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°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°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.     

Heterogeneity of Times Required for Germination and Outgrowth from Single Spores of Nonproteolytic Clostridium botulinum

Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (t_germ), time to emergence (t_emerg), and times to reach the lengths of one (t_C1) and two (t_C2) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (t_germ), emergence (t_emerg − t_germ), cell maturation (t_C1 − t_emerg), and doubling (t_C2 − t_C1) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.     

The Resistance of Spores of Clostridium botulinum Type E to Heat and Radiation

Spores of Clostridium botulinum type E have been screened for resistance to heating in aqueous suspension at 60, 70 and 80°. D ¹⁷⁶ ranged from <0·33 min to 1·25 min. z values of eight strains were between 9·0 and 10·7, and for one strain was 7·4.
Radiation survivor curves have been plotted for nine strains, and a further nine strains screened. The shoulder extends to about 0·4 megarad, whereafter the curve becomes exponential within a D value from 0·065 to 0·16 megarad.     

Heat Resistance of Spores of Marine and Terrestrial Strains of Clostridium botulinum Type C

Resistance to heat of spores of marine and terrestrial strains of Clostridium botulinum type C in 0.067 m phosphate buffer (pH 7.0) was determined. The marine strains were 6812, 6813, 6814, and 6816; the terrestrial strains were 468 and 571. The inoculum level equaled 10(6) spores/tube with 10 replicate tubes for each time-temperature variable. Heating times were run at three or more temperatures to permit survival of some fraction of the inoculum. Survivors were recovered at 85 F (30 C) in beef infusion broth containing 1% glucose, 0.10% l-cysteine hydrochloride, and 0.14% sodium bicarbonate. D values were calculated for each fractional survivor end point after 6 months of incubation. Thermal resistance curves were constructed from the D value data. D(220) (104 C) values for spores of 468 and 571 equaled 0.90 and 0.40 min, respectively. The corresponding values for spores of 6812, 6813, 6814, and 6816 were 0.12, 0.04, 0.02, and 0.08 min. The z values for the thermal resistance curves ranged from 9.0 to 11.5 F (5.0 to 6.2 C).     

Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic Clostridium botulinum Bacteria

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic Clostridium botulinum Eklund 17B. The times to germination (t_germ), one mature cell (t_C1), and two mature cells (t_C2) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the t_germ, the outgrowth time (t_C1 − t_germ), and the first doubling time (t_C2 − t_C1). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (t_C1) and outgrowth (t_C1 − t_germ) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.Clostridium botulinum is a group of four physiologically and phylogenetically distinct anaerobic spore-forming bacteria (known as groups I, II, III, and IV) that produce the highly toxic botulinum neurotoxin (12). The severity of the intoxication, botulism, ensures considerable effort is directed at preventing the growth of this pathogen in food. Nonproteolytic (group II) C. botulinum is one of the two groups most frequently associated with food-borne botulism. It forms heat-resistant spores and can germinate, grow, and produce toxin at 3°C (8); thus, nonproteolytic C. botulinum is a particular concern in mild heat-treated chilled foods (16, 17).Spores formed by pathogens such as C. botulinum are a significant food safety issue since they are able to resist many of the processes, such as cooking, used to kill vegetative cells. Understanding the transformation from a dormant spore to active vegetative cells is an important part of quantifying the risk associated with such organisms. Considerable effort has been targeted at measuring and relating the kinetic responses of populations of C. botulinum to environmental conditions and such data have been used to create predictive models, for example, ComBase (www.combase.cc). Such approaches have made a considerable contribution to ensuring food safety but problems with using population based predictions may arise when an initial inoculum is very small or additional information beyond point values is required. Spores typically contaminate foods at low concentrations so that growth of C. botulinum, when it occurs, is likely to initiate from just a few spores. In these circumstances the distribution of times to growth in packs will reflect the heterogeneity of times to growth from the contaminating individual spores. There is an intrinsic variability between individual spores within a population, and the relationship between population lag and individual lag is complex. Consequently, individual lag times cannot be predicted from population measurements (3). Knowledge of the underlying distribution would allow greater refinement of risk assessments.The lag period between a spore being exposed to conditions suitable for growth and the start of exponential growth will reflect the combined times of germination, emergence, elongation, and first cell division. Currently, very little is known about the variability and duration of these stages and any relationships between them. Measuring the kinetics of spore germination is usually achieved by measuring a population to identify time to percent completion. Such germination curves represent the summation of responses by individual spores. Some authors have measured the biovariability associated with individual spores, but most studies have examined only germination (4-7, 11, 22) and not subsequent outgrowth. More recently, we have used phase-contrast microscopy and image analysis to follow individual spores of nonproteolytic C. botulinum from dormancy, through germination and emergence, to cell division (21, 23). These experiments showed there is very little, or no, relationship between the time spent in each stage by individual spores. We have now extended this work to determine distributions of times for different stages in lag phase as affected by heat treatment and incubation temperature.     

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.     

Survival of Clostridium botulinum Spores

Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a “tail” comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The “tail” was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation “inactivated” spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth.     

Effect of Plating Medium on Heat Activation Requirement of Clostridium botulinum Spores

Clostridium botulinum 62A and ATCC 25763 spores required heat activation for maximum colony formation when plated on reinforced clostridial agar (BBL Microbiology Systems) but not when plated on botulinum assay medium. Spores from strains B-aphis and 53B did not exhibit heat activation when plated on either medium.     

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.     

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg⁻¹. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments.     

Fixation of Mature Spores of Clostridium botulinum

A triple fixation method using a sequential application of 15 or 30% formaldehyde, 6% glutaraldehyde, and 1% osmium tetroxide resulted in excellent fixation of mature spores of Clostridium botulinum.     

