Hydrophobicity of Bacillus and Clostridium spores

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Biochemical properties of Clostridium bifermentans spores.

As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue. As in spores of Bacillus species, germination of C. bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid. In contrast to spores of Bacillus species, dormant spores of C. bifermentans contained little free amino acid.     

Production and counting of spores of Clostridium chauvoei.

The concentration and viability of spores produced by four different strains of Clostridium chauvoei (C. feseri) grown in a modified medium for 18 days are described. The medium yielded enough viable spores for experimental work.     

Resporulation of outgrowing Bacillus subtilis spores.

Germinated spores of Bacillus subtilis were incubated in outgrowth medium and tested periodically for capacity to sporulate when suspended in sporulation medium. Concurrent measurements were made of deoxyribonucleic acid (DNA) content and numbers of cell division septa and nucleoids. Sporulation potential is shown to reach a peak at about 110 min at which time the chromosomes are probably well into the second round of replication. Experiments with nalidixic acid show that sporulation potential can be generated in the outgrowth medium even when DNA synthesis is largely prevented. Further experiments show that nalidixic acid apparently does not prevent the formation of DNA initiation complexes, which can subsequently function after resuspension in the sporulation medium. The results support those previously obtained with a temperature-sensitive DNA mutant which indicated that sporulation could only be induced at a specific stage of chromosome replication, and then only if the cells are in a state of nutritional "step-down".     

Glucose-triggered germination of Bacillus megaterium spores.

Triggering of germination in Bacillus megaterium QM B1551 spores with D-glucose was studied. First, the interaction of glucose with spores for less than 1 min resulted in triggering almost 90% of the spores after the glucose was removed by dilution. Therefore only a brief time is needed for glucose to trigger germination, and then the continuous presence of glucose is not necessary. Detectable uptake of glucose began 2 to 3 min after absorbance loss started, and a non-metabolizable glucose analog, methyl-alpha-D-glucopyranoside, triggered germination in the absence of detectable uptake. Several inhibitors that reduced or eliminated glucose uptake did not block triggering of germination. Therefore, glucose uptake may be a relatively late event and not a prerequisite for triggering of germination.     

Activation and injury of Clostridium perfringens spores by alcohols.

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Red pigment in Bacillus megaterium spores.

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Thermal inactivation and injury of Bacillus stearothermophilus spores.

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Clostridium spores as anti-tumour agents

The successful treatment of cancer remains a huge challenge. Consequently, efforts are being made to develop alternative methods of tumour therapy. One of these is the use of live Clostridium species, based on the observation that obligatory anaerobic bacteria specifically colonize the hypoxic and necrotic regions that are present in solid tumours but normally absent in other parts of the body. Although past results have fuelled scepticism about its clinical use, recent promising findings emphasize the potential of Clostridium-directed tumour therapy. These recent developments are reviewed and the reintroduction of this tumour-targeting protein delivery system into clinical settings is discussed.     

Development and validation of stable reference materials for food microbiology using Bacillus cereus and Clostridium perfringens spores

Aims: To develop a new type of microbiological Reference Materials (RMs), displaying long‐term stability at room temperature. The purpose was to produce and validate two batches of RMs for the enumeration of Bacillus cereus and Clostridium perfringens. Methods and Results: The RMs were based on spores of B. cereus and Cl. perfringens, adsorbed on calcium carbonate pellets. Two batches of 1000 units were manufactured and validated in compliance with ISO guide 35. After verification of their homogeneity, the stability of the ‘RM‐B. cereus’ and ‘RM‐Cl. perfringens’ batches was proven during at least 36 and 9 months, respectively, at room temperature. The validation study was completed by international collaborative trial involving 12 laboratories, allowing the validation of the assigned values. Conclusions: The methodology developed in this work enabled to produce easy‐to‐handle and cost‐effective RMs, displaying an unprecedented stability at room temperature, a good homogeneity and a precise and validated assigned value. Significance and Impact of the Study: This study revealed new paths for the development of stable microbiological RMs. Overcoming the intrinsic instability of the living cells makes it possible to produce valuable tools for the quality assurance of microbiology laboratories.     

UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

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.     

Triple fixation of Bacillus subtilis dormant spores.

A triple-fixation method with a sequential application of 5% glutaraldehyde, 1% osmium tetroxide, and 2% potassium permanganate gave superior preservation of the ultrastructure of Bacillus subtilis dormant spores with a thick spore coat.     

Germination of spores from Clostridium botulinum B-aphis and Ba410.

The germination of spores from Clostridium botulinum B-aphis and Ba410 was examined. In a complex medium, heat activation of spores from both strains doubled the germination rates and was required for germination in the presence of 2% NaCl. In a defined medium (CTB [D. B. Rowley and F. Feeherry, J. Bacteriol. 104:1151-1157, 1970]), the parent strain B-aphis germinated at a rate of 0.77% min-1 in the absence of NaCl and was not affected by 2% NaCl. A salt-tolerant derivative, strain Ba410, germinated at rates of 0.16% min-1 in CTB and 0.04% min-1 in CTB containing 2% NaCl. L-Alanine-triggered spores germinated faster than did L-cysteine-triggered spores from both strains. When both amino acids were present, B-aphis germinated rapidly in the absence of NaCl and had biphasic kinetics in the presence of NaCl. Strain Ba410 had biphasic kinetics in the absence of NaCl and germinated slowly with single-phase kinetics in the presence of NaCl. L-Alanine- and L-cysteine-triggered germinations were each inhibited by both D-alanine and D-cysteine, indicating a common germinant-binding site for both alanine and cysteine. Attempts to select for variants with amino acid-specific germinant-binding sites were unsuccessful. Differences in the germination kinetics of both strains could not be explained by ultrastructural differences. Transmission electron micrographs revealed striking similarities between the strains.     

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.     

Heat resistance of Clostridium sordellii spores

The thermal destruction kinetics of Clostridium sordellii spores was studied in this research. Decimal reduction times (D values) for C. sordellii ATCC 9714 spores ranged between 175.60 min for D₈₀ (the D value for spore suspensions treated at 80 °C) and 11.22 min for D₉₅. The thermal resistance (Z) and temperature coefficient (Q₁₀) values of spores were calculated to be as high as 12.59 °C and 6.23, respectively. At 95 °C, the relative thermal death rate and relative thermal death time of C. sordellii ATCC 9714 spores were found to be 0.0085/min and 118 min, respectively, indicating that the death rate of spores was 118 times lower at 95 °C than at 121.1 °C. Heat treatments at up to 85 °C for 120 min failed to cause a 100-fold destruction in spore populations of C. sordellii ATCC 9714. By contrast, spore counts were reduced by 2log₁₀ cycles within 73 min and 23 min at 90 °C and 95 °C, respectively. This is the first published report of thermal inactivation of C. sordellii spores; however, further studies are needed to confirm these results in real food samples.