- Clostridium are gram-positive, anaerobic, spore-forming bacilli found in soil and intestines. Major pathogenic species include C. perfringens, C. tetani, C. botulinum, and C. difficile. - C. perfringens causes gas gangrene and food poisoning. C. tetani causes tetanus. C. botulinum causes botulism. C. difficile causes antibiotic-associated diarrhea and pseudomembranous colitis. - Laboratory diagnosis involves culturing specimens anaerobically and identifying species by colony morphology, gram stain, and biochemical tests. Toxins are detected to confirm diseases like bot

1. Clostridium
Gram Positive Bacilli
Mr. Mohamed Yusuf Abdi
M.Sc. Medical Microbiology and Immunology
Somali International University
Faculty of Health Sciences
Department of Medical Laboratory

2. Clostridium General Characteristics
• Gram positive bacilli
• Anaerobic and spore-forming bacteria.
• Spore is placed centrally, sub-terminally, or terminally.
• Most of the species are saprophytes that normally occur in soil, water
and decomposing plant and animal matter.
• More than 130 species. Most of the clinically important species
include:
2. Clostridium Tetani
1. Clostridium Perfringens
4. Clostridium Difficile
3. Clostridium Botulinum

3. Clostridium General Characteristics
• On the basis of biochemical reactions, clostridia can be divided into:
(A) Saccharolytic clostridia
(B) Proteolytic clostridia
(C) Proteolytic and saccharolytic clostridia
• A saccharolytic reaction is shown by reddening of the meat with an
unpleasant smell due to carbohydrate decomposition.
• A proteolytic reaction is shown by blackening of the meat with a very
unpleasant smell due to protein decomposition.

4. Resistance of Spores
• Spores of C. botulinum may withstand boiling after 3–4 hours .
• Spores of most strains of C. perfringens are destroyed by boiling for
less than five minutes, but those of some type A strains that cause food
poisoning survive for several hours.
• C.terani spores persist for years in dried earth or dust.
• All species are killed by autoclaving at 121°C within 20 minutes.

5. Clostridium Perfringens
• C. perfringens is a normal inhabitant of the large intestines of human
beings and animals.
• It is capsulated and non-motile
• Five types of C. perfringens (A, B, C, D & E) are recognized.
• Human disease is caused by types A and C (other types cause disease in
animals).

6. Pathogenicity
• The four major toxins, alpha, beta, epsilon and iota, are predominantly
responsible for pathogenicity.
• The alpha (α) toxin is a lecithinase that increases vascular permeability,
resulting in massive hemolysis and bleeding, tissue destruction, hepatic
toxicity, and myocardial dysfunction.
• Beta (b), Epsilon (e) and iota (i) toxins have lethal and necrotizing
properties.
• C. perfringens type A strains produce a potent enterotoxin which causes
diarrhaea and other symptoms of food poisoning.

7. Clinical Manifestations
A. Soft Tissue Infections (Ex. Gas gangrene, clostridial myonecrosis ):
• C. Perfringens is the most common cause of gas gangrene.
• The disease is characterized by rapidly spreading oedema, necrosis of
tissues, gas production and profound toxaemia occurring as a
complication of wound infection.
• Bacterial spores gather in an injury or surgical wound that has no or
poor blood supply (low-oxygen environment).
B. Septicaemia.

8. Clinical Manifestations
C. Food poisoning:
• Usually caused by C. Perfringens type A, which produce enterotoxin
• Pre-cooked meat, chicken, fish and their by-products are the most
common source for closterial food poisoning.
• Incubation period 8–24 hours.
• Clinical presentation that includes abdominal cramps and watery
diarrhea but no fever, nausea, or vomiting.
• The illness is self-limited and recovery occurs in 24–48 hours.

9. • The gas formed in tissues, resulting from fermentation of muscle carbohydrates,
can also destroy muscle structure.
• Initial symptoms of pain, edema, and a bloody exudate in the lesion are followed
by fever, tachycardia, and blackened necrotic tissue filled with bubbles of gas.

10. Laboratory Diagnosis
Specimens: depend on the site of infection
• Material from wounds, necrotic tissue, and exudate to investigate gas
gangrene, and faeces and suspected food to investigate food poisoning.
Culture:
• Cooked meat broth are inoculated and heated at 100°C for 20 minutes,
incubated and subcultured on blood agar plate.
• Optimum temperature range is 37–45 ºC in anaerobic condition.

