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Abstract
A wide variety of foods and food products derived from plants and animals
support the growth of pathogenic and toxigenic bacteria, resulting in foodborne diseases such as food infection and food intoxication or poisoning
which is a major public health problem globally. Bacterial growth and toxin
production in foods are influenced by various intrinsic (e.g. pH, moisture,
redox potential, nutrients) and extrinsic (e.g. temperature) factors. Generally,
food poisoning outbreaks occur as the result of poor food hygiene, and
improper handling, storage and preservation of foods and food products.
Bacterial food intoxication is a food-borne illness caused by the ingestion
of food containing preformed bacterial toxins which are produced as a result
of bacterial growth in the food. The important bacterial species that are most
commonly involved in food poisoning outbreaks are Clostridium botulinum,
Clostridium perfringens, Staphylococcus aureus and Bacillus cerus. Their
toxins mainly act on digestive or nervous systems, leading to severe disorders
and sometimes death. Food poisoning or intoxication can be prevented by
adopting good hygienic practice with proper methods and conditions of food
handling, storage and preservation. Various sensitive and effective
techniques and methods are now available for the detection of food-borne
pathogenic microbes and their toxins.
Key words: Microbial toxin, enterotoxins, botulism, food intoxication,
neurotoxins
1. Centre of Experimental Medicine and Surgery (CEMS), Institute of Medical Sciences,
Banaras Hindu University, Varanasi-221005, Uttar Pradesh
2. Department of Botany, Government Post-Graduate College, Rishikesh-249201, Dehradun,
Uttarakhand
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1. INTRODUCTION
Microorganisms present in foods may cause food spoilage or food-borne
diseases. Various human foods support the growth of pathogenic
microorganisms or act as their carriers for transmission. Food-borne diseases
are a major public health problem globally. Numerous outbreaks of foodborne diseases in various countries have been reported. A food-borne disease
outbreak refers to the occurrence of two or more cases of similar illness
resulting from the ingestion of a common food (Centers for Disease Control
and Prevention, 1996). In USA alone, an estimated 25-81 million cases of
food-borne illness and 9000 deaths are known to occur annually (GernerSmidt and Whichard, 2007). Number of annual cases of food-borne illness
in Australia is about 5.4 million. In France, 75000 cases of food-borne illness
are estimated every year, while in Japan there were 9966 cases per year.
Unfortunately, reliable comparable figures from the under-developed and
developing countries are not available (Granum, 2006; Farthing and Kelly,
2007; IASR, 2008; Hamer and Gorbach 2008; Acheson, 2009; Senior, 2009),
but one can assume that poor hygienic conditions and poor sanitation in
these countries would favour the greater prevalence of food-borne diseases.
In India, food-borne diseases are rarely recorded. Due to high prevalence
of other severe diseases in under-developed and developing countries, foodborne diseases are considered of minor importance. Food-borne illness is
caused by pathogenic organisms such as bacteria, viruses and parasites or
their toxins present in foods. More than 250 food-borne diseases are known.
With the exception certain parasites, food-borne pathogens are microscopic
in size. Food-borne illness caused by microorganisms is generally classified
in to two categories: food infection (food-borne infection) and food intoxication
(food-borne intoxication). The term food poisoning is often used very loosely
and to include food-borne diseases resulting from both food infection and
food intoxication. But in true and strict sense it refers to the diseases caused
by food intoxication.
Bacteria constitute an important group of food-borne pathogens which
are implicated in various food-borne diseases, resulting from both food
infection and intoxication. Bacterial food infection refers to food-borne illness
caused by ingestion of food containing viable (live) bacteria (e.g. Salmonella,
Listeria, pathogenic E. coli) which then grow or multiply in the host, leading
to illness. Bacterial food intoxication, on the other hand, refers to foodborne illness caused by the ingestion of food containing preformed bacterial
toxins (e.g. toxins produced by Staphylococcus aureus and Clostridium
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which vary in nutrient content and other properties. The growth and
survival of microorganisms in human foods are influenced by both extrinsic
(e.g. temperature) and intrinsic (e.g. pH, moisture, redox potential,
nutrients) factors.
2.1. Temperature
Every microorganism has a minimal, a maximal and an optimal temperature
for growth. On the basis of temperature ranges for growth, bacteria can be
grouped as: thermophiles (optimum temperature 55-75 oC), mesophiles
(optimum temperature 30-45 oC), psychrophiles (optimum temperature 515 oC) and psychrotrophs (optimum temperature 25-30oC). Generally, foodspoilage and pathogenic bacteria are mesophiles. However, some food-borne
pathogenic bacteria (e.g. Listeria monocytogenes, Yersinia enterocolitica)
are psychrotrophic which are responsible for disease outbreaks due to the
consumption of food products stored at refrigeration temperature. Decrease
and increase in temperature below and above the optimum growth range
causes the inhibition of bacterial growth in foods. The resistant spores
produced by many bacteria are known to survive both freezing and heating.
