BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

Clostridium Summary

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C. perfringens (see WebLinked image; see WebLinked image) (see WebLinked site)

C. botulinum (see WebLinked site)

C. tetani (see WebLinked image) (see WebLinked site)

C. dificile pseudomembranous colitis (see WebLinked image; see WebLinked image)

1. Histotoxic group --- tissue infections

( C. perfringens type A; exogenously-acquired more commonly than endogenously)

( C. septicum; endogenously-acquired)

a. cellulitis

b. myonecrosis

c. gas gangrene

d. fasciitis

2. Enterotoxigenic group --- gastrointestinal disease

a. clostridial foodborne disease (8-24 h after ingestion of large numbers of organisms on con- taminated meat products, spores germinate, enterotoxin produced ( C. perfringens type A)

b. necrotizing enteritis (beta toxin-producing C. perfringens type C)

( C. difficile endogenously-acquired or exogenously-acquired person-to-person in hospital)

c. antibiotic-associated diarrhea

d. antibiotic-associated pseudomembranous colitis

3. Tetanus (exogenously acquired) --- C. tetani neurotoxin

a. generalized (most common)

b. cephalic (primary infection in head, commonly ear)

c. localized

d. neonatal (contaminated umbilical stump)

4. Botulism (exogenously acquired) --- C. botulinum neurotoxin

a. foodborne (intoxication, 1-2 days incubation period)

b. infant (ingestion of spores in honey)

c. wound (symptoms similar to foodborne, but 4 or more days incubation)

Clostridium perfringens --- histotoxic or enterotoxigenic infections

Morphology and Physiology

• large, rectangular bacilli (rod) staining gram-positive

• spores rarely seen in vitro or in clinical specimens (ovoid, subterminal)

• non-motile, but rapid spreading growth on blood agar mimics growth of motile organisms

• aerotolerant, especially on media supplemented with blood

• grow at temperature of 20-50^oC (optimum 45^oC) and pH of 5.5-8.0

  Pathogenicity Determinants (note that toxins include both cytolytic enzymes and bipartite exotoxins)

• four major lethal toxins (alpha (a), beta (b), epsilon (e), and iota (i) toxins) and an enterotoxin

• six minor toxins (delta(d), theta(q), kappa(k), lambda(l), mu(m), nu(h)toxins) & neuraminadase

• C. perfringens subdivided into five types (A - E) on basis of production of major lethal toxins

• C. perfringens Type A (only major lethal toxin is alpha toxin) responsible for histotoxic and enterotoxigenic infections in humans; Type C causes necrotizing enteritis (not in U.S.)

  Lab Identification

• direct smear and Gram stain, capsules upon direct examination of wound smears

• culture takes advantage of rapid growth in chopped meat media at 45^o C to enrich and then isolate onto blood agar streak plate after four to six hours

• gas from glucose fermentation

• in vivo toxicity testing and identification of the specific toxin types involved

• double zone of hemolysis on blood agar (b-hemolytic theta(q) toxin, a-hemolytic alpha(a) toxin)

• Nagler rxn; precipitation in serum or egg yolk media; a -toxin (phospholipase C) is a lecithinase

• "stormy" fermentation (coagulaltion) of milk due to large amounts of acid and gas from lactose

  Diagnosis/Treatment of systemic infection --- Early diagnosis and aggressive treatment essential

• removal of necrotic tissue (surgical debridement)

• Penicillin G in high doses if more serious infection

• Of poorly defined clinical value are:

• administration of antitoxin

• hyperbaric oxygen (dive chamber) adjunct therapy (??inhibit growth of anaerobe??)

  Clostridium tetani --- agent of tetanus

  Morphology and Physiology--

• long thin gram-positive organism that stains gram negative in old cultures

• round terminal spore gives drumstick appearance

• motile by peritrichous flagella

• grow on blood agar or cooked meat medium with swarming

• beta-hemolysis exhibited by isolated colonies

• spores resist boiling for 20 minutes

  Antigenic Structure--

  flagella (H), somatic (O), and spore antigens. Single antigenic toxin characterizes all strains.

  Pathogenicity Determinants--

• play a role in local infection only in conjunction with other bacteria that create suitable environment for their invasion

• systemic-acting, plasmid-mediated A-B neurotoxin (tetanospasmin) produced intracellularly

• Mode of Action --- one of most poisonous substances

• binds gangliosides in synaptic membranes (synapses of neuronal cells) and blocks release of inhibitory neurotransmitters; continuous stimulation by excitatory transmitters

• muscle spasms (spastic paralysis) (trismus (lockjaw), risus sardonicus, opisthotonos), cardiac arrhythmias, fluctuations in blood pressure

  Lab Identification--

• use characteristics of resistance to heat, motility, and toxin production to help identify

  Diagnosis/Treatment/Prevention

• empirical diagnosis on basis of clinical manifestations

• treat to prevent elaboration and absorption of toxin

• clean wound (debridement), control spasms

• metronidazole administered to eliminate vegetative bacteria that produce neurotoxin

• passive immunity (human tetanus immunoglobulin); vaccination (active) as preventative

• antitoxin administered to bind free tetanospasmin

  C. botulinum --- agent of botulism , a rare, but severe (lethal) neuroparalytic disease

  Morphology and Physiology

• heterogeneous group of fastidious, strictly anaerobic bacilli

• motile by peritrichous flagella

• heat-resistant spores (ovoid, subterminal)

• proteolytic and non-proteolytic

  Antigenic Structure

• species divided into four groups (I-IV) based on type of toxin produced and proteolytic activity

• seven antigenically distinct botulinum toxins (types A to G)

• somatic antigens -- heat stable and heat labile; spore antigens -- more specific

  Pathogenicity Determinants

• lethal foodborne intoxication with toxin types A, B, E, or F; shorter incubation period --->poorer prognosis

• phage-mediated, systemic-acting A-B neurotoxin (botulinum toxin = botulin) released at cell lysis

• Mode of Action -- one of most extremely potent neurotoxins known (1 mg of purified toxin contains about 200,000 minimal lethal doses (MLDs) for a 20g mouse)

• A-B toxin ingested, binds specific receptors on peripheral cholinergic nerve endings (neuromuscular junctions) where it blocks release of presynaptic acetylcholine (excitatory neurotransmitter) blocking muscle stimulation and resulting in flaccid paralysis

• Early: nausea, vomiting, weakness, lassitude (lack of energy), dizziness, constipation

• Later: double vision, difficulty in swallowing and speaking

• Final: death due to respiratory paralysis

  Lab Identification

• microscopic detection or Cx (culture) are often unsuccessful (few organisms and slow growing)

• toxin detected and typed in lab via toxicity and antitoxin neutralization tests in mice or by ELISA

  Diagnosis/Treatment/Prevention

• crucial to rapidly diagnose (symptoms often confusing); note the type of botulinum toxin involved

• Tx (treatment) should be administered as quickly as possible on basis of clinical Dx (diagnosis)

• ventilatory support and trivalent (A, B, E) antitoxin (polyvalent) to bind free toxin in the bloodstream

• administer gastric lavage and metronidazole or penicillin to eliminate the organisms from GI tract

• care in home canning and in heating of home-canned food; toxoid is available

 

 

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