Clostridium botulinum: Properties, Pathogenesis and Laboratory Diagnosis

Clostridium botulinum is gram-positive, endospore-forming bacilli which resemble a tennis racket appearance due to the presence of spores at the sub-terminal end.

Clostridium botulinum cover image

It produces an extremely lethal neurotoxin called botulinum toxin and causes botulism, a rare life-threatening neuroparalytic disease. Botulism is manifested as various clinical syndromes ranging from food poisoning, wound infection to infant botulism.

Botulinum term was derived from Latin word ‘botulus’, meaning sausage; as poorly cooked sausages were formerly associated with food poisoning. Botulinum is also produced by other clostridia such as C. butyricum, C. baratti, and C. argentinense.

Lethal dose of botulinum toxin for a human is 2μg or less, so C. botulinum has the potential to be used as a biological weapon. CDC has listed it as ‘Category A agents’, along with Bacillus anthracis, Yersinia pestis, etc.

Pathogenesis

Homemade canned food source of botulism

Transmission:

It is ubiquitous in nature, widely distributed as a saprophyte in soil, animal manure, vegetables, and sea mud. Homemade canned foods, condiments, and fish products are the most common sources of infection with C. botulinum. Ingestion of contaminated honey is the major cause of infant botulism.

Honey as source of infant botulism

Insufficient cooking temperature followed by packaging in anaerobic conditions facilitates the germination of spores and synthesis neurotoxins.

Mechanism of action of Botulinum toxin (BoNT)

Clostridium botulinum is non-invasive. Its pathogenesis is due to the production of powerful neurotoxin ‘botulinum toxin’ (BoNT), probably the most toxic substance known to be lethal to mankind. It produces flaccid paralysis. There are 7 serological types of botulinum neurotoxin labeled as types A, B, C [C1 C2], D, E, F, and G. Human botulism is caused mainly by types A, B, E and F (rarely).

C. botulinum toxin is categorized as a potential bioterrorism agent but botox is in use to smooth facial wrinkles.

After entry (either ingested, inhaled, or produced in a wound), botulinum toxin is transported via the blood to peripheral cholinergic nerve terminals. The most common nerve terminal sites are neuromuscular junctions, postganglionic parasympathetic nerve endings, and peripheral ganglia. It does not affect the CNS.

In normal condition: Upon stimulation of peripheral and cranial nerves, acetylcholine is normally released from vesicles at the neural side of the motor endplate. Acetylcholine then binds to specific receptors on the muscle, inducing contraction.

Mechanism of Botulinum toxin
Mechanism of Botulinum toxin
(Image source: lumenlearning.com)

Botulinum toxin acts by binding to synaptic vesicles of cholinergic nerves, thereby preventing the release of acetylcholine (Ach) at the peripheral nerve endings, including neuromuscular junctions. This results in a lack of stimulus to the muscle fibers, irreversible relaxation of the muscles, and flaccid paralysis.

As botulinum toxin produces flaccid paralysis it can be used therapeutically for the treatment of spasmodic conditions such as strabismus (misaligned eyes), blepharospasm (uncontrollable blinking), and myoclonus.

Clinical manifestations

The manifestations of botulism are due to decreased acetylcholine in cranial nerve and parasympathetic nerve terminals. Common symptoms include:

  1. Diplopia (double vision) or blurring of vision
  2. Dysphagia (difficulty swallowing)
  3. Dysarthria (difficulty in speech) or slurring of speech
  4. Descending symmetric flaccid paralysis of voluntary muscles.
  5. Decreased deep tendon reflexes
  6. Fatigue
  7. Dizziness
  8. Nausea
  9. Constipation
  10. Respiratory muscle paralysis may lead to death.

There is no sensory or cognitive deficits

Types of Botulism

There are three major types of human botulism based how they are acquired:

  1. Foodborne botulism: It results from the consumption of foods contaminated with preformed botulinum toxin such as homemade canned food.
  2. Wound botulism: It is a systemic intoxication resulting from the growth of C. botulinum and toxin production in the wounds. It presents like foodborne botulism except for the absence of gastrointestinal features.
  3. Infant botulism: Infant botulism is much milder than the adult version. It results from the ingestion of food (usually honey) contaminated with spores of C. botulinum by children ≤1 year of age. Spores germinate in the intestine, and the vegetative cells secrete botulinum toxin. Clinical manifestations include the inability to suck and swallow, weakened voice, ptosis, floppy neck, and extreme weakness hence called floppy child syndrome. It is a self-limiting disease; prognosis is excellent if managed by supportive care and assisted feeding.

Spores do not normally germinate in adult intestine, however may germinate in the intestine of infants.

Laboratory diagnosis

Diagnosis of botulism includes isolation and identification of the bacilli by conventional cultural biochemical procedures and demonstration of the presence of botulinum neurotoxin in a patient sample or in the food (for outbreak investigation) with the toxin neutralization test

Demonstration of botulinum toxin (mouse neutralization test) in serum or feces confirms the clinical diagnosis of botulism.

Sample

Serum, feces, gastric contents, vomitus, wound swab, exudate, or tissues depending on the type of botulism.

Diagnosis of botulism
Diagnosis of Botulism
(Image source- Lindström & Korkeala, 2006)

Direct microscopy

Gram staining of smears made from suspected food or feces-reveals gram-positive, non-capsulated bacilli with subterminal, oval, bulging spores.

Culture

  • Isolation- culture is done on blood agar or Robertson’s cooked meat (RCM) broth.
  • In RCM broth: Turbidity occurs with meat particles turning:
    1. Black and production of foul odor-C. botulinum A, b, f (proteolytic)
    2. Pink- C. botulinum C, D, E (saccharolytic).
  • In blood agar: Colonies are large, irregular, semi-transparent, hemolytic, and fimbriated border.

Growth on culture media may be confirmed by Gram staining, biochemical tests, or molecular assays. Serotyping is done with type-specific antisera.

Identifying features of Clostridium botulinum

  • Motile by peritrichate flagella.
  • Exhibit lipase activity on egg yolk agar.

Toxin Neutralization Test (Mouse Bioassay)

In vivo mouse bioassay is the “gold standard” test to detect active botulinum toxin.  This assay involves the intraperitoneal injection of suspected contaminated food into a mouse and observation for 4-6 days for disease/death.

If that lethal activity can be neutralized (in another set of mice) by injecting antibodies against one of the botulinum toxin serotypes, it confirms the presence of botulinum neurotoxin.

Molecular Diagnosis

Molecular techniques such as polymerase chain reaction (PCR) targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum. Further typing (for example, toxin typing or gene typing) can be done using pulse-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP).

References and further readings

About Acharya Tankeshwar 452 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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