 Gram positive anaerobic or aerotolerant rods ,producing

endospores which are wider than the bacillary bodies giving

the characteristics spindle shape ,hence the name
Clostridium( kloster- spindle)
 Most of them are motile except C. perfringens, C.tetani type
IV
 Noncapsulated except C.perfringens and C. butyricum
 Proteolytic and saccharolytic
 118 species
 Found normally in intestine of human and animals
 Responsible for three major diseases- C. perfringens
C. tetani
C. botulism
C. difficile
1. Based on location and shape of spores:

a) Spindle shaped :central spore.

e.g. C. bifermentans
b) Club shaped: Subterminal round spore.
e.g. C. perfringens
c) Tennis racket shaped: Oval and terminal.
e.g. C. tertium, C. choclearum.
d) Drumstick shaped: spherical and terminal
e.g.C.tetani,C. tetanomorphum,C.sphenoides
2. Based on the site of infection:

a) Histotoxic clostridia
e.g. C. perfringens type A, C. novyi, C.
septicum, .
b) Enteropathogenic clostridia.
e.g. C. perfringens type A2, and type C.
C. difficile
c) Neurotoxic clostridia
1. C. tetani
2.C. botulinum
3. Based on the disease they produced:
a. Gas gangrene group:
e.g. C. perfringens, C. septicum, C. novyi,
C. fallax, C. sordeli, C.
bifermentans.
b. Tetanus: C. tetani
c. Food poisoning
Gastroenteritis C. perfringens type A
Botulism C. botulinum
Necrotising enteritis C. perfringens type C
d. Acute colitis
C. difficile
4. Based on the biochemical reactions:
a. Both proteolytic and saccharolytic
1. Proteolytic predominating
- C. botulinum A,B,F
- Cl. bifermentans
2. Saccharolytic predominating
- Cl. perfringens
- Cl. difficile
b. Slightly proteolytic but not saccharolytic
- Cl. Tetani
c. Saccharolytic but not proteolytic
- Cl. Botulinum C,D,E
d. Neither proteolytic nor saccharolytic
- Cl. cochlearum
 First cultivated by Achalme(1891) and later
described in detail by Welch & Nuttal (1892)
who isolated it from blood and organs of
cadaver.
 commonly known as C. welchi in U.K.
 Most important organism causing gas gangrene
, also produces food poisoning ( C. perfringens
type A2) and necrotic enteritis( C. perfringens
type C).
Kingdom: Bacteria
Division: Firmicutes
Class: Clostridia
Order: Clostridiales
Family: Clostridiaceae
Genus: Clostridium
Species: perfringens
 Normal inhabitant of large intestine of human
and animals. (10 4 /gm faeces).
 Found in faeces and contaminate skin of
perineum, buttocks and thighs.
 Spores are commonly found in soil, dust and
air.
 Gram positive,
 Rod shaped with straight, parallel side and
rounded or truncated ends
( Brick or Box - car shaped).
 4-6 um x 1um, usually occuring singly or in chains or small
bundles.
 Capsulated,
 Non motile
 Pleomorphic, filamentous and involution form are common.
 Spore forming
 Spores are spherical & subterminal but rarely seen in artificial
culture media or in material from pathogenic lesions , and their
absence is one of the characteristic morphological features of Cl.
perfringens
 Moderate obligate anaerobe
 Grows over a pH range 5.5-8.0
 Temperature range of 20 ºC to 50 ºC.
 A temp. of 45˚C is optimal in many strains
where generation time may be as short as ten
minutes- property utilised to obtain pure
culture from mixed growth
 Robertson cooked meat broth- good growth,
meat turned pink but not digested, has acidic
reaction and sour odour
 Blood agar-show characteristics double zone haemolysis
(target haemolysis- inner narrow zone of clear haemolysis
due to theta toxin and outer wider zone of incomplete
hemolysis due to alpha toxin ), flat , spreading , rough ,
translucent colonies with irregular margins
 Produce diffuse opalescent colonies when grown on egg
yolk or serum agar due to lecithinase activity and this can be
inhibited by antitoxin- Nagler test
 Litmus milk medium- ‘stormy fermentation’ Based on
that Saccharolytic Clostridium species ferment Lactose
and produce acid and gas that coagulates casein in litmus
milk medium to form stormy clot.
 Gelatin – hydrolyses gelatin
 Selective media- made by incorporation of
neomycin, polymyxin and crystal violet
 Ferments glucose ,lactose,maltose,sucrose
and other sugars with production of acid and
gas
 Indole negative
 Urease negative
 MR positive, VP negative
 H2S formed abundantly
 Liquefy gelatin
 Most strains reduces Nitrate to Nitrite
 Spores are usually destroyed with in 5
minutes by boiling but those of ‘food
poisoning’ strains of Type A and Type C resist
boiling for 1-3 hrs
 Autoclaving for 121C for 15 minutes is lethal
 Spores resistant to antiseptics and
disinfectant in common use
 On the basis of type of toxin they produce
 Classified into five types- A to E
 Typing based on 4 major toxins- alpha, beta,
epsilon and iota
 Typing done by neutralisation tests with
specific antitoxins by intracutaneous
injection in guinea pig or iv injection in mice
 Gene probes or PCR could be used for typing
 Type A and C- human pathogens
 Type A- gas gangrene, wound infections,
septicemia, food poisoning
 Type C- necrotising enteritis
 Toxins – 12 distinct toxins, four major toxins-
(alpha, beta, epsilon and iota) and
minor toxins
 Enzymes
 Biologically active soluble substances
Major toxins
1. Alpha toxin
- produced by all types
- in large amount by type A
- is phospholipidase or lecithinase C-splits
lecithin into phosphoryl choline and
diglyceride in presence of Mg and Ca ions
- most important toxin biologically
- responsible for profound toxaemia of gas
gangrene .
-is lethal, dermonecrotic,and hemolytic
- resposible for lecithinase reaction in egg
yolk agar and hazy zone of haemolysis on
blood
agar
-- lysis is of hot –cold variety- best seen after
incubation at 37C and chilling at 4C
- relatively heat stable
2. Beta toxin-
- major lethal toxin of type B and C
- responsible for lesion of necrotising
enteritis
3. Epsilon toxin-
- have lethal and necrotising property
- activated by proteolytic enzyme
- produced by type B and D
- increases the permeability of intestine-
thus enhancing its own uptake- and acts
