The combined effect of incubation temperature, pH and sorbic acid on the probability of growth of non-proteolytic, type B Clostridium botulinum

L und , B.M., G raham , A.F., G eorge , S.M. & B rown , D. 1990. The combined effect of incubation temperature, pH and sorbic acid on the probability of growth of non-proteolytic, type B Clostridium botulinum. Journal of Applied Bacteriology 69 , 481–492.
It has been reported that non-proteolytic strains of Clostridium botulinum will grow at 3.3°C, and they are therefore of concern in relation to certain chilled foods. The effects of combinations of inhibitory factors may be used to reduce the risk of growth of these bacteria in foods. The combined effect of pH values between 4.8 and 7.0, temperatures between 6° and 30°C, and sorbic acid concentrations up to 2270 mg/1 on the probability of growth from a single spore of non-proteolytic, type B strains in a culture medium has been determined. A mathematical model has been developed that enables the effect of varying combinations of these factors on the probability of growth of non-proteolytic, type B Cl. botulinum to be predicted.     

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90°C for between 0 and 60 min and subsequently incubated at 5°, 10° or 30°C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90°C for 1 or more min ensured a 10⁶ reduction when spores were subsequently incubated at 5°C for up to 23 weeks. Heating at 90°C for 60 min ensured a 10⁶ reduction over 23 weeks when subsequent incubation was at 10°C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10⁶ when subsequent incubation was at 30°C.     

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum

Heat treatment of spores of non-proteolytic Clostridium botulinum at 85°C for 120 min followed by enumeration of survivors on a medium containing lysozyme resulted in a 4.1 and 4.8 decimal reduction in numbers of spores of strains 17B (type B) and Beluga (type E), respectively. Only a small proportion of heated spores formed colonies on medium containing lysozyme; this proportion could be increased by treatments designed to increase the permeability of heated spores. The results indicate that the germination system in spores of non-proteolytic Cl. botulinum was destroyed by heating, that lysozyme could replace this germination system, and that treatments that increased the permeability of the spore coat could increase the proportion of heated spores that germinated on medium containing lysozyme. These results are important in relation to the assessment of heat-treatments required to reduce the risk of survival and growth of non-proteolytic Clostridium botulinum in processed (pasteurized) refrigerated foods for extended storage.     

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.     

Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature.

Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods. Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium. The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined. No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.     

Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature

The effect of combinations of temperature (2°, 3°, 4°, 5°, 8° and 10°C), pH (5·0–7·2) and NaCl (0·1–5·0% w/w) on growth from spores of non-proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium. Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth. Growth and toxin production were detected at 3°C in 5 weeks, at 4°C in 3/4 weeks and at 5°C in 2/3 weeks. The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time. This is important in assessment of the risk of growth and toxin production by non-proteolytic Cl. botulinum in minimally processed chilled foods.     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH greater than or equal to 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12 degrees and 16 degrees C and pH values between 5.2 and 5.5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5.5 and incubated at 16 degrees C, corresponding to a probability of growth from a single spore within 14 d of 1.6 x 10(-5). The probability of growth in 28 d in the remaining conditions was less than 9 x 10(-6). After transfer of inoculated media from 12 degrees to 30 degrees C growth occurred at pH 5.2-5.5 within 19 d. After transfer of inoculated media from 12 degrees to 20 degrees C growth occurred at pH 5.5 and 5.4 but not at pH 5.3 or 5.2 in 40 d. Growth at pH 5.2-5.5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

The metabolism of pyrimidines by proteolytic clostridia.

Uracil was used by growing cultures of Clostridium sporogenes, and by proteolytic strains of C. botulinum types A and B. Uracil was not used by C. bifermentans; C. botulinum, type B (non-proteolytic); C. botulinum, type F (non-proteolytic); C. botulinum, type E; C. butyricum; C. cochlearium; C. difficile; C. histolyticum; C. oedematiens, type A; C. paraputrificum; C. scatologenes; C. specticum; C. sordellii; C. sticklandii; C. tertium; C. tetani; C. tetanomorphum; C. welchii, types A, B, C, E and 4 untyped strains. The growth of C. sporogenes was not increased by uracil; it was reduced to dihydrouracil. Experiments with washed cells of C. sporogenes showed that the uracil-reducing system was inducible. Washed cell suspensions incubated under hydrogen with uracil, thymine, iso-barbituric acid, 5-amino uracil and cytosine consumed 1 mole H2/mole pyrimidine. The reduction product of cytosine was dihydrouracil indicating that it was deaminated before reduction. The reduction products of the remaining pyrimidines were the corresponding dihydro derivatives. Extracts of C. sporogenes reduced uracil in the presence of NADPH2 but not NADH2.     

Growth and toxin production by non-proteolytic and proteolytic Clostridium botulinum in cooked vegetables

Growth and toxin production by proteolytic and non-proteolytic strains of Clostridium botulinum have been followed in 28 cooked puréed vegetables prepared under strict anaerobic conditions and incubated at 30°C for up to 60 d. Toxin production was confirmed in 25 of the cooked vegetables inoculated with a suspension of spores of proteolytic strains of types A and B, and in 13 inoculated with a suspension of spores of non-proteolytic strains of types B, E and F. For both proteolytic and non-proteolytic strains, a trend was identified correlating growth and toxin production with the pH of the cooked puréed vegetables.     