11. Laboratory Diagnosis
Colony morphology:
• Blood agar: Large beta-haemolytic colonies are produced (most food-
poisoning strains are non-haemolytic).
• Robertson’s cooked meat medium (RCMM): In this medium C.
perfringens is saccharolytic and slightly proteolytic with Gas.
Gram staining: Gram positive thick brick-shaped rod.
Biochemical tests:
• Catalase test is negative.
• Nagler reaction.

12. Gram positive thick brick-shaped rod

13. How can we create anaerobic
atmosphere in microbiology lab?

14. Clostridium Tetani
• C.tetani is worldwide in distribution in the soil and in the feces of
horses and other animals.
• C. tetani produces at least two distinct toxins:
1. Hemolysin (tetanolysin).
2. Neurotoxin (tetanospasmin).

15. Clostridium Tetani
Pathogenicity:
• C tetani infections is localized in the area of devitalized tissue (wound,
burn, injury, umbilical stump, surgical suture) into which the spores
have been introduced.
• The disease is characterized by tonic contraction of voluntary muscles
and muscular spasms. In some cases, difficulty in opening the jaw
(‘lock-jaw’).
• The mortality rate in generalized tetanus is very high.

16. Source of C.tetani

18. Diagnosis
• Most patients with tetanus can be diagnosed clinically.
Laboratory diagnosis:
Specimen:
• Wound exudate or tissue removed from the wound.
Culture:
• Blood agar plate.
• Robertson’s cooked meat medium
• C. Tetani is a strict anaerobe with a temperature (37 ºc optimum).

19. Laboratory diagnosis
Colony morphology:
• Blood agar: C. tetani produces a small haemolytic colonies .
• Robertson’s cooked meat medium: C. tetani is slowly proteolytic.
Gram staining:
• Gram positive ‘drumstick’ bacilli.
Toxigenicity test:
• Toxigenicity is best tested in animals.

20. Gram positive ‘drumstick’ bacilli
drumstick

21. Clostridium Botulinum
• It is a widely distributed saprophyte found in soil, vegetables and
fruits.
• C. botulinum toxins:
• There are seven toxin types (A–G). Human botulism is usually caused
by toxin types A, B, E and rarely type F.
• The toxin is formed in food when C. botulinum spores contaminate
food.
• C.botulinum causes botulism.

22. Clinical Features
Food poisoning:
• The period between ingestion of the toxin and the appearance of signs
and symptoms is usually 1–2 days. There may be initial nausea and
vomiting. There is no fever.
• Visual disturbances (double vision), inability to swallow, and speech
difculty; paralysis are progressive, and death occurs from respiratory
paralysis or cardiac arrest.
• The patient remains fully conscious until shortly before death.
Wound infections.

23. Clinical Features
Infant botulism:
• The ‘floppy child syndrome’ describes a young child, usually less than
6 months old, with flaccid paralysis that is ascribed to the growth of C.
botulinum in the intestine.
• The most common food source in infant botulism is honey
contaminated with botulinum spores.
• Infant botulism may be one of the causes of sudden infant death
syndrome.

24. Floppy Child Syndrome

25. Laboratory diagnosis
• Botulism confirmed by isolating the organism or detecting the toxin in
food products or the patient’s feces or serum.
Specimens:
• Feces, food, vomitus, gastric fluid, serum, environmental samples and
occasionally wound exudate.
Culture:
• Egg-yolk agar, blood agar and CMB.
• C. botulinum is a strict anaerobe.
• Grows best at 30–35 ºC

26. Laboratory diagnosis
Gram staining :
• C. botulinum is a Gram positive, pleomorphic rod with oval sub-
terminal spores.
Note that:
• Presence of bacilli in food or feces in absence of toxin is of no
significance.
• Hence, toxin in culture fluid must be demonstrated by toxigenicity test
in mice.

27. Clostridium Difficile
Pathogenesis:
• C.Difficile produces an enterotoxin (toxin A) and cytotoxin (toxin B).
• It is a proven cause of antibiotic associated diarrhea, and
pseudomembranous colitis a life-threatening condition.
• The disease develops in people taking antibiotics.
• The three drugs most commonly implicated are clindamycin,
ampicillin and the cephalosporins.

28. Laboratory diagnosis
A. Isolation of bacilli:
• C. difficile can be isolated from the feces.
B. Demonstration of toxin.
• The disease is treated by discontinuing the antibiotic that is presumed
to have precipitated the disease and by giving oral metronidazole or
vancomycin.

29. Thank you