(1) The provisional diagnosis is gas gangrene based on the clinical findings of edema, pain, and crepitus around the wound in the setting of crush injury and fracture from a road accident. (2) The likely causative organism is Clostridium perfringens, as it is the most common cause of gas gangrene. (3) The pathogenesis involves contamination of the crushed wound with C. perfringens spores, which then proliferate under anaerobic conditions created by tissue ischemia and produce lethal toxins.

1. By Dr. Rakesh Prasad Sah
Associate Professor, Microbiology
Clostridium Perfringens (Gas gangrene)

2. Clinical Based Question (LAQ)
•A 25 year old man met with a road accident. He had crush injury and
fracture of the right lower leg. He was admitted to the hospital. On loc
al examination, there was oedema and pain at the site of injury. On p
alpitation, crepitus was felt around the wound.
•What is your provisional diagnosis?
•Causative organisms?
•Pathogenesis?
•Lab diagnosis?

3. Introduction
•Anaerobic bacteria  do not have cytochrome system for oxyg
en metabolism  unable to neutralize toxic oxygen metabolites
 either can not grow in presence of oxygen (obligate anaero
bes) or do not utilize oxygen but tolerate its presence of (aerotol
erant anaerobes).
•Needs special requirements for their growth in culture (Anaero
bic Conditions)

4. •Needs special requirements for their growth in culture (Anaero
bic Conditions)
• McIntosh and Filde’s anaerobic Jar
• GasPak system
•Medium with low redox potential: by adding reducing substances
to media e.g.
•Unsaturated fatty acid
•Ascorbic acid
•Glutathione
•Cysteine
•Glucose
•Sulfites
•Metallic iron

5. Clostridium
•Consists of anaerobic, spore forming, Gram positive bacilli.
•Name Clostridium  derived from the word “Kloster” spindle.
•Spores  wider than bacterial bodies  swollen appearance 
resembling a “spindle”.

6. •Genus contains bacteria causes 3 major ds.  due to powerful
exotoxins.
• Tetanus
• Gas gangrene
• Food Poisoning

7. Classification
S. No Disease Organisms
1 Tetanus Cl. tetani
2 Gas gangrene
A) Established Pathogens Cl. perfringens (Cl. welchii), Cl. novyi (Cl. oed
ematiens), Cl. septicum
B) Less Pathogenic Cl. histolyticum, Cl. fallax
C) Doubtful pathogens Cl. bifermentans, Cl. sporogens
3 Food Poisoning
A) Gastroenteritis Cl. perfringens (Type A)
B) Necrotising Enteritis Cl. perfringens (Type C)
C) Botulism Cl. botulinum
4 Acute colitis Cl. difficile

8. Cl. perfringens (Cl. welchii)
•Cultivated by Achalme (1891) and was first described in detail by
Welch and Nuttal (1892).
•Cl. Perfringens most common & impt etiological agent of gas gangren
e (60%), followed by Cl. Novyi (30-40%) and Cl. Septicum (10-20%).
•Cl. Perfringens also produces  food poisoining and
necrotising enteritis in man.
•Commensals of large intestine and spores are found in soil and dust.
•Is invasive as well as toxigenic

9. Morphology
•Large, stout, Gram positive bacillus wit
h sub terminal spores.
•Occurs single or in chains and is
pleomorphic.
•Capsulated and non-motile.
•Spore formation  induce on special
media.

10. Culture
•Grows on
• Blood agar
• Cooked meat broth (CMB)
• Thioglycollate broth
•Grows best in  media containing
carbohydrate  e.g.
glucose blood agar
•Anaerobic, PH  5.5-8.0, tempr  20-
500C, Opt tempr  370C

11. Blood Agar
•Most strains  targeted hemoly
sis  narrow zone of complete h
emolysis  theta toxin and much
wider zone of incomplete haemol
ysis  alpha toxin
Cooked Meat broth
•Meat pieces  pink colour but
are not digested.

