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BOTULISM ⁴

Clinical features Clostridium botulinum Diagnosis Mechanisms of action Pathology Prevention Prognosis Structure Types Clinical Syndromes   Food-borne   Iatrogenic: Generalized   GI colonization   Infantile   Inhalation   Wound

Justinus Kerner 1st Described Botulism

Emile Van Ermengem Isolated Clostridium botulinum

Clostridium botulinum

• Clostridium botulinum
  • Gram positive bacilli
  • Spore producing
  • Produced under anaerobic conditions: Obligate
  • Toxin production
    • May produce all 7 toxin serotypes: A-G
    • Genetic information
      • Type G on plasmids
        
      • Types C1 & D on bacteriophages
        
      • Types A, B, E, and F on the bacterial chromosome
    • Bound to hemagglutinins: ? Inhibits degradation by digestive enzymes
  • Types causing
    • Human disease: A, B, E > F, G
    • Animal disease: C, D, C/D mosaic
• C. baratii
  • Toxin: Produces serotype F
• C. butyricum ¹⁰
  • Strictly anaerobic Gram-positive, spore-forming bacillus
  • Common human & animal gut commensal bacterium
  • Toxin: Produces serotype E
  • Other disease
    • Necrotizing enterocolitis in preterm neonates: 2° to Pathogenic strains
• Other similar Molecules
  • Botulinum type A2
  • Botulinum type X
  • Non-clostridial Botulinum-like proteins
    • Botulinum Wo : Weissella oryzae SG25T
    • Botulinum En : Enterococcus faecalis
    • PMP1: Paraclostridium bifermentans ¹⁴

Botulinum Toxin: Proteins & Transport General: Botulinum toxins Production Only when the spores germinate Germination circumstances Anaerobic conditions Low acidity (pH > 4.5) Low salt & sugar content, Temperatures 37°F–99°F (3°C–37°C) Varies with serotype Most strains produce single serotype Molecular Zinc-endopeptidase protein Produced as Single chain protein (protoxin) Molecular weight ~150 kDa Types share common domain organization Processed into mature toxins by cleavage into 2 subunits 50 kDa light chain (LC) 100 kDa heavy chain (HC) Subunits remain connected through a conserved disulfide bond Disulfide bond is not present in BoNT/Wo Released from bacteria as part of noncovalent multimeric complex Auxiliary proteins Hemagglutinins (HA) Nontoxin, nonhemagglutinin (NTNH) Complex with botulinum May protect toxin at low pH in GI tract Dissociates spontaneously at physiologic pH Some components necessary for toxicity No direct role of auxiliary proteins in toxin-induced Ach blockade Neurotoxic types of botulinum toxin A , A2 , B , C1 , D , E , F , G Type A: Most severe disease & frequent need for ventialtory support Type B: Milder disease Types C & D: Not reported in recent outbreaks Type D: May not be absorbed from human GI tract Type E: Foods of aquatic origin; Varied disease severity; GI symptoms common Type F: Rapid progression & earlier rocovery Tetanus toxin: Sequence homology 30% to 40% Cleavage of Botulinum protoxins: Into 2 chains Heavy (100 kDa) chain C-terminal region (HC) of Heavy (H) chain: Binds to surface of target nerve cells N-terminal region (HN) of H chain: Translocates L chain across membranes Light (L) chain (50 kDa) Light chains have a tetrahedral zinc binding motif: Contains Consensus HELIH amino acid sequence Common to other zinc dependent metalloproteinases 2 histidines, glutamate & water molecule Structure resembling thermolysin-like endoproteases Contain toxic activity Zinc-dependent endopeptidase Cleave proteins forming synaptic vesicle docking & fusion complex 3-D Geometry: Catalytic sites buried deeply within protein Heavy & Light subunits linked by disulfide bond Double chain product is active form: Inhibits cholinergic transmission Protease producing cleavage Often contained by producing organism May also occur in GI tract Toxin passes from GI tract to vasculature: Mechanisms Transport cell: Absorptive enterocyte Binding to the apical surface of epithelial cells Receptor-mediated endocytosis Transcytosis through cells Delivery to the basolateral surface of cells Ability of toxin to cross cells depends on Toxin type Able to cross cells: Botulinum type A & B Not able to cross cells: Botulinum type C & Tetanus Heavy chain of botulinum toxin: C-terminus Toxin passes out of vasculature to presynaptic regions ? Mechanism Does not cross blood-brain barrier Other absorption routes Botulinum toxin may be absorbed from respiratory system General action: Blocks exocytosis Most potent action: Blockade of cholinergic terminals Other blockade of exocytosis: At higher concentrations of toxin Other nerve terminals: Norepinephrine, Serotonin Non-neural cells: If membrane receptors present

