A man was admitted to the hospital with a major soft tissue injury and open leg fracture from a car accident. His leg wound drained a thin, dark fluid. Microbiological examination of the fluid found gram-positive thick rods without spores on gram stain (Clostridium perfringens). C. perfringens is a pathogenic, spore-forming, anaerobic bacterium that can cause gas gangrene, food poisoning, and necrotizing enteritis by producing various toxins. It is commonly found in the intestines but can cause disease when introduced into deep tissue wounds.

1. Clostridium
perfringens
Dr.V.S.Vatkar
Asso Pro
D Y Patil Medical College
Kolhapur

2.  A 28 year old man met with car accident and
suffered major soft tissue injury & open
fracture of his left leg. He was admitted in
the hospital
 next day O/E leg was swollen & tense, with
thin dark fluid draining out form the wound
 Fluid collected from the leg wound & send
for microbiological examination
 Gram stain : GPB (thick rods , no spores)
 Non motile, on B A : large β-hemolytic
colonies seen (anaerobic condition)

3. Anaerobic
Spore forming
Gram positive bacilli
“Kloster” meaning a
spindle

4. Kingdom: Bacteria
Division: Firmicutes
Class: Clostridia
Order: Clostridiales
Family: Clostridiaceae
Genus: Clostridium
Species: perfringens

5. Cl.tetani : Tetanus
Cl.difficle : Acute colitis
Cl.botulinium :Botulism
Cl.septicum : Gas gangrene

6. Synonyms :
• Cl.welchii,
• Bacillus aerogenes
capsulatus
• Bacillus phlegmonis
emphysematosae

7. Originally cultivated by Achalme (1891)
Welch & Nuttall (1892): isolated it form
blood & organs of a cadaver.
Normal inhabitant of the large intestine of
man and the most imp species, causing
diseases like Gas gangrene, food
poisoning and necrotic enteritis.

8.  Gram positive
bacillus, 4-6µ x 1µ
 Straight parallel sides
with rounded ends
 Occurs singly, in
chains or in small
bundles
 spores: central/
subterminal: rarely
seen in artificial
cultures or sample
collected from lesion
• Pleomorphic
• Capsulated, non-motile

9. Anaerobe, can grow under
microaerophilic conditions.
Culture do not die on exposure to air
Ph 5.0 to 8.0
Temp. 20°C to 50°C,opt temp=45 C,
generation time at this temp may be as
short as 10 min
Grows on Blood agar, RCM and on
Thioglycollate broth within 24-48 hrs.

10. In litmus milk : lactose fermentation
leads to acid formation, color of
litmus from blue to red. Acid
coagulates casein(acid clot) &
clotted milk disrupted due to
vigorosus gas production
Stormy fermentation: paraffin plug
pushed up

11. On blood agar
containing rabbit,
sheep or human :
overnight incubation
shows
 ‘Target haemolysis’:
narrow zone of
hemolysis due to θ
toxin
 Zone of hemolysis
fade on prolong
incubation

12.  Ferments glucose,maltose,lactose and sucrose
with production of acid and gas
 Indole : - ve
 M.R. : + ve
 V.P. : - ve
 H2S is formed abundantly
 Most strains reduce nitrates

13.  Spores usually destroyed within 5 minutes by
boiling but those of food poisoning strains of
Type A & Type C ,resist boiling for 1 -3 hours
 Spores are resistant to antiseptics &
disinfectants commonly used
 Autoclaving at 121°C for 15 minutes is lethal

14. Cl.perfringens produces at least
12 distinct toxins
Based on the four major one’s
(alpha,beta,epsilon and iota) , the
strains are classified into 5 types,
from A to E

15. Type A strains : produce α toxin
Type B strains : produce α,β and
epsilon
Type C strains : produce α and β
Type D strains : produce α and
epsilon
Type E strains : produce α and iota
toxins

16. Produced by all strains of Cl.perfringens
but predominantly by type A strains
Most imp. toxin biologically & is
responsible for toxemia of gas gangrene

17. Chemically it is a phospholipidase (lecithinase C),
which, in the presence of Ca++ and Mg++ ions,
splits
Lecithin
Phosphoryl choline Diglyceride

18. Alpha toxin is lethal, heat stable and
dermonecrotic
It is haemolytic for the red cells of most
species
The haemolytic action is due to its action
on phospholipids, situated on RBC
memb

19. Beta(β), epsilon(ε) and iota(ι)
toxins :lethal and necrotising
properties
Gamma(γ) : minor lethal actions
Delta(δ) toxin :lethal effect and is
haemolytic for RBC’s of
sheep,goat,pig and cattle