12. Biochemical Reactions
•Saccharolytic and mild proteolytic action (gelatin liquefaction).
•Ferments
• glucose
• Lactose
• Sucrose
• Maltose
•Litmus milk  lactose fermentation  acid  colour of litmus  blue to red.
•Coagulates casein (acid clot)  clotted milk  disrupted due to vigorous
gas production  stormy fermentation.
•Indole  Negative
•MR  Positive
•VP  Negative
•Reduces Nitrates

14. Classification
•Produces 12 distinct toxins
•On the basis of production of 4 major toxins (alpha, beta, epsilon and iota)
 classified into five types A to E.
S. No. Strain Type Toxin Produced
1 Type A Alpha toxin
2 Type B Alpha, beta and epsilon toxins
3 Type C Alpha and beta toxins
4 Type D Alpha and epsilon toxins
5 Type E Alpha and iota toxins
Type A causes  Gas gangrene and some strains causes  enterotoxins

15. Alpha (α) Toxin
•Produced by all types of Cl. Perfringens  most abundantly by type A strai
ns.
•Chemically it is a  phospholipidase (lecithinase C) and is responsible for
 toxaemia in gas gangrene.
•Is heat stable, lethal, dermonecrotic and hemolytic.
•Hemolysis is best seen  incubation at 370C  followed by chilling at 40C.
•Splits “lecithin” an impt constituents of mammalian cell membrane.
•Used for rapid detection of Cl. perfringens in clinical specimens (Nagler reactio
n).

16. Cl. Perfringens  grown on medium containing  6% agar,5% Fildes peptic digest
of sheep blood and 20% human serum or 5% egg yolk with Neomycin sulphate
anti-toxin is spread to one half of the culture plate
incubated at 370C for 24hrs
Colonies on the half of the plate without antitoxin  surrounded by opacity
while colonies around other half with antitoxin  shows no opacity  due to
neutralisation of alpha toxin.
Nagler Reaction

17. Alpha Toxin
(Lecithinase C)
Lecithin Diglyceride
Phosphoryl
Choline
Deposits around the
colony  Opacity

18. Reverse CAMP Test
•Is similar to the CAMP tests for identifying group B streptococci
•Except that Clostridium sp. Is inoculated in place of Staph. aureus and
a known group B streptococcus is used.
•Group B streptococci show enhanced hemolysis with other clostridia but
only Cl. Perfringens exhibits accenentuated zone of hemolysis as butterfly
appearance.

19. Other Minor Toxins
S.no. Toxin Actions
1 Gamm () and eta () minor lethal actions
2 Delta () lethal and hemolytic
3 Theta () oxygen labile  Agenically related to streptolysin O.
4 Kappa () collagenase
5 Lambda () proteinase and gelatinase
6 Mu () hyaluronidase
7 Nu () deoxyribonuclease
•Beta (), epsilon (), and iota () toxins  lethal and necrotizing pr
operties.

20. Enterotoxin
•Some strains of type A  enterotoxin  diarrhoea & other symptoms
of food poisoning.
Other Soluble Substances
• Haemagglutinins
• Neuraminidase
• Fibrinolysin
• Haemolysin
• Histamine

21. Clinical Manifestations
•Polymicrobial involving other clostridia species.
1. Simple wound contamination
• Involves wound surface contamination without invasion of
underlying tissue  occurs in absence of devitalized tissue
Clostridial Wound infections

22. 2. Anaerobic cellulitis
•Involves facial plane with minimal toxin release, without muscle inv
asion.
3. Anaerobic myositis (gas gangrene)
•Muscle invasion occurs  leads to gas in the muscle compartment
with abundant toxin release.

23. Clostridial Enteric infections
•Caused by C. perfringens type A
enterotoxin.
•Occurs  consumption of impro
perly cooked contaminated meat.
•Enterotoxin  acts by forming
pores in the intestinal mucosal
membrane.
1. Food Poisoning

24. Clostridial Enteric infections
•A life threatining condition
•Characterized by
• Ischemic necrosis of the Jejunum
And
• Gas in the tissue.
•Caused by C. perfringens
type C strains  β-toxin.
Enteritis necroticans (gas gangrene of the bowel)

25. Necrotizing enterocolitis
•Resembles enteritis necroticans but associated with C. perfringens type A.
•Gangrenous appendicitis.

