1 5/9/2015 clostridium: anaerobic endospore formers filename: clostridium.ppt
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Clostridium:Anaerobic
Endospore formers
Clostridium:Anaerobic
Endospore formersFilename: Clostridium.pptFilename: Clostridium.ppt
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Clostridial DiseasesClostridial Diseases
BotulismBotulism C. botulinumC. botulinum TetanusTetanus C. tetaniC. tetani Gas gangreneGas gangrene C. perfringensC. perfringens Food poisoningFood poisoning ClostridiumClostridium spp spp Pseudomembranous colitisPseudomembranous colitis C. C.
difficiledifficile
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ClostridiumClostridium
Gram positiveGram positive RodsRods Endospore formersEndospore formers
asporogenousasporogenous Obligate Anaerobes!!!Obligate Anaerobes!!!
Aerotolerant spp Aerotolerant spp C. perfringens, C. tetani, C. C. perfringens, C. tetani, C. botulinum, C. difficilebotulinum, C. difficile
Soil organismsSoil organisms
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BotulismBotulism
IntoxicationIntoxication FoodsFoods
MeatsMeats FishFish low-medium acid canned foodslow-medium acid canned foods
Wild birds (limberneck)Wild birds (limberneck) ducks, fish, Inuit -- whale blubber, seal finsducks, fish, Inuit -- whale blubber, seal fins
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Botulism: symptomsBotulism: symptoms
AdultsAdults Nerve paralysisNerve paralysis
shorter nerves firstshorter nerves first
BBlurred visionlurred vision Cardiac failureCardiac failure Respiratory failureRespiratory failure
IntoxicationIntoxication
InfantsInfants Failure to ThriveFailure to Thrive DehydrationDehydration PolyneuropathyPolyneuropathy
InfectionInfection Cl. BotulinumCl. Botulinum umbilical cord umbilical cord
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Botulism: SymptomsBotulism: Symptoms
1st symptoms1st symptoms: weakness and dizziness: weakness and dizziness
soon aftersoon after: blurred vision (double vision), : blurred vision (double vision), difficulty swallowing, throat pain, difficulty swallowing, throat pain, constipation, abdominal painconstipation, abdominal pain
Flaccid paralysisFlaccid paralysis: Bilateral, descending, : Bilateral, descending, weakness of the peripheral muscles.weakness of the peripheral muscles.
DeathDeath: respiratory paralysis: respiratory paralysis
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Botulism IntoxicationBotulism Intoxication
EntryEntry Ingestion Ingestion
SpreadSpread absorbed through intestineabsorbed through intestine
spread via blood streamspread via blood stream
moves up nervesmoves up nerves
DiseaseDiseaseIncubation: 1-2 daysIncubation: 1-2 days
Flaccid paralysis, cardiac failure, Flaccid paralysis, cardiac failure, respiratory failurerespiratory failure
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Botulism: TreatmentBotulism: Treatment
Disease is progressive may not respond to Disease is progressive may not respond to treatment.treatment.
-ventilatory support-ventilatory support
-gastric lavage-gastric lavage
-penicillin-penicillin
-antitoxin (polyvalent A,B,E)-antitoxin (polyvalent A,B,E)
Heat food to 80 C to kill toxin and kill spores.Heat food to 80 C to kill toxin and kill spores.
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Infant BotulismInfant Botulism
Infant botulism:Colonizes the GI tract of Infant botulism:Colonizes the GI tract of young infants. Appears as non-specific young infants. Appears as non-specific weakness. weakness.
Flaccid paralysis: respiratory arrest. Flaccid paralysis: respiratory arrest. Mortality = 1-2%Mortality = 1-2% Some cases of sudden infant death syndrome Some cases of sudden infant death syndrome
have proven to be botulism. Eating honey.have proven to be botulism. Eating honey.
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Wound botulism:Wound botulism:
rare -- organisms multiply in the wound.rare -- organisms multiply in the wound. Can occur through umbilical cordCan occur through umbilical cord
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Botulism: Lab Diagnosis Botulism: Lab Diagnosis
Culture:Culture:
culture organisms from feces, food. culture organisms from feces, food.
Heat to 80 C. Food, stool and patient’s serum.Heat to 80 C. Food, stool and patient’s serum.
Toxin Assay:Toxin Assay:
mix one portion of each specimen with mix one portion of each specimen with antitoxin. antitoxin.
Keep one portion antitoxin free.Keep one portion antitoxin free.
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Botulism ToxinBotulism Toxin
Potent neurotoxin regulated by bacteriophage.Potent neurotoxin regulated by bacteriophage.
