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Aims: To investigate effects of the cationic surfactant cetyltrimethylammonium bromide (CTAB), a disinfectant, on spores of Bacillus species., Methods and Results: The ability of CTAB to trigger release of Bacillus spores' large depot of dipicolinic acid (DPA) in a 1 : 1 chelate with Ca 2+ (CaDPA), and to kill spores was investigated. CTAB-triggered CaDPA release from spores of Bacillus subtilis, Bacillus cereus and Bacillus megaterium, but was not followed by completion of germination. CaDPA release triggered by CTAB increased at higher temperatures, and was optimal for B. subtilis spores at pH 9·4 and 30 μg ml -1 CTAB. CTAB also killed Bacillus spores as shown by plate counts and vital staining of treated dormant spores, and after their germination. However, B. cereus and B. megaterium spores were more CTAB-sensitive than were B. subtilis spores. CaDPA release from and killing of CTAB-treated spores of isogenic B. subtilis mutants lacking germination proteins was also examined, and compared with effects of the well-known germinant dodecylamine on spores, to determine how CTAB exerts its effects on spores., Conclusions: The results of this investigation showed that CTAB kills spores of three Bacillus species, perhaps by damaging the spore inner membrane, although it is also possible that some killing by this agent follows its triggering of spore germination., Significance and Impact of the Study: The results of this work indicate that CTAB is also a disinfectant, but also a sporicide, and may be a useful adjunct in spore decontamination, especially at higher temperatures., (© 2019 The Society for Applied Microbiology.)

Aims: To investigate effects of fluoride ion (F - ) on, and kinetics of its movement into and out of, spores and growing cells of Bacillus species., Methods and Results: Effects of F - on Bacillus cell growth, spore germination and outgrowth and heat resistance were investigated, as well as F - movement into and out of spores using 19 F-NMR. F - inhibited Bacillus subtilis spore germination and outgrowth, and YhdU, now named FluC, was crucial to prevent F - accumulation in growing cells and to minimize F - inhibition of spore germination. Dormant wild-type, yhdU and coat defective B. subtilis spores, and Bacillus cereus spores incubated in 40 mmol l -1 NaF for 48 h accumulated 2-2·6 mol l -1  F - and its movement into Bacillus spores was highest at low pH. Bacillus subtilis spores lacking Ca-dipicolinic acid accumulated higher F - levels than wild-type spores., Conclusions: These results are consistent with F - incorporation into the dormant spore core, and as HF and/or NaF, but not CaF 2 . YhdU played no significant role in F - uptake or efflux in dormant spores, but assisted in F - export early in spore germination., Significance and Impact of Study: This knowledge provides new insight into effects of F - on Bacillus cells and spores and how this anion moves into, and out of spores., (© 2018 The Society for Applied Microbiology.)

Aims: To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs., Methods and Results: Spore viability, dipicolinic acid (DPA) release, nucleic acid staining, α-glucosidase activity, protein structure and mutagenesis were measured during autoclaving of G. stearothermophilus spores. Loss of DPA and increases in spore core nucleic acid staining were slower than loss of spore viability. Spore core α-glucosidase was also lost more slowly than spore viability, although soluble α-glucosidase in spore preparations was lost more rapidly. However, spores exposed to an effective autoclave sterilization lost all viability and α-glucosidase activity. Apparently killed autoclaved spores were not recovered by artificial germination in supportive media, much spore protein was denatured during autoclaving, and partially killed autoclave-treated spore preparations did not acquire mutations., Conclusions: These results indicate that autoclave-killed spores cannot be revived, spore killing by autoclaving is likely by protein damage, and spore core α-glucosidase activity is lost more slowly than spore viability., Significance and Impact of the Study: This work provides insight into the mechanism of autoclave killing of spores of an organism used in BIs, and that a spore enzyme in a BI is more stable to autoclaving than spore viability., (© 2016 The Society for Applied Microbiology.)

Aims: To determine how hydrated Bacillus anthracis spores are killed in a high-temperature gas environment (HTGE), and how spores of several Bacillus species including B. anthracis are killed by UV radiation, dry heat, wet heat and desiccation., Methods and Results: Hydrated B. anthracis spores were HTGE treated at c. 220°C for 50 ms, and the treated spores were tested for germination, mutagenesis, rupture and loss of dipicolinic acid. Spores of this and other Bacillus species were also examined for mutagenesis by UV, wet and dry heat and desiccation. There was no rupture of HTGE-treated B. anthracis spores killed 90-99·9%, no mutagenesis, and release of DPA and loss of germination were much slower than spore killing. However, killing of spores of B. anthracis, Bacillus thuringiensis and Bacillus subtilis by UV radiation or dry heat, but not wet heat in water or ethanol, was accompanied by mutagenesis., Conclusions: It appears likely that HTGE treatment kills B. anthracis spores by damage to spore core proteins. In addition, various killing regimens inactivate spores of a number of Bacillus species by the same mechanisms., Significance and Impact of the Study: This work indicates how hydrated spores treated in a HTGE such as might be used to destroy biological warfare agent stocks are killed. The work also indicates that mechanisms whereby different agents kill spores are similar with spores of different Bacillus species., (© 2013 The Society for Applied Microbiology.)

