clostridium.perfringenes

Clostridium
1
Dr . Milad Shaini

Physiology and Structure
 Large Gram positive
 Straight or slightly curved rods with slightly
rounded ends
 Anaerobic bacilli
 Spores are wider than bacillary body, giving bacillus a
swollen appearance resembling spindle; hence named
so (Kolster meaning spindle )
 Spore do not germinate and growth does not normally
proceed unless a suitably low redox potential Eh exists
2

Physiology and Structure
 Saprophytes
 Most clostridia are motile by peritrichous flagella.(except C.tetani Type VI &
C.perfringens)
 C.perfringens & C.butyricum are capsulated; others are non-capsulated
 Some are commensals of the animal & human gut which invade the blood
and tissue when host die and initiate the decomposition of the corpse (dead
body)
 Causes diseases such as gas gangrene, tetanus, botulism & pseudo-
membranous colitis by producing toxins which attack the neurons pathways
3

Spores
The shape & position of spores varies in different spp. & thus useful in their
identification. Spores may be:
o Central or equatorial in C.bifermentans(Spindle shaped)
o Sub terminal in C.perfringens(club shaped)
o Oval and terminal in C.tertium(resembling tennis racket)
o Spherical and terminal in C.tetani( drum sticks )
4

Cultural characteristics
 Clostridia are anaerobic.
 Optimum temp. for growth is 37°C;pH 7-7.4.
 Robertson’s cooked meat broth is useful medium.
 Most species produce gas in this medium
5

Resistance
 Spores of C.botulinum survive boiling after 3-4 hrs.
 Even at 105°C are not killed completely.
 Spores of C.perfringens are destroyed by boiling in 5
minutes.
 Spores of C.tetani persist for years in dry soil, while
few strains resist boiling for 15-90 min.
 All species are killed by autoclaving at 121°C for 20
minutes.
 Halogens are effective;1% aq of Iodine kills spores
within 3 hrs.2% glutaraldehyde kills spores
6

Clostridium perfringens(C.welchii)
7

• Large Gram-positive bacilli with straight, parallel
sides & slightly rounded ends.
• Measure 4-6x1μm 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
Morphology
8

α-Toxin
Produced in large amounts by type A strains, α-toxin is a
phospholipase C (EC 3.1.4.3) and appears to play a major role in gas
gangrene. It hydrolyzes phosphatidylcholine and sphingomyelin,
but not other phospholipids. It is a zinc metal protein and requires
calcium ions for interaction with substrate. It is responsible for the
lecithinase reaction on egg yolk agar and for the hazy zone of
hemolysis on blood agar. The gene encoding α-toxin has been
cloned and sequenced. The results show that the gene product is a
399-amino acid peptide with a molecular mass of 43 kDa;
Immunization of guinea-pigs with α-toxin protects them against gas
gangrene when challenged with C. perfringens and toxin.
10

β-Toxin
β-Toxin is a major lethal toxin produced by type B and
C toxin types. It is responsible for the lesions of
necrotic enteritis of pig-bel. The toxin purified by
affinity chromatography has a molecular mass of 40
kDa and an isoelectric pH of 5.6. Nucleotide
sequencing of the gene encoding the β-toxin reveals
an open reading frame of 1 008 nucleotides that
encodes a protein of 336 amino acids with a molecular
mass of 34.9 kDa
11

ε-Toxin
ε-Toxin is a prototoxin that is activated by proteolytic enzymes and is
produced by both type B and type D strains. It increases the
permeability of the intestine, thus enhancing its own uptake, and acts
systemically as a lethal toxin. In the circulation, it causes swollen,
hyperemic kidneys, edema in the lungs, and excess pericardial fluid. The
nucleotide sequence of the gene encoding ε-toxin shows that the mature
prototoxin consists of 297 amino acids with a molecular mass of 33 kDa.
A 13 amino acid N-terminal peptide is cleaved during activation.
12

