1 Anaerobic Spore Forming Anaerobic Spore Forming Bacilli Bacilli General observations General observations Definition Definition An organism that: An organism that: Requires reduced oxygen Requires reduced oxygen tension for growth tension for growth Fails to grow on the surface Fails to grow on the surface of solid media in 10% carbon of solid media in 10% carbon dioxide in ambient air (18% dioxide in ambient air (18% oxygen) oxygen)
Does not use oxygen for growth Does not use oxygen for growth and metabolism, obtain their and metabolism, obtain their energy from fermentation reactionsenergy from fermentation reactions
Grow at low or negative EhGrow at low or negative Eh Aerobes and facultative anaerobes Aerobes and facultative anaerobes
have the following metabolic have the following metabolic systems which are frequently systems which are frequently absent in anaerobes:absent in anaerobes:
Use of reducing substancesUse of reducing substances Thyoglycolate –Brewer’s brothThyoglycolate –Brewer’s broth Metallic iron filingsMetallic iron filings Cooked meat (Robertson’s cooked Cooked meat (Robertson’s cooked
Exclusion of OExclusion of O22 from part of medium from part of medium Solid medium – shake culturesSolid medium – shake cultures Deep fluid medium with 0.5% agarDeep fluid medium with 0.5% agarUse of anaerobic jarsUse of anaerobic jars Mackintosh jar and fieldes cold catalystMackintosh jar and fieldes cold catalyst Brewer’s and BairdBrewer’s and Baird Gas pak and gas kit systemGas pak and gas kit systemOthersOthers PreReduced Anaerobically Sterilized PreReduced Anaerobically Sterilized
LaboratoryLaboratory Unique morphology on Gram stainUnique morphology on Gram stain Failure of organisms seen on Gram stain Failure of organisms seen on Gram stain
of original sample to grow aerobicallyof original sample to grow aerobically Growth in anaerobic zone of fluid media Growth in anaerobic zone of fluid media
or agar deepsor agar deeps Anaerobic growth on media containing Anaerobic growth on media containing 75 – 100 ul/ml of Kanamycin, neomycin or 75 – 100 ul/ml of Kanamycin, neomycin or
paromycin paromycin
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ClostridiaClostridia
Gram-positive, spore forming, Gram-positive, spore forming, motile or non motile bacillimotile or non motile bacilli
Some species are potentially highly Some species are potentially highly pathogenic to humans or animals pathogenic to humans or animals and produce potent exotoxinsand produce potent exotoxins
Found in soil (especially soil Found in soil (especially soil fertilized with animal excreta) and fertilized with animal excreta) and in the lower intestinal tract of in the lower intestinal tract of humans and animalshumans and animals
botulinumbotulinum Central Central C. perfringens, C. C. perfringens, C.
botulinumbotulinum
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ClostridiaClostridia
Some decomposeSome decompose
– – protein protein proteolytic proteolytic
– – CHO CHO Sacharolytic Sacharolytic
– – neitherneither Some are saprophyticSome are saprophytic
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ClostridiaClostridia
Medically important speciesMedically important species Gas gangrene group:Gas gangrene group:
– C. perfringensC. perfringens ( (C. welchiiC. welchii))– C. septicumC. septicum– C. oedematiensC. oedematiens– C. histolyticumC. histolyticum
C. tetaniC. tetani C. botulinumC. botulinum C. difficileC. difficile
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Clostridia perfringensClostridia perfringens
Morphology and growth Morphology and growth characteristicscharacteristics
Strongly Gram +ve rod, non Strongly Gram +ve rod, non motilemotile
Short, thick, strainght parallel Short, thick, strainght parallel sides and rounded edgessides and rounded edges
Spores rarely seenSpores rarely seen
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Clostridia perfringensClostridia perfringens
Sporulation favoured by alkaline Sporulation favoured by alkaline environment and absence of environment and absence of fermentable CHOfermentable CHO
If spores are produced, they are If spores are produced, they are large, oval and centrallarge, oval and central
Capsulated especially at the site of Capsulated especially at the site of infectioninfection
Capsule disappears when organism Capsule disappears when organism is grown on cultureis grown on culture
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Clostridia perfringensClostridia perfringens
Colonies are 2 – 4 mm in Colonies are 2 – 4 mm in diameter, semitranslucentdiameter, semitranslucent
On BAOn BA Zone of hemolysis caused by Zone of hemolysis caused by θθ
toxintoxin Wider zone of incomplete Wider zone of incomplete
hemaolysis caused by hemaolysis caused by toxin toxin (lecithinase C, phospholipase)(lecithinase C, phospholipase)
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Clostridia perfringensClostridia perfringens