2.2. pH
Like other microorganisms, bacteria have minimum, optimum and maximum
pH for growth. Bacterial growth is significantly affected by the pH of foods.
Most foods are neutral or acidic. In general, fungi (yeasts and molds) are
more acid-tolerant than bacteria. Most bacteria are unable to grow below
pH 3.5 and those implicated in food-borne diseases are unable to grow below
pH 4.5.
2.3. Moisture
The growth of bacteria and other microorganisms in or on food depends on
the available water which is best expressed in terms of water activity (aw).
The water activity of a food or solution is the ratio of the water vapour
pressure of the food or solution (p) to that of pure water (p0) at the same
temperature. Water activity of pure water is assumed to be 1. The value of
aw of a solution decreases below 1 with the increase in concentration of the
solution. Foods exhibit significant variation in aw. Most bacteria and fungi
are unable to grow in foods having aw less than 0.90.
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2.5. Nutrients
Nutritional quality and quantity of a food determines the type of bacteria
that are likely to be present in the food. The nutritional requirements of
bacteria may change depending on the environmental conditions. In
general, most human foods provide adequate nutrients for most
microorganisms. Foods rich in proteins favour the growth of bacteria with
complex nutritional needs. Starchy foods favour the growth of bacteria that
are able to break down the complex carbohydrates. Bacteria producing
lipolytic enzymes are favoured in lipid-containing foods.
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food containing preformed BoNT, (ii) wound botulism, which occurs due to
the production of BoNT in vivo after growth of C. botulinum in an infected
wound, (iii) infant botulism, which results from the production of BoNT in
vivo in the intestinal tract of an infant colonized with C. botulinum, and
(iv) botulism due to the intestinal colonization in children older than infants
and in adults in which no food or wound source is implicated (Hatheway
and Johnson, 1998). There are seven immunologically or antigenically
distinct types of BoNT which are designated as A, B, C, D, E, F and G.
Types A (BoNT/A), B (BoNT/B), E (BoNT/E) and F(BoNT/F) are the principal
causes of human botulism. Among these BoNT/A is most dangerous and is
responsible for most outbreaks of food-borne botulism. Types C (BoNT/C)
and D (BoNT/D) are associated with botulism in birds and mammals. Type
G (BoNT/G) has not been clearly implicated in botulism (Hatheway, 1998).
Irrespective of type, each BoNT is synthesized as a single-chain inactive
polypeptide (150 kDa) that subsequently undergoes proteolytic cleavage,
resulting in the generation of active form of the toxin which consists of two
polypeptide chains linked by a single disulfide bond. The two chains of the
toxin are called light chain (50 kDa) and heavy chain (100 kDa). The toxin
is released by the lysis of bacterial cells. Toxin production depends on the
ability of the bacterial cells to grow in food and to autolyse. Factors such as
composition of the food, temperature of storage and pH of food influence
spore germination, growth and toxin production. Conditions that favour C.
botulinum growth and toxin production include a relatively high moisture,
low salt concentration, low acidic pH (pH >4.6), anaerobic condition and
food stored without refrigeration. The main sources of botulism are canned
meat, fish, string beans, sweet corn, beets, and other low and medium acidic
foods. Spores of C. botulinum can survive long storage periods in raw and
precooked frozen foods and can germinate when conditions become ideal.
Botulinum toxins can be destroyed by heat treatment. However, the
temperature and duration of treatment depend on the type of toxin.
After absorption in the small intestine, the toxin is transported in to
lymphatic channels and then in to bloodstream. The toxin reaches the
cholinergic synapses of peripheral nervous system by blood circulation where
it acts. The botulinum neurotoxins are extremely potent. They inhibit the
release of the neurotransmitter acetylcholine at the neuromuscular junction,
resulting in flaccid muscle paralysis. Their action involves three sequential
steps (Simpson, 1981). In the first step, the toxin binds rapidly and
irreversibly to receptors on the presynaptic nerve surface (Kozaki, 1979;
Murayama et al., 1984). In the second step, which is an internalization
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stage, the toxin crosses the plasmalemma and enters in to the nerve
terminus. It appears that the heavy-chain moiety of the toxin is capable of
inducing channel formation in membranes at low pH (Shone et al., 1987).
It is not known whether the whole toxin or a fragment(s) thereof is/are
delivered to the cytosol by the internalization process, although recent work
suggests that internalization of both the heavy and light chains of
neurotoxin may be required for the inhibition of neurotransmitter release
(Poulain et al., 1989). In the third step, the toxin exerts its action by
inhibiting neurotransmitter release. Normally, the arrival of an action
potential at the nerve ending triggers an influx of calcium ions which itself
promotes the exocytosis of acetylcholine from vesicles at active zones on the
plasmalemma. Several possible mechanisms of inhibition have been proposed
by various workers (Molgo and Thesleff 1984; Sanchez-Prieto et al., 1987;
Shone and Hambleton 1989; Simpson, 1981). The toxins may alter the rate
of calcium ion efflux and influx or act directly on a component of the
acetylcholine release mechanism such as a vesicle protein or cytoskeleton
component.
