Clostridium botulinum

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Clostridium botulinum is clinically significant bacteria, which cause a very rare but a severe disease called “Botulism”. It produces a particular type of exotoxin that affects the nervous system by inhibiting the activity of neuromuscular junction. The botulinum toxin thus behaves as a neurotoxin that blocks the release of a neurotransmitter refers as “Acetylcholine”. Acetylcholine is a chemical compound made by the combination of choline and acetyl Co-A, which gives the chemical signal to perform a physical activity like muscle contraction.

As the ACH or Acetylcholine will not produce, there will be no muscle contraction or movement, which means a body become paralyzed. Clostridium botulinum causes flaccid paralysis, which starts from the upper extremities to the lower extremities of the body.

C. botulinum bacteria are gram-positive, rod shaped and obligate anaerobes (Oxygen acts as a poison). It most usually causes three kinds of botulism, namely foodborne, wound and infant botulism. Among these three types, foodborne botulism is very common.

Content: Clostridium botulinum

  1. Definition of Clostridium botulinum
  2. Distribution
  3. History
  4. Morphology of Clostridium botulinum
  5. Classification
  6. Growth Conditions
  7. Virulence Factors
  8. Characteristics of Botulinum exotoxin
  9. Pathogenesis
  10. Important Facts
  11. Incubation Period
  12. Treatment
  13. Significance

Definition of Clostridium botulinum

Clostridium botulinum belongs to the group of pathogenic bacteria, which produces a highly potent exotoxin that causes botulism disease. These are anaerobic, gram-positive, rod-shaped and spore-forming bacteria. Clostridium botulinum is widely distributed in the world. The spores of C.botulinum are ubiquitous but most commonly found in soil and marine sediments. C.botulinum spores can enter into the body, by two ways:

  • By the ingestion of spores through food contaminated with the presence of C.botulinum.
  • A bacterium can also enter through a wound or the mucosal surface of the skin.

Once, a bacterium enters into the body; it first causes gastrointestinal symptoms after 24-36 hours of ingestion. Then a bacterium grows and produces an exotoxin that causes cranial nerve dysfunction within 3-7 days.

Pathogenesis of Clostridium botulinum

Further, C. botulinum obstructs the muscle contraction by inhibiting the synthesis of acetylcholine and results in paralysis of involuntary muscles. The exotoxin cause “Flaccid paralysis” by blocking the motor nerve terminal to synthesize acetylcholine at the neuromuscular junction. The progression of the disease is in a downward fashion, first effect eyes, face, throat, chest and other lower extremities.

Distribution

Spores of C. botulinum are worldwide in distribution and are saprophytes. The strains of C. botulinum has a wide range of habitat and are commonly present in the soil, the skin of fruits and vegetables, hay, silage, animal manure, sea mud etc.

History

The origin of C. botulinum has been evolved after a major outbreak occurred in the year 1735, after eating German sausages. After its origination, many scientists studied its phenotypes, serotypes, pathogenicity, applications etc.

YearDiscovererDiscovery
1870MullerDerived the name Botulism from the Latin word “Botulus” which means sausage.
1895Emile Van ErmengemFirst isolated C.botulinum from a piece of ham, caused major outbreaks
1944Dr Edward SchantzFirst isolated botulinum neurotoxin
1949-1950-Studied the effect of Botulinum toxin (Botox) on the nervous system, which blocks the neuromuscular junction by blocking acetylcholine.
1980Dr Alan B. ScottUsed neurotoxin to treat strabismus
1989-US, FDA approved the use of Botox for the treatment of strabismus
2002-FDA approved the use of Botox in cosmetic improvement to treat wrinkles, frown lines etc.

Morphology of Clostridium botulinum

The morphological characteristics of C. botulinum include:

Morphology
Shape: C. botulinum posses a rod shape.

Size: A bacterium measures a length of 1.6-22.0 mcm and width of 0.5-2.0 mcm.

Colour: On Gram staining, a bacterium appears violet coloured as C. botulinum gives a positive gram reaction.

Presence of capsule: These lack the presence of an extracellular capsule and are “Non-capsulated”.

Genome size: The genome size of C. botulinum is about 3.89 Mb.

Motility: These are generally motile by having peritrichous flagella.

Spores: C. botulinum produces exospores during reproduction and thus refer as “Spore formers”. The spores of C. botulinum are heat and light-resistant, can survive for several hours at 100 degrees Celsius and 10 min at 120 degrees Celsius. Spores are oval-shaped, bulging, subterminal in position and can remain dormant for 30 years or more.

Classification

Based on serological specificity, C. botulinum produces the following serotypes:

  • A, B, C1, C2, D, E, F and G serotypes.

Except for C2, all the serotypes produce exotoxin, works as a “Neurotoxin” that affects the nervous system. Type C2 acts as an “Enterotoxin” that affects the gastrointestinal system.

Based on physiological and biochemical properties, there are four phenotypic groups of C. botulinum (Group I, II, III and IV).

Group of Clostridium botulinumToxin typesProteolysisLipolysisSaccharolysisMinimum growth temperature ( ֯ C)Optimum growth temperature ( ֯ C)Salt inhibition (in %)Pathogenicity
IA, B, F+++1235-4010Human botulism
IIB, E, F-++3.318-255Human botulism
IIIC1, C2, D-++15403Animal botulism
IVG+--Not known37>3Not known

Growth Conditions

The spores of C. botulinum can resist extreme conditions, and its growth requires the following environmental conditions:

  • C. botulinum does not require oxygen presence.
  • C. botulinum grows within the mesophilic temperature range.
  • Generally grows in a low acidic, low sugar and low salt substrates like meat, fish, egg, beans, apricots etc.

