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.
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.
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.
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.
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.
The lysis is of the hot-cold variety, being best seen after incubation at 37°C followed by chilling at 4°C.
Heat-labile enterotoxin
Murqaha
The incubation period may range from 4 to 5 days.
durbaan
After a period of normal development; the infant develops constipation, listlessness, difficulty in sucking and swallowing, weak or altered cry, muscle weakness, ptosis, and loss of head control.
