Growth and Toxin Production by Clostridium botulinum in Sauteed Onions.

In October, 1983, sauteed onions in "patty-melt" sandwiches were epidemiologically responsible for a large outbreak of botulism in Peoria, Illinois. Spores of strains of Clostridium botulinum type A, recovered from Spanish onions or from patients who consumed sauteed onions, produced high toxin titers within 48 h from 2 spores/g of onions when experimentally inoculated into sauteed onions. Laboratory strains of C. botulinum type A which normally produce high-titered toxin in culture media yielded very low toxin titers and required 3 to 4 d and an extremely high inoculum of spores/g of onions. Five strains of C. botulinum type A were isolated from 75 raw onions obtained from the Peoria restaurant where the outbreak occurred.

Outgrowth and Toxin Production by Clostridium botulinum in Bottled Chopped Garlic.

The ability of Clostridium botulinum types A and B spores to grow and produce toxin in commercially bottled chopped garlic in soybean oil was investigated. Eight type A and seven type B strains of C. botulinum , mostly of vegetable origin, were used as inocula. Various numbers of spores were inoculated directly into the jars containing garlic, incubated at 35°C and sampled for organoleptic acceptance and presence of toxin every 5th d. In parallel studies conducted at room temperature, jars were sampled at 15-d intervals. At 35°C, when 1 spore/g of garlic was used as inoculum, toxin was produced in 15 d by type A and in 20 d by type B strains. At room temperature, five spores of type A or B per g of garlic produced toxin throughout 75 d. Even when highly toxic, garlic looked and smelled acceptable. Five strains of C. botulinum type A were isolated from 115 bulbs of fresh garlic.

Bacillus cereus Contamination of Seeds and Vegetable Sprouts Grown in a Home Sprouting Kit.

Sprouting seeds (alfalfa, mung bean and wheat) were purchased at local health food stores and examined for Bacillus cereus by the official AOAC method. Of 98 units collected, 56 (57%) were positive for B. cereus at levels ranging from 3 to >500 per g. Population levels of B. cereus on sprouts grown from naturally contaminated seeds in a home sprouting kit ranged from a mean of log10 3.72 for alfalfa to 5.39 for wheat; the log10 mean for mung bean sprouts was 4.52. Washing contaminated sprouts for 10 min with warm tap water as recommended by the manufacturer of the sprouting kits reduced the B. cereus count for mung bean sprouts by approximately one log unit but was less effective for wheat sprouts. B. cereus populations large enough to cause food poisoning (>105/g) frequently remained on wheat sprouts even after three wash cycles, and significant numbers of viable B. cereus remained on wheat sprouts even after cooking for 20 min.

Growth and Toxin Production by Clostridium botulinum in Sliced Raw Potatoes Under Vacuum With and Without Sulfite.

The ability of Clostridium botulinum type A or B spores to grow and produce toxin in fresh raw potatoes under vacuum with or without sulfite at 22°C was investigated. Fresh, peeled, sliced potatoes, untreated or dipped for 2 min in sulfite (NaHSO3) and drained, were surface-inoculated at several levels with a mixture of C. botulinum spores, either type A or B, and placed in oxygen-impermeable bags (200 g/bag) that were then vacuum-sealed and incubated at room temperature (22°C). Toxicity was tested on days 0, 3, 4, 5 and 6. After incubation, the potatoes were blended and centrifuged, and the millipore-filtered supernatant fluid was injected intraperitoneally into mice. Sensory evaluation, except taste, was also performed. Potatoes inoculated with C. botulinum type A spores, but untreated with NaHSO3 became toxic in 3 days, which coincided with the sensory evaluation, "Unfit for human consumption." However, despite inoculum size or residual SO2 levels, potatoes treated with NaHSO3 appeared acceptable for human consumption through day 6, even though they were toxic after 4 days of incubation. Toxicity from type B spores occurred later and in fewer test samples than type A. Again, the potatoes appeared acceptable but were toxic. Thus, although NaHSO3 markedly extended the consumer acceptability of peeled, sliced, raw potatoes at the abuse temperature, it did not inhibit outgrowth and toxin production by C. botulinum under these same conditions.

Incidence of Clostridium botulinum in Vegetables Packaged under Vacuum or Modified Atmosphere.

Because modified atmosphere-packaged (MAP) vegetables may provide an anaerobic environment conducive to Clostridium botulinum growth and toxin production, the incidence of C. botulinum spores in commercially available, precut MAP vegetables was determined. One-pound (454-g) packages of MAP vegetables were aseptically opened, added to freshly steamed and cooled sterile trypticase-peptone-glucose-yeast extract broth and incubated at 35°C for 7 days. Positive and negative controls were included with each sampling. After incubation the broth cultures were tested for toxicity by the standard mouse bioassay. Of the 1,118 MAP vegetable packages examined, one package each of shredded cabbage, chopped green pepper, and Italian salad mix contained C. botulinum type A spores. One additional salad mix (main ingredient, escarole) contained both C. botulinum type A and type B spores. Results indicated a low overall incidence rate (0.36%) of C. botulinum spores in commercially available precut MAP vegetables.