Toxins: A -- E, C alpha, C beta, F, GToxins: A -- E, C alpha, C beta, F, G
Humans: A, B, EHumans: A, B, E
150 Kd protein -- cleaved 150 Kd protein -- cleaved
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Botulism Outbreaks by TypeBotulism Outbreaks by Type
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Botulism Intoxication in USABotulism Intoxication in USA
YEAR
INCIDENCE
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Botulism in USA; neonatesBotulism in USA; neonates
YEAR
INCIDENCE
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Botulinum ToxinBotulinum Toxin
The heavy chain attaches to the ganglioside The heavy chain attaches to the ganglioside receptors in nervesreceptors in nerves
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C. botulinum toxin C. botulinum toxin
Synaptic activity at cholinergic synapses is mediated Synaptic activity at cholinergic synapses is mediated by acetylcholine.by acetylcholine.
Acetylcholine is rapidly hydrolysed by acetylcholine Acetylcholine is rapidly hydrolysed by acetylcholine esterase. The result is an electrical stimulus.esterase. The result is an electrical stimulus.
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Sequence of EventsSequence of Events
A. Nerve stimulus -- calcium is stimulatedA. Nerve stimulus -- calcium is stimulated
B. Acetylcholine release into the synaptic B. Acetylcholine release into the synaptic space --space --
moves into post synaptic membrane and moves into post synaptic membrane and acts on specific receptors.acts on specific receptors.
C. Botulinum toxin interferes with the release C. Botulinum toxin interferes with the release of acetylcholine from the synaptic vesicles.of acetylcholine from the synaptic vesicles.
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TetanusTetanus
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C. tetaniC. tetani
C.tetani C.tetani looks like a tennis racket. looks like a tennis racket.
Found in soil, carried by horses.Found in soil, carried by horses.
Toxin: heat labile, 150,000 d peptideToxin: heat labile, 150,000 d peptide
Neurotoxin: splits carboxy terminal to Neurotoxin: splits carboxy terminal to gangliosides on neuronal membranes. gangliosides on neuronal membranes. Moves to CNS by retrograde axonal Moves to CNS by retrograde axonal transport.transport.
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Tetanus IntoxicationTetanus Intoxication
EntryEntry wound wound
SpreadSpread blood streamblood stream
moves up nervesmoves up nerves
DiseaseDiseaseIncubation: 1-2 daysIncubation: 1-2 days
Rigid Paralysis, cardiac failure, Rigid Paralysis, cardiac failure, respiratory failurerespiratory failure
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LockjawLockjaw
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TetanusTetanus
trismus, risus sardonicus, opisthotonos,trismus, risus sardonicus, opisthotonos,
Cephalic -- poor prognosisCephalic -- poor prognosis
Localized -- favourable prognosisLocalized -- favourable prognosis
Prevention: toxoid, 3% formaldehydePrevention: toxoid, 3% formaldehyde
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Risus sardonicusRisus sardonicus
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OpisthotonosOpisthotonos
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Tetanus NeonatorumTetanus Neonatorum
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TetanospasminTetanospasmin
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Toxin similarityToxin similarity
Clostridium botulinumClostridium botulinum and and C. tetani C. tetani are are Zn requiring Endopeptidases that cleave a Zn requiring Endopeptidases that cleave a set of proteins..........set of proteins..........
SynaptobrevinsSynaptobrevins
found in synaptic vesicles of neuronsfound in synaptic vesicles of neurons
Interfere with release of neurotransmitters and Interfere with release of neurotransmitters and the normal inhibitory function.the normal inhibitory function.
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Binding regions of tetanus toxin and botulinum toxin are different in terms of cell specificity.
Binding regions of tetanus toxin and botulinum toxin are different in terms of cell specificity.
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Tetanus DistributionTetanus Distribution
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Incidence of Tetanus in USAIncidence of Tetanus in USA
YEAR
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C.perfringens: DiseasesC.perfringens: Diseases
bacteremiabacteremia myonecrosismyonecrosis gas gangrenegas gangrene cellulitiscellulitis fascitisfascitis food poisoning: food poisoning: enteritis necroticansenteritis necroticans
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C. perfringensC. perfringens
large rectangular, hemolytic, very large rectangular, hemolytic, very distinctive spreading colonies.distinctive spreading colonies.
