Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light.

Bacterial spores are generally more resistant than vegetative bacteria to ultraviolet (UV) inactivation. The UV sensitivity of these spores must be known for implementing UV disinfection of low acid liquid foods. UV inactivation kinetics of bacterial spores in coconut water (CW) and distilled sterile water was studied. Populations of Bacillus cereus and Clostridium sporogenes dormant spores were reduced by more than 5.5 log10 at the UV-C photon fluence of 1142 µE·m-2 and 1919 µE·m-2 respectively. C. sporogenes spores showed higher UV-C resistance than B. cereus, with the photon fluence 300 µE·m-2 required for one log inactivation (D10) and 194 µE·m-2, respectively. No significant difference was observed in D10 values of spores suspended in the two fluid types (p > 0.05). The inactivation kinetics of microorganisms were described by log linear models with low root mean square error and high coefficient of determination (R2 > 0.98). This study clearly demonstrated that high levels of inactivation of bacterial spores can be achieved in CW. The baseline data generated from this study will be used to conduct spore inactivation studies in continuous flow UV systems. Further proliferation of the technology will include conducting extensive pilot studies.

Mechanisms of food processing and storage-related stress tolerance in Clostridium botulinum.

Vegetative cultures of Clostridium botulinum produce the extremely potent botulinum neurotoxin, and may jeopardize the safety of foods unless sufficient measures to prevent growth are applied. Minimal food processing relies on combinations of mild treatments, primarily to avoid deterioration of the sensory qualities of the food. Tolerance of C. botulinum to minimal food processing is well characterized. However, data on effects of successive treatments on robustness towards further processing is lacking. Developments in genetic manipulation tools and the availability of annotated genomes have allowed identification of genetic mechanisms involved in stress tolerance of C. botulinum. Most studies focused on low temperature, and the importance of various regulatory mechanisms in cold tolerance of C. botulinum has been demonstrated. Furthermore, novel roles in cold tolerance were shown for metabolic pathways under the control of these regulators. A role for secondary oxidative stress in tolerance to extreme temperatures has been proposed. Additionally, genetic mechanisms related to tolerance to heat, low pH, and high salinity have been characterized. Data on genetic stress-related mechanisms of psychrotrophic Group II C. botulinum strains are scarce; these mechanisms are of interest for food safety research and should thus be investigated. This minireview encompasses the importance of C. botulinum as a food safety hazard and its central physiological characteristics related to food-processing and storage-related stress. Special attention is given to recent findings considering genetic mechanisms C. botulinum utilizes in detecting and countering these adverse conditions.

The CLO3403/CLO3404 two-component system of Clostridium botulinum E1 Beluga is important for cold shock response and growth at low temperatures.

In order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. Two-component signal transduction systems (TCSs) play a central role in sensing and responding to many different environmental stimuli. Although the nonproteolytic (group II) Clostridium botulinum represents a major hazard in chilled foods, the cold adaption mechanisms of group II C. botulinum organisms are not known. Here, we show that the CLO3403/CLO3404 TCS of C. botulinum E1 Beluga is involved in the cold shock response and growth at 12°C. Cold shock induced the expression of the genes encoding the histidine kinase (clo3403) and the response regulator (clo3404) by more than 100-fold after 5 h relative to their expression in a nonshocked culture at the corresponding time point. The involvement of CLO3403/CLO3404 in growth at low temperature was demonstrated by impaired growth of the insertional clo3403 and clo3404 knockout mutants at 12°C compared to the growth of the wild-type culture. Additionally, the inactivation of clo3403 had a negative effect on motility. The growth efficiency at 12°C of the TCS mutants and the motility of the kinase mutants were restored by introducing a plasmid harboring the operon of the CLO3403/CLO3404 TCS. The results suggest that the CLO3403/CLO3404 TCS is important for the cold tolerance of C. botulinum E1 Beluga.

Important role of class I heat shock genes hrcA and dnaK in the heat shock response and the response to pH and NaCl stress of group I Clostridium botulinum strain ATCC 3502.

Class I heat shock genes (HSGs) code for molecular chaperones which play a major role in the bacterial response to sudden increases of environmental temperature by assisting protein folding. Quantitative reverse transcriptase real-time PCR gene expression analysis of the food-borne pathogen Clostridium botulinum grown at 37°C showed that the class I HSGs grpE, dnaK, dnaJ, groEL, and groES and their repressor, hrcA, were expressed at constant levels in the exponential and transitional growth phases, whereas strong downregulation of all six genes was observed during stationary phase. After heat shock from 37 to 45°C, all HSGs were transiently upregulated. A mutant with insertionally inactivated hrcA expressed higher levels of class I HSGs during exponential growth than the wild type, followed by upregulation of only groES and groES after heat shock. Inactivation of hrcA or of dnaK encoding a major chaperone resulted in lower maximum growth temperatures than for the wild type and reduced growth rates under optimal conditions compared to the wild type. The dnaK mutant showed growth inhibition under all tested temperature, pH, and NaCl stress conditions. In contrast, the growth of an hrcA mutant was unaffected by mild temperature or acid stress compared to the wild-type strain, indicating that induced class I HSGs support growth under moderately nonoptimal conditions. We show that the expression of class I HSGs plays a major role for survival and growth of C. botulinum under the stressful environmental conditions that may be encountered during food processing or growth in food products, in the mammalian intestine, or in wounds.

