Clostridium
Botulism
Botulism is a disease caused by the ingestion of food containing the neurotoxin produced by Clostridium botulinum.
Organism This rod-shaped soil bacterium is saprophytic, spore forming, gas forming, and anaerobic.
Seven types are distinguished on the basis of the serological specificity of their toxins; Type A is the one commonly causing human botulism. It is more toxic than type B. Type B is found more often than type A in most soils of the world and is less toxic to human beings. Type C causes botulism of fowls, cattle, mink, and other animals. Type D is associated with feed poisoning of cattle. Type E, which is toxic for humans, has been obtained chiefly from fish and fish products. Type F, which except for its toxin is similar to types A and B, produces human botulism. Type G has been isolated from the soil in Argentina but has not been implicated in human botulism.
Not all types produce a single toxin. Type A strains and most cultures of type B are proteolytic and are putrefactive enough to give an obnoxious odor to proteinaceous foods, but some strains of type B and those of type E are not.
C. botulinum strains frequently are divided into three general groups, based on cultural and physiological characters, as follows: Group I includes all type A strains (proteolytic) and the proteolytic strains of B and F. Group II includes all type E strains (nonproteolytic) and the nonproteolytic strains of B and F Group Ill includes types C and D; they are nonproteolytic and share a common metabolic pattern.
Growth and Toxin Production Toxin production by C. botulinum depends on the ability of the cells to grow in a food and to autolyze. The types A, B, E, and F toxins are synthesized as large, comparatively inactive proteins which become fully toxic after some hydrolysis.
The factors that influence spore germination, growth, and hence toxin production are the composition of the food or medium, especially its nutritive properties (e.g., glucose or maltose is known to be essential for toxin production), moisture content, pH, O-R potential, and salt content, and the temperature and time of storage of the food. It is the combination of these factors that determines whether growth can take place and the rate and extent of that growth.
Media containing milk or casein, glucose or maltose, and corn-steep liquor yield more potent type A toxin than other media. The concentrations of sodium chloride necessary to prevent growth and toxin production in foods depend on the composition of the food and the temperature. A pH near neutrality favors C. botulinum. The minimal pH at which growth and toxin production will take place depends on the kind of food and the temperature. A pH of 4.5 or lower will prevent toxin production in most foods.
There have been outbreaks of botulism from inadequately heat-processed canned high-acid foods (pH less than 4.5) including tomatoes, tomato juice, and blackberries.
Temperature is an important factor in determining whether toxin production will take place and what the rate of production will be. Vegetative growth will take place at a lower temperature than the minimum for spore germination.
Toxin The toxin of C. botulinum is so powerful that only a tiny amount is sufficient to cause death. It is absorbed mostly in the small intestine and paralyzes the involuntary muscles of the body. The heat treatment necessary to destroy it depends on the type of organism producing the toxin and the medium in which it is heated.
The growth of C. botulinum in foods results in such a foul, rancid odor. Meats and proteinaceous, low-acid vegetables develop an especially obnoxious odor. More acid foods, however, and those low in proteins may become just as toxic without much evidence of putrefaction. The nonproteolytic strains of C. botulinum give less evidence of spoilage than do the proteolytic ones.
Gas production is not always evident and is not a reliable indication of spoilage by this organism. It is advisable to reject all foods, raw or canned, that give evidence of spoilage and to reject canned foods that exhibit any pressure in the container. The toxin persists in foods for long periods, especially when storage has been at low temperatures. It is unstable at pH values above 6.8.
Toxicity and Bacteriophages
The production of the toxin is coded for by the genome of a temperate bacteriophage in the bacteria. This is responsible for the occasional loss of toxigenicity by some strains.
One strain may harbour more than one temperate bacteriophage (multiple infection), and some strains produce more than one type of toxin. The spores C. botulinum, have a high resistance to heat.
The heat treatment necessary to destroy all the spores in a food depends on the kind of food, the type and strain of C. botulinum, the medium in which the spores were formed, the temperature at which they were produced, the age of the spores, and the numbers of spores present.
The habitat of C. botulinum is believed to be the soil. Plant crops may become contaminated from the soil, and intestinal contents and hence manure of animals after consumption of such plants.
Botulism occurs only rarely, but has high mortality.
Foods involved Inadequately processed home-canned foods, preserved meats and fish are most often the cause of botulism. There have been exceptional instances of poisoning from acid foods, such as tomatoes, apricots, pears, and peaches. These acid foods had been grossly underprocessed, and the underprocessing had permitted the growth of other microorganisms to aid growth and toxin production by C. botulinum.
The spores of C. botulinum will survive long storage periods in raw and precooked frozen foods and can grow and produce toxin if these foods are held long enough at a high enough temperature after thawing. Outbreaks have been traced to tomato relish, chili peppers, chili sauce, salad dressing, mutton, corn and chicken mash.
