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Clostridium botulinum may be found in any region of the world. Since it is found mostly in the soil, it may contaminate vegetables cultivated in or on the soil. It also colonizes the gastro-intestinal tract of fishes, birds and mammals. C. botulinum lies dormant in the form of spores until the right environmental conditions are met. These spores are very resistant to adverse environmental effects, making them amenable to most environments and very hard to kill. The spores will grow under favorable conditions (anaerobiosis and substrate-rich environments) and will begin to produce their toxins as they rapidly propagate.The endospores that are produced from C. botulinum can survive in a vast array of environments until it meets the correct conditions to proliferate. In order for proliferation to occur, the spores need to be present in non-halophilic salinity and anaerobic conditions. C This bacterium is also mesophilic, with an optimal temperature of around 37 degrees celsius.

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Clostridium botulinum is a gram-positive rod-shaped bacteria that forms vegetative endospores. A typical prokaryote, Clostridium botulinum, has no nucleus or other membrane enclosed organelles. The bacteria reproduces through binary fission. The bacteria is a obligate anaerobe and ferments carbohydrates and nitrogenous compounds such as amino acids.

C. botulinum uses the Strickland Reaction to ferment amino acids. The amino acids it needs include typtophane, threonine, valine, leucine, isoleucine, methionine, argininie, phenylalanine, and tyrosine.

C. botulinum is often found in improperly canned goods; the improper preparation does not heat the cans to a high enough temperature to denature the C. botulinum endospores, allowing the bacteria to survive leading to potential ingestion by human consumers. Foods which cannot be heated to temperatures high enough to kill the endospores should take other precautions to prevent colonization of the Clostridium botulinum, such as maintaing a pH below 4.6 which will not harbor the bacteria.

Nowadays, the radiation able to kill the spore of C. botulinum. Thus, the formation of toxin will definitely been reduced. Kempe & Graikoski (1961) state that the presence of type A toxin in can of meat that have been inoculated with spore of C. botulinum sterile with gamma radiation and incubated results in no growth of spores.

The Stickland Reactionis the a chemical reaction that involves the coupled oxidation and reduction of amino acid to organic acids. The electron donor amino acid is oxidised to a volatile carboxylic acid one carbon atom shorter than the original amino acid. For example, alanine with a three carbon chain is converted to acetate with two carbons. The electron acceptor amino acid is reduced to a volatile carboxylic acid the same length as the original amino acid. For example, glycine with two carbons is converted to acetate.

In this way, amino acid fermenting microbes can avoid using hydrogen ions as electron acceptors to produce hydrogen gas. Amino acids can be Stickland acceptors, Stickland donors, or act as both donor and acceptor. Only histidine cannot be fermented by Stickland reactions, and is oxidised. With a typical amino acid mix, there is a 10% shortfall in Stickland acceptors, which results in hydrogen production. Under very low hydrogen partial pressures, increased uncoupled anaerobic oxidation has also been observed.

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CAUSES OF CLOSTRIDIUM BOTULINUM GROWTH IN FOOD

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CAUSES OF CLOSTRIDIUM BOTULINUM GROWTH IN FOOD

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C. botulinum spores are often found on the surfaces of fruits and vegetables and in seafood. The organism grows best under low-oxygen conditions and produces spores and toxins. The toxin is most commonly formed when food is improperly processed (canned) at home. C. botulinum cannot grow below a pH of 4.6, so acidic foods, such as most fruits, tomatoes, and pickles, can be safely processed in a water bath canner. However, foods with a higher pH (most vegetables and meats) must be processed under pressure. Therefore, a pressure cooker should be used. The pressure cooker will reach high enough temperatures to destroy the C. botulinum spores.

For example, if a low-acid food, such as green beans, is canned improperly (not canned under pressure or improperly canned using a pressure canner), C. botulinum bacteria and other bacteria present will be destroyed by the boiling of water and food, but the C. botulinum spores will not be destroyed. The canning process will remove the oxygen from the jar, creating a low-oxygen environment that is will allow the spores to grow into active bacteria. When the jars are stored at room temperature, the spores can germinate and produce the toxin. However, the toxin is sensitive to heat and can be destroyed if the food in question is boiled for 10 minutes (longer at high altitudes).

Botulism is a paralyzing disease affecting the body's nervous system that is caused by the ingestion of one of the potent neurotoxins produced by C. botulinum bacterium. This neurotoxin is among the most toxic substances known; even microscopic amounts can cause illness.

Pickled food   favored by Clostridium botulinum

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1. Culture Methods: C. botulinum requires strict anaerobic conditions for growth. All culture media must be deoxygenated and all laboratories equipment must be deoxygenated before contact with sample culture. conventional detection and isolation of C. botulinum are based on culturing in liquid medium and subsequent detection of botulism toxin in the culture by mouse bioassay. Example of liquid media that can be used are tryptone-peptone-glucose-yeast extract medium, reinforced clostridial medium and fastidious anaerobes broth. Blood agar and egg yolk agar(EYA) serve as the most common unselective plating media.
2. Mouse bioassay: the standard method used in detection of the neurotoxin present in the patients serum and/or feces.
a) Mouse lethality assay: This test is carried out by intraperitoneal injection into the laboratory mice of sample that has been diluted in phosphate buffer. If the sample contain toxin, the mouse will develop the typical sign of botulism which are fuzzy hair, muscle weakness, respiratory failure. The symptoms will usually develop within a day or may take several days. the toxin type is determined by neutralizing the toxin with specific anti-toxin. briefly, the mice injected with anti-toxin will survive while others develop botulism. By using this method, all seven types of toxin can be identified. This method is very sensitive but it is laborious and expensive and naturally presents an ethical dilemma due to the use of laboratory animals.
b) Nonlethal mouse assay: The nonlethal mouse assay is equal to the conventional bioassay in sensitivity and specificity, but it does not cause signs of distress or impaired movements in the animals. However, the assay has been targeted to potency testing of purified neurotoxins, and thus it has not been validated for microbiological laboratories investigating complex sample matrices.

