Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens

Heat resistance at 95 C, heat activation at 75 C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains. The D95-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively. The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively. Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KC1 medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl2 in the presence of CO2 without heat activation (A-type). The strains were tested for enterotoxigenicity by a reversed passive latex-agglutination (RPLA) test. All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative. A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation. All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative.     

The Effect of Sodium Chloride on Heat Resistance and Recovery of Heated Spores of Bacillus stearothermophilus

Increasing concentrations (2, 4 and 8% w/v) of sodium chloride in the heating medium progressively reduced the heat resistance of spores of Bacillus stearothermophilus. Storage at 4° in water or in sodium chloride solutions had little effect on viable counts of unheated spores, but with the increase in sodium chloride concentration there was a reduction in the heat activation effect and a small decrease in heat resistance of the spores. Increasing the severity of heat treatment rendered spores increasingly sensitive to sodium chloride in the plating medium.     

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D_120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.     

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.     

Mechanism of the Heat Sensitization of Bacillus subtilis Spores by Ethidium Bromide

Pretreatment with ethidium bromide (5 μg/ml) followed by a water wash had no effect on unheated Bacillus subtilis spores, but the viability of these spores after heating was much lower than that of similarly heated spores exposed to water alone. The fate of water- or ethidium bromide-treated spores, unheated or heated, was followed by allowing them to germinate and outgrow in a minimal or a complex liquid medium. Spores exposed to ethidium bromide and then heated (85°C, 10 min) exhibited a developmental block during germination and outgrowth. Many of them were blocked at the stage when the bacterium emerged from the germinated spore. When 0.35 μg of ethidium bromide per ml was added to heated spores in the germination-growth medium, the outgrowth of heated spores was inhibited to the same extent as were pretreated spores. Ethidium bromide acted in the first hour of germination of heated spores since addition after this time was ineffective in inhibiting recovery events. Repair of heat-damaged spore DNA was detected during the first 2 h of germination. The addition of ethidium bromide (final concentration, 0.35 μg/ml) inhibited DNA repair during early outgrowth. Increased sensitivity of spores to heat after pretreatment with sublethal concentrations of ethidium bromide was due to the inhibition of the repair of heat-damaged DNA.     

Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores

Assessing true numbers of viable anthrax spores is complex. Optimal heat activation conditions vary with species, media and germinants. Published time/temperature combinations for Bacillus anthracis spores range from 60 degrees C for 1, post-heating counts were less than their pre-heating counterparts on between 71% and 88% of occasions. A high probability was found of viable spore counts differing significantly from counts determined microscopically, with differences of almost 1 log possible. Viable counts were lower than microscopic counts in 15 of 18 tests.     

Role of Curing Agents in the Preservation of Shelf-stable Canned Meat Products

Experiments were conducted to gain a better understanding of the mechanism by which sodium chloride, sodium nitrate, and sodium nitrite supplement the action of heat in preserving canned cured meat products. Heated spores of putrefactive anaerobe 3679h were less tolerant of all three curing agents in the outgrowth medium than were unheated spores. When the curing agents were added to the heating menstruum, but not to the outgrowth medium, sodium chloride and sodium nitrate tended to protect the spores against heat injury, but sodium nitrite did not. When the spores were both heated and cultured in the presence of the curing agents: (i) nitrate and salt increased the apparent heat resistance at low concentrations (0.5 to 1%) but decreased it at concentrations of 2 to 4%; (ii) nitrite was markedly inhibitory, especially at pH 6.0. At the normal pH of canned luncheon meats (approximately 6.0), nitrite appears to be the chief preservative agent against spoilage by putrefactive anaerobes.     

Factors Affecting the Rate of Heat-induced Spore Germination in Dictyostelium discoideum

Washed spores of Dictyostelium discoideum, strains NC-4H, NC-4D, and V-12, germinated rapidly after being heat shocked at or near 45.0 C for 30 min. Cultures of the slime molds were grown in association with Escherichia coli B/r as the host bacterium; spores taken from plates of synthetic medium had a higher final germination value than spores from complex medium containing peptone and yeast extract. Young spores germinated more rapidly than older spores. Optimal germination occurred between pH 6.0 and 7.0, and, of the buffers tested, potassium phosphate allowed the most rapid germination. After heat shocking, spores were diluted into fresh oxygenated buffer to provide enough oxygen for completion of germination. Germination occurred most rapidly between incubation temperatures of 22 and 25 C.     

