CLOSTRIDIUM
Department of Microbiology,AIMSR
Dr.I.Wani
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The genus consists of
G+ve ,anaerobic, Spore
forming bacilli.
Spores are wider than
bacillary body, giving
bacillus a swollen
appearance resembling
spindle; hence named
so (Kolster meaning
spindle )
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MORPHOLOGY
-Highly pleomorphic,straight or slightly curved
rods with slightly curved ends.
-G +ve , 3-8 x 0.4-1.2 m in size
-Motile (except Cl.tetani Type VI & Cl.perfringens)
- Cl.perfringens & Cl.butyricum are capsulated;
others are non-capsulated
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SPORES
The shape & position of spores varies in different spp.
& thus useful in their identification. Spores may be;
Central or equatorial in Cl.bifermentans(Spindle shaped)
Sub terminal in Cl.perfringens(club shaped)
Oval or terminal in Cl.tertium(resembling tennis racket)
Spherical and terminal in C.tetani( drum sticks )
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CULTURAL CHARACTERISTICS
-Clostridia are anaerobic.
-Optimum temp. for growth is 37C;pH 7-7.4.
-Robertsons cooked meat broth is useful medium.
-Most species produce gas in this medium
-Saccharolytic species turn meat pink.
-Proteolytic species turn meat black with foul smell
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RESISTANCE
-Spores of Cl.botulinum survive boiling after 3-4 hrs.,
even at 105C are not killed completely.
-Spores of Cl.perfringens are destroyed by boiling in
5 minutes.
-Spores of Cl.tetani persist for years in dry soil, while
few strains resist boiling for 15-90 min.
-All species are killed by autoclaving at 121C for 20
minutes.
-Halogens are effective;1% aq. Soln .of Iodine kills
spores within 3 hrs.2% glutaraldehyde kills spores
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Clostridia of medical importance
Clostridium
Causing
Antibiotic
Tetanus Gas gangrene Botulism
associated diarrhea
e.g. Cl. tetani e.g.Cl.perfringens. e.g. Cl. botulinum
e.g. Cl. difficille
Saccharolytic
Proteolytic Mixed
e.g. Cl. perfringens
e.g. Cl. sporogenes Cl. histolyticum
&Cl. septicum
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1.Clostridium perfringens(Cl.welchii)
Morphology
-Large Gram-positive bacilli with straight, parallel
sides & slightly rounded ends.
-Measure 4-6x1m in size,occuring singly or in chains
-Pleomorphic,capsulated & non-motile.
-Spores are central or sub terminal. Spores are
rarely seen in culture media or material from
pathogenic lesions, a characteristic morphologic
feature
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CULTURAL CHARACTERISTICS
-Robertsons cooked meat broth is ideal; meat is
turned pink but not digested with sour odor.
-Stormy fermentation of lactose in litmus milk; the
acid coagulates casein-acid clot.
-On BAM: Target haemolysis
BIOCHEMICAL REACTIONS:
Glucose Indole -ve
Lactose Fermented with MR +ve
Maltose A & G production VP -ve
-H2S prodn. test & Nitrate redn. test - +ve
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Left to right:
a. RCM: Meat turned pink but not digested
b. Litmus Milk: Stormy fermentation & acid clot in Litmus milk
c. BAM: Target hemolysis
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RESISTANCE: Boiling for 5 min. & autoclaving at
121C for 15 minutes kills all spores.
TOXINS: 12 distinct types of toxins elaborated;
toxin-lethal,dermonecrotic & hemolytic
, & toxins- lethal & necrotizing
& toxins- minor lethal action
toxin-lethal & hemolytic for red cells
toxin-lethal & cytolytic
toxin- collagenase
toxin-proteinase & gelatinase
toxin- hyaluronidase & toxin-deoxyribonuclease
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ENZYMES
-Neuraminidase-destroys myxovirus receptors on red
cells
-Hemagglutinin-active against red cells of humans
-Fibrinolysin
-Hemolysin
-Bursting factor- has specific action on muscle tissue
& responsible for muscle lesions in gas gangrene.
-Circulatory factor-causes an increase in adrenaline
sensitivity of capillary bed ,also inhibits phagocytosis
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PATHOGENICITY
-Three Clinical conditions produced include;
1.Simple wound contamination: Slow wound healing
2.Anaerobic or clostridial cellulitis:
-Clostridiae invade fascial planes(fasciitis) with minimal
toxin production but no invasion of muscle tissue.