Botulinum Toxin Type B

• Type A ¹¹
  • Epidemiology: Most common outbreaks in Rocky Mountains & West
  • Cleavage target: SNAP-25 (t-snare)
  • Physiology
    • Miniature endplate potentials (MEPPs)
      • Frequency: Reduced
      • Amplitude: Usually Reduced
    • EPPs: Quantal content reduced
    • Elevated Ca⁺⁺ level in synaptosomes: Overcomes blockade
    • Spontaneous transmitter release: More blockade than Type B
    • Repetitive stimulation evokes post-synaptic response
  • Clinical
    • More severe & long-lasting paralysis: 67% with intubation
• Type B
  • Epidemiology: Most common outbreaks in East, especially Allegheny range
  • Bot B protein
    • Has most structural homology to tetanus toxin
    • Cleavage target: Synaptobrevin 1 (VAMP) (v-snare)
    • Requires assembled intracellular microtubules for action
  • Physiology
    • Miniature endpale potentials (MEPPs): Normal or Moderate reduction in frequency
    • EPPs: Quantal content reduced; Variable latencies
    • Blocks effect of α-Latrotoxin
    • Repetitive stimulation does not evoke post-synaptic response
  • Clinical: Somewhat less severe paralysis than Type A
• Types C & D
  • Toxin targets
    • C1: SNAP 25 & Syntaxin
    • D: Synaptobrevin
  • Poorly absorbed from intestine
  • Cause disease in animals
    • Avian botulism: Type C; "Limber neck" ⁷
    • Equine: Forage (adult) poisoning; Shaker Foal Syndrome
  • Only rarely implicated in human intoxication
  • Botulinum C3: Ribosylates ADP
    • Substrates: RAC1 ; RAC2 ; RAC3
• Type E
  • Found in fresh water sediment
    • Most common outbreaks in Baltic, Alaska & Great Lakes states
      
    • Associated with fish ingestion
      
  • Toxin target: SNAP 25
• Type F ⁹
  • Produced by: C. baratii
  • Toxin target: Synaptobrevin
  • Possible predisposing factors
    • Sources: Food in some patients
    • Antimicrobials before disease onset
    • GI disease
  • Clinical
    • Frequency: 1% of botulism cases
    • Onset age: Adults; Mean 54 years; Few infants
    • GI colonization syndromes
    • Rapid course
      • Symptom onset to hospitalization: Often < 1 day
      • Progression to: Respiratory failure & Severe weaknes
    • Ophthalmoplegia
    • Asymmetry: Some patients
• Type G
  • Toxin target: Synaptobrevin
• Type H
  • Hybrid-like structure
  • Regions of Similarity to serotypes A1 & F5

• Time course
  • Incubation period
    • Average: 18 to 38 hours
      
    • Extremes: 2 hours to 1 week
• Afebrile
• Early symptoms
  • Mouth: Dysphagia; Dry
  • Speech: Slurred; Difficult; Hoarse voice
  • Eye: Blurred vision; Diplopia
  • Shortness of breath
  • GI
    • Nausea & vomiting: More with food bourne botulism
    • Constipation: Childhood botulism
• Weakness
  • Diffuse
    • Usually symmetric
    • Proximal > Distal
    • "Descending paralysis"
  • Bulbar: Dysphagia; Dysarthria
  • Extraocular: Ptosis; Extraocular muscle weakness
  • Respiratory
    • Usually with other signs
    • Isolated involvement: Uncommon
• Sensory loss: Never prominent
• Tendon reflexes
  • Reduced
  • ? Less involved with type E
• Autonomic
  • Cholinergic
    • Pupils: Many, but not all, patients
    • Dilated
    • Unreactive
    • Bradycardia
    • Hypotension
    • Skin: Hypohidrosis
    • Urinary retention
    • Gastrointestinal
      • Nausea & vomiting associated with contaminated food
      • Constipation: Frequently first sign, especially in infants; Common late
      • Diarrhea may occur early
  • Lab
    • Sympathetic skin response: Absent
    • Plasma norepinephrine: Reduced
• Treatment
  • Supportive care
  • Anti-toxin: Human
    • Usual type used: Trivalent vs A, B, E
    • May be most effective for Type E
    • US Army anti-toxin: Heptavalent; For bioterrorist attack
  • Botulism immune globulin
    • For: Infant botulism
    • Reduces time in hospital, on ventilator & tube feeding

• Electrodiagnostic ¹
  • Repetitive nerve stimulation
    • Increment: With (40 Hz) or paired stimuli
      • Occurs in 50% to 60% of patients
      • Testing of multiple muscles may be needed to see increment
      • More common with type B botulism
    • Decrement on slow RNS: Occasional patients
  • CMAPs: Small
  • Motor unit potentials: Short
  • Recruitment: Normal, or Mildly reduced
  • SNAPS: Normal
  • EMG: Muscle denervation; Fibrillations & Positive sharp waves
• Muscle biopsy
  • Muscle fiber atrophy
  • Target fibers
• Analysis of serum, feces & implicated food
  • Passive transfer of serum or other body fluid to mice
    • Toxicity to mice
      
    • Selectively prevented by anti-toxin
  • Stool or wound culture: Not useful in Iatrogenic or Inhalation syndromes

• Food-borne botulism ⁸
  • Epidemiology
    • Frequency: 25 cases in US in 2012; 16% of US botulism cases
    • Intoxication most common in: Alaska, California, Colorado, New Mexico, Illinois
    • Associated foods
      • Alaska
        • Marine animals: Fish, whales, seals, walruses, beavers
        • Botulinum toxin type E
      • Home canned
        • Foods: Vegetables; Potatoes; Sea food, preserved
        • Botulinum toxin types A & B more common
      • Recent epidemics
        • Peyote cactus tea
        • Mascarpone cream cheese
        • Pruno (Prison homemade alcohol) ⁶
        • Bamboo shoots
        • Carrot juice
        • Tofu
  • Disease mechanism: Related to ingestion of toxin
    • Food
      • Contaminated with Clostridium spores
      • Clostridium grows in Anaerobic conditions: Produces toxin
    • Absorption
      • Stomach: Not absorbed; Toxin complex with accessory proteins is resistant to proteolysis
      • Intestine
        • Alkaline pH of intestine dissociates toxin from associated proteins
        • Dissociated toxin is absorbed into circulation by endocytosis & translocation
  • Clinical
    • Onset
      • Age: Usually adults
      • Time: 12 to 36 hours after ingestion
      • Course: May be rapid
      • Cranial distribution: Diplopia; Dysphagia; Dizziness
    • Examination: Descending paralysis
      • Ptosis
      • Gag reflex: Absent
      • Respiratory failure
      • Limb strength: May be preserved
    • Respiratory failure (40%)
      • May occur early in diseasse course
      • Intubation often on 1st or 2nd hospital day
    • GI: Nausea & Vomiting
    • Pregnant & Postpartum women
      • Source: Foodbourne toxin most common
      • Clinical syndrome
        • Similar to others
        • Common symptoms or signs
          • Weakness
          • Dry mouth
      • Little or no risk of congenital botulism to fetus
    • Prognosis
      • Mortality in past 50 years: 7% for foodborne botulism
      • Probably worse prognosis in older patients
  • Treatment
    • Supportive care: Respiratory; Wound debridement
    • Early
      • Emetics: Avoid magnesium containing
      • Lavage
      • Enemas: Not when paralytic ileus
    • Antitoxin
      • Most useful in 1st 24 hours
      • Trivalent ABE
      • Obtain from
        • CDC: 404-639-3753 (day); 404-639-2888 (night)
        • Also available from: California & Alaska Departments of Health
      • Use on clinical diagnosis: Without waiting for lab confirmation
      • Effects
        • Mortality reduced
        • Shortens illness (For Type A)
      • Complications (immune): 9%
    • ? 3,4-diaminopyridine
• Wound botulism ¹²
  • Epidemiology
    • Most common in California
    • Botulinum types A > B
  • Disease mechanism: Toxin is produced locally by infecting organisms in wound
    • In wound or abscess
    • After proliferation of bacterial spores
  • Causes
    • Drug abuse most common cause
      • Intramuscular or subcutaneous ("Skin-popping")
      • "Black tar" heroin: Preparation for injection does not destroy spores
      • Subcutaneous abscesses (50%)
      • Most common in California
    • Injuries: Crush; Compound fractures
  • Clinical
    • Incubation period 4 to 14 days, longer when wound is debrided
    • Onset
      • Dysarthria (75%)
      • Dysphagia (50%)
      • Blurred vision (40%)
    • Weakness: Progression
      • Generalized weakness: Descending
      • Respiratory failure (40%)
        • May occur early in diseasse course
        • Intubation often on 1st or 2nd hospital day
      • Bulbar
        • Dysarthria
        • Dysphagia
        • Face weak
      • Eye
        • Pupillary reactivity: Reduced
        • Ptosis
        • Vision: Blurred
    • Systemic
      • Skin: Cellulitis & Abscesses
      • No GI motility change
    • Prognosis
      • Worse with
        • Longer time to antitoxin & wound debridement
        • Female sex
      • Ventilator dependence frequency: Type A > B > E
      • Slow improvement in strength over weeks to months
      • 1 year: Most near normal ± fatigue
      • Autonomic function may improve later than weakness
      • Mortality in past 50 years: 5% to 10%
    • Toxicology: Positive for opiods
    • Treatment
      • Antitoxin: Bivalent (A, B) botulinum equine (from CDC)
        • Most useful in 1st 24 hours
      • Wound debridement
      • Antibiotic
        • Penicillin G: Three million units of Penicillin G IV every 4 hours
        • Metronidazole: 500 mg every 8 hours for penicillin-allergic patients
  • Electrophysiology
    • CMAP amplitude: Reduced (50%)
    • RNS
      • 2 Hz: Decrement in 50%;
      • 30 Hz: Increment in 50%
    • Motor unit potentials: Small
  • Diagnosis: Wound cultures positive in 35%

