CLOSTRIDIUM

1. Clostridium perfringens ( Kloster-Spindle )
Clostridia →
 Gram positive, anaerobic, spore forming bacilli:
 Major diseases → Gas gangrene, food poisoning &
tetanus
 Spores →
a) Central or equatorial → giving the bacillus a
spindle shape ( Cl. bifermentans )
b) Subterminal → the bacillus appearing club
Shaped ( Cl. perfringens )
c) Oval & terminal, resembling a tennis racket
( Cl. tertium )

2. d) Spherical & terminal, giving a drumstick
appearance. ( Cl. tetani )
 Motile with peritrichate flagella except Cl.
perfringens & Cl. tetani type VI which are nonmotile.

3. Infections
Clostridial infections
A. The Gas gangrene group
1. Established C l. perfringens
Pathogens C l. septicum
C l. novyi
2. Less pathogenic C l. histolyticum
C l. fallax
3. Doubtful pathogens C l. bifermentans
C l. sporogenes
B. Tetanus C l. tetani.

4. C. Food poisoning
1. Gastroenteritis C l. perfringens ( Type A)
2. Necrotising enteritis C l. perfringens ( Type C )
3. Botulism C l. botulinum
D. Acute colitis C l. difficile

5. Clostridium perfringens
( C l. welchii , Bacillus aerogenes capsulatus,
B. phlegmonis emphysematosae )
• Bacillus was originally cultivated by Achalme (1891)&
was first described in detail by Welch & Nuttall (1892)
who isolated it from the blood & organs of a cadaver
• Causes Gas gangrene, food poisoning & necrotic
enteritis in humans & serious diseases in animals.
• Normal inhabitant of the large intestines of human
being & animals. The spores are commonly found in
soil, dust & air.

6. Morphology :-
– Plump, Gram positive bacillus with straight,
parallel sides & rounded or truncated ends, about
4-6 µm x 1 µm, occuring singly or in chains or
small bundles.
– Pleomorphic, filamentous & involution forms are
common.
– Capsulated & nonmotile.
– Spores are central or subterminal, rarely seen in
lesions & their absence is one of the characteristic
morphological features of Cl.perfringens.

8. Cultural Characteristics :-
• Anaerobe, but can grow under microaerophilic
conditions.
• PH range 5.5-8.0. Temperature range 20ºc-50ºc
optimum is 37ºc & 45ºc is optimal for many strains.
• Generation time: 10 minutes
: RCM inoculated with mixtures of Cl. Perfringens &
other bacteria & incubated at 45ºc for 4-6 hrs serves
as an enrichment.
• Growth in RCM => meat turned pink, but not
digested culture has an acid
reaction & a sour odour

9. In Litmus milk :
Fermentation of lactose → Acid formation
( colour change of blue litmus to red )
↓
ACID coagulates the casein ( acid clot )
↓
clotted milk disrupted due to vigorous gas production.
↓
Stormy Fermentation.
→ On blood agar ‘target hemolysis’ =>Narrow zone of
complete hemolysis due to theta toxin & a much wider
zone of incomplete hemolysis due to the alpha toxin

10. Biochemical reactions :
→ Glucose, maltose, lactose & sucrose are
fermented with the production of acid & gas.
→ MR positive & VP negative. Indole Negative,
→ Abundant H2S produced.
→ Most strains reduce nitrates

11. Resistance :
→ Spores are destroyed within 5 minutes by boiling
but,food poisoning strains of Type A & certain
Type C strains resist boiling for 1-3 hours.
→ Autoclaving at 121ºc for 15 minutes is lethal
→ Spores are resistant to the antiseptics &
disinfectants in common use.
Classification :
→ A to E based on the toxins they produce
→ Typing done by neutralization test with specific
antitoxins by intracutaneous injection in guinea
pigs or intravenous injection in mice

12. Toxins :
→ 12 distinct toxins, enzymes & biologically active
soluble substances.
→ Four ‘Major toxins’, alpha, beta, epsilon & iota are
responsible for pathogenicity.
→ Alpha (α) toxin ) => ( Phospholipidase)
( leccithinase c )
: produced by all types of Cl. Perfringens
( Abundantly by type A strains ).
: Lethal, dermonecrotic & hemolytic
: Lecithin α toxin Phosphoryl choline + diglyceride
Ca++, Mg++ions (opalescence in serum or egg
yolk containing media ).

13. Nagler reaction :
:Antitoxin- No opalescence around colony in serum
containing media. ( 6% agar, 5% fildes, peptic
digest of sheep blood & 20% human serum).
Other lecithinase, forming bacteria → Cl. novyi,
Cl. bifermentans, some vibrios, some aerobic spore
bearers.
: Alpha toxin is hemolytic due to its action on the
phospholipid on the erythrocyte membrane.
Hot-cold variety → best seen after incubation at 37ºc
followed by chilling at 4ºc.