Differences and Similarities Among Proteolytic and Nonproteolytic Strains of Clostridium botulinum Types A, B, E and F: A Review.

Cultures of Clostridium botulinum types A, B, E and F, which are responsible for human botulism, fall into two groups with different characteristics unrelated to toxin type. These groups differ primarily with respect to proteolysis, but also have different somatic and spore antigens and DNA; the heat resistance of their spores, their growth at low temperatures and their salt tolerance also differ. All known type A strains are proteolytic and all type E strains are non proteolytic, but types B and F have some proteolytic and some nonproteolytic strains. Although proteolytic strains can activate their own toxins, nonproteolytic strains cannot do so and therefore require trypsinization for maximum toxicity. Proteolytic strains are unable to grow at temperatures below 10 C, but have relatively high salt tolerance and spores of high heat resistance. Nonproteolytic strains can grow at 3.3 C and have a lower salt tolerance; their spores have a much lower heat resistance than those of proteolytic strains.

Heat Resistance of Clostridium botulinum Type G in Phosphate Buffer.

The heat resistance of two strains of Clostridium botulinum type G in phosphate buffer was studied by the thermal death time (TDT) tube method and the thermal destruction rate (TDR) method. The strains were estimated to have one highly heat-resistant spore among approximately 100 spores or 10,000 relatively heat-labile spores. The heat-labile spores were studied by the TDR method and the heat-resistant spores by the TDT tube method. Decimal reduction times (D) for the heat-labile spores were determined by the slopes of the survivor curves. D values for strain 89 ranged from 0.6 min at 190°F to 6.9 min at 170°F and for strain 2739 from 0.9 min at 200°F to 5.9 min at 180°F. Thermal destruction curves for the heat-labile spores gave z values of 24.0 and 17.5 for two spore stocks of strain 89 and 26.0 for strain 2739. D values for the heat-resistant spores, calculated from the combined data of replicate experiments by the Schmidt probability method, ranged from 0.29 min at 240°F to 1.51 min at 210°F for strain 89 and from 0.25 min at 240°F to 1.48 min at 210°F for strain 2739. Extrapolated to 250°F, the thermal destruction curves of the heat-resistant spores gave D250 values of 0.14 to 0.19 min. The thermal destruction curves of the heat-resistant spores were very flat, however, with z values of 37.9 and 49.1 for the two spore stocks of strain 89 and 37.7 for strain 2739. Low-acid canned food processes will provide the same margin of safety for type G as for other proteolytic strains of C. botulinum but ultra high processing temperatures probably will not.

Common Characteristics of the Swiss and Argentine Strains of Clostridium botulinum Type G 1.

Five strains of Clostridium botulinum type G of human origin, from Switzerland, were compared with two strains isolated from soil in Argentina. The Swiss and Argentine strains are the only type G strains isolated to date. Characteristics compared were toxigenicity, sporulation, proteolysis and carbohydrate fermentation. High toxin titers were produced in trypticase-peptone-glucose-yeast extract broth incubated anaerobically at 30°C for 10 d. Sporulation occurred in three strains incubated anaerobically on soil extract agar at 35°C for 15 d. Different concentrations of soil extract in the medium promoted sporulation of different strains. Toxins of the Swiss and Argentine strains showed identical patterns for trypsin activation, reaction to A-F antitoxin and neutralization by antitoxin prepared from strain 89G. All seven strains showed delayed proteolytic activity but failed to ferment any of the sugars tested.

Clostridium botulinum Spores in Corn Syrup and Other Syrups.

A total of 738 bottles of corn syrup and other products containing corn syrup (e.g., pancake, maple, waffle, and table syrups) was examined by a membrane filtration procedure for the presence of Clostridium botulinum spores. One each of 354 light and 271 dark corn syrups was presumptively positive for C. botulinum type A spores, but subsequent testing of the entire contents of both bottles proved negative. All other 113 syrups were negative. Results showed that corn syrup and other syrups currently on the market are not food sources of C. botulinum spores for infants.

Evaluation of Unacidified Products Bottled in Oil for Outgrowth and Toxin Production by Clostridium botulinum.

A variety of unacidified products bottled in oil or water were investigated for their ability to support growth and toxin production by Clostridium botulinum . The products were inoculated with a mixture of five strains of C. botulinum type A spores (about 50 spores/g or ml) and incubated at room temperature (23°C). At monthly intervals the organoleptic acceptability of the products, as determined by appearance, odor, and texture, was evaluated and a portion of each sample was removed, diluted 1:2 in gel-phosphate buffer, and injected intraperitoneally into mice. At the end of 4 months the drained solids of each sample were macerated with a minimal amount of buffer and centrifuged; the clear extracts were then injected into mice. None of the products tested supported growth and toxin production by C. botulinum .