Disease People are so susceptible to botulism that if appreciable amounts of toxin are present, everyone who eats the food becomes ill and consumption of very small pieces of food, a pod of a string bean or a few peas, can cause illness and death.
The typical symptoms of botulism usually appear within 12 to 36 hr, although a longer or shorter time may be required. The earliest symptoms usually are nausea and vomiting and possibly diarrhea, together with fatigue, dizziness, and a headache. Later there is constipation. Double vision may be evident early, and difficulty in swallowing and speaking may be noted. Patients may complain of dryness of the mouth and constriction of the throat, and the tongue may become swollen and coated. The temperature of the victim is normal or subnormal. Involuntary muscles become paralyzed, paralysis spreads to the respiratory system and heart, and death usually results from respiratory failure.
Symptoms are similar for types A, B, and E poisoning, although nausea, vomiting, and urinary retention usually are more severe with type E toxin. In fatal cases, death usually comes within 3 to 6 days after the poisonous food has been ingested, but the period may be shorter or longer.
The only known method for the successful treatment of botulism is the administration of antitoxin. It should always be used at the earliest possible moment since it may prove and is not successful after the symptoms of botulism have appeared. Other treatments include artificial respiration, keeping the patient quiet, maintaining the fluid balance in the body, and, perhaps, elimination treatments.
Conditions Necessary for an Outbreak
The following conditions are necessary for an outbreak of botulism: (1) presence of spores of C. botulinum of type A, B, or E in the food being canned or being processed in some other way, (2) a food in which the spores can germinate and the clostridia can grow and produce toxin, (3) survival of the spores of the organism, e.g., because of inadequate heating in canning or inadequate processing otherwise, (4) environmental conditions after processing that will permit germination of the spores and growth and toxin production by the organism, (5) insufficient cooking of the food to inactivate the toxin, and (6) ingestion of the toxin-bearing food.
Prevention of Outbreaks The methods and precautions for the prevention of botulism that have been mentioned in the preceding discussion include (1) use of approved heat processes for canned foods, (2) rejection of all gassy (swollen) or otherwise spoiled canned foods, (3) refusal even to taste a doubtful food, (4) avoidance of foods that have been cooked, held, and not well reheated, and (5) boiling of a suspected food for at least 15 min.
Avoid raw or precooked foods that have been frozen, thawed, and held at room temperatures.
To prevent botulism from smoked fish it has been recommended that (1) good sanitation be maintained throughout production and handling, (2) during smoking or thereafter the fish be heated to at least 82 C for 30 min in the coldest part, (3) the fish be frozen immediately after packaging and kept frozen, and (4) all packages be marked “Perishable-Keep Frozen.”
Infant botulism results from colonization of the infant’s gut with C. botulinum and production of toxin in situ. It occurs mostly in infants aged 2 weeks to 6 months, particularly around the time that non-milk feeds are introduced. At this stage the infant’s gut microflora is not fully developed and is less able to outcompete and exclude C. botulinum. Since it only requires the ingestion of viable spores, environmental sources other than food may be involved. Honey has been associated with several cases of infant botulism in the USA. Consequently, it is thought inadvisable to feed honey to children less than a year old.
The illness is characterized by neuromuscular symptoms related to those of classical botulism and diagnosis can be confirmed by the isolation of the organism and its toxin from the faeces.
Wound botulism is caused by a subcutaneous infection with C. botulinum. This can result from trauma, but in recent years is more commonly associated with intravenous drug use. Accidental overdoses of botulinum toxin during its cosmetic use to remove facial wrinkles have also caused occasional cases.
C. botulinum will often constitute only a small proportion of the total microflora so enrichment or pre-incubation is necessary to improve the chances of isolation. Sometimes enrichment cultures are heated prior to incubation to eliminate non-spore forming anaerobes.
After enrichment in a medium such as cooked meat broth, the culture is streaked on to fresh horse-blood or egg yolk agar and incubated anaerobically for 3 days. Characteristic smooth colonies, 2–3 mm in diameter with an irregular edge and showing lipolytic activity on egg-yolk agar (type G excepted) are transferred into a broth medium to check for toxin production.
Clostridium Perfringens
Organism: First reported in the United States in 1945, Clostridium perfringens gastroenteritis is being detected and reported more frequently. The species is classified into five types, designated A–E, based on the production of four major exotoxins, α, β, €, ŧ, and eight minor ones.
C. perfringens (welchii), type A, a gram-positive, nonmotile, anaerobic, spore-forming rod. Maximal temperature for growth is about 55oC, and optimal temperature is about 43 to 47oC. Growth is restricted at 15 to 20oC.
