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S3 Lec 5: Clostridium by Dr. Maria Cielo B. Malijan, MD, DPPS, FPSDBP
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November 5, 2010
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is Clostridium  Unique to C. perfringens
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M CHARACTERISTICS  A streak of C. perfringens produces a lollipop
vs shape on this medium.
• Gram (-), spore-forming bacilli
GAS GANGRENE
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ai • Anaerobes
M • Spores (resistant to heat, disinfection and to air; • Clostridial myonecrosis , Gas Gangrene
n they do not germinate) o Rapidly spreading edema, myositis,
Re • Saprophytic tissue necrosis, gas production and
• Some are commensals in humans and animals toxemia as a complication of wound
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• Opportunistic pathogens infection
• Causes diseases by toxins produced
Ri

• •
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Employ butyric fermentation pathways to Infection maybe:
1. Endogenous clostridia (bowel)
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generate energy  causing foul odors
2. exogenous clostridia (soil)
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vs • Includes the ff. species
o Clostridium perfringens
•
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If tissue has been traumatized by foreign body
e  Gas gangrene and is devitalized (anaerobic environment)
en  Food poisoning clostridial spores in the injured muscle germinate
Arl o Clostridium tetani • Toxin production occurs as bacteria multiply
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 Tetanus • Characterized by accumulation of gas and fluid
Ni o Clostridium botulinum and the extensive destruction of muscle and
 Botulism
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connective tissue.
o Clostridium difficile
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• PMNs are absent from the site
Pseudomembranous enterocolitis
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 • Organisms spread to new areas behind
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destructive action of toxins
Clostridium perfringens
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ac • Untreated mortality 15%

• Most common sp of clostridia isolated from

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C. perfringens GAS GANGRENE
clinical specimens
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• Widely distributed in soil and sewage

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• Trauma, surgery, etc.,  poor blood supply
•
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Ri Commensal of lower GIT of animals and humans anaerobic tissue environment  gas gangrene
ay • Large gm (+) “box car” shaped rod
ck • Capsulate, nonmotile PATHOGENESIS
Ni • Grow quickly on selective media
ad • can be identified by Nagler reaction which • Trauma (1-6 days) Fever and pain  Release of
Gl exploits the action of its phospholipase on egg bacterial exotoxins(α toxin -phospholipase C or
Je yoke medium lecithinase, Hemolysins,,Collagenases, Proteases,
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• colonies are surrounded by zones of turbidity and Lipases)  Muscle necrosis  spreading infection
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the effect is specifically inhibited if C.perfringens  gas gangrene
antiserum containing alpha antitoxin is present
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on the medium
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Nagler reaction
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releasing enterotoxins  Cramps in 8 to 12 hours

A = macroscopic  Diarrhea
B = microscopic
DIAGNOSIS
• Gas gangrene is a severe form of gangrene • isolation from feces for typing
(tissue death) caused by the bacterium
Clostridium perfringens. PREVENTION
• It generally occurs at a wound or surgical site,
causing painful swelling and destruction of • thoroughly reheating foods to a T > 75ºC so
involved tissue. Gas gangrene is rapidly that vegetative cells and toxin are destroyed
progressive and often fatal.
Clostridium tetani

• Spherical terminal spores, “drumstick “ or tennis

racket” appearance
• Gm(+) but gm(-) forms encountered
• No capsule
• Obligate anaerobe
• Motile (peritrichous flagella)
• Grows well in cooked meat broth; produces a thin
spreading film on enriched blood agar

