Tailing of survivor curves of clostridial spores heated in edible oils

Tailing of survivor curves was observed for Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores heated whilst suspended in edible oils, but not for the same spores suspended in buffer (pH 7˙2) or mineral oil or for Bacillus cereus F4165/75 spores suspended in buffer or oils. The tailing cannot be ascribed to a genetic or developmental heterogeneity in the resistance of the spore population or to a heterogeneity of the treatment severity during heating. Heat adaptation due to the release of protective factor(s), to the selection for resistant spores or to the diffusion of oil constituents inside the spore protoplast to protect key molecules from heat denaturation was also ruled out. The tailing can be ascribed to spore clumping during the course of heating or to a heterogeneity in heat resistance of germination system(s) within spores, concurrently with the activation of a dormant germination system. It is probably caused by some oleic acid containing triglycerides.     

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low a _w of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105°C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7.2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of aw during heating. Spore resistance to heat increased in the order: buffer much less than commercial oil less than olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low aw (0.479 and 0.492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values (ca 28 degrees C in oils and 10 degrees-12 degrees C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7˙2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of a_w during heating. Spore resistance to heat increased in the order: buffer ⋖ commercial oil < olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low a_w (0˙479 and 0˙492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values ( ca 28°C in oils and 10°-12°C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

Soybean and sunflower oils increase the infectivity of Colletotrichum gloeosporioides f. sp. aeschynomene to Northern Jointvetch

Soybean and sunflower oils increased the level of infection of northern jointvetch, Aeschynomene virginica, plants by Colletotrichum gloeosporioides f. sp. aeschynomene. Inoculation of seedlings with spore suspensions containing 10% (v:v) soybean oil or 10% sunflower oil resulted in more disease than when inoculated with suspensions of spores in water alone. The lengths of the dew periods required to establish equivalent levels of disease by spore suspensions containing 10% soybean or 10% sunflower oil were approximately 4–8 h less compared to aqueous suspensions. Incubation of spores in 10% soybean oil followed by removal and resuspension in water did not affect the infectivity of spores when compared to spores incubated in aqueous suspensions. Spore germination and appressoria formation were unaffected by either of the oils tested in in vitro assays; however, in in vivo assays, 10% soybean oil and 10% sunflower oil increased spore germination in comparison to spores that were suspended in water.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Streptomyces viridochromogenes spore germination initiated by calcium ions.

Initiation of germination of heat-activated Streptomyces viridochromogenes spore occurs in media containing only calcium ions and organic buffer. The calcium-induced initiation of germination was accompanied by a decrease in absorbance of the spore suspension, an increased rate of endogenous metabolism, the loss of spore carbon, and the loss of heat resistance. Calcium amounts to 0.28% of the dry weight of freshly harvested spores. The amount of calcium remained the same after incubation of spores in water after heat activation. The spore content of calcium doubled after incubation in 0.5 mM CaCl2 for 5 min at 4 degrees C and during calcium-induced germination. Nearly all of the calcim appears to be bound to sites external to the spore membrane, since the chelating agents (ethylenedinitrilo) tetraacetic acid and arsenazo III removed virtually all of the calcium ions. The calcium ions must be present during the entire initiation of germination period. Germination ceases after an (ethylenedinitrilo) tetraacetic acid wash and begins again immediately after addition of calcium ions.     

Activation of Clostridium perfringens spores under conditions that disrupt hydrophobic interactions of biological macromolecules

The effect of hydrophobic interactions on the activation of C. perfringens NCTC 8679 spores was examined by heating spores under conditions that modify the hydrophobic properties of biological macromolecules. After the activation treatment and a washing procedure, germination was determined by measuring the decrease in optical density of spores suspended in an enriched germination medium. Activation was inhibited for spores that were treated under conditions that strengthen hydrophobic interactions, i.e., a decrease in pH or the presence of structure-stabilizing neutral salts. Activation was enhanced by treatment under conditions that disrupt hydrophobic interactions, i.e., an increase in pH or the presence of urea, dibucaine, or denaturing neutral salts. A deactivation treatment with the antichaotropic salt (NH4)2SO4 reversed activation by the chaotropic salt CaCl2 and to a lesser extent reversed activation by sublethal heat (75 degrees C) or urea. Most treatments that enhanced activation increased spore injury at higher temperatures, which resulted in decreased germination. However, (NH4)2SO4 and a decrease in pH from 5.6 to 3.8, which inhibited activation, also favored injury. The results suggest that activation involves a conformational change of a spore protein(s) through weakening of hydrophobic molecular forces and that activation and injury occur at different spore sites.     

Activation of Clostridium perfringens spores under conditions that disrupt hydrophobic interactions of biological macromolecules.

