Clostridium difficile

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Clostridium difficile
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Clostridium difficile

• Humans
• Pseudomembranous colitis
• High prevalence among hospital patients
• Antibiotic associated
• Horses
• Colitis - X
• Also antibiotic associated
• Pigs
• Neonatal typhlocolitis
• Others
• hamsters, guinea pigs, rabbits, primates

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General Characteristics

• Source
• feces of nondiarrheic humans 5 - 10
• hospital environment up to 25 of patients
• soil, marine sediments
• dogs and cats up to 35
• wide variety of other animals
• food
• beef
• pork
• poultry

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Clinical Disease

• Antibiotics disrupt normal flora
• ampicillin, cephalosporins most commonly
  associated (more widely used)
• clindamycin
• Loss of competitive exclusion
• Uncontrolled proliferation of C. difficile
• Onset 4 - 10 days after start of antibiotic, up
  to 2 weeks after termination

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Clinical Disease

• Transmission via spores (vegetative cells oxygen
  sensitive)
• fecal-oral
• contaminated clothing, surfaces
• aerosols
• Pass through stomach, bile acids
• induce germination?
• Antibiotics - compromised normal flora
• C. difficile grows rapidly in unoccupied niches
• Vegetative cells produce toxins

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Piglet Disease Model

• Dilute spore preparation in sodium bicarbonate
• Sodium bicarbonate will neutralize stomach acid
  which may contribute to spore survival in the
  stomach
• Administer to piglets intragastrically
• Or use alternate diluent

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In Vitro Germination
2 hr in media
1 hr in media
0 hr spores In media
Over-night in Sodium bicarbonate
5 hr in media
Over-night in media
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Clinical Disease

• Damage to colonic mucosa
• pseudomembrane
• separate lesions coalesce
• Symptoms
• severe abdominal pain
• watery diarrhea
• possibly ileus
• high number of neutrophils in stool

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Lesions of C. difficile
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Pathogenesis

• Large clostridial cytotoxins
• AB-like cytotoxins
• A- enzymatic portion
• B- functions as a ligand
• Endocytosed via coated pits
• Endosomal acidification - conformational change

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Toxin Processing
A-fragment toxic portion
B-fragment attachment
Host cell
Middle translocation
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Toxin Processing
Host cell
endosome
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Toxin Processing
Host cell
Endosome Low pH
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Toxin Processing
Proteolytic cleavage of N-terminal fragment
Host cell High pH
Endosome Low pH
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Toxin Processing
Free to attack Rho and Rac GTPases
Proteolytic cleavage
Host cell
Endosome Low pH
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Pathogenesis

• Toxin A
• 308 kDa (largest known exotoxin)
• Enterotoxic in vivo
• cells can no longer control water movement
• fluid accumulation with tissue damage (blood and
  mucus)
• Causes diarrhea w/ intense inflammation
• Chemotactic for neutrophils
• Must be internalized for toxic effect

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Pathogenesis

• Toxin A
• C-terminal 1/3 (B fragment)
• host cell binding
• recognizes a carbohydrate moiety on the apical
  surface
• not toxic but required for toxic effect
• N-terminal 1/3 (A fragment) toxin-domain
• Glycosyltransferase activity in the cytosol
• glycosylation of rho-subfamily proteins
• rho-type proteins are signal transduction
  molecules
• formn of actin filaments cell adhesion
  structures
• inactivates these proteins ? breakdown of actin
  filament network ? opening of tight
  junctions/apoptosis/necrosis

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Actin is fundamental to maintenance of cell
shape, polarity and intracellular adhesions.
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Pathogenesis

• Toxin B
• 269 kDa
• Has no enterotoxic activity in vivo
• Trace amounts of toxin A or mucosal damage
  necessary for toxic effect in rodent bowel
• Cytotoxic in vitro ( 1000 times more active than
  toxin A)
• 63 amino acid homology with toxin A (gene
  duplication)