The organism will not grow below pH 5.0 or above pH 9.0. It is inhibited by 5 % NaCI (aw = 0. 97), and some strains inhibited by 2.5 % sodium nitrate. The spores of food-poisoning strains differ considerably in their heat resistance.
Foods Involved: The spores have been found in samples of most raw foods examined as well as in soil, sewage, and animal faeces. Most commonly involved are meats that have been cooked, allowed to cool slowly, and then held for some time before consumption. Meat and poultry products account for about three-quarters of outbreaks attributed to C. perfringens. Fish paste and cold chicken also have been incriminated. Since the spores are fairly common in raw foods and are heat-resistant, their presence in many foods may be unavoidable. Cooking of foods will destroy the vegetative cells and the spores of some strains; however, germination and out growth of surviving spores are possible in cooked foods which are inadequately refrigerated. A typical outbreak might involve a large cut of roasted meat. The oven cooking would not kill all the spores but might create favorable reduced conditions. Following cooking and in the absence of proper cooling, the spores of C. perfringens germinate and their numbers increase. The presence of a large number of C. perfringens in a cooked food is indicative of mishandling.
Disease: Gastroenteritis
The symptoms, which appear usually in 8 to 24 hr following ingestion include acute abdominal pain, diarrhea, and gas; fever, nausea, and vomiting are rare. The ingestion of millions of viable cells of C. perfringens per gram of food are required for symptoms to occur. A toxin (enterotoxin) is released in the gut during sporulation of the cells and results in excessive fluid accumulation in the intestinal lumen. The enterotoxin is relatively heat-sensitive, being inactivated at 60oC for 10 min.
Conditions necessary for an outbreak:
(1) The food contains or becomes contaminated with C. perfringens
(2) usually the food is cooked and reduced conditions develop
(3) the food is inadequately cooled, and favorable temperatures and enough time are allowed for appreciable growth
(4) the food is consumed without reheating so that large numbers of viable cells are ingested,
(5) the cells sporulate in vivo and elaborate the enterotoxin.
Isolation and Identification: In the examination of foods, the total count (vegetative cells plus spores) is determined but with faecal specimens a spore count, obtained after heating a suspension at 80oC for 10 min, is also performed. The most commonly employed selective plating media used to enumerate C. perfringens employ antibiotic(s) as the selective agent and sulfite reduction to produce black colonies as the differential reaction. The most popular combinations are tryptose/sulfite/cycloserine (TSC) medium and oleandomycin/polymyxin/sulfadiazine/perfringens (OPSP), incubated anaerobically for 24 h at 37oC. Pour plate technique is preferred. Suspect colonies can be confirmed by the absence of motility, their ability to reduce nitrate to nitrite, lactose fermentation, and gelatin liquefaction. Serotyping based on capsular antigens is employed for epidemiological purposes, supplemented by typing with bacteriocins, particularly when serotyping is not possible. A number of methods are available for the detection of enterotoxin
Prevention of Outbreaks
Means of prevention of outbreaks of C. perfringens food infection include (1) adequate and rapid cooling of cooked meats and other foods, (2) holding hot foods above 60oC, (3) reheating of leftover foods, and (4) good personal hygiene.
Bacillus cereus and other Bacillus species
Organism: B. cereus is a gram-positive, aerobic, spore-forming rod. Its optimal temperature for growth is 30oC, with a minimal temperature for growth at 10oC and a maximum of 49oC. The pH range for growth is 4.9 to 9.3. Numerous surveys on foods and ingredients have indicated a high percentage of samples containing B. cereus. It is undoubtedly widely distributed in nature and our food supply. Extremely large numbers (108 per gram) of viable cells of B. cereus must be ingested to develop signs and symptoms of the syndrome. Two syndromes are recognized: the relatively late-onset, diarrheal syndrome and a rapid-onset emetic syndrome.
B. cereus gastroenteritis: Diarrheal syndrome due to diarrheagenic toxin. Symptoms include nausea, abdominal cramps, watery diarrhoea, vomiting after an incubation period of 8-16 h or 1.5-5 h. Food items implicated are custards, cereal products, puddings, sauces, meat loaf etc. Control measures include chilling of foods rapidly in small quantities, holding hot foods at 65oC or above, practising personal hygiene, processing and preparing food in sanitary manner and reheating leftover foods to 71.1oC.
Emetic syndrome is due to emetic toxin, After an incubation period of 1-6 h, (or 2-5 h minimum), Symptoms include nausea and vomiting predominantly. Similar to staphylococcal intoxication, of short duration- lasts for a day or less.
A number of other Bacillus species such as B. licheniformis and B. pumilis or B. subtilis and B. thuringiensis also have been associated with foodborne illness.
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