3. Immunological Method (ELISA): A wealth of immunoassay formats for the detection of botulinum neurotoxins have been reported. Compared with the mouse test, the immunoassays are technically simple and fast to perform and interpret. ELISA is by far the most commonly applied immunoassay format in the detection of botulinum neurotoxins. Briefly, the neurotoxin in a sample binds to a solid test matrix that is usually precoated with polyclonal or monoclonal capture antibodies against one or more toxins (sandwich ELISA). A second toxin antibody is then used to bind the toxin. Finally, an antiantitoxin molecule, carrying an enzyme such as alkaline phosphatase or horseradish peroxidase, is used to produce a signal through enzymatic cleavage of a chromogenic substrate.

4. Endopeptidase assay: The endopeptidase assays are based on specific cleavage of synaptic proteins (SNARE complex proteins) by different botulinum neurotoxins, combined with immunological detection of the cleaved peptide or detection of fluorescence released when a peptide labeled with a quenched chromophore is cleaved. The endopeptidase assays are highly specific, and no cross-reactivity between different botulinum toxins or with tetanus toxin has been reported.

5. Molecular detection of C. botulinum: DNA-based detection method include PCR and Southern hybridization which are both sensitive and rapid compared to culture technique and mouse bioassay. Molecular detection are merely based on detection of the botulinum neurotoxin gene, bot, in the sample. This technique does not need laboratory animals.

6. Genetic characterization of C. botulinum:  International molecular typing libraries with genetic fingerprints of C. botulinum strains isolated from botulism epidemics would serve as valuable source of information when investigating new outbreaks. Example of the techniques are  DNA sequencing, pulsed-field gel electrophoresis (PFGE), and ribotyping, as well as PCR-based techniques such as AFLP, randomly amplified polymorphic DNA analysis (RAPD), and repetitive element sequence-based PCR (Rep-PCR)

REFERENCE: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1471988/

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Botulism is caused by Clostridium botulinum bacteria and other Clostridium species that have synthesized and produced neurotoxin in foods. The toxin causes the disease by blocking the release of acetylcholine from motor nerve endings.
There are 4 kinds of botulism:

1.  Foodborne botulism occurs when foods that have been contaminated with toxin is consumed. Most foodborne botulism is attributed to home-canned foods. However, outbreaks occur in foods that are prepared commercially.

2. Infant botulism occurs when infants under 6 months old eat spores of C. botulinum rather than the toxin. The spores will grow in infants' intestines where they produce toxin. Honey may contain spores of C. botulinum and should not be given to children under 6 months old. Nevertheless, infant botulism is rare.

3. Adult intestinal colonization botulism occurs when C. botulinum spores are ingested. The spores will grow and produce toxin in the digestive system. People with altered gastrointestinal systems and local microbiologist are often affected.

4. Wound botulism occurs when live bacteria infect an open cuts and carried the toxin throughout the body by the blood.

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Symptoms of botulism generally appear 12 to 72 hours after eating contaminated food.  With treatment, illness lasts from 1 to 10 days.  Full recovery from botulism poisoning can take weeks to months. Symptoms of botulism are

Difficulty swallowing or speaking.

Dry mouth.

Facial weakness on both sides of the face.

Blurred or double vision.

Drooping eyelids.

Trouble breathing.

Nausea, vomiting and abdominal cramps.

Paralysis.

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People that commonly affected by this kind of microorganism are children and adults. It is due to the home-canned foods that has a low acid content, improperly canned commercial foods, home-canned or fermented fish, herb-infused oils, baked potatoes in aluminum foil, cheese sauce, bottled garlic, foods held warm for extended periods of time

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Treatment or cure of Botulism can be treated with an antitoxin that blocks circulation of the toxin in the bloodstream. This prevents the patient’s case from worsening, but recovery still takes several weeks.

Since botulism poisoning most commonly comes from foods improperly canned at home or food industry, the most important step in preventing botulism is to follow proper canning procedure and HACCP plan. Ohio State University’s Extension Service provides a useful guide to sanitary canning techniques. Further botulism prevention techniques include not eating canned food if the container is bulging or if it smells bad, although not all strains on Clostridium Botulinum smell storing garlic or herbinfused oil in the refrigerator. Not storing baked potatoes at room temperature to prevent infant botulism, do not give even a small amount of honey to an infant, as honey is one source of infant botulism.

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Clostridium botulinium is commonly found in oxygen poor environment with low acidity level. Hence, they are more likely to be found in soil and marine sediments throughout the world. Due to this reason, Clostridium botulinium may be found in food that are in contact with soil such as vegetables, fruits and also meat. They can also be found in the gastrointestinal track of birds and fish.

Clostridium botulinium can also be found in preserved food like canned food. This may happen if there is contamination during preservation process. Regular spoilage bacteria may be killed, however leaving a favorable and uncompetitive condition for Clostridium botulinium to grow and cultivate in food.

The picture below shows countries ( in black ) that encountered outbreak of Clostridium botulinium , such as North America, Australia and Europe. However we must aware that this bacteria can actually be found anywhere in the world.

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