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Pathogenesis

• Toxin B
• N-terminal domain highly conserved, same
  activity as in toxin A
• C-terminal domain quite different, may recognize
  different receptor
• Toxins act synergistically
• Toxin A damage to mucosal cells allows toxin B
  maximal effect

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PathogenesisReceptor Mediated Disease
Host cell
Receptor
TcdA
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PathogenesisReceptor Mediated Disease
Host cell
Receptor
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Clostridium difficileReceptor Mediated
DiseaseTcdA and TcdB in Pigs
Biotinylated TcdA binds to neonatal colonic
epithelial cells
Biotinylated TcdB does not bind to neonatal
colonic epithelial cells
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Clinical Disease

• Diagnosis
• Detection of the organism
• culture of feces for C. difficile (48-72 h)
• immunoassay
• Detection of toxins
• tissue culture
• immunoassay

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Outcome and Treatment

• Affects 3.5 x 106 North Americans per year
• Fatality rate 27 - 44 if untreated
• Treatment
• cessation of antibiotic, if possible
• treatment with anti-C. difficile-drugs
  vancomycin, metronidazole
• extended course to prevent recurrence
• restoration of normal intestinal flora
• fecal enema from family member

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Prophylaxis

• Feeding of Saccharomyces boulardii (yeast)
• Administration of toxin-neutralizing antibodies
• Parenteral immunization against toxins

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Disease of Neonates

• Human infants are not affected
• lack toxin receptors
• What about neonates of other species?

? An emerging disease of suckling pigs
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Porcine CDAD

• Nursing piglets 1-7 DOA
• High morbidity, low mortality
• Pasty-to-watery diarrhea
• Constipation, obstipation
• Lesions
• Multifocal fibrinous typhlocolitis epithelial
  erosions/ulcers
• PMN exudation (volcano lesion)
• Virulence TcdA only

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(No Transcript)
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Significance to the Swine Industry

• Mean morbidity 20
• range 10 to 90
• Mean case fatality rate 20
• can be as high as 50
• Prevalence of disease?
• Significance of low mortality disease to swine
  units
• increased time to weaning
• even 1 or 2 days can be very significant
• possibly variation in condition of pigs

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Summary

• G, spore forming rod - facultative anaerobe.
• Severe disease in humans, horses, guinea pigs,
  rabbits.
• Mild disease in others (newborn pigs foals).
• Spores must be ingested for transmission.
• Disruption of commensal flora ? prolif of C
  difficile.
• Antibiotics or neonates

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Summary

• Toxins A and B - largest known exotoxins.
• A-B-like toxins
• COOH-terminus
• Binding to receptors
• NH3-terminus
• Enzymatic activity
• Dysfunction of intracellular signaling molecules
• Disrupts actin filaments
• ? loss of cell adhesions ? loss of mucosal
  integrity
• Inflammation
• Toxin A is the key player in disease
• Receptor Mediated Disease

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Clostridium difficile
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The Important Stuff

• What are the phenotypic characteristics and an
  essential growth requirement of C. difficile?
• Gram-positive, spore-forming rods
• Grows only in anaerobic conditions

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The Important Stuff

• What are the phenotypic characteristics and an
  essential growth requirement of C. difficile?
• Gram-positive, spore-forming rods
• Grows only in anaerobic conditions
• What individuals are at greatest risk of dz?
• Humans - hospital patients with altered normal
  flora
• Also affects adult horses, hamsters, guinea pigs,
  primates
• Mild disease in foals or neonatal pigs.
• Receptor mediated disease

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The Important Stuff

• What are the toxins of C. difficile and their
  characteristics?
• Toxins A and B
• A-B-type toxins
• Very large
• Toxin A is most clinically significant
• receptors

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The Important Stuff

• What is the molecular pathogenesis of C
  difficile-associated diarrhea?
• Disrupt intracellular signalling molecules
  essential for actin polymerization.
• Disturbs cell junctions, cell shape and polarity.
• Destroys mucosal integrity.
• Contributes to cytokine release ? inflammation.