target hemolysistarget hemolysis found in soil and intestines, man and found in soil and intestines, man and
animalsanimals
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C. perfringens toxinsC. perfringens toxins
Alpha toxin is a lecithinase (phospholipase C)Alpha toxin is a lecithinase (phospholipase C) lyses erythrocytes, platelets, leucocytes, lyses erythrocytes, platelets, leucocytes,
endothelial cellsendothelial cellsMassive hemolysis and tissue destructionMassive hemolysis and tissue destruction
Theta Toxin- Beta hemolysis- increases Theta Toxin- Beta hemolysis- increases permeability-- necrotizing enterocolitispermeability-- necrotizing enterocolitis
Delta -- hemolysisDelta -- hemolysis Kappa -- collagenase Kappa -- collagenase
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C. perfringens toxinsC. perfringens toxins
Mu -- hyaluronidaseMu -- hyaluronidase Nu -- DNAaseNu -- DNAase
Lambda toxin -- proteaseLambda toxin -- protease
Neuraminidase -- hydrolyses serum Neuraminidase -- hydrolyses serum glycoproteinsglycoproteins
Enterotoxin -- reverses water, sodium and Enterotoxin -- reverses water, sodium and chloride transport in the intestine (like V. chloride transport in the intestine (like V. cholerae) Produced by Group A.cholerae) Produced by Group A.
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Nagler ReactionNagler Reaction
Presumptive identification Presumptive identification of of C. perfringensC. perfringens
alpha toxin (lecithinase) alpha toxin (lecithinase) hydrolyses phospholipidshydrolyses phospholipids
egg yolk agar becomes egg yolk agar becomes turbidturbid
specifically blocked by specifically blocked by antitoxinantitoxin
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Nagler Reaction blocked by Antibodies
Nagler Reaction blocked by Antibodies
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Clinical Syndromes:Clinical Syndromes:
Bacteremia - usually transient, only diagnostic Bacteremia - usually transient, only diagnostic with otherwith other
clinical symptomsclinical symptoms
Myonecrosis - gas gangreneMyonecrosis - gas gangrene
- trauma or surgical - trauma or surgical contaminantcontaminant
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Gas GangreneGas Gangrene
EntryEntry PenetratingWound PenetratingWound
Multiplication Multiplication in dead anaerobic tissuein dead anaerobic tissue
Toxin productionToxin productionhemolysin, proteases, lipase, collagenasehemolysin, proteases, lipase, collagenase
DiseaseDiseasenecrosis, edema, gasnecrosis, edema, gas
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C. perfringens Gas gangreneC. perfringens Gas gangrene
Incubation: <1 week Incubation: <1 week pain severepain severe
muscle necrosismuscle necrosis
shock shock
renal failurerenal failure
deathdeath
--crepitantcrepitant
-cellulitis & fascitis (no muscle)-cellulitis & fascitis (no muscle)
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C perfringens Food poisoningC perfringens Food poisoning
incubation 8-24hrs.incubation 8-24hrs.
Abdominal cramps, watery diarrhea Abdominal cramps, watery diarrhea
lasts less than 24hrs.lasts less than 24hrs.
Contaminated meat (left overs)Contaminated meat (left overs)
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C. difficileC. difficile
Gram + anaerobic rod.Gram + anaerobic rod.
-found in normal flora-found in normal flora
-ultimate opportunistic pathogen-ultimate opportunistic pathogen
-difficult to determine cause as the organism -difficult to determine cause as the organism is ubiquitousis ubiquitous
-not difficult to culture-not difficult to culture
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C. difficile Diagnosis:C. difficile Diagnosis:
cytotoxin - stoolcytotoxin - stool
cultureculture
C.difficile C.difficile antigen -- latex agglutinationantigen -- latex agglutination
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CytotoxinCytotoxin
slurry of stool centrifugedslurry of stool centrifuged
filter through 0.45 u filterfilter through 0.45 u filter
0.1ml - supernatant to buffer at pH 7.20.1ml - supernatant to buffer at pH 7.2
WI-38 tissue cells human diploid lung WI-38 tissue cells human diploid lung fibroblastsfibroblasts
Add supernatant to tissue culture.Add supernatant to tissue culture.
Observe for cytotoxicity 24 hrs.Observe for cytotoxicity 24 hrs.
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Mechanism of pathogenicity:Mechanism of pathogenicity:
Toxin AToxin A enterotoxin enterotoxin
hypersecretion of fluidhypersecretion of fluid
Toxin BToxin B cytotoxincytotoxin
cytopathic to tissue monolayerscytopathic to tissue monolayers
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C. difficileTreatment:C. difficileTreatment:
stop antibiotic causing diseasestop antibiotic causing diseasemetronidazole, vancomycinmetronidazole, vancomycin
Relapses due to resistant spores.Relapses due to resistant spores.retreatment with same antibioticretreatment with same antibiotic
neutralization with specific antitoxin obtained neutralization with specific antitoxin obtained commerciallycommerciallyamount of toxin present can be determined by a amount of toxin present can be determined by a
dilution series of the stool sample.dilution series of the stool sample.
