Inactivation of Bacillus spores by gaseous ozone

The sporicidal activity of ozone in the gas phase was investigated. Spores of six strains of Bacillus species deposited on filter paper or glass fibre filter were conditioned at different relative humidities (r.h.), and then exposed to ozone ranging in concentration from 0.5 to 3.0 mg/l at different r.h. There was a lag phase in the initial stage of exposure followed by an exponential decrease in the number of survivors with time, although no lag phase was observed with one strain. Inactivation rates increased with increasing exposure r.h. while no significant inactivation was attained at a r.h. of 50% or below. The conditioning r.h. influenced the duration of the lag phase. The D-values (decimal reduction time) in the logarithmic phase varied roughly in inverse proportion to the ozone concentration.     

Inactivation of Bacillus spores by gaseous ozone

The sporicidal activity of ozone in the gas phase was investigated. Spores of six strains of Bacillus species deposited on filter paper or glass fibre filter were conditioned at different relative humidities (r.h.), and then exposed to ozone ranging in concentration from 0.5 to 3.0 mg/1 at different r.h. There was a lag phase in the initial stage of exposure followed by an exponential decrease in the number of survivors with time, although no lag phase was observed with one strain. Inactivation rates increased with increasing exposure r.h. while no significant inactivation was attained at a r.h. of 50% or below. The conditioning r.h. influenced the duration of the lag phase. The D-values (decimal reduction time) in the logarithmic phase varied roughly in inverse proportion to the ozone concentration.     

Inactivation of Bacillus spores in reconstituted skim milk by combined high pressure and heat treatment

AIMS: To determine the resistance of a variety of Bacillus species spores to a combined high pressure and heat treatment; and to determine the affect of varying sporulation and treatment conditions on the level of inactivation achieved. METHODS AND RESULTS: Spores from eight Bacillus species (40 isolates) were high pressure-heat treated at 600 MPa, 1 min, initial temperature 72 degrees C. The level of inactivation was broad (no inactivation to 6 log10 spores ml(-1) reduction) and it varied within species. Different sporulation agar, high pressure equipment and pressure-transmitting fluid significantly affected the response of some isolates. Varying the initial treatment temperature (75, 85 or 95 degrees C) shifted the relative order of isolate high pressure-heat resistance. CONCLUSIONS: The response of Bacillus spores to combined high pressure-heat treatment is variable and can be attributed to both intrinsic and extrinsic factors. The combined process resulted in a high level of spore inactivation for several Bacillus species and is a potential alternative treatment to traditional heat-only processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Sporulation conditions, processing conditions and treatment temperature all affect the response of Bacillus spores to the combined treatment of high pressure and heat. High levels of spore inactivation can be achieved but the response is variable both within and between species.     

Dynamic light-scattering studies on the effect of heat and disinfectants on spores of Bacillus cereus.

The relative stability of spores of Bacillus cereus grown at three different temperatures was examined by using quasi-elastic light scattering (q.l.s.) in conjunction with turbidity and scanning electron microscopy (s.e.m.). Cultures grown at 20, 30 and 40 degrees C (BC20, BC30 and BC40 respectively) were compared in terms of (i) their effective hydrodynamic radius, rH, as determined from q.l.s. and (ii) their gross morphology, as determined from s.e.m. The effects of autoclaving at 121.1 degrees C on both these properties was also examined. We observed (1) that cultures BC20 and BC30 appeared to have similar values for rH, whereas that of BC40 appeared some 50% higher, and (2) BC40 had a correspondingly much lower heat resistance (its structural integrity was lost after about 20 min autoclaving, whereas that of BC20 and BC30 was retained even after 80 min autoclaving). These data were in good agreement with independent measurements of heat-resistance coefficients. Changes in the hydrodynamic radius, polydispersity (both using q.l.s.) and turbidity were monitored with time on addition of the disinfectants sodium hypochlorite and peracetic acid; again BC40 appeared to have a lower resistance.     

Inactivation of Bacillus thuringiensis spores by ultraviolet and visible light.

The inactivation of Bacillus thuringiensis spores and spores treated with two protectants, one proteinaceous and the other a commercial product, Shade, at wavelengths of the near-ultraviolet and visible spectra and at 254 nm is described. Determination of the inactivating wavelengths may be used to establish an efficient sunlight protective system for B. thuringiensis when used as a microbial insecticide.     

