a_w of the core, cortex, and coat of spores in water, as well as the masses of the core, cortex, and coat plus exosporium in the dry state, are calculated from volumes, dry densities, and water absorption isotherms of the sporal components. From data presented here for spores ofBacillus subtilis var.niger andBacillus cereus T, and from previously published data forBacillus stearothermophilus, the logarithm of the heat resistance of the spores in water is linearly related to the effective a_w of their core and cortex.