Haim M. Solomon

Inactivation of Clostridium botulinum Type A Spores by High-Pressure Processing at Elevated Temperatures

The effects of high-pressure treatments at various temperature-time combinations on the inactivation of spores of Clostridium botulinum type A strains 62-A and BS-A in phosphate buffer (0.067 M, pH 7.0) and in a crabmeat blend were investigated. The log unit reduction of strain 62-A spores increased significantly as the processing pressure increased from 417 to 827 MPa (from 60,000 to 120,000 lb/in2) at 75 degrees C. The reduction of BS-A and 62-A spores in either medium increased as processing temperatures increased from 60 to 75 degrees C and processing times increased from 5 to 15 or 20 min at a maximum pressure of 827 MPa. Approximately 2- and 3-log reductions of BS-A and 62-A spores, respectively, in phosphate buffer were obtained at the maximum pressure-maximum temperature combination of 827 MPa and 75 degrees C for a processing time of 20 min. Processing for 15 min at the maximum pressure-maximum temperature combination resulted in maximum reductions of 3.2 and 2.7 log units for BS-A and 62-A spores, respectively, in the crabmeat blend. Results obtained in this study indicate that the crabmeat blend did not protect BS-A and 62-A spores against inactivation by high-pressure processing.

A Large Outbreak of Botulism: The Hazardous Baked Potato

In April 1994, the largest outbreak of botulism in the United States since 1978 occurred in El Paso, Texas. Thirty persons were affected; 4 required mechanical ventilation. All ate food from a Greek restaurant. The attack rate among persons who ate a potato-based dip was 86% (19/22) compared with 6% (11/176) among persons who did not eat the dip (relative risk [RR] = 13.8; 95% confidence interval [CI], 7.6-25.1). The attack rate among persons who ate an eggplant-based dip was 67% (6/9) compared with 13% (241189) among persons who did not (RR = 5.2; 95% CI, 2.9-9.5). Botulism toxin type A was detected from patients and in both dips. Toxin formation resulted from holding aluminum foil-wrapped baked potatoes at room temperature, apparently for several days, before they were used in the dips. Consumers should be informed of the potential hazards caused by holding foil-wrapped potatoes at ambient temperatures after cooking.