Partial hemolysis becomes Partial hemolysis becomes complete if the plate is refrigerated complete if the plate is refrigerated and rewarmedand rewarmed
On egg yolk agar it produces diffuse On egg yolk agar it produces diffuse opalescence due to phospholipaseopalescence due to phospholipase
In cooked meat it produces gas but In cooked meat it produces gas but being predominantly saccharolytic being predominantly saccharolytic it does not digest meatit does not digest meat
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Clostridia perfringensClostridia perfringens Ferments lactose with acid and Ferments lactose with acid and
gas production, thus in litmus gas production, thus in litmus milk the organism breaks down milk the organism breaks down lactose producing acid which lactose producing acid which makes the milk clotmakes the milk clot
The clot is ruptured and broke The clot is ruptured and broke down into pieces (stormy clot)down into pieces (stormy clot)
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Clostridia perfringensClostridia perfringens
Antigenic structureAntigenic structure
SerotypesSerotypes Types A – E according to Types A – E according to
combination of exotoxins combination of exotoxins producedproduced
The exotoxins are antigenicThe exotoxins are antigenic ToxinsToxins
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Clostridia perfringensClostridia perfringens
-toxin (phospholipase, lecithinase C)-toxin (phospholipase, lecithinase C) hydrolyses lecithin and sphengomyelin hydrolyses lecithin and sphengomyelin
thus disrupts the cell membranes of thus disrupts the cell membranes of various host cells,including erythrocytes, various host cells,including erythrocytes, leucocytes and muscle cellsleucocytes and muscle cells
θθ-toxin oxygen labile hemolysin; alters -toxin oxygen labile hemolysin; alters capillary permeability and toxic for heart capillary permeability and toxic for heart musclemuscle
Responsible for Responsible for -hemolysis on blood -hemolysis on blood agaragar
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Clostridia perfringensClostridia perfringens
Enterotoxin – produced by some Enterotoxin – produced by some strains of strains of C. perfringens C. perfringens type A; type A; responsible for food poisoning, responsible for food poisoning, released into the upper GIT when released into the upper GIT when ingested vegetative forms undergo ingested vegetative forms undergo sporulation, causes diarrhoea by sporulation, causes diarrhoea by reversing the flow of water and reversing the flow of water and electrolytes in the small intestine, electrolytes in the small intestine, precise mechanism not yet definedprecise mechanism not yet defined
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Clostridia perfringensClostridia perfringens
-toxin, an enterotoxin produced by -toxin, an enterotoxin produced by some strains of C. perfringens type C, some strains of C. perfringens type C, induces intestinal paralysis, inflammation induces intestinal paralysis, inflammation and gangrenous necrosis – necrotizing and gangrenous necrosis – necrotizing enteritis (enteritis necroticans)enteritis (enteritis necroticans)
Morphology and Growth characteristicsMorphology and Growth characteristics Widely distributed in nature: soil, GIT Widely distributed in nature: soil, GIT
of man and other animalsof man and other animals Gram +ve rods, delicate, may appear Gram +ve rods, delicate, may appear
filamentous after 48 hrs incubation at filamentous after 48 hrs incubation at 37 37 CC
Clostridia tetaniClostridia tetani Strictly anaerobicStrictly anaerobic Swarming growth on BA; swarming Swarming growth on BA; swarming
prevented by firm agar (2-3%) or prevented by firm agar (2-3%) or tetanus antitoxintetanus antitoxin
Tiny colonies with rhizoids (medusa Tiny colonies with rhizoids (medusa head appearance)head appearance)
On horse BA they are On horse BA they are -hemolytic -hemolytic which tends to change into which tends to change into -hemolysis -hemolysis after prolonged incubationafter prolonged incubation
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Clostridia tetaniClostridia tetani
Hemolysis is due to tetanolysinHemolysis is due to tetanolysin No effect on egg yolkNo effect on egg yolk Grows in cooked meat but no Grows in cooked meat but no
effect on the meateffect on the meat Can be identified by gas Can be identified by gas
chromatographychromatography Isolation difficult in the lab Isolation difficult in the lab
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Clostridia tetaniClostridia tetani
Antigenic structureAntigenic structure 10 serotypes on the basis of specific 10 serotypes on the basis of specific
flagellar antigensflagellar antigens All share a common O antigenAll share a common O antigen ToxinsToxins Tetanospasmin (exotoxin), polypeptide, Tetanospasmin (exotoxin), polypeptide,