20. Theta (θ) toxin : an O2 labile
haemolysin and antigenically related to
streptolysin O, shows a cytolytic activity
Kappa(κ) toxin: a collagenase
Lambda(λ) toxin : a protienase &
gelatinase
Mu(μ) toxin : a hyaluronidase
Nu (v) toxin : a deoxyribonuclease

21. 1) Enzymes which destroy the blood group
subs. A and H
2) A neuraminidase which destroys myxovirus
receptors on the RBC’s
3) A substance which renders the RBC’s
panagglutinable
4) A haemagglutinin active against human
RBC’s
5) Fibrinolysin
6) Haemolysin – distinct from α, ϴ and ɗ toxins

22. 7) Histamine
8) A bursting factor with specific action
on muscle tissues
9) A circulatory factor which can cause an
increase in adrenaline sensitivity of the
capillary bed & can also inhibit
phagocytosis

23.  produced by some strains of type A causing
diarrhoea and other symptoms of food
poisoning
 It interacts with epithelial cell tight junction
proteins, leading to diarrhea and intestinal
cramping caused by leakage of water and ions
 Production of enterotoxin is co-regulated with
sporulation, the toxin is released when
vegetative cells undergo lysis.

24. The diseases caused ,range from simple
wound contamination to disease like Gas
gangrene
3 imp. pathogenic lesions caused are:
a. Gas gangrene
b. Food poisoning
c. Necrotising enteritis

25. As defined by Oakley in 1954 :
A rapidly spreading ,oedematous
myonecrosis,occuring in association
with severe wounds of extensive muscle
masses that have been contaminated
with pathogenic
Clostridia,esp.Cl.perfringens

26. Came into prominence in World war I
(1914), Disease of the war
In civilian life, the disease follows road
accidents and crush injuries
Rarely it may follow surgical operations
Clostridia enter the wounds along with
foreign particles like soil, dust, bits of
clothing and shrapnel
Cl.novy & Cl.septicum, Cl.histolyticum,
Cl.bifermentans also causes inf.

27.  MacLennan – 3 types of wound
infections
a. Simple wound contamination : no
invasion of underlying tissues
b. Anaerobic cellulitis : Cl. invade the
tissues but with min. toxin production
and without invasion of the muscles
c. Gas gangrene : Cl. invade the healthy
muscle tissues with abundant
formation of exotoxins

28. Crushing or tearing of an artery
Muscle anoxia
Capillary pressure increases
Blood supply decreases
Ph of damaged tissue falls
Breakdown of carbohydrates & liberation of amino acids
Ideal platform for proliferation of anaerobes

29. The Clostridia multiply and elaborate
toxins which further cause tissue damage
Muscle anoxia is due to increased
capillary permeability which is caused by
Lecithinases ,by damaging the cell
membranes
Haemolytic anaemia and haemoglobinuria is
due to the lysis of RBC’s by the alpha toxin

30. - High grade fever
- I P: 7 hrs to 6 wks after wounding
- brownish pus
- discolouration
- foul odour
- watery discharge from the wound
- Underlying tissue becomes crepitant
- death occurs due to circulatory failure

32.  Caused by some strains of type A which are
called as food poisoning strains (cooked
meat/poultry)
Ingestion of contaminated food
liberation of heat labile enterotoxin in
small intestine
After an IP of 8-24 hours, abdo.pain, diarrhoea
and vomiting set in

34. Necrotising enteritis
A severe and fatal necrotising jejunitis is caused by type C
strains of Cl.perfringens

35. Gangrenous appendicitis
Caused by Cl.perfringens type A and sometimes by
type D strains
Necrotising colitis
A rare but fatal disease in which large segments of
the colon become gangrenous
Biliary tract infections
Cl.perfringens has been reported to produce 2
rare but serious infections of the biliary tract :
1) Acute emphysematous
cholecystitis
2) Post- cholecystectomy
septicaemia

36. Panophthalmitis
occasionally following penetrating eye injuries
Thoracic infections
seen commonly as battle wound injuries than in
civilian ones
Urogenital infections
*infection of the urinary tract may sometimes
follow a surgical procedure like nephrectomy
*Cl.Infection of the uterus is a serious condition
asso.with septic abortion . Septicaemia is common
in this condition

37. Lab. diagnosis of gas gangrene :
Made primararily on clinical grounds.
The lab. provides only the confirmation
of clinical diagnosis, identification and
enumeration of the infecting organisms.