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°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°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°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 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°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 processes rainbow trout were observed.     

Effect of Temperature of Liquid Nitrogen on Radiation Resistance of Spores of Clostridium botulinum

An apparatus consisting of a Dewar flask and a relay system controlling the flow of liquid nitrogen permitted the irradiation of samples in tin cans or Pyrex tubes at temperatures ranging from 0 ± 1.5 C to -194 ± 2 C. An inoculated pack comprising 320 cans of ground beef containing 5 × 10⁴ spores of Clostridium botulinum 33A per can (10 cans per radiation dose) was irradiated with Co⁶⁰ at 0 and -196 C. Incubation was carried out at 30 C for 6 months. Approximately 0.9 Mrad more radiation was required to inactivate the spores at -196 C than at 0 C. Cans irradiated at -196 C showed partial spoilage at 3.6 Mrad and no spoilage at 3.9 Mrad; the corresponding spoilage-no spoilage doses at 0 C were 2.7 and 3.0, respectively. The majority of positive cans swelled in 2 to 14 days; occasional swelling occurred as late as 20 days. At progressively higher doses, swelling was delayed proportionally to the radiation dose received. The remaining nonswollen cans had no toxin after 6 months of storage, although occasional cans contained very low numbers of viable spores comprising on the average 0.1% of the original spore inoculum. The D₁₀ values in phosphate buffer were 0.290 Mrad for 0 C and 0.396 Mrad for -196 C; in ground beef, the corresponding D₁₀ values were 0.463 Mrad and 0.680 Mrad, respectively. These D₁₀ values indicate that the lethal effect of γ rays decreased at -196 C as compared with 0 C by 13.5% in phosphate buffer, and by 47% in ground beef.     

Antigenic Relationships Among the Proteolytic and Nonproteolytic Strains of Clostridium botulinum

Relationships of the somatic antigens among Clostridium botulinum strains have been investigated by tube agglutination and agglutinin absorption tests. Results revealed a relationship by which strains of C. botulinum are grouped by their proteolytic capacity rather than by the type of specific toxin produced. Thus, C. botulinum type E and its nontoxigenic variants, which are nonproteolytic, share common somatic antigens with the nonproteolytic strains of types B and F. Absorption of antiserum of a strain of any one type with antigen of any of the others removes the antibody to all three types. In the same manner, C. botulinum type A shares somatic antigens with the proteolytic strains of types B and F, and absorption of any one antiserum with an antigen of either of the other two types removes the antibody to all three types. Partial cross-agglutination of C. sporogenes, C. tetani, and C. histolyticum with the somatic antisera of the proteolytic group was also observed.     

Resistance of Spores of Clostridium botulinum 33A to Combinations of Ultraviolet and Gamma Rays

Spores of Clostridium botulinum 33A exhibit a sigmoidal survival curve if subjected to gamma radiation. The present investigation was concerned with two questions: (i) what is the form of an ultraviolet (UV)-survival curve and (ii) what is the combined effect of UV- and gamma radiation? The UV-survival curve was found to be of sigmoidal type with a "shoulder" width of 675 ergs/mm(2) and a D(10) (exp) of 2,950 ergs/mm(2). To test the combination effect, spores were subjected to UV doses of 225, 450, 675, and 900 ergs/mm(2) followed by a series of increasing doses of gamma rays from 200 to 2,000 krad in 200-krad steps. The gamma ray-survival curves showed that increasing UV pretreatment caused a gradual loss of the "Prodiginine" yielding straight line exponential survival curves after preirradiation with UV doses of 675 ergs/mm(2) and above. Simultaneously the D(10) value for gamma-ray irradiation was reduced, e.g. UV preirradiation with 900 ergs/mm(2) reduced the D(10) by 40%. This observation emphasizes the potential practical advantage of combining UV and gamma rays for sterilization of heat-sensitive commodities.     

Radiation Injury of Clostridium botulinum Spores in Cured Meat

Cans of chopped ham, inoculated with spores of Clostridium botulinum strains 33A and 41B at levels of 2,500 and 250 per gram, were subjected to an enzyme-inactivating heat treatment and irradiation with 0.5, 1.5, 2.5, or 3.5 Mrad of Co(60). A portion of the pack was not irradiated, and received a commercial thermal process (F(0) = 0.2). Viable spores were enumerated after treatment and after 6 months of incubation at 30 to 37.7 C. Toxic spoilage occurred at 0 and 0.5, but not at 1.5, 2.5, or 3.5 Mrad. More spoilage and toxin formation occurred in the product irradiated at 0.5 Mrad than in identical product receiving no radiation treatment. Confirmed botulinal spores were isolated from all of the radiation variables of 2,500 per gram-inoculated product and from all but the 3.5 Mrad low-inoculum cans. However, neither growth nor toxin was observed in unspoiled product. The "injury" phenomenon previously described in thermally processed cured meats (survival of botulinal spores without capacity for multiplication or toxigenesis) apparently occurs also in irradiated cured meats.     

Hypochlorite Injury of Clostridium botulinum Spores Alters Germination Responses

[This corrects the article on p. 1361 in vol. 45.].     

Controlling the Sporulation of Clostridium sporogenes and the Heat Resistance of the Spores

S ummary . During growth of Clostridium sporogenes in tryptone-salt-peptone-glucose medium the pH value of the medium varies due to formation of acid and CO₂ and to subsequent production of NH₃. Glucose concentrations of 0·2, 0·5 or 1·0% result in increasing sporulation times and in spores of low, extremely high ( D ₁₁₀ c . 80 min) and negligible heat resistance, respectively. When the pH value is maintained at 7, a reproducible sporulation time of a few hours is observed and the resulting spores have a heat resistance ( D ¹¹⁰) of 13 min, regardless of the glucose concentration.