Nisin Resistance in Clostridium botulinum Spores and Vegetative Cells

The frequencies at which vegetative cells and spores of Clostridium botulinum strains 56A, 62A, 17409A, 25763A, 213B, B-aphis, and 169B formed colonies on agar media containing 0, 10(sup2), 10(sup3), and 10(sup4) IU of nisin per ml at 30(deg)C were determined. Strain 56A had the highest frequencies of nisin resistance, while strains 62A, 169B, and B-aphis had the lowest. For most strains, spores were more resistant than vegetative cells. One exposure to nisin was sufficient to generate stable nisin-resistant isolates in some strains. Stepwise exposure to increasing concentrations of nisin generated stable resistant isolates from all strains. Spores produced from nisin-resistant isolates maintained their nisin resistance. The frequency of spontaneous nisin resistance was reduced considerably by lowering the pH of the media and adding 3% NaCl. Nisin-resistant isolates of strains 56A and 169B also had increased resistance to pediocin PA1, bavaricin MN, plantaricin BN, and leuconocin S.     

Heat resistance of the chemical resistance forms of Clostridium botulinum 62A spores over the water activity range 0 to 0.9.

Having available the separate chemical resistance forms of Clostridium botulinum 62A spores from an investigation of the effect of spore form on wet heat resistance and also a method for measuring heat resistance at known water activities over the whole water activity (aw) range, we measured the heat resistance of these preparations at four different temperatures at each aw interval of 0.1 from aw 0 to aw 0.9. The required temperature dependence of resistance was calculated for each aw increment. The spore forms showed a low resistance at aw values of 0 and 0.7 of 0.9, with a rise in resistance in the range aw 0.1 to 0.5. The temperature dependence values behaved similarly.     

Thermal destruction of Clostridium botulinum spores suspended in tomato juice in aluminum thermal death time tubes.

The heat destruction characteristics of Clostridium botulinum spores suspended in tomato juice and phosphate buffer were determined by the survivor curve method with aluminum thermal death time tubes. Two type A strains of C. botulinum and a type B strain were evaluated. Strains A16037 and B15580 were implicated in outbreaks of botulism involving home-canned tomato products. Strain A16037 had a higher heat resistance than either 62A or B15580. The mean thermal resistance (D-values) for A16037 in tomato juice (pH 4.2) were: 115.6 degrees C, 0.4 min; 110.0 degrees C, 1.6 min; and 104.4 degrees C, 6.0 min. The mean D-values for A16037 in Sorensen 0.067 M phosphate buffer (pH 7) were: 115.6 degrees C, 1.3 min; 110.0 degrees C, 4.4 min; and 104.4 degrees C, 17.6 min. At each test temperature, the D-values were approximately three times higher in buffer than in tomato juice. The z-value for C. botulinum A16037 spores in tomato juice was 9.4 degrees C, and in buffer the z-value was 9.9 degrees C. The use of aluminum thermal death time tubes in a miniature retort system makes it possible to determine survivor curves for C. botulinum spores at 121.1 degrees C. This is possible because the lag correction factor for the aluminum tubes is only about 0.2 min, making possible heating times as short as 0.5 min.     

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.     

Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food

The TaqMan real-time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence-specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10-fold dilution series of DNA and spores . The DNA and spores were detected up to level of 0.1 ng/ml and 10(2)spores/ml, respectively. Spore spiked food sample preparation prior to the real-time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 10(2) spores/ml for spiked sausage slurry, and 10(3) spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 10(2) spores/ml in both sausage and canned corn. Therefore the real-time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real-time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.     

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.     

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.     

Life from the ashes: survival of dry bacterial spores after very high temperature exposure

We found that spores of Bacillus amyloliquefaciens rank amongst the most resistant to high temperatures with a maximum dry heat tolerance determined at 420 °C. We found that this extreme heat resistance was also maintained after several generations suggesting that the DNA was able to replicate after exposure to these temperatures. Nonetheless, amplifying the bacterial DNA using BOXA1R and (GTG)₅ primers was unsuccessful immediately after extreme heating, but was successful after incubation of the heated then cooled spores. Moreover, enzymes such as amylases and proteases were active directly after heating and spore regeneration, indicating that DNA coding for these enzymes were not degraded at these temperatures. Our results suggest that extensive DNA damage may occur in spores of B. amyloliquefaciens directly after an extreme heat shock. However, the successful germination of spores after inoculation and incubation indicates that these spores could have a very effective DNA repair mechanism, most likely protein-based, able to function after exposure to temperatures up to 420 °C. Therefore, we propose that B. amyloliquefaciens is one of the most heat resistant life forms known to science and can be used as a model organism for studying heat resistance and DNA repair. Furthermore, the extremely high temperature resistivity of these spores has exceptional consequences for general methodology, such as the use of dry heat sterilization and, therefore, virtually all studies in the broad area of high temperature biology.