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C. difficileC. difficile
Culture: standard test for Clostridia include: Culture: standard test for Clostridia include: indole, sugars, lecithinase, catalase (usually indole, sugars, lecithinase, catalase (usually neg.)neg.)
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C. difficile: Latex agglutinationC. difficile: Latex agglutination
stool buffered and centrifugedstool buffered and centrifuged
drop on slide of stool supernatantdrop on slide of stool supernatant
add 1 drop latex detection reagent. Latex add 1 drop latex detection reagent. Latex particles coated with rabbit antibody to particles coated with rabbit antibody to C.difficile C.difficile antigen.antigen.
In presence of In presence of C.difficile C.difficile clumps can be seen clumps can be seen by eye.by eye.
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Culture: Culture:
InoculateInoculateanaerobe blood agar -- 2-3 days anaerobe blood agar -- 2-3 days
egg yolk medium -- 2-3 days egg yolk medium -- 2-3 days
Incubation temp. = 30 C except Incubation temp. = 30 C except C.perfringensC.perfringens
Inoculate cooked meat medium - (broth with Inoculate cooked meat medium - (broth with meat particles) meat particles)
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C. difficile: CultureC. difficile: Culture
a/ heat to destroy vegetative cellsa/ heat to destroy vegetative cells
b/ alcohol spore selection for heat labile spores.b/ alcohol spore selection for heat labile spores.
Clinical syndromes: most serious is (PMC)Clinical syndromes: most serious is (PMC)
Pseudomembranous colitis brought about by Pseudomembranous colitis brought about by destruction of the other indigenous intestinal destruction of the other indigenous intestinal flora. Ranges from mildflora. Ranges from mild
to serious. PMC self-limiting.to serious. PMC self-limiting.
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Differential diagnosis: Differential diagnosis:
S.aureus, E.coli, S.aureus, E.coli, shigellosis,shigellosis,
acute ulcerative colitis, acute ulcerative colitis, Campylobacter jejuniCampylobacter jejuni
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The EndThe End
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Key TermsKey Terms
Obligate anaerobeObligate anaerobe endosporeendospore asporogenousasporogenous botulismbotulism tetanustetanus gas gangrenegas gangrene pseudomembranous pseudomembranous
colitiscolitis myonecrosismyonecrosis
Botulism toxinBotulism toxin tetanus toxintetanus toxin trismustrismus opisthotonusopisthotonus risus sardonicusrisus sardonicus C. perfringens C. perfringens
enterotoxinenterotoxin aerotolerantaerotolerant Nagler reactionNagler reaction
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Key TermsKey Terms
Enteritis necroticansEnteritis necroticans tetanospasmintetanospasmin
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Key OrganismsKey Organisms
ClostridiumClostridium C tetaniC tetani C botulinumC botulinum C. perfringensC. perfringens C. difficileC. difficile
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Key ConceptsKey Concepts
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Epidemiology of BotulismEpidemiology of Botulism
Disease/bacterial factorsDisease/bacterial factors TransmissionTransmission who is at riskwho is at risk geography/ seasongeography/ season modes of controlmodes of control
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Epidemiology of C. difficile infections
Epidemiology of C. difficile infections
Disease/bacterial factorsDisease/bacterial factors TransmissionTransmission who is at riskwho is at risk geography/ seasongeography/ season modes of controlmodes of control
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Epidemiology of C. tetani infections
Epidemiology of C. tetani infections
Disease/bacterial factorsDisease/bacterial factors TransmissionTransmission who is at riskwho is at risk geography/ seasongeography/ season modes of controlmodes of control
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Epidemiology of C. perfringens infections
Epidemiology of C. perfringens infections
Disease/bacterial factorsDisease/bacterial factors TransmissionTransmission who is at riskwho is at risk geography/ seasongeography/ season modes of controlmodes of control
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Short AnswersShort Answers
Construct a table of the virulence factors Construct a table of the virulence factors associated with associated with C. tetaniC. tetani and the biological and the biological activity of eachactivity of each
Use a series of no more than four diagrams Use a series of no more than four diagrams to describe the mechanism of to describe the mechanism of tetanospasmin activitytetanospasmin activity
Describe the clinical manifestions of Describe the clinical manifestions of generalized, cephalic and localized tetanusgeneralized, cephalic and localized tetanus
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Short AnswersShort Answers
Construct a table listing the common clostridial Construct a table listing the common clostridial species and the associated human diseases.species and the associated human diseases.
Construct a table listing 5 virulence factors Construct a table listing 5 virulence factors associated with associated with C. difficileC. difficile and the biological and the biological activity of eachactivity of each
Construct a table of the virulence factors Construct a table of the virulence factors associated with associated with C. perfringensC. perfringens and the and the biological activity of eachbiological activity of each