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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