     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

A method for the assessment of safe rehydration regimes for pasta

It is well-known that bacterial spores are more resistant to dry heat than moist heat. Therefore, in order to ensure the safe application of thermal processes aimed at the destruction of Clostridium botulinum it is essential that all the ingredients of a food product are fully rehydrated. A simple microbiological method, based on the immobilization of spores of known moist heat resistance in a product, has been developed to evaluate the processing conditions required to give full rehydration of pasta. The method is compared to traditional approaches.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Vegetable juice aids the recovery of heated spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic Clostridium botulinum at 85C for 2 min followed by plating on a standard laboratory medium reduced the count of viable spores by a factor of greater than 10⁴. A similar result was obtained when the plating medium was supplemented with juice from courgette, carrot or mung bean sprout. When plating was on media supplemented with hen egg white lysozyme or juice from turnip, swede, flat bean, cabbage or potato, heating at 85C for 10 min did not reduce the viable count by a factor of 10⁴. Thus these vegetable juices increased the measured heat resistance of spores of non-proteolytic Cl. botulinum . These findings are relevant to the safety of minimally processed (e.g. sous-vide ) foods.     

Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates carrying plasmid genes versus chromosomal enterotoxin genes

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringens isolates associated with food poisoning carry a chromosomal cpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (D values) at 55 degrees C than vegetative cells of plasmid cpe isolates exhibited. Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100 degrees C than the spores of plasmid cpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomal cpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.     

Heat resistance of Paenibacillus polymyxa in relation to pH and acidulants

The efficacy of different organic acids in decreasing the heat resistance of Paenibacillus polymyxa spores was assessed. The relationship between concentration of the undissociated form of different organic acids and decrease in heat resistance was also investigated. The heat resistance of P. polymyxa spores was tested in distilled water at 85, 90 and 95 degrees C, at pH4 and in the presence of 50, 100 and 200 mmol l(-1) of the undissociated form of lactic, citric or acetic acid and sodium citrate or acetate. The undissociated form of organic acids was responsible for increasing the heat sensitivity of spores. The most effective acid was lactic acid. The D values of the spores decreased rapidly (between 74 and 43%) in the presence of 50 mmol l(-1) of the undissociated form of organic acid, and increasing concentrations of these forms affected the heat resistance of spores less than proportionally. The heat resistance of the spores in milk was approximately threefold lower than in distilled water. This work has shown that the undissociated fraction of organic acids increases, albeit non-linearly, the sensitivity of spores to heat, even in complex substrates such as milk. By knowing the amount of organic acids added to a given substrate, their dissociation constants and the final pH, it could be possible to estimate the concentration of undissociated forms and the corresponding increase in lethality of heat treatments. This would help the food industry to maximize the lethality achieved by heat processes and/or safely reduce the heat treatments already in use.     

A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates

Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.     

Effect of initiated spores on the resistance of nongerminated resting forms of Bacillus cereus remaining in the suspension to the action of damaging agents

The elevated resistance of a Bacillus cereus spore suspension against the action of UV was found to depend on the quantity of resting forms initiated in the suspension prior to an irradiation. The resistance against UV increased 80-50 times if 60-90% of spores were initiated in the suspension as compared to that of the original resting forms. When suspensions containing 40% of non-germinated B. cereus spores were kept at 4 degrees C for 14 days, the latter became 10 and 14 times more resistant to elevated temperature (90 degrees C) and chloramine (2.5%), respectively, as compared to control intact spores. The higher resistance of non-germinated spores against the action of physical and chemical damaging agents was registered within the entire period of experiments (over three months). This phenomenon was not observed if ca. 100% of spores were initiated in a suspension. The resistance of initiated spores against the action of UV was 40 times lower than that of B. cereus resting forms.     

Examination of Prepared Foods in Plastic Packages for Clostridium botulinum

The incidence of Clostridium botulinum organisms was determined in a variety of plastic-packaged "vulnerable" foods (food requiring little or no heating prior to consumption). A total of 113 foods were examined by use of an enrichment recovery procedure followed by toxin testing in animals. Results of the survey indicate that the incidence of C. botulinum organisms in these vulnerable foods is extremely low. The ability of inoculated food products to support growth and toxigenesis of C. botulinum type E was then tested. The 64 packaged foods were inoculated with type E spores and incubated anaerobically at 30 C for 11 days. A slurry of each food was prepared, smears for fluorescent-antibody testing were made, and animal tests were performed for toxin. If the animal tests were negative, enrichment cultures were prepared from the slurry and incubated at 30 C. On direct examination of the slurries for toxin, only samples of turkey roll and soybean cake supported growth and toxigenesis by C. botulinum type E. However, the enrichment culture method was able to induce growth and toxin production in 60 of the remaining 62 samples.