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; the predominant (or only) toxin from these types
is designated by the same capital letter. 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 but not of human
beings so far as is known. 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. The nutritive properties of
the food are likely to determine the minimal pH or temperature and the maximal
concentration of sodium chloride for growth and toxin production.
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. The presence of
sodium nitrate in sausage or of disodium phosphate in cheese spread reduces the
level of sodium chloride necessary to prevent toxin production. 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. A maximal pH of
8.89 was found for vegetative growth.
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.
It is presumed that (1)
growth of other organisms which could raise the pH of a food so that C.
botulinum could grow, (2) growth of C. botulinum followed by growth of
other organisms which lowered the pH of a food that originally had a pH higher
than 4.5, and (3) variation or stratification of the pH in an acidulated
product to permit growth of C. botulinum.
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.
Experimentally, types C and D can be “cured” of their temperate bacteriophage
and become nontoxigenic. Types A, B, and F have been “cured” of bacteriophages
also but have remained toxigenic. 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. Type E spores are found in soil, in sea and lake mud, and in fish,
primarily in their intestinal tracts. 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.
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.”
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.
A technique has been
described that simplifies this procedure by incorporating antitoxin into the
agar medium so that toxin-producing colonies are surrounded by a zone of
toxin–antitoxin precipitate.
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.