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 degrees 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 degrees C of spores that had previously been heated at 80 degrees 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 degrees 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.

Continuous flow microwave-assisted processing and aseptic packaging of purple-fleshed sweetpotato purees.

Pumpable purees from purple-flesh sweetpotatoes (PFSP) were subjected to microwave heating using a 60 kW, 915 MHz continuous flow system, followed by aseptic packaging in flexible containers to obtain a shelf-stable product. Initial test runs were conducted using a 5 kW 915 MHz microwave system to measure dielectric in-line properties and examine the puree temperature profiles. The results demonstrated uniformity in heating of the puree at sterilization temperatures (>121 degrees C), and the dielectric constants and loss factors were within the range of published values for orange-fleshed sweetpotato purees. The pilot-scale test runs in a 60 kW microwave unit produced shelf-stable puree packages stable at room temperature. Polyphenolic content of the PFSP purees were evaluated and the results showed that while total phenolics increased (5.9%) and total monomeric anthocyanins slightly decreased (14.5%) with microwave application, antioxidant activity determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC) assays did not significantly change as a result of microwave processing. Color values showed that microwave-processed samples differed from fresh puree in saturation and hue angle, but not in overall color change. PFSP purees increased in gel strength when microwave processed, packaged, and stored, but the gel could be easily disrupted into flowable purees. Overall, high-quality retention can be obtained by microwave processing and aseptic packaging of PFSP purees so that they can be used as functional food ingredients.

Combined effects of ionizing-irradiation and different environments on Clostridium botulinum type E spores.

We examined the combined effects of gamma-radiation (24 degrees C) on spores of Clostridium botulinum-type Eklund strain suspended in different gas-saturated Na-phosphate buffer in absence or presence of protectors or sensitizers. Response surface methodology (RSM) was also used to ascertain the effects of radiation on the recovery of spores using a medium containing various levels of NaCl or Na-thioglycollate. The former (< 0.5%) decreased viable spore counts, but the latter (0.15%) did not. Irradiation inactivation of Eklund spores was most effective in air-saturated buffers compared to N2O and N2 gas. The Na2-EDTA (0.01 M) was the most efficient radioprotector of spores due to its reactivity toward hydroxy radicals, followed by t-butanol (0.1 M) in NO2 or N(2)-saturated buffers, respectively. Catalase (10.0 mg ml(-1)) and DL-cysteine (0.1 mM) sensitized the spores during irradiated N2O or N(2)-saturated buffers, and NaCl (0.01 M) only sensitized spores in N2 environment. Spores frozen at -75 degrees C for 30 days and thawed prior to use were more sensitive to radiation damage compared to freshly prepared spores. Glycerol (15%), in Na-phosphate buffer (pH 7.0, 0.06 M), protected Eklund spores and increased the number of spores from 10(6) to 10(11) colony forming unit (CFU) ml(-1), and enhanced their radiosensitivities. Seven strains of C. botulinum type E were screened for plasmids and strain BL764 had two plasmids (15.8 and 46.8 mDa), BL4028 also had two (4.4 and 13.2 mDa), BL4850 contained only one (4.9 mDa), whereas EQA, BL211, Eklund, and Beluga had none. Gamma-Radiation (10 kGy, absorbed dose) cured the 15.8-mDa plasmid in strain BL764, but its absence yielded no changes in toxigenicity.

The sterilization of honey with cobalt 60 gamma radiation: a study of honey spiked with spores of Clostridium botulinum and Bacillus subtilis.

Unprocessed honey is a recognized wound-healing remedy. However, to make clinical use of honey acceptable, it should be sterile. To find the lowest dose of irradiation needed for sterilization, six batches of honey (a-f) were gamma irradiated with 6, 12, 18, 22 and 25 kGy Cobalt-60. After a dose of 25 kGy the antibacterial activity was not altered. Presumably glucose oxidase (EC 1.1.3.4), which produces hydrogen peroxide, is not easily damaged by irradiation. Amylase activity on the other hand was significantly reduced to 19%, 19%, 21%, 22%, 43% in batches a), b), c), d) and f) respectively, whereas no decrease was observed in batch e). All batches spiked with approximately 10(6) spores from Cl. botulinum or B. subtilis per 50 g honey proved to be sterile after irradiation with a dose of 25 kGy. Honey was also spiked with Cl. botulinum at up to 5000 spores per 50 g honey, which is the upper limit of natural contamination. The sterilizing dose in this case was 18 kGy.

