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Clostridium:
Anaerobic
Endospore formers
Filename: Clostridium.ppt
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Clostridial Diseases
Botulism C. botulinum
 Tetanus C. tetani
 Gas gangrene C. perfringens
 Food poisoning Clostridium spp
 Pseudomembranous colitis C.
difficile

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Clostridium
Gram positive
 Rods
 Endospore formers



asporogenous
Obligate Anaerobes!!!
 Aerotolerant
spp C. perfringens, C. tetani,
C. botulinum, C. difficile

Soil organisms
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Botulism
Intoxication
 Foods

 Meats
 Fish
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
low-medium acid canned foods
Wild birds (limberneck)
 ducks,
fish, Inuit -- whale blubber, seal fins
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Botulism: symptoms

Adults
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Nerve paralysis
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Infants
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shorter nerves first
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Blurred vision
Cardiac failure
Respiratory failure
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Failure to Thrive
Dehydration
Polyneuropathy
Infection Cl. Botulinum

umbilical cord
Intoxication
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Botulism: Symptoms
1st symptoms: weakness and dizziness
soon after: blurred vision (double vision),
difficulty swallowing, throat pain,
constipation, abdominal pain
Flaccid paralysis: Bilateral, descending,
weakness of the peripheral muscles.
Death: respiratory paralysis
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Botulism Intoxication
Entry Ingestion
Spread
absorbed through intestine
spread via blood stream
moves up nerves
Disease
Incubation: 1-2 days
Flaccid paralysis, cardiac failure,
respiratory failure
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Botulism: Treatment
Disease is progressive may not respond to
treatment.
-ventilatory support
-gastric lavage
-penicillin
-antitoxin (polyvalent A,B,E)
Heat food to 80 C to kill toxin and kill spores.
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Infant Botulism
Infant botulism:Colonizes the GI tract of
young infants. Appears as non-specific
weakness.
 Flaccid paralysis: respiratory arrest.
 Mortality = 1-2%
 Some cases of sudden infant death
syndrome have proven to be botulism.
Eating honey.

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Wound botulism:
rare -- organisms multiply in the wound.
 Can occur through umbilical cord
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Botulism: Lab Diagnosis
Culture:
culture organisms from feces, food.
Heat to 80 C. Food, stool and patient’s serum.
Toxin Assay:
mix one portion of each specimen with
antitoxin.
Keep one portion antitoxin free.
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Botulism Toxin
Potent neurotoxin regulated by bacteriophage.
Toxins: A -- E, C alpha, C beta, F, G
Humans: A, B, E
150 Kd protein -- cleaved
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Botulism Outbreaks by Type
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Botulism Intoxication in USA
YEAR
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Botulism in USA; neonates
YEAR
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Botulinum Toxin

