Growth from spores of nonproteolytic Clostridium botulinum in heat-treated vegetable juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80 degrees C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80 degrees C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.     

Germination and outgrowth of single spores of Clostridium botulinum and putrefactive anaerobes

Single spores of putrefactive anaerobes (PAs) germinated more slowly than those of Clostridium botulinum types A & B, with calculated times for 10% germination from 2 to 302 d. If PA spores were used in inoculated pack studies to simulate the response of Cl. botulinum , the likelihood of growth of Cl. botulinum would be underestimated.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Differing L-alanine germination requirements of hypochlorite-treated Clostridium botulinum spores from two crops

Similar populations of hypochlorite-treated spores were enumerated from two crops of Clostridium botulinum 12885A produced by the same procedure; however, germination required different L-alanine concentrations. Lactate permitted the germination of spores from both crops with suboptimal L-alanine concentrations. The data suggest that the spores differ slightly in chemical or structural composition.     

The effect of temperature on the germination of single spores of Clostridium botulinum 62A

Phase-contrast microscopy coupled with image analysis has been used to study the germination of single spores of Clostridium botulinum and to investigate the variation of germination lag of individual spores in a population (biovariability). The experiment was repeated at five different temperatures between 20°C and 37°C to look at the effect of temperature on the biovariability of the spore germination. Data analysis shows that the germination lag distribution is skewed, with a tail, and that its shape is affected by the temperature. The origin of this biovariability is not exactly known, but could be due to a distribution of characteristics (e.g. permeabilities) or molecules (e.g. lytic enzymes) in the spore population. The method developed in this study will help us to describe and better understand the kinetics of spore germination and how this is influenced by different environmental factors such as temperature and other factors that influence germination.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses.

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Differing L-alanine germination requirements of hypochlorite-treated Clostridium botulinum spores from two crops.

Similar populations of hypochlorite-treated spores were enumerated from two crops of Clostridium botulinum 12885A produced by the same procedure; however, germination required different L-alanine concentrations. Lactate permitted the germination of spores from both crops with suboptimal L-alanine concentrations. The data suggest that the spores differ slightly in chemical or structural composition.     

Proposed role of lactate in germination of hypochlorite-treated Clostridium botulinum spores

Clostridium botulinum 12885A spores treated with hypochlorite required added DL-calcium lactate for L-alanine germination. Lactate was the active component of calcium lactate. Equimolar concentrations of L-malate, but not of DL-propionate, could replace lactate, suggesting that the alpha-hydroxy acid structure is important. Neither lactate nor malate was an effective germinant for buffer-treated or hypochlorite-treated spores. If the L-alanine concentration was increased 100-fold (to 450 mM), the lactate germination requirement was overcome. The data suggest that the L-alanine germination sites were modified by hypochlorite so that a higher concentration of alanine was required for activity. Lactate appeared to be an activator of modified or non-hypochlorite-modified L-alanine germination sites.     

Historical and contemporary NaCl concentrations affect the duration and distribution of lag times from individual spores of nonproteolytic clostridium botulinum

In this study we determined the effect of NaCl concentration during sporulation (0 or 3.0% [wt/vol] added NaCl) and subsequent growth (0 or 2.0% [wt/vol] added NaCl) on the distributions of times associated with various stages of the lag phase of individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B. The effects of NaCl on the probability of germination and the probability of subsequent growth were also determined. Spore populations exhibited considerable heterogeneity at all stages of lag phase for each condition tested. Germination time did not correlate strongly with the times for later stages in the lag phase, such as outgrowth and doubling time. Addition of NaCl to either the sporulation or growth media increased the mean times for, and variability of, all the measured stages of the lag phase (germination, emergence, time to one mature cell, and time to first doubling). There was a synergistic interaction between the inhibitory effects of NaCl in the sporulation medium and the inhibitory effects of NaCl in the subsequent growth medium on the total lag time and each of its stages. Addition of NaCl to either the sporulation medium or the growth medium reduced both the probability of germination and the probability of a germinated spore developing into a mature cell, but the interaction was not synergistic. Spores formed in medium with added NaCl were not better adapted to subsequent growth in suboptimal osmotic conditions than spores formed in medium with no added NaCl were. Knowledge of the distribution of lag times for individual spores and quantification of the biovariability within lag time distributions may provide insight into the underlying mechanisms and can be used to improve predictions of growth in food and to refine risk assessments.     

Effects of potassium sorbate and other antibotulinal agents on germination and outgrowth of Clostridium botulinum type E spores in microcultures.

The effects of potassium sorbate, sodium hypophosphite, sodium tripolyphosphate, sodium nitrite, and linoleic acid on the germination and outgrowth of Clostridium botulinum type E spores were studied in microcultures. At pH 5.8 to 6.0 in liver veal agar, the germination rate was decreased to nearly zero with 1.0, 1.5, or 2.0% sorbate. At pH 7.0 t 7.2, these levels of sorbate afforded germination and outgrowth of abnormally shaped cells that were defective in cell division. At the high pH range, 0.5 or 1.0% hypophosphite had effects similar to those of sorbate. The use of 0.05% sodium nitrite with sorbate enhanced the lysis of outgrowing cells at pH 7.2 or lower. Emergence and elongation were inhibited by 0.05% linoleic acid with or without 1.0% sorbate at pH 7.0 to 7.2. The addition of 0.5% tripolyphosphate to media containing 1.5% sorbate at pH 7.1 prevented normal cell growth to an extent greater than with sorbate alone.     

Proposed role of lactate in germination of hypochlorite-treated Clostridium botulinum spores.

Clostridium botulinum 12885A spores treated with hypochlorite required added DL-calcium lactate for L-alanine germination. Lactate was the active component of calcium lactate. Equimolar concentrations of L-malate, but not of DL-propionate, could replace lactate, suggesting that the alpha-hydroxy acid structure is important. Neither lactate nor malate was an effective germinant for buffer-treated or hypochlorite-treated spores. If the L-alanine concentration was increased 100-fold (to 450 mM), the lactate germination requirement was overcome. The data suggest that the L-alanine germination sites were modified by hypochlorite so that a higher concentration of alanine was required for activity. Lactate appeared to be an activator of modified or non-hypochlorite-modified L-alanine germination sites.     

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)     

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

Germination of spores from Clostridium botulinum B-aphis and Ba410

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