Effects of irradiation on growth and toxigenicity of Clostridium botulinum types A and B inoculated onto chicken skins.

This study was conducted to examine the effects of 0.3-Mrad irradiation on growth and toxigenicity of Clostridium botulinum types A and B on chicken skins. Irradiation followed by aerobic or anaerobic incubation at 30 degrees C extended the shelf life of skin samples and delayed growth and toxin production by C. botulinum. During 2 weeks of incubation at 10 degrees C, the irradiated and nonirradiated C. botulinum spores failed to grow or produce toxin.

Relation between radiation resistance and salt sensitivity of spores of five strains of Clostridium botulinum types A, B, and E.

The NaCl tolerance of different strains of Clostridium botulinum varies over a wide range, and the patterns of NaCl inhibition differ distinctly and characteristically from strain to strain. The more radiation-resistant strains, such as 33A, 62A, and 7272A, are more resistant to NaCl, whereas the more radiation-sensitive strains, such as 51B and 1304E, are more sensitive to NaCl. This rule appears to hold irrespective of whether the spores were unirradiated controls or whether they were radiation damaged prior to exposure to NaCl in the recovery media. The data seem to indicate that radiation doses in the shoulder portion of the radiation survival curves did not noticeably sensitive the spores to NaCl, whereas radiation doses in the exponential-decline portion of the survival curve invariably produced a distinct sensitization. Thus, strains 33A and 62A were not sensitized to NaCl by 0.3 to 0.4 Mrad, i.e., in the shoulder portion of the survival curve. Radiation-sensitive strain 51B, which shows no distinct shoulder in its survival curve, was sensitized to NaCl by 0.1 Mrad, the lowest radiation dose employed in this study. These observations seem to suggest a possible relationship between deoxyribonucleic acid repair capacity and salt tolerance.

Cryogenic gamma irradiation of prototype pork and chicken and antagonistic effect between Clostridium botulinum types A and B.

Inoculated, irradiated pork (2,300 cans) and chicken (2,000 cans) pack studies were performed to establish the 12D dose for these foods. Each can was inoculated with a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (five type A and five type B), or a total of 10(7) spores. The cans received a series of increasing doses of gamma rays (60Co) at -30 +/- 10 degrees C; they were incubated for 6 months at 30 +/- 2 degrees C and examined for swelling, toxicity, and recoverable botulinal cells. The highest rate of swelling for both foods occurred within the first week of incubation, and maximum swelling was observed within 4 to 5 weeks. The minimal experimental sterilizing dose (ESD) based on flat, nontoxic sterile cans was 3.0 less than ESD less than or equal to 3.2 Mrad for pork and 4.0 less than ESD less than or equal to 4.2 Mrad for chicken. An analysis of the partial spoilage data by extreme-value statistics indicated with 90% confidence that the rate of spore death in the two foods was not a normal distribution, but appeared to favor a shifted exponential function. Based on the latter distribution, and assuming one most resistant strain in the mixture of 10 used, the 12D dose computed to 4.37 Mrad, with a shoulder of 0.11 Mrad, for pork and to 4.27 Mrad, with a shoulder of 0.51 Mrad, for chicken. An assumption that there were two or more most resistant strains in the inoculum progressively lowered the 12D dose. There was an apparent antagonism between the irradiated type A and B viable strains in the two foods. Cans with type B cells and toxin predominated over cans with type A cells and toxin, but cans with a mixture of type A and B toxins predominated over cans with a mixture of Type A and B cells. At the highest sublethal doses, only type A cells survived in pork, but in chicken there was a least one type B strain that was at least as resistant as type A strains.

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.

Influence of postirradiation incubation temperature on recovery of radiation-injured Clostridium botulinum 62A spores.