MW 160,000, released in lysis; MW 160,000, released in lysis; production under control of a plasmid production under control of a plasmid genegene
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Clostridia tetaniClostridia tetani
Neurotoxin,inhibits release of Neurotoxin,inhibits release of acetylcholine, thus interfering with acetylcholine, thus interfering with neuromuscular transmissionneuromuscular transmission
Inhibits post synaptic spinal neurons by Inhibits post synaptic spinal neurons by blocking the release of an inhibitory blocking the release of an inhibitory mediator = generalized muscular spasms, mediator = generalized muscular spasms, hypereflexia and seizureshypereflexia and seizures
0.1 ug lethal to humans, treatment with 0.1 ug lethal to humans, treatment with formaldehyde formaldehyde toxoid toxoid
Tetanolysis = hemolysinTetanolysis = hemolysin
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Clostridium botulinumClostridium botulinum
Morphology and growth Morphology and growth characteristicscharacteristics
Widely distributed in nature, Widely distributed in nature, present in soil, vegetables and gut present in soil, vegetables and gut of manof man
Gram +ve, straight rounded edges Gram +ve, straight rounded edges with oval or rounded centrally with oval or rounded centrally placed or subterminal sporesplaced or subterminal spores
Strict anaerobeStrict anaerobe
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Clostridium botulinumClostridium botulinum
Antigenic structureAntigenic structure 8 types (A – H) based on serological 8 types (A – H) based on serological
tests of the neurotoxintests of the neurotoxin A, B, E and F = cause botulism in A, B, E and F = cause botulism in
manman C and D = cause botulism in animals C and D = cause botulism in animals
and birdsand birds G = pathogenicity unknownG = pathogenicity unknown
types A, B and E toxinstypes A, B and E toxins MW 150,000, highly toxic, lethal dose MW 150,000, highly toxic, lethal dose
for humans < 1 for humans < 1 µµgg Heat labile, destroyed rapidly at 100 Heat labile, destroyed rapidly at 100
C, resistant to GIT enzymesC, resistant to GIT enzymes Production under control of a Production under control of a
temperate phagetemperate phage
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Clostridium botulinumClostridium botulinum
Acts on neuromuscular junctions by Acts on neuromuscular junctions by inhibiting release of acetylcholine, inhibiting release of acetylcholine, resulting in muscular paralysisresulting in muscular paralysis
Voluntary and autonomic cholinergic Voluntary and autonomic cholinergic nervous systems affcted nervous systems affcted
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Clostridium difficileClostridium difficile
Morphology and growth Morphology and growth characteristicscharacteristics
First isolated from stools of healthy First isolated from stools of healthy newborns over three decades agonewborns over three decades ago
Role as an enteric pathogen recently Role as an enteric pathogen recently documenteddocumented
Widespread in environment, occurs in Widespread in environment, occurs in GIT flora of 2-4% of healthy adults in GIT flora of 2-4% of healthy adults in the communitythe community
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Clostridium difficileClostridium difficile Many hospitals heavily contaminated by Many hospitals heavily contaminated by
spores of this organismspores of this organism Slow growing in culture, hence its nameSlow growing in culture, hence its nameAntigenic structureAntigenic structure ToxinsToxins Medically important strains produce two Medically important strains produce two
distinct large polypeptide toxins, A and Bdistinct large polypeptide toxins, A and B Toxins encoded in chromosomal genes Toxins encoded in chromosomal genes
and released during stationary or post and released during stationary or post stationary growth phases of the vegetative stationary growth phases of the vegetative organisms (or during cell lysis) organisms (or during cell lysis)
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Clostridium difficileClostridium difficile Toxin A – primarily an enterotoxin; has Toxin A – primarily an enterotoxin; has
cholera toxin-like activity and also causes cholera toxin-like activity and also causes extensive mucosal damage leading to extensive mucosal damage leading to formation of hemorrhagic fluid rich in formation of hemorrhagic fluid rich in albuminalbumin
Toxin B – highly potent cytotoxin, decreases Toxin B – highly potent cytotoxin, decreases cellular protein synthesis and disrupts the cellular protein synthesis and disrupts the microfilament system of cells (like diphtheria microfilament system of cells (like diphtheria toxin)toxin)
Animal studies suggest that toxin A induced Animal studies suggest that toxin A induced mucosal damage allows absorption of toxin B mucosal damage allows absorption of toxin B from the human gutfrom the human gut
