Donald A. Kautter

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.

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.

Incidence and growth potential of Bacillus cereus in ready-to-serve foods.

To simulate temperature abuse, 106 test portions of ready-to-serve moist foods, 12 test portions of rehydrated powdered infant formula, and 18 test portions of nonfat dry milk were incubated for 20 and 24 h at 26°C, and then examined for Bacillus cereus . Of the ready-to-serve moist foods, 88 of 106 were positive for B. cereus at levels ranging from 0.25 to 8.5 × 106/g after 20 h of incubation and from 0.1 to 58 × 106/g after 24 h. All of the powdered milk and 12 of the 15 units of infant formula, representing five brands, were positive, with counts ranging from 0.15 to 5.0 × 106/g in 20 h and 5.0 to 49 × 106 after 24 h. B. cereus counts in the powdered products were low, ranging from 0.09/g for one of two soy-based products to an average of 0.29/g for milk-based products. However, these levels were sufficient to initiate growth of B. cereus in almost every 2-oz serving. Similar results were obtained for rehydrated nonfat milk, with initial B. cereus counts ranging from 0.29 to 1.5/g; at 26°C the counts averaged 3.3 × 107 after 20 h and 5.5 × 107 after 24 h. Counts ranged from 2.0 × 104 to 1.1 × 105 after 9 h in milk and were in excess of 106/g after 10.5 h.

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.

Effect of Low Temperatures on Growth of Nonproteolytic Clostridium botulinum Types B and F and Proteolytic Type G in Crabmeat and Broth1

The ability of unheated and heated spores of nonproteolytic Clostridium botulinum types B and F, and of the weakly proteolytic type G, to grow and produce toxin in crabmeat and broth at low temperatures was investigated. Sterilized crabmeat or broth was inoculated with 103 spores/g or ml and incubated anaerobically at 4, 8, 12 and 26 C for 180 days. Both heated and unheated spores of all three types grew and produced toxin at 26 C in broth and crabmeat. Types B and F grew in broth at 12, 8 and 4 C when unheated but only at 12 and 8 when heated; they did not grow in crabmeat at any of these temperatures, heated or not. Heated and unheated type G grew at 12 C in both broth and crabmeat but not at lower temperatures.

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.

Evaluation of a Reversed Passive Latex Agglutination Test Kit for Clostridium perfringens Enterotoxin

A reversed passive latex agglutination (RPLA) test kit for Clostridium perfringens enterotoxin (CPE) marketed by the Denka-Seiken Co., Tokyo, Japan, was evaluated by using culture supernatant fluids and extracts from feces of food poisoning patients. Nanograms of CPE were detectable with the assay and the reaction was specific, as shown by parallel activity in a double antibody enzyme-linked immunosorbent assay (ELISA). Although less sensitive, the RPLA method is easier to perform than the ELISA and counterimmunoelectrophoresis, both of which require special test reagents and equipment not generally available.

Botulism in Commercially Canned Foods

Occasionally, a problem thought to be well under control returns to plague us. This is the case with botulism in commercially canned foods. These foods have had a remarkably good record over the last 45 years with approximately 775 billion cans of commercially canned foods being consumed with only four known deaths through 1971. Beginning in 1971, however, botulinal toxin and/or Clostridium botulinum has been found in commercially canned vichyssoise, chicken vegetable soup, peppers, marinated mushrooms, tuna fish, beef stew, and in 41 cans of mushrooms from 20 lots packed by seven domestic and two foreign producers. The typical cause of botulism in canned foods is underprocessing which may result from inadequate equipment, improper operating procedures, and scheduled processes which are not appropriate for the actual operating conditions being used.

Improved Media for Sporulation and Enterotoxin Production by Clostridium perfringens

Sporulation of 24 strains of Clostridium perfringens isolated from stools of food poisoning patients and normal controls was improved by adding sodium carbonate to Duncan-Strong (DS) sporulation medium and replacing starch with raffinose in Tanigutis AEA medium. Viable counts of heat-tolerant spores (75°C for 20 min) were 2 to 186 times greater in modified AEA medium and 2 to 169 times greater in modified DS than in DS medium. Reversed passive latex agglutination assays revealed a corresponding increase in enterotoxin titers in supernatant fluids of the 12 enterotoxigenic strains grown in modified AEA medium and in modified DS medium.

Effect of Low Temperatures on Growth of Clostridium botulinum Spores in Meat of the Blue Crab

The ability of unheated and heated spores of Clostridium botulinum types B. E, and F to grow and produce toxin in crabmeat from the blue crab at low temperatures was investigated. Sterilized crabmeat was seeded with 103 unheated spores/g or 104 heated spores/g and incubated anaerobically at 4, 8, 12, and 26 C. Broth cultures served as controls. Both unheated and heated spores of the three strains grew and produced toxin in crabmeat at 26 C in 3 and 6 days, respectively. In addition, unheated spores of the nonproteolytic type E strain grew and produced toxin in crabmeat at 12 C in 14 days. Neither heated spores of type E nor heated or unheated spores of types B and F grew in crabmeat at any refrigerated temperature within 180 days.