Sensitivities of germinating spores and carvacrol-adapted vegetative cells and spores of Bacillus cereus to nisin and pulsed-electric-field treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

The crystalline layers in spores of Bacillus cereus and Bacillus thuringiensis studied by freeze-etching and high resolution electron microscopy

The comparative morphology of spores from Bacillus cereus, B. thuringiensis S-9 (wildtype), B. thuringiensis HB 9-1 (acrystaliferous mutant) and B. finitimus, was studied by the freeze-etching technique. Particular attention was given to the three crystalline layers found in these spores, the pitted layer, the parasporal layer, and the basal layer of the exosporium with lattice constants of 9.2 nm, 5.8 nm and 8.0 nm respectively. The parasporal layers, corresponding to the fraction of Fl of Scherrer and Somerville [18] were found to be membraneous sheets laying between spore and exosporium. Analysis of the isolated exosporium by negative staining and digital image processing revealed a lattice structure belonging to the p 6 hexagonal space group with a repeat of 8.0 nm. The unit cell contains 6 subunits, each 3 nm in diameter, which are centered around a hypothetical channel readily penetrated by the stain.     

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.     

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.     

Isolation, purification and identification of protective substances from a culture broth of germinating Bacillus cereus spores

A fraction increasing the resistance of resting spores to UV-irradiation and high temperature has been isolated from the culture medium at the stage of B. cereus st. 96 spore initiation. Amino acid analysis, gas chromatography, electrophoresis, and TLC of the products of acidic and alkaline hydrolysis of the isolated fraction demonstrated that the active component of the fraction was the lipoteichoic acid.     

Protein synthesizing systems from spores and vegetative cells of Bacillus cereus

A system of polyphenylalanine synthesis was optimized for a comparison of the polymerizing activities of ribosomes from spores and vegetative cells of Bacillus cereus T. Ribosomes of both types react similarly, showing a magnesium optimum of about 6 mM and spermidine optima of about 5 mM and 4 mM for vegetative and spore ribosomes, respectively. These lead to optimum mono- to multivalent cation rations of 9 and 10 respectively at 100 mM ammonium ion. A comparison of the response of these ribosomes to suboptimal concentrations of magnesium and spermidine show that they differ qualitatively from each other, suggesting that they possess different structure, macromolecular or ionic components.Abbreviations DFP diisopropylfluorophosphate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

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.     

Ribonucleic acid polymerase of germinating Bacillus cereus T.

It appears that a de novo synthesis of the deoxyribonucleic acid-dependent ribonucleic acid-polymerase in Bacillus cereus T takes place fairly late in outgrowth, at the onset of the vegetative cycle. Therefore, the ribonucleic acid-polymerase used by germinating spores is the one carried on from sporulating cells. However, the sporal enzyme is less soluble that the vegetative one, and its "core" is bound to two extra peptides. This complexing to other molecules could play a role in the regulation of gene expression during germination.     

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.