The effect of hydrophobic interactions on the activation of C. perfringens NCTC 8679 spores was examined by heating spores under conditions that modify the hydrophobic properties of biological macromolecules. After the activation treatment and a washing procedure, germination was determined by measuring the decrease in optical density of spores suspended in an enriched germination medium. Activation was inhibited for spores that were treated under conditions that strengthen hydrophobic interactions, i.e., a decrease in pH or the presence of structure-stabilizing neutral salts. Activation was enhanced by treatment under conditions that disrupt hydrophobic interactions, i.e., an increase in pH or the presence of urea, dibucaine, or denaturing neutral salts. A deactivation treatment with the antichaotropic salt (NH4)2SO4 reversed activation by the chaotropic salt CaCl2 and to a lesser extent reversed activation by sublethal heat (75 degrees C) or urea. Most treatments that enhanced activation increased spore injury at higher temperatures, which resulted in decreased germination. However, (NH4)2SO4 and a decrease in pH from 5.6 to 3.8, which inhibited activation, also favored injury. The results suggest that activation involves a conformational change of a spore protein(s) through weakening of hydrophobic molecular forces and that activation and injury occur at different spore sites.     

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.     

Properties of spores of Bacillus subtilis strains which lack the major small, acid-soluble protein.

Bacillus subtilis strains containing a deletion in the gene coding for the major small, acid-soluble, spore protein (SASP-gamma) grew and sporulated, and their spores initiated germination normally, but outgrowth of SASP-gamma- spores was significantly slower than that of wild-type spores. The absence of SASP-gamma had no effect on spore protoplast density or spore resistance to heat or radiation. Consequently, SASP-gamma has a different function in spores than do the other major small, acid-soluble proteins.     

Effects of inactivation or overexpression of the sspF gene on properties of Bacillus subtilis spores.

Inactivation of the Bacillus subtilis sspF gene had no effect on sporulation, spore resistance, or germination in a wild-type strain or one lacking DNA protective alpha/beta-type small, acid-soluble proteins (SASP). Overexpression of SspF in wild-type spores or in spores lacking major alpha/beta-type SASP (alpha- beta- spores) had no effect on sporulation but slowed spore outgrowth and restored a small amount of UV and heat resistance to alpha- beta- spores. In vitro analyses showed that SspF is a DNA binding protein and is cleaved by the SASP-specific protease (GPR) at a site similar to that cleaved in alpha/beta-type SASP. SspF was also degraded during spore germination and outgrowth, and this degradation was initiated by GPR.     

Sequence of events during Bacillus megaterim spore germination

Levinson, Hillel S. (U.S. Army Natick Laboratories, Natick, Mass.), and Mildred T. Hyatt. Sequence of events during Bacillus megaterium spore germination. J. Bacteriol. 91:1811-1818. 1966.-An integrated investigation of the sequence of events during the germination of Bacillus megaterium spores produced on three different media-Liver "B" (LB), synthetic, and Arret and Kirshbaum (A-K)-is reported. Heat-activated spores were germinated in a mixture of glucose and l-alanine. For studies of dipicolinic acid (DPA) release and increase in stainability and phase-darkening, germination levels were stabilized by the addition of 2 mm HgCl(2). Heat resistance was measured by conventional plating techniques and by a new microscopic method. The sequence (50% completion time) of LB spore germination events was: loss of resistance to heat and to toxic chemicals (3.0 min); DPA loss (4.7 min); stainability and Klett-measured loss of turbidity (5.5 min); phase-darkening (7.0 min); and Beckman DU-measured loss of turbidity (7.2 min). The time difference between 50% completion of stainability and complete phase darkening was 1.5 min, in excellent agreement with the microgermination time of 1.49 min as determined by observation of spores darkening under phase optics. Alteration of the sporulation medium modified the 50% completion times of these germination events, and, in some cases, their sequence. In the A-K spores, the rates of loss of heat resistance and DPA were substantially higher than those of the other germination events, whereas in spores produced in the LB and synthetic media all germination events followed an approximately parallel time course. This is discussed from the point of view of spore population heterogeneity and germination mechanisms.     

Initiation of Germination and Inactivation of Bacillus pumilus Spores by Hydrostatic Pressure

The effect of hydrostatic pressures as high as 1,700 atm at 25 C on the heat and radiation resistance of Bacillus pumilus spores was studied. Phosphate-buffered spores were more sensitive to compression than spores suspended in distilled water. Measurements of the turbidity of suspensions, the viability, refractility, stainability, dry weight, and respiratory activity of spores, and calcium and dipicolinic acid release were made for different pressures and times. Initiation of germination occurred at pressures exceeding 500 atm and was the prerequisite for inactivation by compression. The rate of initiation increased with increasing pressure at constant temperature. This result is interpreted as a net decrease in the volume of the system during initiation as a result of increased solvation of the spore components.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.     

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207-217. 1963.-It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates.     

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.