The heavy chain attaches to the ganglioside
receptors in nerves
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C. botulinum toxin
Synaptic activity at cholinergic synapses is mediated
by acetylcholine.
Acetylcholine is rapidly hydrolysed by acetylcholine
esterase. The result is an electrical stimulus.
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Sequence of Events
A. Nerve stimulus -- calcium is stimulated
B. Acetylcholine release into the synaptic
space -moves into post synaptic membrane and
acts on specific receptors.
C. Botulinum toxin interferes with the release
of acetylcholine from the synaptic vesicles.
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Tetanus
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C. tetani
C.tetani looks like a tennis racket.
Found in soil, carried by horses.
Toxin: heat labile, 150,000 d peptide
Neurotoxin: splits carboxy terminal to
gangliosides on neuronal membranes.
Moves to CNS by retrograde axonal
transport.
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Tetanus Intoxication
Entry wound
Spread
blood stream
moves up nerves
Disease
Incubation: 1-2 days
Rigid Paralysis, cardiac failure,
respiratory failure
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Lockjaw
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Tetanus
trismus, risus sardonicus, opisthotonos,
Cephalic -- poor prognosis
Localized -- favourable prognosis
Prevention: toxoid, 3% formaldehyde
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Risus sardonicus
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Opisthotonos
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Tetanus Neonatorum
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Tetanospasmin
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Toxin similarity
Clostridium botulinum and C. tetani are Zn
requiring Endopeptidases that cleave a set
of proteins..........
Synaptobrevins
found in synaptic vesicles of neurons
Interfere with release of neurotransmitters and
the normal inhibitory function.
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Binding regions of tetanus toxin
and botulinum toxin are different
in terms of cell specificity.
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Tetanus Distribution
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Incidence of Tetanus in USA
YEAR
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C.perfringens: Diseases
 bacteremia
 myonecrosis
 gas
gangrene
 cellulitis
 fascitis
 food poisoning: enteritis necroticans
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C. perfringens
large rectangular, hemolytic, very
distinctive spreading colonies.
 target hemolysis
 found in soil and intestines, man and
animals
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C. perfringens toxins
Alpha toxin is a lecithinase (phospholipase C)
 lyses erythrocytes, platelets, leucocytes,
endothelial cells
 Massive hemolysis and tissue destruction
 Theta Toxin- Beta hemolysis- increases
permeability-- necrotizing enterocolitis
 Delta -- hemolysis
 Kappa -- collagenase
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C. perfringens toxins
Mu -- hyaluronidase
 Nu -- DNAase
Lambda toxin -- protease
Neuraminidase -- hydrolyses serum
glycoproteins
Enterotoxin -- reverses water, sodium and
chloride transport in the intestine (like V.
cholerae) Produced by Group A.
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Nagler Reaction
Presumptive
identification of C.
perfringens
 alpha toxin (lecithinase)
hydrolyses phospholipids
 egg yolk agar becomes
turbid
 specifically blocked by
antitoxin
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Nagler Reaction blocked by
Antibodies
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Clinical Syndromes:
Bacteremia - usually transient, only diagnostic
with other
clinical symptoms
Myonecrosis - gas gangrene
- trauma or surgical contaminant
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Gas Gangrene
Entry PenetratingWound
Multiplication
in dead anaerobic tissue
Toxin production
hemolysin, proteases, lipase,
collagenase
Disease
necrosis, edema, gas
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C. perfringens Gas gangrene
Incubation: <1 week
pain severe
muscle necrosis
shock
renal failure
death
-crepitant
-cellulitis & fascitis (no muscle)
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C perfringens Food poisoning
incubation 8-24hrs.
Abdominal cramps, watery diarrhea
lasts less than 24hrs.
Contaminated meat (left overs)
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C. difficile
Gram + anaerobic rod.
-found in normal flora
-ultimate opportunistic pathogen
-difficult to determine cause as the organism
is ubiquitous
-not difficult to culture
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C. difficile Diagnosis:
cytotoxin - stool
culture
C.difficile antigen -- latex agglutination
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Cytotoxin
slurry of stool centrifuged
filter through 0.45 u filter
0.1ml - supernatant to buffer at pH 7.2
WI-38 tissue cells human diploid lung
fibroblasts
Add supernatant to tissue culture.
Observe for cytotoxicity 24 hrs.
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Mechanism of pathogenicity:
Toxin A
enterotoxin
hypersecretion of fluid
Toxin B
cytotoxin
cytopathic to tissue monolayers
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C. difficileTreatment:
stop antibiotic causing disease
metronidazole, vancomycin
Relapses due to resistant spores.
retreatment with same antibiotic
neutralization with specific antitoxin obtained
commercially
amount of toxin present can be determined by a
dilution series of the stool sample.
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C. difficile
Culture: standard test for Clostridia include:
indole, sugars, lecithinase, catalase (usually
neg.)
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C. difficile: Latex agglutination
stool buffered and centrifuged
drop on slide of stool supernatant
add 1 drop latex detection reagent. Latex
particles coated with rabbit antibody to
C.difficile antigen.
In presence of C.difficile clumps can be seen
by eye.
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Culture:
Inoculate
anaerobe blood agar -- 2-3 days
egg yolk medium -- 2-3 days
Incubation temp. = 30 C except C.perfringens
Inoculate cooked meat medium - (broth with
meat particles)
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C. difficile: Culture
a/ heat to destroy vegetative cells
b/ alcohol spore selection for heat labile spores.
Clinical syndromes: most serious is (PMC)
Pseudomembranous colitis brought about by
destruction of the other indigenous intestinal
flora. Ranges from mild
to serious. PMC self-limiting.
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Differential diagnosis:
S.aureus, E.coli, shigellosis,
acute ulcerative colitis, Campylobacter jejuni
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The End
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Key Terms
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Obligate anaerobe
endospore
asporogenous
botulism
tetanus
gas gangrene
pseudomembranous
colitis
myonecrosis

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

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Botulism toxin
tetanus toxin
trismus
opisthotonus
risus sardonicus
C. perfringens
enterotoxin
aerotolerant
Nagler reaction
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Key Terms
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Enteritis necroticans
tetanospasmin
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Key Organisms
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Clostridium
C tetani
C botulinum
C. perfringens
C. difficile
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Key Concepts
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Epidemiology of Botulism
Disease/bacterial factors
 Transmission
 who is at risk
 geography/ season
 modes of control
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Epidemiology of C. difficile
infections
Disease/bacterial factors
 Transmission
 who is at risk
 geography/ season
 modes of control
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Epidemiology of C. tetani
infections
Disease/bacterial factors
 Transmission
 who is at risk
 geography/ season
 modes of control
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Epidemiology of C. perfringens
infections
Disease/bacterial factors
 Transmission
 who is at risk
 geography/ season
 modes of control

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Short Answers
Construct a table of the virulence factors
associated with C. tetani and the biological
activity of each
 Use a series of no more than four diagrams
to describe the mechanism of
tetanospasmin activity
 Describe the clinical manifestions of
generalized, cephalic and localized tetanus

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Short Answers
Construct a table listing the common
clostridial species and the associated human
diseases.
 Construct a table listing 5 virulence factors
associated with C. difficile and the
biological activity of each
 Construct a table of the virulence factors
associated with C. perfringens and the
biological activity of each
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