38.  Specimens to be collected :
1.Films from the muscles from the edge of the
affected area & from the tissues from the
necrotic area
2. A swab from the exudate in the deeper parts of
the wound
3. Necrotic tissue & muscle fragments

39. a) Direct microscopy :
Gram stained smears give presumptive
diagnosis about Clostridial species & their relative
numbers
Accordingly -
* Presence of large no. of regularly shaped gram + bacilli,
WITHOUT SPORES ,is strongly suggestive of
Cl.perfringens infection
* Citron bodies and boat or leaf shaped pleomorphic bacilli
with irregular staining – Cl.septicum
* Large bacilli with oval or sub terminal spores –
Cl.oedematiens
* Slender bacilli with round terminal spores – Cl.tetani or
Cl.tetanomorphum

40.  Microscopy also helps to distinguish gas
gangrene from anaerobic streptococcal
myositis – both are almost similar clinically in
the early stages of the disease.
In Strept.myositis ,large no. of pus cells &
streptococci are seen but not the bacilli while in
gas gangrene scanty pus cells & diverse
bacterial flora is seen

41. b) Culture
The specimens are inoculated on fresh and
heated blood agar and on RCM
. On blood agar – target haemolysis is seen
. after 24-48 hours .Most strains produce beta
haemolysis and few are nonhaemolytic

42. Nagler’s reaction :
A useful test for the rapid detection of Cl.perfringens
from clinical specimens
A plate of serum or egg yolk is used
Cl.perfringens is grown on a medium with
6% agar
5% Filde’s peptic digest of sheep blood
20 % Human serum

43.  On one half of the plate,
antitoxin is spread on the
surface & is kept for
incubation for 24 hours
 The colonies, on the side
with the antitoxin, show no
opacity, due to specific
neutralization of the alpha
toxin
 The colonies, on the side without
the antitoxin , will be surrounded
by an opacity

44. To differentiate from group B streptococci
- A blood agar plate is inoculated with single streak of
suspected Cl.perfringens
- Inoculate a gr.B strep. with a single streak at an angle
of 90°, within few mm from Cl.perfringens
Incubate the plate anaerobically for 24-48 hours
Positive : Arrow head of synergistic haemolysis
is seen

46.  Toxigenicity of the strain can be analyzed by animal
inoculation
0.1 ml of a 24 hr.growth in RCMM ,is injected IM into
a healthy guinea pig. The animal dies within 24 hours.
A control animal, protected with the antiserum, is also
included.
On autopsy, bacteria can be recovered from the heart
and spleen of the test animal.

47. Made by isolating heat resistant Cl.perfringens type A
from faeces & from food
RCM is inoculated and heated at 100°C for 30 mins.
After cooling it is incubated at 37°C for 18 hours.
Subcultured on selective medium and incubated
anaerobically
The isolates are identified by morphology ,c/c,
biochem.reactions and by Nagler’s reaction

48. 1) Surgery :
Most imp. prophylactic & therapeutic method in
gas gangrene.
All damaged tissues should be removed & the
wound is flushed with antiseptic solution to remove
blood clots, necrotic tissues and foreign materials

49. 2) Antibiotics :
Penicillin's are known to be particularly active
against C. perfringens. Resistance to penicillin
is very rare and β- lactamase has not been
demonstrated in C. perfringens.
A low degree of resistance to ampicillin has been
reported in several studies

50. Tetracycline resistance is the most common antibiotic
resistance trait found in C. perfringens . It has been
reported that C. perfringens can carry the tetracycline-
resistance genes tetA(P), tetB(P), tetM and tetQ
Resistance to MLS antibiotics is also a common trait. It
is caused by methylation of the 23S rRNA gene
Chloramphenicol resistance in C. perfringens is not
common, though several resistant isolates have been
identified

51. 3) Antitoxins :
Passive immunization with polyvalent
antitoxin (anti gas gangrene serum) is given as
a prophylactic measure in gas gangrene.
The dose given –
10,000 U of Cl.per
10,000 U of Cl.oede I.M.or I.V
5,000 U of Cl.sep

52. 4) Hyperbaric oxygen :
Introduced in the depth of the wound
to reduce anaerobiosis
5) Active immunization :
with toxoids, to induce antitoxic response

53. HEPTAVAC
Contains 7:1 Clostridial vaccine
It is an opaque fluid vaccine containing purified toxoids
of Cl.perfringens,types B,C and D , together with
formalin killed cells and a purified formal toxoid
The antigen is absorbed into aluminum hydroxide & is
preserved with 0.013 % thiomersal BP

54. A multiplex PCR which can detect all the
major toxin genes of Cl.perfringens is
developed
Also PCR system for the detection of 2
other imp. toxin genes ,cpe and cpb2 is
also available

55.  For more detailed characterization of
Cl.perfringens isolates, diff.methods
have been used .
1. Plasmid profiling
2. Multilocus enzyme electrophoresis
(MLEE)
3. Ribotyping
4. Amplified fragment length polymorphism
(AFLP)

56. THANK YOU