Heat activation of Streptomyces viridochromogenes spores.

The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.     

Requirement for and Sensitivity to Lysozyme by Clostridium perfringens Spores Heated at Ultrahigh Temperatures

The requirement of ultrahigh temperature (UHT)-treated Clostridium perfringens spores for lysozyme and the sensitivity of heated and unheated spores to lysozyme were studied. The UHT-treated spores requiring lysozyme for germination and colony formation originated from only a small portion of the non-UHT-treated spore population. This raised a question of whether the requirement for lysozyme was natural to the spores or was induced by the UHT treatments. However, these spores did not require lysozyme for germination before UHT treatment, which confirmed that the requirement for lysozyme had been induced by the UHT treatment. Only 1 to 2% of the spores were naturally sensitive to lysozyme; therefore, the mere addition of lysozyme to the plating medium did not permit the enumeration of all survivors. Treatment of UHT-treated spores with ethylenediaminetetraacetate (EDTA) sensitized the spores to lysozyme and increased by 10- to 100-fold the number of survivors that were detected on a medium containing lysozyme. Under the heating conditions used, spores that were naturally sensitive to lysozyme and spores that required EDTA treatment were equally heat resistant.     

Activation and germination of Bacillus thuringiensis spores in Manduca sexta larval gut fluid

Incubation of Bacillus thuringiensis HD-1 spores in the larval gut fluid of Manduca sexta (tobacco hornworm) resulted in increased viable counts, conversion to phase-dark spores, and a loss of absorbance in spore suspensions, indicative of spore germination. Heat-activated and untreated spores incubated in water did not exhibit these changes. Only when spores were heat activated and incubated in germinants L-alanine and adenosine did changes in the spores approximate those observed in gut fluid. These data suggest that M. sexta larval gut fluid induces the activation and germination of B. thuringiensis spores.     

Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues.

Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate--200 mM 2-mercaptoethanol--0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process.     

pH值对Gigaspora margarita孢子萌发、菌丝生长与聚磷酸盐含量的影响

土壤pH值是影响AM真菌的生理与生态过程的重要因子之一,本试验在培养基上接种Gigasporamargarita的孢子,研究了pH值分别为5.2、6.0和6.8时孢子萌发率、菌丝生长和菌丝中聚磷酸盐(polyP)的含量。结果表明,不同pH条件下的孢子萌发率没有明显差异,培养12d后的萌发率为70%左右;随着pH的升高,菌丝的长度逐渐增加,表明低pH对菌丝的生长有一定的抑制效应;培养12d后,孢子中polyP含量低于菌丝中polyP含量,pH6.0和pH6.8的条件下菌丝中polyP含量明显高于pH5.2的含量,表明低pH也能降低菌丝中的聚磷酸盐含量。认为低pH对菌丝生长和polyP含量的抑制可能是其限制AM真菌功能发挥的重要机制之一。     

Recovery of Viability and Radiation Resistance by Heat-Injured Conidia of Penicillium expansum Lk. ex Thom

Spores heated in water at 54 C for up to 1 hr were plated on nutrient agar immediately or held for 3 days in aerated water at 23 C and then plated. Under these conditions, holding was optimal for recovery, increasing survival percentage up to 20-fold over values for immediate plating. Recovery was prevented partially or completely, however, when spores were held in any of the following solutions: glucose, potassium phosphate, ammonium or sodium acetate, sodium azide, or 2,4-dinitrophenol, or in the sodium or potassium salts of pyruvate, and tricarboxylic acid cycle acids. Both anaerobiosis and incubation at 0 C prevented recovery. Survivors of a heat treatment were more sensitive to gamma radiation than were unheated spores. Conditions which affected the recovery of viability had the same effect on restoration of radiation resistance. Thus, many of the processes for restoration of radiation resistance seem involved also in recovery of viability after heating. After a 99% inactivating treatment (about 30 min at 54 C), heated spores respired as fast as unheated spores, or faster. Malate, citrate, succinate, and acetate stimulated respiration in unheated spores and inhibited it in heated spores.     