ι-Toxin
ι-Toxin is a binary toxin consisting of two subunits, ι-a and ι-b, which are immunologically and
biochemically distinct. The gene sequences encoding these subunits have been determined
and the deduced amino acid sequences for the functionally active proteins correspond to
peptides with molecular masses of about 40 and 81 kDa, respectively. A mixture of both
components is needed to demonstrate biological activity by mouse lethality or dermonecrosis.
The light chain (ι-a) is an enzyme that ADP-ribosylates polyarginine and skeletal muscle and
nonmuscle actin. ι-a must gain entrance into the target cell before it can have any effect, but
cannot do this of itself; ι-b recognizes a binding site on the cell membrane, binds to the site,
and interacts with ι-a to facilitate its entry. ι-Toxin of C. perfringens is similar to ι-toxin of C.
spiroforme in serological, biological, and enzymatic activities.
13

Minor Toxins
θ toxin
δ toxin
κ toxin
λ toxin
μ toxin
ν Toxin
14

Enterotoxin
Enterotoxin produced by C.perfringens is responsible for
food-borne diarrhea which occurs after consumption of
foods containing large numbers of the vegetative
organism. It has been found in toxin types A, C, and D,
but type B and E strains have not been sufficiently
tested to establish whether they also produce it.
15

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
16

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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Pathogenicity
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,C.perfringens is most frequently
encountered(60%) followed by C.Novyi & C.septicum(20-40%).
Three Clinical conditions produced include:
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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.
19

Pathophysiological events of gas gangrene
 The Clostridia multiply & elaborate different toxins.
 Lecithinases damage cell membranes.
 α-toxin causes lysis of erythrocytes-hemolytic anemia
 Collagenases destroy collagen barriers in the tissue
 Hyaluronidases breakdown intercellular substance
 Abundant gas production reduces blood supply
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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.
21

Other infections
 Food poisoning: usually caused by Type A strains
 Gangrenous appendicitis: C.perfringens Type A & occasionally by Type D
 Necrotizing enteritis: caused by Type C strains
 Biliary tract infection: Rare but serious
 Endogenous gas gangrene of intra-abdominal origin
 Brain abscess & meningitis: Rare
 Panophthalmitis: Rare
 Thoracic infections
 Urogenital infections- usually follow UT surgery
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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 positive, non-motile, capsulated bacilli.
• Spores are rarely observed in C.perfringens
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2.Culture:
On RCM(Robertson’s cooked meat broth)→
meat turned pink but not digested
On blood agar → target hemolysis
Stormy fermentation of lactose in litmus milk;
the acid coagulates casein-acid clot.
25

3. Biochemical reactions
Glucose Indole Negative
Lactose Fermented MR Positive
Maltose VP Negative
H2S prodn & Nitrate redn Positive
26

4.Nagler’s Reaction
 Rapid detection of C.perfringens from clinical
sample
 Done to detect the lecithinase activity of alpha
toxin
 Characteristics opalescence is produced around
colonies in positive test due to breakdown of
lipoprotein complex in the medium
27

5.Reverse CAMP Test
 Used for differentiation of C.perfringens from other
clostridium species.
 CAMP positive Group B Streptococcus is streaked in
SBA & C.perfringens is streaked perpendicular to it
“arrowhead”(enhanced) hemolysis is seen between
growth of C.perfringens & Group B streptococcus
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Prophylaxis and 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 three doses
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Other Clostridia associated with GG
1.C.septicum also called as Vibrion septique
Produces 4 distinct toxins;
 α-toxin: hemolytic & dermonecrotic
 β-toxin: leucotoxic
 γ-toxin: hyaluronidase
 δ-toxin: hemolysin
It also produces fibrinolysin
2.C.novyi: 4 types recognized(A-D),Type A causes GG
3.C.histolyticum: Produces 5 distinct toxins Infrequently
associated with GG
30

clostridium.perfringenes

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