An Outbreak of Type A Botulism Associated with a Commercial Cheese Sauce

Botulism is a rare disease; between 1983 and 1992, an average of only 22 cases of food-borne botulism were reported to the Centers for Disease Control and Prevention each year (Unpublished data). Nevertheless, a reported case of food-borne botulism represents a public health emergency because many persons may be affected if the contaminated food is not identified. This is especially true of outbreaks linked to commercial products or restaurants. However, because few clinicians have ever seen a case of botulism, the diagnosis may be delayed or not even considered. Diagnosing botulism is a special challenge when patients present with mild symptoms and do not have a history of exposure to typical food vehicles, such as home-canned vegetables. We describe an outbreak of botulism that was characterized by relatively mild symptoms and subtle physical findings. The outbreak was caused by a food vehicle that was initially considered to be unlikely. The investigation shows the importance of considering the diagnosis of botulism soon after patients present with acute cranial nerve dysfunction and of promptly reporting suspected cases to public health officials. The Outbreak On 4 October 1993, a 42-year-old woman (patient 1) visited her family physician in a small town in southern Georgia. She had had nausea, blurred vision, and loss of balance for 2 days. Results of physical examination were normal except for a possible sixth-nerve palsy. Labyrinthitis was diagnosed, and the patient was sent home. When her physician contacted her the next day, she was too weak to come to the telephone. Her husband reported that her speech was slurred and that she was having difficulty swallowing. He mentioned that their 21-year-old daughter (patient 2) also had nausea and difficulty swallowing. Both patients were referred to a neurologist, who recognized this unusual clustering of neurologic symptoms as possible botulism. The patients were admitted to a hospital, and public health officials were notified. That same day, a 38-year-old woman with a history of hypertension (patient 3) was seen in the emergency department of the same hospital because of blurred vision, slurred speech, and weakness in her right arm. She was admitted to the medical ward with a diagnosis of transient ischemic attack. By coincidence, the family physician of patients 1 and 2 was also attending on the medical ward that night; he recognized that patient 3 might be another case of botulism. Patient 3 mentioned that her friend, patient 4, was having similar symptoms. Patient 4 was notified that her illness might also be botulism, and she too was hospitalized. Two days earlier, her new symptoms had been diagnosed as an allergic reaction to a tranquilizer. Patient 5 had visited an optometrist on 5 October with fatigue and blurred, double vision. She received a diagnosis of mild glaucoma and astigmatism and was given a prescription for eyeglasses. She presented to the emergency department on 7 October after hearing about the outbreak on the radio. None of the patients had eaten any home-canned foods. However, on 1 October, all of them had eaten food from a delicatessen that had re-opened on 23 September after having been closed for 6 months because of the owners family obligations. Local health officials closed the delicatessen on 6 October and seized leftover foods. Methods Clinical and Epidemiologic Investigation Hypothesis-generating interviews were done with the hospitalized patients and the owner of the delicatessen. Each step in the preparation and storage of foods was reviewed. After a standardized questionnaire that addressed food histories and symptoms was developed, investigators attempted to interview (either by telephone or in person) all persons who had eaten food from the delicatessen in the 6 days it was open between 23 September and 2 October 1993. For the purposes of the investigation, a case of botulism was defined as dysphagia, dysphonia, dysarthria, or diplopia that developed after 23 September in any person who had eaten food purchased at the delicatessen. A press release was issued to identify patrons of the delicatessen. The press release asked all persons who had eaten at the delicatessen to call the local health department. The owner and known patrons of the delicatessen were asked to name other patrons, and businesses in the neighborhood around the delicatessen were surveyed as to whether workers had eaten food from the delicatessen. In an attempt to find additional cases, 50 physicians in the area were called and asked whether they had seen any patients since 23 September who reported blurred or double vision, dry mouth, difficulty swallowing, change in voice, or muscle weakness. To identify any cases that may have been mistakenly diagnosed as other conditions, physicians were also asked if they had recently seen any patients with a diagnosis of stroke, transient ischemic attack, the Guillain-Barre syndrome, or myasthenia gravis. Logs from the emergency department of the local hospital were reviewed for these symptoms and diagnoses. All hospitalized patients were examined by the same neurologist, and their hospital and outpatient records were reviewed. No neurologic examination was done on three persons who met the case definition for botulism but did not seek medical attention. These patients were identified by their responses to the standard questionnaire. The delicatessen was inspected by officials of the Georgia Department of Agriculture. Officials of the Food and Drug Administration inspected the canning facility and searched for unused cans of the same batch of cheese sauce. Laboratory Investigation Samples of food taken from the delicatessen were assayed for botulinum toxin and were cultured for Clostridium botulinum as described elsewhere [1]. All persons who ate the implicated food were asked to submit serum and stool specimens. Gastric aspirate specimens were obtained from two hospitalized patients. Serum, stool, and gastric aspirate specimens were assayed for C. botulinum toxin, and stool specimens were cultured for C. botulinum. Inoculation experiments were done in the Food and Drug Administration botulism laboratory to determine the time and temperature needed for C. botulinum to grow and for toxin to be produced in the implicated brand of cheese sauce. Spores harvested from cultures of the outbreak strain of C. botulinum were heat-shocked at 80 C for 10 minutes and then diluted with sterile water to a concentration of 104 spores/mL. Twenty g of the cheese sauce was then added to sterile test tubes that contained 0.1 mL of inoculum; the final concentration was 103 spores/20 g of cheese. The tubes were incubated at 22 C and 5 C; they were then assayed for toxin on day 8 and every 3 to 4 days for 2 months. Toxin testing was also done before incubation to ensure that no toxin was transferred with the inoculum. Toxin was measured in mouse minimum lethal doses using the mouse bioassay [2]. Results Epidemiologic Findings The delicatessen first opened in August 1992 and then closed for 6 months from March to September 1993. It reopened on 23 September 1993, serving lunch 3 days a week (Thursday through Saturday). Food was served at the delicatessen from 23 to 25 September and from 30 September to 2 October (Figure 1). Routine inspections of the delicatessen done on 30 September and after the outbreak showed no violations of state standards for retail food sale establishments. Figure 1. Date of meals eaten by all 52 patrons of the delicatessen and dates of symptom onset in 8 patrons with botulism. Fifty-two persons who ate food from the delicatessen in the 6 days it was open between 23 September and 2 October 1993 were identified and interviewed. Eight (15%) met the case definition for botulism. Their ages ranged from 20 to 48 years; 6 were women. No additional cases were identified through the review of emergency department logs or the physician survey. The owner of the delicatessen estimated that she served about 20 meals each day. Many of the patrons were friends of the owner or members of the owners family and had eaten there more than once. Eight (36%) of 22 persons who ate food from the delicatessen on Friday, 1 October, met the case definition compared with none of the 30 who ate the food only on other days. Among the 22 persons who ate food from the delicatessen on 1 October, all 8 ill persons but none of 14 well persons had eaten a barbecue stuffed potato. Six other persons had eaten barbecue stuffed potatoes before 1 October but remained well. The owner did not recall selling any stuffed potatoes on 2 October. Clinical Findings and Laboratory Confirmation of Botulism The illnesses ranged from mild to severe. Most patients had few, subtle objective neurologic findings despite having many symptoms characteristic of botulism. Symptoms developed a median of 2.5 days after exposure (range, 1 to 6 days). All ill persons had 3 or more symptoms consistent with botulism (median, 8.5 symptoms; range, 3 to 14 symptoms) and, by definition, at least 1 symptom that suggested a cranial nerve abnormality. The most common symptoms were dry mouth, difficulty speaking and swallowing, and change in voice quality (Table 1). All ill persons had neurologic and gastrointestinal symptoms. Three persons had illnesses so mild that they did not seek medical care. Five persons were hospitalized in an intensive care unit after botulism was suspected; in four of these persons, an illness other than botulism was initially diagnosed (Table 2). Patient 3, the most severely affected, developed complete bilateral ptosis, markedly dysarthric speech, weakness of the tongue and palate, arm and leg weakness, and respiratory failure. She died of a pulmonary embolism after being supported by mechanical ventilation for 18 days. Patient 1 was noticeably dysarthric and required nasogastric intubation because of difficulty swallowing. Patients 2, 4, and 5 had few object