Aim: Heat resistance, germination and outgrowth capacity of Bacillus cereus spores in processed foods are major factors in causing the emetic type of gastrointestinal disease. In this study, we aim to identify the impact of different sporulation conditions on spore properties of emetic toxin-producing B. cereus strains., Methods and Results: Spore properties of eight different emetic toxin-producing strains were tested, with spores produced in five different sporulation conditions: aerated liquid cultures, air-liquid biofilms, 1.5% agar plates, 0.75% agar plates and swarming colonies. Model food studies revealed spores from emetic toxin-producing strains to germinate efficiently on meat broth- and milk-based agar plates, whereas germination on rice-based agar plates was far less efficient. Notably, spores of all strains germinated efficiently when 0.1% meat broth was added to the rice plates. Analysis of spores derived from different environments revealed large diversity and showed biofilm spores for the strains tested to be the largest in size, the most heat resistant and with the lowest germination capacity., Conclusions: Sporulation in complex conditions such as biofilms and surface swarming colonies increases heat resistance and dormancy of spores., Significance and Impact of the Study: The results obtained imply the importance of sporulation conditions on spore properties of emetic toxin-producing B. cereus strains, as occur for instance in food processing., (© 2012 The Society for Applied Microbiology.)

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 MnCl(2) and up to 1 mmol l(-1) MnCl(2). 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 H(2)O(2). 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., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Aims: A general protocol was derived for optimizing the production of pure, high concentration Clostridium endospore suspensions., Methods and Results: Two sporulation methods were developed that yielded high concentrations of notably pure Clostridium sporogenes, C. hungatei and C. GSA-1 (Greenland ice core isolate) spore suspensions (10 ml of 10(9) spores ml(-1) with >99% purity each). Each method was derived by evaluating combinations of three sporulation conditions, including freeze drying of inocula, heat shock treatment of cultures, and subsequent incubation at suboptimal temperatures that yielded the highest percentage of sporulation. Pure spore suspensions were characterized in terms of dipicolinic acid content, culturability, decimal reduction time (D) value for heat inactivation (100 degrees C) and hydrophobicity., Conclusions: While some Clostridium species produce a high percentage of spores with heat shock treatment and suboptimal temperature incubation, other species require the additional step of freeze drying the inocula to achieve a high percentage of sporulation., Significance and Impact of the Study: Pure Clostridium spore suspensions are required for investigating species of medical and environmental importance. Defining the conditions for optimal spore production also provides insight into the underlying mechanisms of Clostridium sporulation.

Aims: To compare the properties of the spores of Bacillus subtilis prepared in liquid and on plates., Methods and Results: The spores of B. subtilis were prepared at 37 degrees C using a nutrient exhaustion medium either in liquid or on agar plates. The levels of core water, dipicolinic acid (DPA) and small, acid-soluble spore proteins (SASP) were essentially identical in spores made in liquid or on plates. Spores prepared in liquid were killed approximately threefold more rapidly at 90 degrees C in water than the spores prepared on plates, and the spores prepared in liquid were more sensitive to nitrous acid and a diluted stable superoxidized water. Spores prepared in liquid also germinated more rapidly with several agents than those prepared on plates. Pellets of spores prepared on plates were darker than spores prepared in liquid, and spores prepared in liquid had more readily extracted coat protein. However, there were no major differences in the relative levels of individual coat proteins or the cross-linking of the coat protein GerQ in the two types of spores, although the inner membrane of spores prepared on plates had a higher ratio of anteiso- to iso-fatty acids., Conclusions: The preparation in liquid yielded spores with some different properties than those made on agar plates. Spores made in liquid had lower resistance to heat and several chemicals, and germinated more readily with several agents. There were also differences in the composition of the inner membrane of spores prepared under these two conditions. However, there were no major differences in the levels of DPA, core water, SASP and individual coat proteins or the cross-linking of a coat protein in spores made in liquid and on plates., Significance and Impact of the Study: This work demonstrates that the preparation method can affect the resistance and germination properties of bacterial spores, even if an identical medium and temperature are used. Evidence was also obtained consistent with the role of the inner membrane in spore resistance and germination, and that some factor in addition to core water, DPA and SASP content plays a role in spore resistance to wet heat.

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.

Aims: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them., Methods and Results: Spores of B. subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective alpha/beta-type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway. Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins. Spores prepared at a higher temperature were also much more resistant to these agents. Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite- and chlorine dioxide-treated spores much more readily released DPA upon a subsequent normally sub-lethal heat treatment than did untreated spores. Hypochlorite-killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+-DPA, and these spores could not be recovered by lysozyme treatment. Chlorine dioxide-treated spores also did not germinate with Ca2+-DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients. However, while germinated chlorine dioxide-killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties., Conclusions: Hypochlorite and chlorine dioxide do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat. Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane. While chlorine dioxide-killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage., Significance and Impact of the Study: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide.