Clostridium:Anaerobic Endospore formers

Filename: Clostridium.ppt

Clostridial Diseases

• Botulism C. botulinum

• Tetanus C. tetani

• Gas gangrene C. perfringens

• Food poisoning Clostridium spp

• Pseudomembranous colitis C. difficile

Clostridium

• Gram positive

• Rods

• Endospore formers
  • asporogenous

• Obligate Anaerobes!!!
  • Aerotolerant spp C. perfringens, C. tetani, C. botulinum, C. difficile

• Soil organisms

Botulism

• Intoxication

• Foods
  • Meats
  • Fish
  • low-medium acid canned foods

• Wild birds (limberneck)
  • ducks, fish, Inuit -- whale blubber, seal fins

Botulism: symptoms

• Adults
  • Nerve paralysis
    • shorter nerves first
  • Blurred vision
  • Cardiac failure
  • Respiratory failure

• Intoxication

• Infants
  • Failure to Thrive
  • Dehydration
  • Polyneuropathy

• Infection Cl. Botulinum
  • umbilical cord

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

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

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.

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.

Wound botulism:

• rare -- organisms multiply in the wound.

• Can occur through umbilical cord

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.

Botulism Toxin

Potent neurotoxin regulated by bacteriophage.

Toxins: A -- E, C alpha, C beta, F, G

Humans: A, B, E

150 Kd protein -- cleaved

Botulism Outbreaks by Type

Botulism Intoxication in USA

Botulism in USA; neonates

Botulinum Toxin

• The heavy chain attaches to the ganglioside receptors in nerves

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.

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.

Tetanus

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.

Tetanus Intoxication

Entry wound

Spread
blood stream

moves up nerves

Disease
Incubation: 1-2 days

Rigid Paralysis, cardiac failure, respiratory failure

Lockjaw

Tetanus

trismus, risus sardonicus, opisthotonos,

Cephalic -- poor prognosis

Localized -- favourable prognosis

Prevention: toxoid, 3% formaldehyde

Risus sardonicus

Opisthotonos

Tetanus Neonatorum

Tetanospasmin

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.

Binding regions of tetanus toxin and botulinum toxin are different in terms of cell specificity.

Tetanus Distribution

Incidence of Tetanus in USA

YEAR

C.perfringens: Diseases

• bacteremia

• myonecrosis

• gas gangrene

• cellulitis

• fascitis

• food poisoning: enteritis necroticans

C. perfringens

• large rectangular, hemolytic, very distinctive spreading colonies.

• target hemolysis

• found in soil and intestines, man and animals

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

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.

Nagler Reaction

• Presumptive identification of C. perfringens

• alpha toxin (lecithinase) hydrolyses phospholipids

• egg yolk agar becomes turbid

• specifically blocked by antitoxin

Nagler Reaction blocked by Antibodies

PPT Slide

Clinical Syndromes:

Bacteremia - usually transient, only diagnostic with other

clinical symptoms

Myonecrosis - gas gangrene

- trauma or surgical contaminant

Gas Gangrene

Entry PenetratingWound

Multiplication
in dead anaerobic tissue

Toxin production
hemolysin, proteases, lipase, collagenase

Disease
necrosis, edema, gas

PPT Slide

C. perfringens Gas gangrene

Incubation: ə week

pain severe

muscle necrosis
shock
renal failure
death

-crepitant

-cellulitis & fascitis (no muscle)

PPT Slide

C perfringens Food poisoning

incubation 8-24hrs.

Abdominal cramps, watery diarrhea

lasts less than 24hrs.

Contaminated meat (left overs)

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

C. difficile Diagnosis:

cytotoxin - stool

culture

C.difficile antigen -- latex agglutination

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.

Mechanism of pathogenicity:

Toxin A

enterotoxin

hypersecretion of fluid

Toxin B
cytotoxin

cytopathic to tissue monolayers

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.

C. difficile

Culture: standard test for Clostridia include: indole, sugars, lecithinase, catalase (usually neg.)

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.

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)

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.

Differential diagnosis:

S.aureus, E.coli, shigellosis,

acute ulcerative colitis, Campylobacter jejuni

The End

Key Terms

• Obligate anaerobe

• endospore

• asporogenous

• botulism

• tetanus

• gas gangrene

• pseudomembranous colitis

• myonecrosis

• Botulism toxin

• tetanus toxin

• trismus

• opisthotonus

• risus sardonicus

• C. perfringens enterotoxin

• aerotolerant

• Nagler reaction

Key Terms

• Enteritis necroticans

• tetanospasmin

Key Organisms

• Clostridium

• C tetani

• C botulinum

• C. perfringens

• C. difficile

Key Concepts

Epidemiology of Botulism

• Disease/bacterial factors

• Transmission

• who is at risk

• geography/ season

• modes of control

Epidemiology of C. difficile infections

• Disease/bacterial factors

• Transmission

• who is at risk

• geography/ season

• modes of control

Epidemiology of C. tetani infections

• Disease/bacterial factors

• Transmission

• who is at risk

• geography/ season

• modes of control

Epidemiology of C. perfringens infections

• Disease/bacterial factors

• Transmission

• who is at risk

• geography/ season

• modes of control

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

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