systemically as a lethal toxin
4. Iota toxin (i toxin)
- have lethal and necrotising property
- binary toxin consist of two subunits-a and
b (immunologically and biologically distinct
- a- ADP ribosylates
- b- recognises the binding site on a cell
membrane- binds to the site-interact with
a unit to facilitate its entry
Enterotoxin
- protein in nature
- responsible for food borne diarrhoea
- occurs after consumption of food containing
large number of vegetative organism
- found in type A,C and D
Enzymes
1. Neuraminidase or sialidase
- pathogenecity factor
- acts on erytrocytes – makes them
panagglutinable-increases blood viscosity-
promotes capillary thrombosis
- causes modification of gangliosides on host
cell membrane- allow more direct contact
of pathogens with host and provide suitable
receptor for other toxins produced by same
or other microorganism
2. Fibrinolysin
3. Histamine
4. Bursting factor- has specific action on
muscle, responsible for characteristic muscle
lesion in gas gangrene
5. Circulating factor- cause increase in
adrenaline sensitivity of capillary bed and
also inhibit phagocytosis
Minor Major
 Wound and soft tissue infections
-Cellulitis
-Clostridial myonecrosis ( gas gangrene)
 Food poisoning
 Uterine infections
 Gangrenous appendicitis
 Necrotising enteritis
 Biliary tract infection
 Endogenous gas gangrene of intra abdominal in origin
 Brain abscess and meningitis(rare)
 Panopthalmitis
 Thoracic infections
 Clostridial septicemia
 3 types with increasing severity
1.Simple contamination or colonization- clostridia are
present in injured tissue but without evidence of
infection. Such contamination is common(30-80%) cases
and wound heals by first intention without sequelae.
2. Clostridial cellulitis- infection is limited to local fascia,
muscle not involved and toxemia is minimal
3. Clostridial myonecrosis or gas gangrene- muscle is
involved and toxemia is severe
 In the presence of tissue anoxia simple colonization may
rapidly progress to cellulitis and gas gangrene
 Factors which favours the active multiplication of
Clostridial species.
1. Disrupted or impaired blood supply to the tissues
due to trauma, pressure of tourniquets, casts or dressings,
presence of foreign bodies in wound, presence of necrotic
tissues , haemorrhage in wound due to trauma. action of
necrotizing agents found in soil e.g.CaCl2 etc.
2.Lowering of PH to about 6.8.
3.Utilisation of Oxygen by co -infecting aerobic
bacteria.
4. Autolysis of some tissue proteins making
aminoacids e.g. cysteine , tryptophan available – most
necessary for initiation of growth.
Clostridial cellulitis
 Mild sequelae of wound contamination or
colonization
 infection is limited to local fascia without involvement
of muscle.
 Characterized by a foul, seropurulent infection of the
depths of the wound with insignificant toxaemia.
 No bacteremia and invasion of healthy tissues.
 Infections locally and mortality is generally nil.
 Proteolytic and non- toxigenic clostridia and strains of
C. perfringens of low toxigenicity involved.
 Defined as arapidly spreading edematous myonecrosis occuring
chracteristically in association with severe wound of extensive
muscle masses that have been contaminated with pathogenic
Clostridia, particularly Cl perfringens
 Usually polymicrobial etiology.
 6 major Clostridial spp. involved:
C. perfringens(90 % alone or with others)
C. novyi( 8 %)
C. septicum( 4 %) ( usually spontaneous or non
traumatic)
C. bifermentans
C. sordelli less than 1 %
C. fallax
 Non clostridial anaerobes and various facultative aerobes also
involved which have no direct role in gas gangrene however
facilitate tissue invasion by synergy with histotoxic Clostridia.
Pathogenesis
 Route of entry
a. Exogenous infection- organism enters along
with implanted foreign material, soil, dust,
bits of clothing etc.
 Endogenous infection-
 Clostridia cannot multiply and produce disease in
normal tissues because the high oxidation–reduction
potential (Eh) of the circulating blood (+126–+246 mV)
and of the tissues is above that necessary for the
initiation of anaerobic bacterial growth (+74 mV for
C. perfringens)
 A series of changes occurs in the damaged and anoxic
tissues that lead to a rapidly falling Eh and establish
an ideal environment for the growth of clostridia
 Clostridia multiply and produce toxins
 The production of bacterial toxins and products of
bacterial metabolism promotes the growth of the
organisms so that gas gangrene becomes established.
 Defenses are further compromised, since
neither phagocytes nor antibodies can
enter the necrotic zone, and absence of
perfusion prevents antimicrobial agents
from reaching the affected tissues
 Infection spread from the original site
 Rapid invasion and destruction of the
healthy tissue
 If untreated release of toxin in circulation
 Toxaemia and circulatory failure
 Death
Clinical Presentation
Incubation period - 7 h to 7 days.
Symptoms –
 H/O trauma
 Pain develops early in the region of the wound and
increases in intensity
 progressive swelling and edema.
 The wound is edematous, tender, and exudes a profuse
serous or serosanguinous discharge which is foul smelling
 Muscle becomes black and noncontractile
 As the disease progresses, bubbles of gas appear in the
discharge, crepitus may become evident in the tissues, and
 the skin becomes white and marbled.
 The pulse rate increases markedly, is feeble,
and often impalpable and there is a mild to
moderate pyrexia.
 The patient is collapsed, profoundly toxemic,
and shocked, but remains
mentally alert and anxious.
 The blood pressure falls and peripheral
venous collapse often makes venepuncture
impossible.
 The syndrome usually terminates with
sudden death due to circulatory failure.
 Food borne gastroenteritis due to
consumption of food contaminated with large
number of vegetative cells of
enteropathogenic Clostridia.
 Caused by C. perfringens type A
 Spores of these are markedly heat reistance
 Produces heat labile enterotoxin