Differences in susceptibility to peroral inoculation with Bacillus piliformis spores in rats and mice

The median liver lesion producing doses of peroral inoculation with the spores of Tyzzer's organism RJ strain were 10(4. 3) in rats and 10(2. 7) in rats receiving prednisolone treatment for the provocation of Tyzzer's disease. In contrast to rats, liver lesions were detected in few mice inoculated perorally with 10(7) spores. In mice inoculated perorally with 10(7) spores, excretion of infective spores in the feces was detected only on day 1 postinoculation. On the other hand, no difference in susceptibility between rats and mice was detected upon intravenous inoculation with vegetative cells of the RJ strain. These results suggest that germination of the spores in the intestinal tract causes the difference in the susceptibility in rats and mice.     

Inactivation of Bacillus anthracis spores in murine primary macrophages

The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of d-alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of l-alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.     

Demonstration of antibodies to Bacillus piliformis in SPF colonies and experimental transplacental infection by Bacillus piliformis in mice.

Antibodies to Bacillus piliformis were demonstrated by the immunofluorescence antibody technique in sera from mice and rabbits from SPF breeding colonies. Mice in various stages of pregnancy were experimentally infected with Bacillus piliformis and killed 2 to 3 days later. The organism was demonstrated in the uterus, foetal membranes and in the liver of the foetuses. Infection was not limited to any particular stage of pregnancy.     

Mechanism of killing of spores of Bacillus cereus and Bacillus megaterium by wet heat

Aims: To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. Methods and Results: Bacillus cereus and B. megaterium spores wet heat‐killed 82–99% gave two bands on equilibrium density gradient centrifugation. The lighter band was absent from spores that were not heat‐treated and increased in intensity upon increased heating times. These spores lacked dipicolinic acid (DPA) were not viable, germinated minimally and had much denatured protein. The spores in the denser band had viabilities as low as 2% of starting spores but retained normal DPA levels and most germinated, albeit slowly. However, these largely dead spores outgrew poorly if at all and synthesized little or no ATP following germination. Conclusions: Wet heat treatment appears to kill spores of B. cereus and B. megaterium by denaturing one or more key proteins, as has been suggested for wet heat killing of Bacillus subtilis spores. Significance and Impact of the Study: This work provides further information on the mechanisms of killing of spores of Bacillus species by wet heat, the most common method for spore inactivation.     

Inactivation of spores of Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis by chlorination

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.     

Inactivation of thymidylate synthase by chlorine disinfectants

The effects of two chlorine disinfectants, calcium hypochlorite (HTH) and 3-chloro-4,4-dimethyl-2-oxazolidinone (Agent I), on the activity of thymidylate synthase have been investigated. Although both disinfectants inactivated the enzyme, the following differences were observed: When the two disinfectants were used at the same total chlorine concentration, the rate and extent of inactivation were greater with Agent I than HTH. The substrate dUMP partially protected thymidylate synthase from inactivation by Agent I, but it did not appreciably protect against inactivation by HTH. Large changes in the ultraviolet spectrum of the enzyme occurred when it was treated with HTH, which suggests reactions with aromatic amino acid side chains; no spectral changes occurred when thymidylate synthase was treated with Agent I. Blocking the sulfhydryl groups of thymidylate synthase with sulfhydryl reagents prevented the irreversible inactivation of the enzyme by Agent I, but not by HTH.     

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.     

Inactivation of spores by nonthermal plasmas

Bacterial and fungal spore contamination in different industries has a greater economic impact. Because of the remarkable resistance of spores to most physical and chemical microbicidal agents, their inactivation need special attention during sterilization processes. Heat and chemical sporicides are not always well suited for different sterilization/decontamination applications and carries inherent risks. In recent years, novel nonthermal agents including nonthermal plasmas are emerging as effective sporicides against a broad spectrum of bacterial and fungal spores. The present review discusses various aspects related to the inactivation of spores using nonthermal plasmas. Different types of both low pressure plasmas (e.g., capacitively coupled plasma and microwave plasma) and atmospheric pressure plasmas (e.g., dielectric barrier discharges, corona discharges, arc discharges, radio-frequency-driven plasma jet) have been successfully applied to destroy spores of economic significance. Plasma agents contributing to sporicidal activity and their mode of action in inactivation are discussed. In addition, information on factors that affect the sporicidal action of nonthermal plasmas is included.     

How moist heat kills spores of Bacillus subtilis

Populations of Bacillus subtilis spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but ≥98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.