Gram-positive anaerobic bacteria which form heat-resistant spores and also the causative organisms in cases of gas gangrene, tetanus, botulism, food poisoning and pseudomembranous colitis.

1. CLOSTRIDIUM
DR. NAGENDRA KUMAR
DEPARTMENT OF MICROBIOLOGY
GMC, JAMMU

2. CLOSTRIDIA
 GRAM-POSITIVE ANAEROBIC BACTERIA WHICH FORM HEAT-
RESISTANT SPORES
 COMMENSAL FLORA IN GUT OF HUMANS AND ANIMAL
 CAUSE GAS GANGRENE, TETANUS, BOTULISM, FOOD
POISONING AND PSEUDOMEMBRANOUS COLITIS
 PATHOGENICITY
 EXOTOXIN: PATHOGENIC CLOSTRIDIA PRODUCE
POWERFUL EXOTOXINS WHICH ARE RESPONSIBLE FOR
THE PATHOGENESIS AND DISEASE, E.G., TETANUS,
BOTULISM AND GAS GANGRENE
 INVASIVE TOXIN: C. PERFRINGENS, BESIDES BEING
TOXIGENIC, IS ALSO INVASIVE AND CAN SPREAD ALONG
THE TISSUES AND EVEN CAUSE SEPTICEMIA

3.  GRAM-POSITIVE, RODS,
HIGHLY PLEOMORPHIC, 3–
8 MICRON, SPORE-
FORMING, SHAPE AND
POSITION VARY
 SPHERICAL OR OVAL;
TERMINAL,
SUBTERMINAL, CENTRAL
 MOTILE, STATELY
MOTILITY
MORPHOLOGY
Universities Press Pvt Ltd

4. CULTURE
Anaerobic
Aerotolerant
Low redox potential by adding
reducing substances – unsaturated fatty
acids, ascorbic acid, glutathione,
cysteine

5. GROWTH
REQUIREMENTS
pH 7–7.4
Temperature 37°C
Some are thermophillic, psychrophilic
BA hemolytic
Robertson’s cooked meat – turbidity, gas
Saccharolytic and proteolytic
Litmus milk medium, production of acid, gas, clot
detected

6. SPORE
CENTRAL – SPINDLE – C. BIFERMENTANS
SUBTERMINAL – CLUB – C. PERFRINGENS
OVAL TERMINAL – TENNIS RACQUET – C. TERTIUM
SPHERICAL TERMINAL – DRUMSTICK – C. TETANI
EXHIBIT VARIABLE RESISTANCE TO HEAT, DRYING AND DISINFECTANTS

7. SPORE
ARRANGEMENT
S

8. CLINICAL CASE
1
• A 50-YEAR-OLD MAN MET WITH A ROAD TRAFFIC ACCIDENT
IN WHICH HE SUSTAINED MULTIPLE FRACTURES WITH OPEN
WOUNDS AND A CRUSH INJURY OF THE LEG. HE WAS TAKEN
TO THE NEAREST HOSPITAL TWO DAYS LATER, AT WHICH
TIME, HE WAS FOUND TO BE IN SHOCK.
• HE WAS STARTED ON SUPPORTIVE THERAPY AND
ANTIBIOTICS. THERE WAS EDEMA AND PAIN AT THE SITE OF
INJURY WITH INCREASED DISCOLOURATION AND A SEROUS
DISCHARGE. THE AREA AROUND THE WOUND HAD
CREPITUS ON PALPATION.
• MICROSCOPIC EXAMINATION OF THE WOUND DISCHARGE
SHOWED THE PRESENCE OF THICK, BRICK-SHAPED, GRAM-
POSITIVE BACILLI ALONG WITH GRAM-POSITIVE COCCI.
BASED ON A PROVISIONAL DIAGNOSIS OF GAS GANGRENE,
IMMEDIATE SURGICAL TREATMENT WITH EXTENSIVE
EXCISION OF THE LOCAL AREA (TO PREVENT FURTHER
SPREAD) AND INTRAVENOUS PENICILLIN WITH CLINDAMYCIN
WERE GIVEN.
• THE EXUDATE WAS ALSO INOCULATED INTO ROBERTSON’S
COOKED MEAT MEDIUM. CLOSTRIDIUM PERFRINGENS AND
PEPTOSTREPTOCOCCI GREW IN THE CULTURE.

9. CLOSTRIDIUM
PERFRINGENS
LARGE RECTANGULAR, STOUT, GRAM-
POSITIVE, CAPULATED, NON-MOTILE
BACILLUS, 4–6 X L ΜM.
PLEOMORPHIC, SINGLE, CHAINS
SPORES: SUBTERMINAL, NOT
PRODUCED IN ARTIFICIAL MEDIA

10. CULTURE
MICROPHILIC – AEROPHILIC ANAEROBES
PH – 5–8, TEMPERATURE 20–50°C (45°C)
RCM (PINK) – 4–6 HRS
MEDIA: NA, BA, THIOGLYCOLATE BROTH, RCM
BA: POLYMYXIN, NEOMYCIN, IRON, CITRATE (BLACK COLONIES)
BA: RABBIT, SHEEP, HUMAN COLONIES SHOW TARGET HEMOLYSIS
LITMUS MILK – FERMENTATION OF LACTOSE, CHANGE OF LITMUS – BLUE TO RED
ACID COAGULATES CASEIN – CLOTTED MILK DISRUPTED – STORMY FERMENTATION