26. Other Clostridial infections
•Bacteremia (C. perfringens, C. tertium and C. septicum)
•Skin and soft tissue infections
• C. perfringens, C. histolyticum, C. septicum, C. novyi and C. sord
ellii  cause necrotizing infections of skin and soft tissues
• Infection of the endometrium  toxic shock syndrome
• Meningitis and brain abscess
• Panophthalmitis (C. sordellii or C. perfrngens)
(rapidly progressing (choroid, retina, vitreous fluid, aqueous fluid, cornea, sclera,
and conjunctiva) with extension into the orbital structures.)

27. Gas Gangrene
Definition
•A rapidly spreading, edematous myone-
crosis occuring in association with severly
crushed wounds contaminated with
pathogenic clostridia, particularly with
C. perfringens.
•Previously the disease was called
Malignant edema or
Clostridial myonecrosis.

28. Pathogenesis
Anaerobic Environment
Contamination of wounds
Non-traumatic Gas gangrene

29. Development of gas gangrene requires
RTA (Road traffic accident)
Crushing injuries of muscles
lead to interruption in blood supply and tissue ischemia
causing liberation of large or medium sized arteries
open fractures of long bones or foreign bodies (bullet injuries) or devitalized tissues
Anoxic muscle
Anaerobic environment
Starts utilizing pyruvate anaerobically to produce lactic acid

31. Contamination of wound
•Contamination of wound  with Clostridial spores present In the s
oil or clothes.
•Rarely non-traumatic gas gangrene  hematogenous seeding of
normal muscle with bowel clostridia  occurs in people with GIT
pathologies (e.g. colonic malignancy)

32. Clinical Manifestations of Gas Gangrene
•I.P.  10-48hrs for C. perfringens
• 2-3 days for C. septicum
• 5-6 days for C. novyi
•Various manifestation include
• Sudden onset of excruciating pain at the affected site.
• Rapid development of a foul-smelling thin serosanguineous
discharge.
• Gas bubbles (Crepitus) in the muscle planes

34. Clinical Manifestations of Gas Gangrene
Continue….
•Various manifestation include
• Brawny edema and induration.
• Gangrenous tissues  become liquefied and sloughed off.
• Shock and organ failure develop later.
• High mortality rate (50%).

35. Laboratory Diagnosis
•Specimens
• Necrotic tissues
• Muscle fragments
• Exudates from deeper parts of the wound

36. Laboratory Diagnosis
•Direct Microscopy
• Thick, stubby, boxcar-shaped grampositive bacilli without spore
are suggestive of C. perfringens.

37. Laboratory Diagnosis
•Culture
• Media RCM (Robertson cooked meat broth, egg yolk, agar etc)
• Incubation  anaerobically by GasPak

38. Identification
Target hemolysis (double zone hemolysis)
Nagler’s reaction: Opalescence surrounding the streak line on egg yolk
agar

39. Identification
Reverse CAMP test: Positive
 Heat tolerance test: Positive
In litmus milk: Produces stormy clot reaction.

40. Treatment
Early surgical debridement - most crucial step in the management o
f gas gangrene.
All devitalized tissues should be widely resected so as to remove con
ditions that produce anaerobic environment.
Closure of wounds – delayed for 5–6 days until the sites are free from
infection.

41. Antibiotics: Combination of penicillin and clindamycin is recom
mended for 10–14 days
Hyperbaric oxygen: It may kill the obligate anaerobic clostridia
- C. perfringens; however, it has no effect on aerotolerant clostri
dia (C. septicum)
Passive immunization with anti-α-toxin antiserum.

42. Prophylaxis
•Surgery
•Antibiotics: Metronidazole, penicillin, sulphonamide, tetracycl
ine, and amoxycillin.
•Antitoxin
•Hyperbaric oxygen
•Active immunisation  Toxoids.

43. Clinical Based Question (LAQ)
•A 25 year old man met with a road accident. He had crush injury and
fracture of the right lower leg. He was admitted to the hospital. On loc
al examination, there was oedema and pain at the site of injury. On p
alpitation, crepitus was felt around the wound.
•What is your provisional diagnosis?
•Causative organisms?
•Pathogenesis?
•Lab diagnosis?