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.

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.

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.

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.

Shelf life and toxin development by Clostridium botulinum during storage of modified-atmosphere-packaged fresh aquacultured salmon fillets

Shelf life (onset of sensory spoilage) and the potential for toxin production by Clostridium botulinum type E in retail-type packages of fresh aquacultured salmon fillets packaged in high-barrier film bags under selected atmospheres (100% air, a modified atmosphere containing 75% CO2:25% N2, and vacuum) and stored under refrigeration (4°C) and temperature-abuse conditions (8 and 16°C) were investigated. Chemical spoilage indicators (trimethylamine and surface pH) and microbial populations were compared with sensory spoilage characteristics. Storage temperature influenced the time to onset of both sensory spoilage and toxin development in salmon fillets packaged in all atmospheres. The shelf life of fillets packaged in all atmospheres decreased with increase of storage temperature from 4 to 16°C. Trimethylamine content associated with the onset of spoilage for 100% air-packaged fillets increased as storage temperature increased. However, for modified-atmosphere-packaged fillets, the trimethylamine content associated with the onset of spoilage increased as storage temperature decreased from 8 to 4°C. Surface pH was not a good spoilage indicator for modified-atmosphere-packaged fillets. Toxin development preceded sensory spoilage at 16°C storage for fillets packaged in modified atmospheres. Toxin development coincided with sensory spoilage or was slightly delayed for the fillets packaged in all the atmospheres at 8°C storage. At 4°C none of the fillets packaged in either of the atmospheres developed toxin, even 20 days after spoilage as determined by sensory characteristics.

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.