Spores & vegetative cells

• Occurrence of tetanus bacilli:
o Intestine of humans and animals
o Derived from animal feces and indirectly
via soil
• Specially prevalent in manured soil
• Spores R to adverse conditions
LABORATORY DIAGNOSIS • Some resist boiling in water for up to 3 hrs
• May resist dry heat at 160º for 1 hr and 5%
Specimen : phenol for 2 wks or more
• sloughs of necrotic tissue • Iodine (1%) in water kill spores within a few hrs
• exudates : deeper areas • Toxin : 130 ng –lethal dose for humans
• Produces 2 toxins
Clostridium perfringens o oxygen labile hemolysis (tetanolysin)
• hemolytic on sheep blood agar spores and o neurotoxin (tetanospasmin) - pathogenic
vegetative cells ( not toxic by oral route)
• produces double hemolytic rings • With natural occurrence, bacilli stay at the site of
infection but toxin diffuses to the spinal cord then
TREATMENT the entire system
• Toxin absorbed and transmitted to the CNS via
• Prompt surgical wound care motor nerves
o Debridement (fascial compartments are
incised) PATHOGENESIS
o Removal of FB
Trauma or punctured wound  tissue contamination 
• High dose Antibiotics
exotoxins  exotoxins  gangliosides block release of
o PCN, Metronidazole + Aminoglycoside
or inhibitory neurotransmitters ( glysine and GABA) 
o Clindamycin ; Cefotaxime or Imipenem prevents contraction of antagonistic muscles  muscle
spasms
• NO innate immunity
• α antitoxin and use of hyperbaric oxygen MODE OF ACTION
therapy controversial

C. perfringens FOOD POISONING

• Meat cooked in bulk  Heat penetration and

cooking is slow 
During the cooling period, the spores germinate
in anaerobic environment and multiply Ingested
(protected from the gastric juice in the protein of
the meat)Passes to the intestine, sporulate,