 Theenterotoxin causes marked

hypersecretion in jejunum and ileum.
 Food
poisoning usually caused by a cold or
warmed up meat dish
 When contaminated meat is cooked,spores in
the interior may survive-germinate and
multiply during storage or warming in the
anaerobic environment in the cooked meat-
produces enterotoxin- food poisoning
 Incubation period: 8-24 hours after ingestion
 Characterized by –abdominal pain diarrhaoea
and vomitting
 Short duration of illness and symptoms
usually disappear within 10-24 hrs. May be
severe in debillitated person.
 by β-toxin of C. perfringens type C.
 Sporadically occurs among populations in New Guinea
known as “Pig bell” and was recognized as “Drambrand”in
Germany.
 Organism acquired from consumption of pork from animals
carrying it.
 Increase consumption of sweet potato and yam inhibits
protease in GI tract and β-toxin by bacteria got protected
and responsible for disease.
 The disease is characterized by acute abdominal pain,
abdominal diarrhoea, vomiting, ulceration of the small
intestine and perforation of the intestine and acute toxemia.
 Common cause of death in the native children of Papua
New Guinea.
 in most of the cases , consequences of gas
gangrene following trauma or injury to soft
tissues.(exogenous)
 In non penetrating injuiry it may be associated
with intestinal malignancy and involve a
localized myonecrosis in addition to a
fulminating septicemia.
 Apparently migrate out of the patient intestine
as a consequence of malignant process or any
surgery. (Usually endogenous)
• C perfringens is a rare cause of
endophthalmitis,
• causes rapid destruction of the ocular tissues.
• result from perforating injury
 Oftenpresent in vagina as flora and may
contaminate perineum, buttocks, thigh
leading to infections under certain
predisposing factors such as malignancy,
threatened abortion etc.
 Laboratory diagnosis of gas gangrene
 Laboratory diagnosis of food poisoning
 Specimen collection:
- collect the exudate from the deeper part of wound
- Necrotic tissues, pus or exudate should place in
sterile screw capped bottle.
- Two or three swabbed specimen should be collected
. One for film preperation, other for anaerobic culture.
 Transport of specimen:
- cooked meat broth
- Thioglycollate broth
-if collect in syringe then probe the needle into
rubber cork or bend it.
- transported anaerobically on commercially available
anaerobic transport devices.
 Direct examination
- gram staining
 Culture
-Media used: Cooked meat broth, Blood agar, Lactose
egg yolk agar, Litmus milk agar
- Identification- microscopy, cultural character,
biochemical reaction
 Nagler reaction
 Rapid enzymological test- for the
identification of clostridial products in wound
exudates
a. A species-specific sialidase-inhibition test gives
results in 2–6 h and agrees well with the results of
bacteriological examination for C. perfringens
infection,
1. Semi quantitative Culture:
- can be done on faeces and food in the basis -
- >10⁵cfu /g of C. perfringens in implicated
food
- >10⁶ /g of C. perfringens in faeces
2) Direct detection of enterotoxin in faeces, food
or culture supernates.
-reversed passive latex agglutination test.
- ELISA
- CIE
- western immunoblotting