Typical symptoms of botulism usually occur within 12-36 hours after
consumption of the contaminated food. Early symptoms are digestive
disturbances followed by nausea, vomiting, diarrhoea, together with
dizziness and headache. Double vision may occur early and there may be
difficulty in speaking. Mouth may become dry, throat constricted and tongue
may get swollen and coated. Involuntary muscles become paralysed and
paralysis spreads to the respiratory system and to the heart. Death normally
results from respiratory failure. In fatal cases, death occurs within 3-6 days
after the poisonous food has been ingested. Information regarding the
outbreaks and occurrence of botulism is available for few countries. In USA,
160 outbreaks of food-borne botulism were reported during 1990 to 2000
and food items involved in these cases were home-canned (Sobel et al.,
2004). A total 91 cases of botulism were reported in Alaska alone between
1990 and 2000. Botulism among Alaska natives was associated with the
eating of whale meat. Adoption of food hygiene and increased awareness
about disease resulted in decreased mortality rate in natives in later decades
(Chiou et al., 2002; Eisenberg and Bender, 1976). In United Kingdom, an
outbreak of 27 cases was reported in 1989 which was linked to the
consumption of commercial hazelnut yogurt. Botulism is a major public
health problem in Argentina, which is due to consumption of improperly
processed meats and vegetables as well as home-canned food. Between 1992
and 2004, forty-one food-borne botulism cases were reported (Tornese et
al., 2008).
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growth of B. cereus in the small intestine (Beecher, 1997; Lund and Granum,
1997; Granum, 1994), while the emetic toxin is produced by growing cells
in the food (Kramer and Gilbert, 1989). The emetic toxin has been named
cereulide and consists of a ring structure of three repeats of four amino
and/or oxy acids: [D-O-Leu-D-Ala-L-O-Val-L-Val]3. This ring structure has a
molecular mass of 1.2 kDa and is chemically analogue to the potassium
ionophore valinomycin. The emetic toxin is resistant to heat, pH and
proteolysis.
Among the three different enterotoxins of B. cereus, two have been
shown to be involved in food poisoning (Agata, 1995; Beecher, 1994, 1995;
Lund and Granum, 1996). B. cereus produces different components of the
enterotoxins (Beecher, 1994, 1995, 1997; Lund and Granum, 1996, 1997).
A three-component hemolysin (HBL) consisting of three proteins (B, L1
and L2) with enterotoxin activity have been reported (Beecher, 1994, 1995,
1997). A non-hemolytic three-component enterotoxin (NHE) was also
identified (Lund and Granum, 1996). The three components of this toxin
were different from the components of HBL. One enterotoxin protein had a
molecular mass of about 57 kDa and another about 100 kDa (Kramer and
Gilbert, 1989). The largest could be the 105 kDa component of the NHE.
The third enterotoxin T is a single component protein, but has not been
shown to be involved in food poisoning. It has been suggested, from studies
of interactions with erythrocytes, that the B protein is the component that
binds HBL to the target cells, and that L1 and L2 have lytic functions
(Beecher and Macmillan, 1991). Another proposed model for the action of
HBL, suggests that the components of HBL bind to target cells independently
and then constitute a membrane-attacking complex resulting in a colloid
osmotic lysis (Beecher, 1997). Studies of interactions between NHE and
Vero cells have shown that a protein of the 105 kD might be the binding
component of that complex (Lund and Granum, 1997). The two other
components are probably not able to bind to these cells alone.
Temperature is one of the most important factors that can be controlled
to reduce or eliminate B. cereus growth as well as food poisoning. The
temperature required to prevent the growth of B. cereus in food is greater
than 60C (140F) until served, or rapidly cooling foods to below 10C (50F)
for storage. Ideally, foods should be cooled to below 15C (59F) within two
to three hour after cooking. Disease caused by B. cereus differs from country
to country. In Japan the emetic type is reported about 10 times more
frequently than the diarrhoeal type, while in Europe and North America
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the diarrhoeal type is the most frequently reported (Kramer and Gilbert,
1989). B. cereus food poisoning has been reported in relatively few countries
of Europe. B. cereus is the cause of 33% of the total cases of food poisoning
(excluding virus) in Norway (1988-1993) (Granum, 1994), 47% in Iceland
(1985-1992), 22% in Finland (1992), 8.5% in The Netherlands (1991), and
5% in Denmark (1990-92) (Schmidt, 1995). Much lower numbers have been
reported from other countries, such as England and Wales (0.7%), Japan
(0.8%), USA (1.3%) and Canada (2.2%) (Kramer and Gilbert, 1989).
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4. CONCLUSIONS
Bacterial food poisoning or intoxication is a major public health problem
world-wide. The majority of food poisoning outbreaks occur as the result of
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