The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Inactivation of Bacillus piliformis spores by heat and certain chemical disinfectants

The inactivation of Tyzzer's organism (Bacillus piliformis) spore isolated from rats by heat and various chemical disinfectants was studied. The spores were from B. piliformis-infected rat liver tissues. The spore suspension (10(4) 50% of rat liver lesion producing dose with prednisolone treatment/ml) was treated with heart or disinfectants. Inactivation of the spores was examined in experimentally infected rats. Rats were inoculated perorally with a treated spore suspension and injected subcutaneously with prednisolone. On the sixth day after inoculation, rats were examined grossly for liver lesions. Spores were inactivated at 80 degrees C for 15 min but not at 60 degrees C for 30 min. Spores were inactivated by 0.4% peracetic acid, 0.015% sodium hypochrolite, 1% iodophol, 5% phenol. Alcide and 0.37% formaldehyde solution, but not by 0.037% formaldehyde solution, 70% ethanol, 0.3% benzethonium chloride solution, 3% cresol and soap solution, or 4% chlorhexidine digluconate. These findings suggest that B. piliformis spores are relatively sensitive to heat and certain chemical disinfectants.     

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.     

Microgermination of Bacillus cereus spores

The biphasic nature of germination curves of individual Bacillus cereus T spores was further characterized by assessing the effects of temperature, concentration of germinants, and some inorganic cations on microgermination. Temperature was shown to affect both phases of microgermination as well as the microlag period, whereas the concentration of l-alanine and supplementation with adenosine exerted a significant effect only on the microlag period. The germination curves of individual spores induced by inosine were also biphasic and resembled those of spores induced by l-alanine. High concentrations (0.1 m or higher) of calcium and other inorganic cations prolonged both phases of microgermination, particularly the second phase, and had a less pronounced effect on the microlag period. The second phase of microgermination was completely inhibited when spores were germinated either in the presence of 0.3 m CaCl(2) or at a temperature of 43 C; this inhibition was reversible. Observations on the germination of spore suspensions (kinetics of the release of dipicolinic acid and mucopeptides, loss of heat resistance, increase in stainability, decrease in turbidity and refractility) were interpreted on the basis of the biphasic nature of microgermination. Dye uptake by individual spores during germination appeared also to be a biphasic process.     

Protease associated with spores of Bacillus cereus.

A proteolytic activity is associated with the dormant spores of Bacillus cereus T and can be solubilized by washing the spores with 1 M KCl. This proteolytic activity is responsible for the attack of beta chains of ribonucleic acid-polymerase in extracts of dormant spores of this organism.     

Thermal analysis of the spores of Bacillus cereus with special reference to heat activation.

The heat activation of bacterial spores was studied by means of differential thermal analysis in the temperature range 30-110 degrees C using the spores of Bacillus cereus. The thermogram showed three endothermic peaks at 56, 95, and 103 degrees C with one exothermic peak at 105 degrees C during the heating process. The spore coat separated from the native spores also showed a peak at 56 degrees C on its heating thermogram. The peak at 56 degrees C was reversible for both native spores and the spore coat. It was suggested that this peak at 56 degrees C might be related to the heat-activation process that takes place in the spore-coat region. It seems that the peak is due to the denaturation or the structural change of the spore-coat protein that might facilitate either the permeation of germination stimulators or the release of some germination inhibitor into or out of the spores.     

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores

Aims: To compare physical properties of spores that were produced in broth sporulation media at greater than 10⁸ spores ml⁻¹. Methods and Results: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0·68 ± 0·11 μm wide and 1·21 ± 0·18 μm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0·48 ± 0·38 μm³ and 0·97 ± 0·07 μm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l⁻¹ glutamate and antifoam. Spores were 0·95 ± 0·11 μm wide and 1·31 ± 0·17 μm long. Spore volumes were 0·78 ± 0·61 μm³ and volume-equivalent spherical diameters were 1·14 ± 0·11 μm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. Conclusions: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. Significance and Impact of the Study: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.     

Low-pH activation of Bacillus cereus spores

Heat activation of bacterial spores at low pH was investigated in detail. Unlike activation of spores in distilled water at a neutral pH, activation at low pH involves two superimposed processes: enhanced activation and death. Low-pH-activated spores failed to germinate in d-alanine, in contrast to spores activated at neutral pH, owing to the abolition of alanine-racemase activity. Morphological and permeability changes such as release and partial disruption of spores were dipicolinic acid-observed during low-pH activation. The kinetics pattern of low-pH activation, as well as the change in properties of the spores thereafter, suggest that the mechanism of low-pH activation differs from that of other kinds of heat-activation.     

Effect of relative humidity of hot air on the heat resistance of Bacillus cereus spores

The heat resistance to hot air of spores of Bacillus cereus (ATCC 14579) attached to carriers of stainless steel or silicone rubber was investigated in a range from 1% to 100% relative humidity (RH). Apart from an initial stage, linear survivor curves were obtained for all relative humidities. Neither the attachment itself nor the material of the carrier had an influence on the resistance. A distinct maximum of heat resistance was found at 40% RH. At 122°C the rate constants at 40% RH were five orders of magnitude smaller than at 100% RH and two orders of magnitude smaller than at 1% RH. At relative humidities of more than 40% the rate constants were strongly temperature dependent, whereas at lower relative humidities they were less temperature dependent. No significant influence of the relative humidity on the Arrhenius activation energy was found within each humidity range. The mean values were 295 kJ mol^-1 for relative humidities of 60% to 100% RH and 165 kJ mol^-1 for 1% to 20% RH. The occurrence of a maximum is ascribed to the existence of two inactivation mechanisms, the first is retarded and the second is accelerated by a reduction of relative humidity. It is assumed that the first mechanism is a protein denaturation. The second mechanism may be an oxidative process.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores.

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Isolation and properties of pili from spores of Bacillus cereus.

Structures whose morphology is identical to that of bacterial pili have been isolated from spores of Bacillus cereus. The structures are absent from log-phase and sporulating cells. The pili are 6.8 nm in diameter, are of variable length, and appear to emanate randomly from the exosporium. Examination of spores from 12 Bacillus species showed that only those from B. cereus and B. thuringiensis have pili. Although isolated spore pili were shown to be composed of protein, their subunit nature was not discernible due to the extreme insolubility of the structure. An antiserum to spore pili was labeled with ferritin and used to examine the distribution of pilus antigen on the outer spore surface.