The number of colonies formed by unirradiated Clostridium botulinum 62A spores was independent of temperature, in the range from 20 to 45 degrees C (in 5 degrees C increments); no colonies developed at 50 degrees C. Spores irradiated at 1.2 or 1.4 Mrads produced more macrocolonies at 40 degrees C than at higher or lower temperatures. Apparently, radiation-injured spores were capable of repair of 40 degrees C than at the other temperatures studied. More than 99% of the radiation (1.2 Mrads) survivors were injured and were unable to form macrocolonies in the presence of 5% NaCl. The germinated radiation-injured spores were also sensitive to dilution, resulting in the loss of viability of 77 to 79% of the radiation survivors. At 30 and 40 degrees C, the irradiated spores did not differ significantly in the extent of germination (greater than 99% at both 30 and 40 degrees C), emergence (64% at 30 degrees C and 67% at 40 degrees C), and the maximum number of emerged cells that started to elongate (69% at 30 degrees C and 79% at 40 degrees C). However, elongation was remarkably more extensive at 40 degrees C than at 30 degrees C. Many elongated cells lysed within 48 h at 30 degrees C, indicating an impaired repair mechanism. If the radiation-injured spores were incubated at 40 degrees C in the recovery (repair) medium for 8 to 10 h, they germinated, emerged, and elongated extensively and were capable of repair. If, after 8 to 10 h at 40 degrees C, these cultures were shifted to 30 degrees C, the recovery at 30 increased by more than eightfold, resulting in similar colony counts at 30 and 40 degrees C. Thus, repair appeared to be associated with outgrowth. Repair did not occur in the presence of chloramphenicol at 40 degrees C, whereas penicillin had no effect, suggesting that the repair involved protein synthesis but did not require multiplication.

Low-temperature irradiation of beef and methods of evaluation of radappertization process.

An inoculated, irradiated beef pack (1,240 cans) was conducted for the determination of microbiological safety for unrestricted human consumption. Each can contained a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (5 type A and 5 type B), or a total of 10(7) spores/can. The cans were irradiated to various doses (100 cans/dose) with 60Co gamma rays at -30 +/- 10 C, incubated at 30 +/- 2 C for 6 months, and examined for swelling, toxicity, and recoverable botulinal cells. The minimal experimental sterilizing dose based on nonswollen, nontoxic sterile cans were 2.2 less than experimental sterilizing dose based on nonswollen, nontoxic sterile cans was 2.2 less than experimental sterilizing dose less than or equal to 2.6 Mrad. Using recoverable cells as the most stringent criterion of spoilage, and assuming the conventional simple exponential (without an initial shoulder) rate of spore kill, the "12D" dose was 3.7 Mrad when estimated on the basis of mixture of 10 strains totaling 10(7) spores/can, and 4.3 Mrad if it is assumed that each can of beef contained 10(6) spores of a single most resistant strain and all of these spores were of identical resistances. However, an analysis of the data by extreme value statistics indicated with 90% confidence that the spore death rate was not a simple exponential but might be a shifted exponential (with an initial shoulder), Weibull, lognormal, or normal, with a "12D" equivalent of about 3.0 Mrad regardless of the initial spore density per can. There was an apparent antagonism between the irradiated type A and B strains in the cans. Some of the cans contained type B toxin but did not include type B viable cells. Other cans had a mixture of type A and B toxins, but a large number of these cans did not yield recoverable type B cells. However, type A viable cells could always be demonstrated in those cans containing type A toxin.

[Detection of atoxigenic variants in Cl. botulinum type A and B cultures exposed to different treatments]. / Vyiavlenie atoksigennykh variantov v kul'turakh Cl botulinum tipov A i B pod vliianiem razlichnykh vozdeîstviî

A possibility of detection of atoxigenic clones in the cultures of Cl. botulinum, types A and B, under conditions of treatment with acridine strains, UV-irradiation and heating of spores was studied. Of the 10 strains investigated, atoxigenic variants were obtained only in 3, belonging to type B; the frequency of formation of atoxigenic clones under the effect of ethidium bronide constituted 36--45.7%, and of the UV-irradiation-4.2%; heating of spores proved to be ineffective. A frequent loss of the toxigenicity sign coincided with the view on the elimination of the episomal determinant of the toxin-formation, not with the mutational nature-of this change. An association between the differences in stability of retention of the toxigenicity signs in various strains and types of Cl. botulinum and possible differences of the corresponding genetic determinants of the toxin formation is discussed.

Observations on nonconverting phage, c-n71, obtained from a nontoxigenic strain of Clostridium botulinum type C.

A nontoxigenic mutant (C-N71) obtained from a toxigenic strain of Clostridium botulinum type C, Stockholm, with nitrosoguanidine treatment was found to be lysogenic by the lysis test. Although the filtrate of a passaged lysate of this nontoxigenic but lysogenic strain, C-N71, lysed cells of the nontoxigenic strain C-AO2 equally as well as the converting phage c-st obtained from the strain C-Stockholm, it did not convert C-AO2 to the toxigenic state. The lysis spectrum of this filtrate was the same as that of the c-st phage. The ability of the filtrate to lyse the indicator cells, C-AO2, was destroyed neither by trypsin nor DNase but was inactivated by heat treatment at 80 C for 10 min. This suggested that the agent which caused lysis was not boticin but probably a phage. An electron micrograph of the complete phage, c-n71, which was similar in morphology to that of the c-st phage was obtained from the filtrate of strain C-N71. Anti-c-n71 phage rabbit serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggest that the c-n71 phage is a mutant of the c-st phage which lacks the gene controlling production of botulinum type C toxin.