    From: A Kornberg

    Infantile Botulism Hypotonia; Enlarged pupils

• GI colonization syndromes
  • Epidemiology
    • Occurs in areas with botulinum spores in soil
    • Age
      • Usually Infants (Infant botulism)
      • Adults: Type F
    • US: Common in
      • California (50% of cases in US), Utah, Pennsylvania & Hawaii
      • Utah soil features
        • Alkaline, Low organic content, Sagebrush grasslands
        • C botulinum type A
      • Eastern US soil features
        • Slightly acidic, Higher organic content, Cultivated fields
        • C botulinum type B
  • Disease mechanism: Toxin is produced locally by organisms infecting GI tract
  • Exposure: Ingested Clostridium botulinum spores
    • Raw honey may be a source of type B organisms
    • Clostridium grows better in GI tract without normal flora
      • Young infants: Before normal flora has developed
      • After treatment with antibiotics: Reduces normal flora
      • Less competition from normal GI organisms
    • Organisms produce toxin in the GI tract
  • Clinical
    • Onset
      • Age: 1 to 6 months; Median 4 months
      • Adults: ~ 1 to 2 months after ingestion of spores
        • Predisposing factors
          • Achlorhydria
          • Chronic antibiotics
          • Gastrectomy
          • Intestinal surgery
        • Toxin types: A; B; F (produced by C. baratii)
    • Onset: Clinical syndrome
      • Course: Acute
      • Initial signs: Constipation & Weakness
    • Weakness
      • Eye: Ptosis; Ophthalmoplegia; Mydriasis
      • Bulbar
        • Dysphagia
        • Poor suck & feeding
        • Reduced gag
      • Weak cry
      • Hypotonia
      • Respiratory distress
    • Parasympathetic changes
      • Hypotension
      • Tachycardia
      • Mydriasis
    • Diagnosis
      • Stool: Culture & Analysis for toxin
      • Repetitive nerve stimulation: Increment
    • Treatment: Infants

      Increment on rapid repetitive nerve stimulation

      • Botulism immune globulin ⁵
        • Shortens hospital stay by ~17 days
        • Cost: $45,000 per patient
      • Supportive care: Respiratory; Nutrition; Fluid
      • Anti-toxin & Antibiotics: Not effective
    • Prognosis
      • Improve spontaneously without treatment
      • Time course: Weeks to months
      • Deaths: Rare with adequate support
• Iatrogenic: Generalized signs after local botulinum injection
  • Dose used: Within therapeutic guidelines
  • Weakness: Generalized
  • Autonomic: May be involved
  • Lab: Abnormal single fiber EMG
• Inhalation botulism syndrome ³
  • Exposure
    • Aerosolized botulinum toxin
    • Sources: Bioterrorism; Veterinary
  • GI prodrome: None
  • Early signs: Dysphagia; Dilated pupils; Ophthalmoparesis
  • Likely Epidemiology in Bioterrorism
    • Several cases: Multiple simultaneous groups
    • Common temporal & geographical, but not food, exposure
    • Probably not waterbourne
    • Latency: 12 to 80 hours
    • Unusual toxin type for geographic area
  • Treatment
    • Respiratory support
    • Antitoxin: As early as possible

• Canning or preserving foods with appropriate heat, pressure, & low pH
  • Spores
    • Survive 2 hr at 100 °C
    • Inactivated at 120 °C
    • High elevations: Boiling food before canning may not inactivate the spores
    • Factors favoring spore germination
      • Low acidity (pH > 5.0)
      • Low O₂;
      • High water content
  • Toxin: Inactivated after 1 minute at 85 °C, or 5 minutes at 80 °C
• Avoid exposure of infants to honey (may contain Clostridium botulinum spores)
• Botulinum intoxication: No immunity from subsequent episodes
• Immunization
  • Pentavalent toxoid
  • Used only for high risk personnel: Laboratory workers; Military

Botulism: Pathology

Botulinum toxin treatment: Terminal axons at Neuromuscular junctions (Rat) Botulinum toxin induces: Enlargement of NMJs (middle) & Sprouting of nerve terminals (middle & top) Normal NMJ Terminal axon branches at NMJ (Surrounded by esterase stain (Blue))

H&E stain

H&E stain

NADH stain

NADH stain

VvG stain

VvG stain

ATPase pH 9.4 stain

ATPase pH 9.4 stain

ATPase pH 4.3 stain

Esterase stain

Esterase stain

Botulinum Toxin, Type B