14. Opacity around
Colonies
No opacity
around
colonies
(containing
antitoxin)
Nagler reaction

15.  Beta (β), epsilon (ε) & iota (ι) → lethal &
necrotising property.
 Gamma (γ) & eta (η) → minor lethal action.
 Delta ( δ) toxin → lethal & hemolytic
 Theta (θ) toxin is oxygen labile. Lethal and
general cytolytic.
 Kappa (κ) toxin is collagenase.
 Mu (µ) toxin is a hyaluronidase.
 nu (v) toxin is deoxyribonuclease.
Enzymes :
→ A & H, a neuraminidase → destroy myxovirus
receptors on RBC.
→ fibrinolysin,histamine ( bursting factor),
(circulating factor).

16. Pathogenicity :
1. Gas gangrene →
Type A is the predominant agent.
2. Food poisoning
→ some strains of type A. ( Heat resistant spores)
→ Heat labile enterotoxin.
→ Caused by cold or warmed up meat dish.
→ Incubation period 8-24 hrs. Abdominal pain
diarrhoea & vomiting .Self limited illness. Recovery
in 24-48 hrs.
→ Diagnosis by isolating heat resistant Cl. perfringens
Type A from the feces & food.

17. 3. Gangrenous appendicitis
C l. perfringens type A (& occasionally Type D )
4. Necrotising enteritis
C l. perfrigens type C strains with heat resistant
spores.
→ Abdominal pain & diarrhoea following
unaccustomed feasting on pig meat along with
trypsin inhibitors like sweet potatoes. Immunisation
with type c toxoid is protective
5. Biliary tract infection ( Rare & serious )
Acute emphysematous cholecystitis
postcholecystectomy septicemia

18. 6. Endogenous gas gangrene of intra-abdominal origin
complication of abdominal surgery.
7. Brain abscess & Meningitis .
8. Panophthalamitis
9. Thoracic infections → follow penetrating wounds of
thorax, battle casualties.
10. Urogenital infections.
→ Follow surgical procedure such as nephrectomy.
→ Infection of uterus associated with septic abortion.
septicemia is common.

19. Gas gangrene :-
• Rapidly spreading , odematous myonecrosis in
association with severe wounds of extensive muscle
masses contaminated with pathogenic clostridia .
• Disease of war in civilian life, the disease generally
follows road accidents or injury involving crushing of
large muscle masses Rarely, follow surgical operations.
• Clostridia enter the wounds with impure foreign
particles such as soil, road dust, bit of clothing or
shrapnel
• Three types of anaerobic wound infections
1. Wound contamination with no invasions of the
underlying tissue → delay in wound healing

20. 2. Anaerobic cellulitis
Invasion of fascial planes with minimal toxin
production & no invasion of muscle tissues.
3. Anaerobic myositis or gas gangrene
Clostridia invasion of healthy muscle tissues &
abundant formation of exotoxins
→ Conditions favoring clostridial multiplication
→ Low oxygen tension
→ Ionised calcium salts & silicic acid in the soil causes
necrosis.
→ Crushing tissue or tearing of the arteries produces
anoxia of the muscle.

21. → Extravasations of blood increases pressure on the
capillaries, reducing blood supply still further.
→ EH & Ph of the damaged tissues fall. These changes
along with the chemical changes in damaged &
anoxia muscles, including breakdown of
carbohydrates of & liberation of amino acids from
proteins favour growth of anaerobes
→ Extravasated hemoglobin & myohemoglobin are
reduced & cease to act as oxygen carriers → so,
aerobic oxidation halted & anaerobic reduction of
pyruvate to lactate leads to a further fall in EH.

22. → Incubation period 7 hrs to six week after wounding
coverage. 10-48 hrs with Cl. perfringen 2-3 days
with Cl. septicum & 5=6 days with Cl. novyi
→ Pain, tenderness & edema of the affected part with
systemic signs of toxemia, thin watery discharge ,
which later becomes profuse & serosanguinons,
accumulation of gas (crepitus)
→ profound toxemia & prostration develop & death
occurs due to circulatory failure

23. Laboratory diagnosis :
→ Specimens to be collected → films from the muscles
at the edge of the affected
area.
→ Exudates from the active
part of capillary pipette
or a swab
→ Necrotic tissue & muscle
fragments.
→ Gram stain
→ Aerobic & anaerobic culture
→ four tubes of RCM broth are inoculated & heated at
100ºc for 5,10,15 & 20 mins to differentiate organisms
with heat resistant spores

24. → Blood cultures are often positive in Cl. perfringens
& Cl. septicum infections.
→ The isolates are identified based on their
morphological, cultural, biochemical & toxigenic
characters.
Prophylaxis & therapy :
Surgery
Hyperbaric oxygen
Prophylaxis → Antibiotic’s in combination with surgical
methods.
Passive immunisation with ‘anti-gas gangrene serum
( 10,000 IU Cl. perfringens, 10,000 IU Cl.novyi & 5,000
IU Cl. septicum antitoxin given Im or in emergencies IV
)