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• Toxin moves retrograde along nerve fibers, ≈
20 mm/day
• Main target: anterior horn cells of spinal cord,
brain stem
• Blockade of neurotransmitter (glycine and
GABA) release in the CNS
CLINICAL FEATURES
TETANUS :
• Incubation period variable (10-14 days)
• Usual sequence: Local injury  muscle
stiffness  mild intermittent muscular
contraction near the wound  trismus
(lockjaw)  generalized rigidity  violent
and painful spasms and rigidity of voluntary
muscles arching of back
NEONATAL TETANUS
• Follows infection of the umbilical stump in
infants born to non - immune mothers
• usually results from a failure of aseptic
technique during birthing and some cultural
practices
• Risus Sardonicus – tetanus
• Trismus or lock jaw
DIAGNOSIS
• Cultures
• Clinical manifestations
TREATMENT
• Tetanus human immunoglobulin
o to neutralize unbound toxin
• PCN or Metronidazole
o to kill vegetative cells and prevent further
elaboration of toxin
• Local debridement of the wound
• Supportive
PREVENTION
• Immunization with tetanus toxoid
• Booster immunization at 10 year intervals
Clostridium botulinum
• Strict anaerobic gm(+) bacillus
• Motile with peritrichous flagella
• Spores are oval and subterminal
• Occur in soil, vegetables, fruits, leaves, silage,
manure, mud of lakes and sea mud
• Optimum growth in 35°
• Widespread occurrence in nature,
• ability to produce a neurotoxin in food and R of
its spores to inactivation
• Causation : insufficient heating in the process of
food preservation
TOXINS
• Released by vegetative cells ( not spores) by cell
lysis
• Botulinal toxins among the most poisonous
• Phage encoded toxin genes :
o ( 7, A-G)
 Types A* (NG), B, E, F
PATHOGENESIS
• Uncooked food with spores germinate 
ingestion of preformed neurotoxins released in
inactive form  activated by proteolytic cleavage
 toxins block release of excitatory
neurotransmitter ACH at neural synapses 
flaccid paralysis + respiratory failure
• Acetylcholine in nerve terminals is packaged in
vesicles. Normally, vesicle membranes fuse with
those of the nerve terminals, releasing the
transmitter into the synaptic cleft. The process is
mediated by a series of proteins collectively
called the SNARE proteins.
• Botulinum toxin, taken up into vesicles, cleaves
the SNARE proteins, preventing assembly of the
fusion complex and thus blocking the release of
acetylcholine.
Clostridium botulinum can cause: (1) Human Botulism
and (2) Infant Botulism
HUMAN BOTULISM
• Severe, fatal form of food poisoning
• Caused by eating preserved hams, large
sausages, home preserved meats and
vegetables, canned products (fish - sushi), liver
paste, green beans, hazel nut puree, honey)
• Acidic food better / safer : no bacterial growth at
low pH (<4.5)
• Foods responsible may not exhibit spoilage
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 • Preformed toxin (heat labile)absorbed from
intestinal tract, but not inactivated by gastric acid
or proteolytic enzymes
• Toxin primarily affects cholinergic system, blocks
release of ACH
• Incubation Period :1-2 days or longer
• Initially :
o Nausea and vomiting
o Cranial nerves are first affected
o oculomotor muscles affected
o ( diplopia, drooping eyelids with squint)
o descending symmetric paralysis of motor
nerves
o Progressive descending motor loss with
flaccid paralysis,
o no loss of consciousness or sensation
o (+) weakness, sleepiness
o CLINICAL FEATURES
 Thirsty, difficulty in speech and
swallowing
 Problems of breathing and
despair
 Abdominal pain and restlessness
 Death ( respiratory of cardiac
failure)
INFANT BOTULISM
• CLINICAL FEATURES :
o Infants 5-20 wks of age
o Ingestion of honey contaminated with
botulinal spores implicated
o Susceptibility due to less well established
competing intestinal flora
o Constipation and weak sucking ability and
generalized weakness
o can progress to flaccid paralysis and
respiratory arrest
o Called “floppy baby syndrome”
o Mortality rate 1-2 %
DIAGNOSIS
• Organism or toxin is suspected food, vomitus or
feces
• toxin in patient’s blood by toxin-antitoxin
neutralization test in mice
TREATMENT
• Remove unabsorbed toxin from stomach and
intestinal tract
• Neutralize unfixed toxin by giving polyvalent
antitoxin (trivalent toxins A,B,E) IV for foodborne
or wound botulism
• Supportive care
PREVENTION
• Avoid home canning of foodstuffs ( use pressure
cooker), control commercial canning
• Acid fruits must be bottled safely at home and
heated at 100°
• Food containers that bulge may contain gas
produced by C. botulinum and should not be
opened or tasted
• Prophylactic dose of polyvalent antitoxin by IM to
all suspects
• Immunization for laboratory staff
BOTULINUM TOXIN (BOTOX)
Bilateral 6th CN palsy Bilateral 6th
CN palsy
Without the botulinum toxin With the
botulinum toxin
Clostridium difficile
• Motile, slender, gm(+) rods
• Large, oval,subterminal spores
• Common in feces of neonates
• commensal in gut of 3-10% of healthy adults
TRANSMISSION
• diarrhea
• results in contamination of environment with
organisms that form spores
• hospital personnel often responsible for
transmission
PSEUDOMEMBRANOUS COLITIS
• results from elimination of normal intestinal flora
through antibiotic treatment
• Oral antibiotics ( clindamycin, ampicillin,
cephalosporins, tetracyline , Chloramphenicol or
antineoplastic agents) disturb the gut microflora
and make the host susceptible to colonization by
pathogens and to overgrowth by commensal sp.
VIRULENCE FACTORS
• Toxin A ( enterotoxin)
• Toxin B (cytotoxin)
• Adhesin factor
Toxin A (enterotoxin)
• damages the intestinal mucosa
• responsible for leakage of fluids
• chemotactic for neutrophils, resulting in their
release ofcytokines and subsequent inflammation
• causes fluid accumulation by damaging
o mucosal cells (disrupts cytoskeleton)
• they cannot control water movement
Toxin B (cytotoxin)
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 • depolymerizes actin, destroying the cellular
cytoskeleton
• treated cells form membrane protrusions or blebs Clostridium difficile: large oval subterminal spores
Adhesin factor

• allow binding to human colonic cells

• Hyaluronidase enzyme

PATHOGENESIS

• Characterized by a leukocytic infiltrate into

the lamina propria and by elaboration of a
mixture of fibrin, mucus, necrotic epithelial
cells and leukocytes.

DIAGNOSIS

• Isolation from feces by enrichment and

selective media
• Toxin detection by testing extracts against
monolayers of human embryo fibroblast or
ELISA

TREATMENT
• discontinue antibiotics – to restore the normal
flora
• Oral Vancomycin or Metronidazole
• Supportive (fluid and electrolyte losses)

Comparison of Clostridia sp.

Clostridium perfringens: large rectangular spores; non

motile; associated with wound infections

Clostridium tetani: terminal spores ( squash racket)

Clostridium botulinum: oval subterminal spores

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