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).     

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).     

Amino Acid Enhancement of Recovery in Dry-heat Damaged Spores of Bacillus subtilis

Spores of Bacillus subtilis MD2 and var. niger were dry-heat damaged at 150°, 160° and 170°C and recovered on media of increasing complexity. The greater the heat dose the more marked was the effect of amino acid supplements on recovery. For strain MD2 maximum germination and outgrowth of unheated spores could be obtained on a minimal salts + glucose medium with alanine, aspartic acid, glycine and methionine; the latter three amino acids served to enhance growth, not germination. The recovery of heat-damaged spores was significantly increased by adding valine plus isoleucine or arginine or glutamine. The increase was probably due to the use of valine and isoleucine as substrates of NAD-linked dehydrogenases to generate reducing power and serve as NH₃-donor, initiating germination in spores which were unable to germinate as a result of inactivation of alanine dehydrogenase. Valine or isoleucine added singly suppressed recovery by feedback inhibition of the pathways to both these amino acids during outgrowth.     

Oxidative Activation of Bacillus cereus Spores

A study was made of the activation of Bacillus cereus strain T spores by using the oxidizing agent sodium perborate. The degree of activation was measured with constant germination conditions by using L-alanine, inosine, adenosine, and L-alanine plus adenosine as germination stimulants. The germinal response following the various treatments was compared with the responses obtained with heat activation. It was concluded that the optimal time for activation with 30 mM sodium perborate at room temperature was about 4 hr. If the exposure time was greatly extended, the spores would germinate spontaneously. When the perborate treatment followed heat activation, the germinal response to L-alanine was stimulated, to inosine retarded and without apparent effect for adenosine or L-alanine plus adenosine. Results of experiments designed to demonstrate deactivation by slow oxidation showed that spores activated with sodium perborate were not deactivated by slow oxidation, whereas those activated by heat were. A deactivation study using mercaptoethanol as the deactivation agent showed that both methods of activation could be deactivated after a 24-hr exposure, but this deactivation was reversible by extending the exposure to mercaptoethanol. The results of heat-sensitivity studies revealed that about 70% of the sodium perborate-activated spores were heat sensitive after 60 min in a germination menstruum of L-alanine plus adenosine, whereas similarly treated heat-activated and nonactivated spores were about 99.99% heat sensitive, respectively.     

Conditions Affecting Germination of Clostridium botulinum 62A Spores in a Chemically Defined Medium

Spores of Clostridium botulinum type 62A were germinated in a chemically defined medium (8 mm l-cysteine, 11.9 mm sodium bicarbonate, 4.4 mm sodium thioglycolate; buffered with 100 mm TES, pH 7.0). The rate and extent of germination were increased when an aqueous spore suspension was heated sublethally (80 C, 60 min) before addition to the germination medium. Neither sublethal nor lethal doses of gamma radiation had any marked effect on subsequent germination. Maximum germination (>90% in 2 hr) in the defined medium occurred in the pH range of 6.5 to 7.5, at 30 to 37 C, with an l-cysteine level of 8 mm. Increasing l-cysteine to 32 mm increased the rate (over that with 8 mm l-cysteine) but not the extent of germination. The rate and extent of germination increased with NaHCO(3) addition to 8.3 mm, but increasing levels to 11.9 mm had no further effect. For maximum germination, 2.2 mm sodium thioglycolate was required and higher levels (to 8.8 mm) had no further enhancing or inhibitory effect. Under optimal conditions for germination, 97% of the spores had become heat sensitive; 98% had become sensitive to radiation; 88 and 91% had become phase dark and stainable, respectively, and the spore suspension had lost 46% of its initial optical density by 2 hr. Loss of heat resistance preceded loss of radiation resistance, acquisition of stainability, and phase darkening by about 12 min.