-Lesions vary from limited gas abscess to extensive
involvement of limbs.
-Seropurulent discharges with offensive odor produced
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3.Anaerobic myositis or myonecrosis or gas gangrene
-Most serious complication of clostridial invasion of
healthy muscle tissue .
-Abundant formation of exotoxin & production of gas.
-GG is disease of war. In civilian life it follows road
accidents or injuries with crushing of muscle mass.
-GG is rarely infection of single clostridium; several
species found in association with anaerobic
streptococci & facultative anaerobes (E.coli,Stap,Proteus)
-Among pathogenic clostridiae,Cl.perfringens is most
frequently encountered(60%) followed by Cl.Novyi &
Cl.septicum(20-40%). 14
PATHOPHYSIOLOGICAL EVENTS OF GAS GANGRENE
-Calcium salts & salicic acid in soil cause necrosis.
-Crushing tissue/tearing of arteries produce anoxia of
muscle.
-Extravasation of blood increase pressure on capillaries
reducing further blood supply.
-Eh & pH of damaged tissues falls.
-Carbohydrates in tissue are fermented producing gas.
-Proteins are broken down into amino acids.
-Extravasated hemoglobin & myohemoglobin are
reduced & they cease to act as oxygen carriers.
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-The Clostridiae multiply & elaborate different toxins.
-Lecithinases damage cell membranes.
--toxin causes lysis of erythrocytes-hemolytic anemia
-Collagenases destroy collagen barriers in the tissue
-Hyaluronidases breakdown intercellular substance
-Abundant gas production reduces blood supply
CLINICAL PRESENTATION: (IP 6 hrs. to 6 weeks)
-Increasing pain, tenderness & edema of affected part
with blackening of tissue & foul smelling serous exudes
-Crepitus due to accumulation of gas bubbles
-Death occurs due to circulatory failure. 16
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OTHER INFECTIONS:
-Food poisoning: usually caused by Type A strains
-Gangrenous appendicitis: Cl.perfringens Type A
& occasionally by Type D
-Necrotizing enteritis: caused by Type C strains
-Biliary tract infection: Rare but serious -EC & PCS
-Endogenous gas gangrene of intra-abdominal origin
-Brain abscess & meningitis: Rare
-Panophthalmitis: Rare
-Thoracic infections
-Urogenital infections- usually follow UT surgery18
LABORATORY DIAGNOSIS
A. Hematological investigation: Not significant
B. Bacteriological Investigation:
Specimen: Wound swabs,necrosed tissue, muscle
fragments, exudates from active parts etc.
1.Microscopy:Gram +ve, non-motile, capsulated
bacilli.
-Spores are rarely observed in Cl.perfringens
2.Culture:
On RCM meat turned pink but not digested
On blood agar target hemolysis
3. Biochemical reactions: As discussed above
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4.Naglers Reaction
-Rapid detection of Cl.perfringens from clinical sample
-Done to detect the lecithinase activity of alpha toxin
-Characteristics opalescence is produced around
colonies in +ve test due to breakdown of
lipoprotein complex in the medium
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5.Reverse CAMP Test:
-Used for differentiation of Cl.perfringens from other
clostridium species.
-CAMP +ve Group B Streptococcus is streaked in SBA
& Cl.perfringens is streaked perpendicular to it
arrowhead(enhanced) hemolysis is seen between
growth of Cl.perfringens & Group B streptococcus
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PROPHYLAXIS & TREATMENT
1.Surgery: All damaged tissue should be removed,
wounds irrigated to remove clots, necrotic tissue &
foreign materials, excision of affected parts in EGG.
2.Antibiotics: Metronidazole given intravenously
before surgery & repeated 8 hourly for 24 hrs.
-Broad spectrum antibiotics in combinations (like
metronidazole+gentamycin+amoxicillin)are effective.
3.Antitoxins: Passive immunization with AGS
3 doses- 1 intravenous dose followed by
2 intramuscular doses at 6hrs. interval
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OTHER CLOSTRIDIAE ASSOCIATED WITH GG
1.Cl.septicum also called as Vibrion septique
-Produces 4 distinct toxins;
-toxin: hemolytic & dermonecrotic
-toxin: leucotoxic
-toxin: hyaluronidase
-toxin: hemolysin
-It also produces fibrinolysin
2.Cl.novyi: 4 types recognized(A-D),Type A causes GG
3.Cl.histolyticum: Produces 5 distinct toxins
Infrequently associated with GG
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