Identification of Clostridium botulinum with API 20 A, Rapid ID 32 A and RapID ANA II

Three commercially available test systems for the identification of anaerobic bacteria were evaluated for the identification of 18 proteolytic group I and 69 non-proteolytic group II Clostridium botulinum, four Clostridium sporogenes and 18 non-toxigenic group II C. botulinum-like strains. All proteolytic C. botulinum strains were misidentified by the Rapid ID 32 A and RapID ANA II, while 14 strains and all C. sporogenes strains were identified as C. botulinum or C. sporogenes by the API 20 A. Reversely, all non-proteolytic C. botulinum strains were misidentified by the API 20 A while the Rapid ID 32 A recognized 67 and RapID ANA II 68 strains. All C. sporogenes strains were recognized by the RapID ANA II, while the Rapid ID 32 A recognized one strain. All non-proteolytic non-toxigenic strains were identified as C. botulinum group II by the Rapid ID 32 A, 17 strains by the RapID ANA II, and one strain by the API 20 A. The results show that these test systems do not provide a reliable method for identification of C. botulinum.     

Effect of heat treatment on survival of, and growth from, spores of nonproteolytic Clostridium botulinum at refrigeration temperatures.

Spores of five type B, five type E, and two type F strains of nonproteolytic Clostridium botulinum were inoculated into tubes of an anaerobic meat medium plus lysozyme to give approximately 10(6) spores per tube. Sets of tubes were then subjected to a heat treatment, cooled, and incubated at 6, 8, 10, 12, and 25 degrees C for up to 60 days. Treatments equivalent to heating at 65 degrees C for 364 min, 70 degrees C for 8 min, and 75 degrees C for 27 min had little effect on growth and toxin formation. After a treatment equivalent to heating at 85 degrees C for 23 min, growth occurred at 6 and 8 degrees C within 28 to 40 days. After a treatment equivalent to heating at 80 degrees C for 19 min, growth occurred in some tubes at 6, 8, 10, or 12 degrees C within 28 to 53 days and at 25 degrees C in all tubes within 15 days. Following a treatment equivalent to heating at 95 degrees C for 15 mine, growth was detected in some tubes incubated at 25 degrees C for fewer than 60 days but not in tubes incubated at 6 to 12 degrees C. The results indicate that heat treatment of processed foods equivalent to maintenance at 85 degrees C for 19 min combined with storage below 12 degrees C and a shelf life of not more than 28 days would reduce the risk of growth from spores of nonproteolytic C. botulinum by a factor of 10(6).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of temperature on the growth of non-proteolytic type B Clostridium botulinum

The effect of temperature between 4 and 35°C on the growth rate of a non-proteolytic type B strain of Clostridium botulinum was examined. Growth was in culture media at pH 6.7 and was measured by viable counts using the Most Probable Number (MPN) method. Doubling times were derived from the curve fitting model of Baranyi et al. (1992) and ranged from 42.3 h at 3.9°C to 22 min at 35°C.     

A Pyrolysis Gas-liquid Chromatography Study of Clostridium botulinum and Related Organisms

Low resolution pyrolysis gas-liquid chromatography could differentiate the following groups of Clostridium botulinum and related organisms: (1) Cl. botulinum type A. proteolytic types B and F and Cl. sporogenes ; (2) Cl. botulinum types C and D. and (3) Cl. botulinum type E and non-proteolytic types B and F. Toxin types A and B could be distinguished from type E and from type F.     

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.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Identification and grouping of Clostridium botulinum strains by numerical analysis of their electrophoretic protein patterns

Strains of Clostridium botulinum type A, type E and both non-proteolytic and proteolytic types B and F were characterized by their electrophoretic protein patterns. As the protein pattern changes during sporulation, special attention was paid to the prevention of sporulation by selecting an appropriate medium (Strasdine's medium plus 1% w/v glucose) and a scheme of repeated subculturing. Ribosomal proteins, evolutionarily conservative and hence relatively similar in all types of bacteria, were removed to optimize the resolving power of the electrophoretic technique. Protein patterns were compared by computing correlation coefficients of normalized densitometric tracings. The method is highly reproducible and its resolving power is high: all protein patterns found were specific. The strains tested fall into two main groups: the proteolytic and the non-proteolytic cluster. Type A strains form a separate subgroup within the proteolytic cluster, the same applies to type E strains within the non-proteolytic group. Although time-consuming for spore-forming bacteria, this method is, to our knowledge, the only technique that recognizes individual strains of Cl. botulinum . For non-spore-forming micro-organisms the method is certainly much simpler and hence even more valuable.     

Identification and grouping of Clostridium botulinum strains by numerical analysis of their electrophoretic protein patterns

Strains of Clostridium botulinum type A, type E and both non-proteolytic and proteolytic types B and F were characterized by their electrophoretic protein patterns. As the protein pattern changes during sporulation, special attention was paid to the prevention of sporulation by selecting an appropriate medium (Strasdine's medium plus 1% w/v glucose) and a scheme of repeated subculturing. Ribosomal proteins, evolutionarily conservative and hence relatively similar in all types of bacteria, were removed to optimize the resolving power of the electrophoretic technique. Protein patterns were compared by computing correlation coefficients of normalized densitometric tracings. The method is highly reproducible and its resolving power is high: all protein patterns found were specific. The strains tested fall into two main groups: the proteolytic and the non-proteolytic cluster. Type A strains form a separate subgroup within the proteolytic cluster, the same applies to type E strains within the non-proteolytic group. Although time-consuming for spore-forming bacteria, this method is, to our knowledge, the only technique that recognizes individual strains of Cl. botulinum. For non-spore-forming micro-organisms the method is certainly much simpler and hence even more valuable.