Thus, the effect of botulinum exotoxin can be detoxified by subjecting it to high heat (100degrees Celsius for 10 min), high acidity, high sugar and high oxygen environment.

Virulence Factors

Clostridium botulinum is classified among the group of pathogenic bacteria because it produces an exotoxin that causes botulism. The exotoxin acts as a virulence factor which can cause disease in mammals like humans, animals etc. In the absence of exotoxin, Clostridium botulinum considers being non-invasive or non-infectious.

Botulinum Toxin

Initially when an exotoxin synthesized by the different groups of Clostridium botulinum, acts as non-toxic protoxin or progenitor toxin. The exotoxin activates by the enzyme trypsin and the other proteolytic enzymes.

In the pure crystalline state, the botulinum exotoxin is considerably the most potent or fatal substance known to humankind. The exotoxin has a molecular weight of 150 kDa. Generally, botulinum exotoxin is a neurotoxin that disrupts the normal functioning of the nervous system and results in paralysis of involuntary muscles. A lethal dose for mice is 0.000,000,033 mg, and 1-2 µg for human.

Characteristics of Botulinum exotoxin

Stability: A toxin produced by Clostridium botulinum is a relatively stable compound. Botulinum exotoxin is stable under acidic condition (>4.6) and dissociates under alkaline conditions.

Chemistry of Botulinum exotoxin: Initially produced as a single inactive polypeptide chain of molecular weight 150 kDa, where it lacks a leader sequence. The protease enzyme functions to cleave the neurotoxin into active di-chain neurotoxins.

An exotoxin consists of one heavy and one light chain of molecular weight 100 kDa and 50 kDa respectively. The heavy and light chain attaches by the di-sulphide bond and also contains one atom of zinc. The di-sulphide bond between the heavy and light chain can help the neurotoxin in cell penetration.

Absorption: An exotoxin mainly absorbs in the small intestine (In an active form).

Uniqueness: It is unique than the other exotoxin, as it produces after the cell lysis.

Isolation: An exotoxin can isolate by the method of ion-exchange chromatography.

Effect: Botulinum neurotoxin blocks the synthesis of ACH (Acetylcholine) at the synapses and neuromuscular junction and results to cause paralysis. In severe illness, the exotoxin leads to respiratory paralysis, which eventually causes death. A neurotoxin is zinc endopeptidase which cleaves the membrane protein of synaptic vesicles refers as “Synaptobrevin”.

Toxin inactivation: Toxin inactivation can achieve by subjecting it to the temperature of 80degrees Celsius for 30-40 minutes and at 100degrees Celsius for at least 10 minutes.

Pathogenesis

Botulinum toxin follows the following pathogenesis pathway:

botulinum neurotoxin

  1. Botulinum toxin is a neurotoxin, which absorbs in the bloodstream.
  2. A toxin releases at the time of attachment of the neuron with the muscle cell. It releases out from the cytoplasmic membrane of the muscle cells.
  3. Then the exotoxin binds to the motor nerve terminal and does not allow the fusion of synaptic vesicles. Thus, a toxin blocks the release of acetylcholine that is necessary for the nerve impulse and muscle contraction.
  4. There will be no muscle contraction, which consequently causes paralysis of the muscle fibre.

Botulinum toxin first affects the cranial nerves and then cause symmetrical descending flaccid paralysis from upper to lower extremities.

Important Facts

Some interesting facts of Clostridium botulinum are:

  1. Clostridium botulinum produces a protein with specific neurotoxicity refers to as “Neurotoxin”.
  2. C. botulinum causes a severe disease “Botulism” by the cause of ingested food contaminated with a bacterium.
  3. It is a causative agent of botulism disease, which is a type of food intoxication.
  4. C. botulinum causes botulism, which is rare, but its mortality rate is very high.
  5. Among all the serotypes of botulinum neurotoxin, type-A is more toxic.
  6. Antigenicity of C. botulinum has been studied by the complete neutralization of toxins by homologous antitoxins.
  7. Serotypes C and D produce more than one toxin and can cross-react with antitoxins to each other.
  8. Exotoxin produces at the time of sporulation only, i.e. if the organism does not grow, and no toxin will produce.
  9. C. botulinum requires an optimum temperature of 35 degrees Celsius to produce proteolytic exotoxins and requires 26-28 degrees Celsius to produce non-proteolytic type.

Incubation Period

C. botulinum starts showing neurological symptoms after 12-36 hours of ingestion. Sometimes, it may take several days to produce visible signs. Shorter in the incubation period, more severe is the illness and high mortality rates.

Treatment

Foodborne botulism: To treat foodborne botulism, antitoxin should be administered to block the activity of exotoxin. In case of respiratory failure, a mechanical ventilator is suggested.

Infant botulism: For infant botulism, enemas are recommended to remove the contaminated food.

Wound botulism: To treat this, antitoxin should be administered to neutralize the effect of exotoxin. In addition to this, surgical debridement and excision of the affected area can also be employed.

Significance

Clostridium botulinum is a medically important bacteria, which cause a severe disease ‘Botulism’. It also uses in the preparation or production of medicaments like Botox, Dysport etc. to relieve muscle pain. Botulinum exotoxin can use as a bioweapon for Biowarfare and Bioterrorism, due to its high potency to kill a person and also use in the cosmetic industry to remove the glabellar lines.

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