Mechanism of the Heat Sensitization of Bacillus subtilis Spores by Ethidium Bromide

Pretreatment with ethidium bromide (5 μg/ml) followed by a water wash had no effect on unheated Bacillus subtilis spores, but the viability of these spores after heating was much lower than that of similarly heated spores exposed to water alone. The fate of water- or ethidium bromide-treated spores, unheated or heated, was followed by allowing them to germinate and outgrow in a minimal or a complex liquid medium. Spores exposed to ethidium bromide and then heated (85°C, 10 min) exhibited a developmental block during germination and outgrowth. Many of them were blocked at the stage when the bacterium emerged from the germinated spore. When 0.35 μg of ethidium bromide per ml was added to heated spores in the germination-growth medium, the outgrowth of heated spores was inhibited to the same extent as were pretreated spores. Ethidium bromide acted in the first hour of germination of heated spores since addition after this time was ineffective in inhibiting recovery events. Repair of heat-damaged spore DNA was detected during the first 2 h of germination. The addition of ethidium bromide (final concentration, 0.35 μg/ml) inhibited DNA repair during early outgrowth. Increased sensitivity of spores to heat after pretreatment with sublethal concentrations of ethidium bromide was due to the inhibition of the repair of heat-damaged DNA.     

Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores.

Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.     

Ethidium Bromide-Resistant Mutant of Bacillus subtilis

An ethidium bromide-resistant mutant (EB8) derived from a Marburg strain of Bacillus subtilis was found to be conditionally resistant to 10 mug of ethidium bromide per ml. Expression of resistance is complete only during vegetative growth at incubation temperatures above 30 C in complex medium or minimal medium supplemented with Casamino Acids. Strain EB8 is cross-resistant to acriflavine and proflavine. The ethidium bromide resistance marker is co-transduced with hisA1 at a frequency of 6% and is located to the right of hisA1 on the B. subtilis chromosome as it is usually represented on the map. Incorporation of [5-(3)H] uridine by strain EB8 showed that ribonucleic acid synthesis in both whole cells and protoplasts is ethidium bromide-resistant.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

The sporicidal activity and inactivation of chlorhexidine gluconate in aqueous and alcoholic solution

The sporicidal activity of chlorhexidine gluconate in aqueous and alcoholic solution against spores of Bacillus subtilis was examined over a broad temperature range. Activity was not observed at 20 degrees C even with concentrations as high as 10% chlorhexidine. Temperatures of 37 degrees-70 degrees C in combination with such high concentrations were required for reductions in spore viability. No viable spores were recoverable after 4 h contact at 55 degrees C with 10% aqueous chlorhexidine and none after 3 h contact with the alcoholic solution. Because of the high concentrations necessary for activity and the possibility of sporostasis occurring from inefficient chlorhexidine inactivation, existing inactivation systems were examined and modified to obtain satisfactory results. The spores of other Bacillus species examined (B. cereus, B. megaterium and B. stearothermophilus) proved to be considerably less resistant than those of B. subtilis. Presence of organic matter had little effect on the activity.     

Genotypic and phenotypic characteristics of L-line cells resistant to ethidium bromide

Stable mutants resistant to ethidium bromide in concentrations of 1 and 3 micrograms/ml have been selected in a single step in L cells. The frequency of spontaneously occurring ethidium bromide resistant clones after the exposure to 1 microgram/ml of the drug has been established as 5.10(-5). Resistant variants were induced following treatment with mutagen N-methyl-N-nitro-N-nitrosoguanidine. The resistant clones were shown to be resistant to higher concentration of the agent then which was used for selection. In multistep selection, a number of clones resistant to ethidium bromide in concentration up to 50 micrograms/ml was obtained. The alteration in the permeability of plasma membrane to the drug is the clue mechanism of the resistance.     

A note on the effect of sporulation conditions on the resistance of Bacillus spores to heat and chemicals

Spores of Bacillus subtilis SA22 harvested after 22 d incubation on nutrient agar at 30°C were more resistant to 0–04% peracetic acid at 20°C than spores harvested following 2 d incubation. Similarly, spores of B. subtilis globigii B17, harvested after 7 d incubation on a sporulation agar were up to 10 times less resistant to 0.04% peracetic acid at 20°C than spores harvested after 35 d incubation. An increase in resistance to heating at 100°C and to exposure to 17.7% hydrogen peroxide at 20°C occurred as the age of B. subtilis SA22 spores prior to harvesting increased, whereas differences in resistance were not observed with spores of B. subtilis globigii B17.     

Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Heterologous transformation in Bacillus subtilis. 1. Transmission of DNA of the R1drd19 plasmid]

Bac. subtilis 168 (BD-25) cells were infected with DNA of plasmide R1drd19 isolated from E. coli strain; transformants resistant to streptomycin (500 microgram/ml) and kanamycin (40 microgram/ml) appeared with the frequency of 2.10(-6). These transformants retained resistance to the mentioned antibiotics stably. A satellite DNA peak was revealed in centrifugation in the density gradient of cesium chloride with ethidium bromide. It was possible to infect cells of Bac. subtilis 168 (BD-25) with plasmide DNA isolated from the transformants. Plasmide transduction with the aid of phages AR9 and PBSI multiplied on the transformant strains was also effected. Physico-chemical analysis of the transformed plasmide DNA was conducted; its molecular weight was determined.     

Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays

DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 microgram/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 microgram/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT I. EXPERIMENTS WITH SUSPENSIONS OF SPORES IN WATER

SUMMARY: A method of assessing the sporicidal efficiency of a UHT milk sterilizing plant operating on water is described. Water heavily contaminated with spores of a strain of Bacillus subtilis was filtered, after treatment in the plant, through membrane filters and the surviving spores estimated by incubation of the membranes in nutrient agar. With this plant a temperature of c . 135° caused a 99·99999% kill of B. subtilis spores. Confirmation of the lethal effects of temperatures above 135° was obtained by passing treated water into 10 gal churns containing sterile concentrated nutrient broth and incubating the churns.     

Sensitivity of Encephalitozoon cuniculi to various temperatures, disinfectants and drugs.

Spores of Encephalitozoon cuniculi were exposed to various temperature or to disinfectants, and their infectivity was then tested on monolayer cultures of canine kidney cells. The maximum survival time for spores suspended in medium 199 was 1 day at -20 degrees C, 98 days at 4 degrees C, 6 days at 22 degrees C, and 2 days at 37 degrees C. Only 2.5% survived 30 min at 56 degrees C. Boiling for 5 min or autoclaving at 120 degrees C for 10 min killed all spores. Dry spores survived less than a week at 4 degrees C but at least 4 weeks at 22 degrees C. Exposure for 30 min to recommended working concentrations of 9 of the 11 disinfectants tested killed all spores. The growth-inhibition effect of 7 antibiotics and chemotherapeutics was studied on canine kidney cell culture inoculated with E. cuniculi. None could completely inhibit growth. The most effective was chloroquine phosphate which, at a concentration of 12.5 mg per 1000 ml culture medium and during a test period of 8 weeks, reduced the harvest of E. cuniculi to 31% of that from inoculated, untreated cultures.     

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.     

Application of ethidium bromide for high-resolution banding analysis of chromosomes from human malignant cells

Ethidium bromide was added to cultured human leukemic bone marrow and solid tumor cells to evaluate its inhibitory effect on mitotic chromosome condensation and its possible application to high-resolution banding analysis. In most experiments ethidium bromide treatment resulted in a high proportion of mitotic cells having elongated chromosomes, without remarkable reduction in either the mitotic index or quality of metaphase chromosomes. Optimal effect on chromosome length was obtained by adding 10 micrograms/ml of ethidium bromide during the final 2 hr of culture. Because of the simplicity and reproducibility of the technique involved, ethidium bromide can be used routinely to extend the length of chromosomes for fine-banding analysis of malignant cells.     

Effects of three oxidizing biocides on Legionella pneumophila serogroup 1.

A study was conducted to determine the bactericidal effects of ozone and hydrogen peroxide relative to that of free chlorine on Legionella pneumophila serogroup 1. In laboratory batch-type experiments, organisms seeded at various densities were exposed to different concentrations of these biocides in demand-free buffers. Bactericidal effects were measured by determining the ability of L. pneumophila to grow on buffered charcoal-yeast extract agar supplemented with alpha-ketoglutarate. Ozone was the most potent of the three biocides, with a greater than 99% kill of L. pneumophila occurring during a 5-min exposure to 0.10 to 0.30 micrograms of O3 per ml. The bactericidal action of O3 was not markedly affected by changes in pH or temperature. Concentrations of 0.30 and 0.40 micrograms of free chlorine per ml killed 99% of the L. pneumophila after 30- and 5-min exposures, respectively. A 30-min exposure to 1,000 micrograms of H2O2 per ml was required to effect a 99% reduction of the viable L. pneumophila population. However, no viable L. pneumophila could be detected after a 24-h exposure to 100 or 300 micrograms of H2O2 per ml. Attempts were made to correlate the biocidal effects of O3 and H2O2 with the oxidation of L. pneumophila fatty acids. These tests indicated that certain biocidal concentrations of O3 and H2O2 resulted in a loss or severe reduction of L. pneumophila unsaturated fatty acids.     

Effects of three oxidizing biocides on Legionella pneumophila serogroup 1

A study was conducted to determine the bactericidal effects of ozone and hydrogen peroxide relative to that of free chlorine on Legionella pneumophila serogroup 1. In laboratory batch-type experiments, organisms seeded at various densities were exposed to different concentrations of these biocides in demand-free buffers. Bactericidal effects were measured by determining the ability of L. pneumophila to grow on buffered charcoal-yeast extract agar supplemented with alpha-ketoglutarate. Ozone was the most potent of the three biocides, with a greater than 99% kill of L. pneumophila occurring during a 5-min exposure to 0.10 to 0.30 micrograms of O3 per ml. The bactericidal action of O3 was not markedly affected by changes in pH or temperature. Concentrations of 0.30 and 0.40 micrograms of free chlorine per ml killed 99% of the L. pneumophila after 30- and 5-min exposures, respectively. A 30-min exposure to 1,000 micrograms of H2O2 per ml was required to effect a 99% reduction of the viable L. pneumophila population. However, no viable L. pneumophila could be detected after a 24-h exposure to 100 or 300 micrograms of H2O2 per ml. Attempts were made to correlate the biocidal effects of O3 and H2O2 with the oxidation of L. pneumophila fatty acids. These tests indicated that certain biocidal concentrations of O3 and H2O2 resulted in a loss or severe reduction of L. pneumophila unsaturated fatty acids.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses.

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.