11. VIRULENCE
FACTORS
 STRAINS – FIVE TYPES A–E
 TOXINS – MOST PROLIFIC OF TOXIN PRODUCING BACTERIA
 FOUR MAJOR TOXINS – ALPHA, BETA, EPSILON AND IOTA
 ALPHA TOXIN – MOST IMPORTANT BIOLOGICALLY, LETHAL,
DERMONECROTIC AND HEMOLYTIC
Table 28.6 Toxins produced by C. perfringens types

12. CLOSTRIDIUM
PERFRINGENS
ENZYMES
NEURAMINIDASE DESTROYS
MYXOVIRUS RECEPTORS
HEMAGGLUTININ – ACTIVE AGAINST
RBCS
FIBRINOLYSIN
BURSTING FACTOR – MUSCLE
LESIONS IN GAS GANGRENE
CIRCULATING FACTOR – INCREASE IN
ADRENAL SENSITIVITY

13. GAS GANGRENE
TYPE A
COMMONLY SEEN IN ASSOCIATION
WITH OTHER CLOSTRIDIA
WOUND CONTAMINATION
ANAEROBIC CELLULITIS
MUSCLE TISSUES ARE INVADED –
ANAEROBIC MYOSITIS Fig. 28.10 Gas gangrene of the lower limb showing
edema and discoloured skin

14. PATHOGENESIS OF GAS
GANGRENE
Exogenous: Clostridia usually enter a wound along
with implanted foreign particles such as soil, road dust,
bits of clothing or shrapnel. They may also be present on
skin, especially that of the perineum and thighs
Endogenous: Infection may also develop after surgical
procedures (especially amputations for vascular disease)
and even injections (especially adrenaline)

15. CLOSTRIDIUM SEPTICUM
Pleomorphic bacillus
Oval, central/subterminal spores
Anaerobic. saccharolytic, abundant gas
Four distinct toxins
Alpha toxin is hemolytic, demonecrotic and lethal
Gas gangrene in humans

16. CLOSTRIDIU
M NOVYI
Large, pleomorphic bacillus
Oval, subterminal spores
Strict anaerobe
Type A – causes gas gangrene
Large amounts of edema
fluid, little or no observable
gas, high mortality

17. CLOSTRIDIUM HISTOLYTICUM
Oval, subterminal, bulging spores
Proteolytic
Gas gangrene in humans
Infection – exogenous/endogenous
Exogenous – implanted foreign particles
Endogenous – clean surgical procedures

18. ANAEROBIC
WOUND
INFECTIONS
Simple wound
contamination – no invasion
of tissue
Anaerobic cellulitis –
invasion of fascial planes,
minimal toxin production,
no invasion of muscle tissue
Anaerobic myositis – gas
gangrene, invasion of
muscle tissue, abundant
formation of exotoxins

19. CLINICAL PRESENTATION
Incubation period – 7 hours to 6 weeks
C. perfringens – 10–48 hours
C. septicum – 2–3 days
C. novyi – 5–6 days
Increasing pain, tenderness and edema over the affected part
Accumulation of gas – crepitus
Untreated – profound toxemia and prostration

20. LABORATORY DIAGNOSIS
Specimen
Films – edge of affected area, tissue from necrotic
area, exudate from deeper part of wound
Exudate collected from depth of wound –
collected by capillary pipette or swab
Necrotic tissue/muscle fragments
Blood cultures in C. perfringens and C. septicum
infections
However, C. perfringens bacteremia may occur
without gas gangrene

21. MICROSCOPY
C. perfringens – Gram-positive
bacilli without spores
C. septicum – boat- or leaf-
shaped pleomorphic bacilli
C. novyi – large bacilli with oval
or subterminal spores
Fig. 28.13 Methylene blue-stained culture
specimen revealing the presence of numerous
Clostridium septicum bacteria (note the
absence of spores) (Source: CDC, PHIL, Image
ID 217)

22. CULTURE
Robertson’s cooked meat medium:
Fresh and heated blood agar
Target hemolysis resulting from a
narrow zone of complete hemolysis due
to theta toxin and a much wider zone of
incomplete hemolysis due to the alpha
toxin
This double zone pattern of hemolysis
may fade on longer incubation

23. NAEGLER’S REACTION
C. perfringens grown on
media containing Fildes
peptic digest of sheep blood
and human serum with
antitoxin on one half of the
plate
Colonies on the half
without antitoxin will be
surrounded by a zone of
opacity
No opacity around the
colonies on the half of the
Fig. 28.14 Nagler’s reaction—on the right, the
presence of toxin produces opacity in the serum
due to the breakdown of lecithin

24. CULTURE
FIG. 28.15 REVERSE CAMP TEST

25. TREATMENT AND PROPHYLAXIS
Surgery: Most important therapeutic and prophylactic
Damaged tissue removed extensively and promptly
Hyperbaric oxygen
Antibiotics: Metronidazole I/V – before surgery and every 8 hours
Mixed aerobic and anaerobic infection: combination of metronidazole with
amoxicillin and gentamicin.
Passive prophylaxis – anti-gas gangrene serum given I/M
10,000 IU – C. perfringens
10,000 IU – C. novyi
5000 IU – C. septicum

26. THANK YOU….