Clostridium botulinum Spores in Infant Foods: A Survey.

In an examination of 10 categories of infant foods obtained in the Washington, D.C. area, Clostridium botulinum spores were detected in 2 of 100 samples of honey and 8 of 40 samples of corn syrup. This is the first report of the occurrence of C. botulinum spores in retail samples of corn syrup. In an ensuing nationwide survey of corn syrup, C. botulinum spores were detected in 5 of 961 bottles examined.

Outgrowth of Clostridium botulinum in Shredded Cabbage at Room Temperature Under a Modified Atmosphere.

The ability of Clostridium botulinum types A and B spores to grow and produce toxin in shredded cabbage at room temperature under a modified atmosphere was investigated. Seven type A and seven type B strains of C. botulinum , mostly of vegetable origin, were used as inocula. Shredded cabbage in high barrier bags, 250 g/bag, was inoculated with various numbers of spores, sealed under a modified atmosphere of 70% CO2 and 30% N2 and incubated at room temperature. Duplicate bags were examined for organoleptic acceptability and the presence of toxin from day 3 by blending the entire contents of each bag and injecting mice with dilutions of the extracts. Toxic extracts were typed with appropriate antitoxins. Only type A spores grew and produced toxin in the cabbage. An inoculum of approximately 100-200 type A spores/g of cabbage, whether in single strains or in various combinations, produced toxin on days 4, 5, and 6, while the cabbage was still organoleptically acceptable, as determined by appearance, odor, and texture.

Immunodiffusion Detection of Clostridium botulinum Colonies.

Toxic colonies of Clostridium botulinum types A, B, E and F were detected by precipitating toxin around the colonies on agar containing antitoxin. Incorporating antitoxin in a gel diffusion agar overlay after colonies had developed was unsatisfactory for detection of type E, but worked well for all types when added directly to the plating medium. Addition of 0.6 IU of antitoxin per ml of agar gave satisfactory results with all types except type E. Zones of precipitation were produced by proteolytic strains of types A, B and F incubated at 35°C for 3 d and by nonproteolytic strains of types B and F incubated at 28°C for 4 d; type E required 1.2 IU of antitoxin per ml of agar and 5 d of incubation at 35°C. Nontoxigenic putrefactive anaerobes produced no zones of precipitation with any of the antitoxins, and toxic colonies of C. botulinum mixed among them were easily distinguished. This method was used successfully for selecting type B colonies from plates containing toxic enrichment cultures of tomato juice.

Fate of Listeria monocytogenes in Shredded Cabbage Stored at 5 and 25°C under a Modified Atmosphere.

Shredded cabbage was inoculated with Listeria monocytogenes Scott A cells and stored in normal air or a modified (70% carbon dioxide and 30% nitrogen) atmosphere at 5 and 25°C. Under the normal atmosphere at 25°C, colony counts increased by 2 logs within 2 d of storage but then decreased to undetectable levels within 6 d of storage. In the modified atmosphere at 25°C, numbers also decreased to undetectable levels within 6 d, but with a less marked initial increase and a decline that was more rapid than in the unmodified atmosphere. In the cold (5°C), the counts increased gradually, but only by about 1 log, in both atmospheres. In the normal atmosphere at 5°C, however, colony counts decreased sharply after 13 d of storage. Reductions in colony counts coincided with decreases in cabbage pH and development of spoilage. The increased level of carbon dioxide was ineffective in controlling L. monocytogenes at 5°C. At 25°C cabbage spoilage was rapid and colony counts declined under both atmospheres of storage.

Amplified Immunoassay ELISA-ELCA for Measuring Clostridium botulinum Type E Neurotoxin in Fish Fillets.

The measurement of Clostridium botulinum type E toxin in fish was accomplished using an amplified immunoassay (enzyme-linked immunosorbent assay-enzyme-linked coagulation assay [ELISA-ELCA]) based on the coagulation cascade. Fresh catfish fillets inoculated with a mixture of spores from five strains of C. botulinum type E were packaged in high barrier film with air, vacuum and modified atmosphere and stored at 4, 8 or 16°C for up to 75 days. Toxin production was monitored during storage by both mouse bioassay (trypsin and non-trypsin treated) and ELISA-ELCA on the non-trypsinized samples. All 26 inoculated products that were positive by the mouse bioassay were also positive by ELISA-ELCA. Of 35 uninoculated samples which were not toxic in mouse bioassay, none were positive by ELISA-ELCA; of 73 inoculated samples which were not toxic by mouse bioassay, 14 had toxin measurable by the ELISA-ELCA. The position of these immunoassay-positives in the sampling sequence indicated that the toxin was identified by the immunoassay before it was found in the mouse bioassay. These results suggest that the ELISA-ELCA technique is a usable alternative to the mouse bioassay for monitoring C. botulinum type E toxin production in fish challenge studies.