Bacterial agents of
bioterroism
Laboratory network for
biological terrorism
Bacillus anthracis
Anthrax
Anthrax: Overview
• Primarily disease of
herbivores
• Humans usually
infected by contact with
infected animals or
contaminated animal
products
• Soil reservoir
• Woolsorter’s disease
(inhalation anthrax)
• No person-to-person
transmission of
inhalational anthrax
CDC
ANTHRAX
• Three forms of human anthrax occur:
1. Cutaneous
2. Gastrointestinal
• Oropharyngeal
• Abdominal
3. Inhalation (Woolsorter’s Disease)
Cutaneous anthrax
Vesicle development, day 2
Eschar formation, day 4
Inhalation Anthrax
• Infective dose = 8,000 - 15,000 spores
• Incubation period = 1-6 days
• Duration of illness = 3-5 days
• Fever, malaise, and fatigue
• Short period of improvement = up to 2
days
• Abrupt respiratory distress…death <24hrs
• Person to person transmission = no
Anthrax: Specimen Selection
• Inhalation: Sputum and Blood
• Cutaneous: Vesicles and Eschar
• Gastrointestinal: Stool and Blood
Bacillus anthracis
Key Sentinel Lab Tests
•
•
•
•
•
Gram stain
Growth characteristics on agar
Sporulation, in air
Motility
Capsule by India Ink
Bacillus anthracis
Gram Stain Morphology
• Broad gram-positive rod: 1-1.5 X 3-5 µ
• Oval, central - subterminal spores: 1 X 1.5 µ
with no significant swelling of cell
• Spores are NOT usually present in clinical
specimens unless exposed to atmospheric O2
B. anthracis, Gram stain
demonstrating spores
B. anthracis
Colonial Morphology
• Colonial morphology of 18-24hr @ 35 C:
– Well isolated colonies are 2-5 mm in diameter
– Flat or slightly convex, irregularly round
– Edges: slightly undulate, often curly tailing
edges
– Ground glass appearance
– “Sticky” consistency….stands up like beaten
egg whites
B. anthracis,
colony on SBA
“STICKY” consistency of B.
anthracis’ colony on SBA
Bacillus anthracis
Presumptive Identification
• Gram-positive, broad rod, catalasepositive, spore-positive, aerobe:
Bacillus sp.
• Spores are oval and nonswelling with
ground glass colony appearance:
Bacillus morphology group 1, includes
B. anthracis, B. cereus, B cereus var
mycoides, and B. thuringiensis
Bacillus anthracis
Presumptive Identification, con’t
• Nonmotile: B anthracis and B
cereus var mycoides (and B.
megaterium)
• Nonhemolytic, forms capsule:
Presumptive B. anthracis
• Refer to state lab for testing
Yersinia pestis
Plague
Plague: Overview
• Natural vector - Rodent flea
• Mammalian hosts
– rats, squirrels,
chipmunks, rabbits, and
carnivores
• Enzootic or Epizootic
Plague Epidemiology
• U.S. averages 13 cases/yr
• 30% of cases are in Native Americans in the
Southwest. 15% case fatality rate
• Most cases occur in summer and near the
patient’s residence
– bubonic (infected lymph nodes)
– septicemic (blood-borne organisms)
– pneumonic (transmissible by aerosol;
deadliest)
Yersinia pestis
Specimen Selection
• Specimen selection is important
– Bubo - lymph node aspirate
– Blood - organisms may be intermittent. Take
three specimens 10-30 minutes apart
– Pneumonic
• Sputum/throat - use Wayson stain
• Bronchial washings - Wayson stain
• Inoculate routine plating media
Sentinel Lab Procedures
Yersinia pestis
• Gram stain
• Wayson stain
• Growth characteristics on agar
• Growth characteristics in broth
Yersinia pestis
Gram stain
• Small, gram-negative coccobacilli
Yersinia pestis
Wayson Stain
• Used for rapid assessment
– when it is a part of the identification process
• Best with tissue, sputum, blood
• Stains of pure culture isolates tend to lose bipolarity
• Pink-blue cells with polar granules (safety pin
appearance)
Yersinia pestis
Wayson Stain
• Pink-blue cells with a closed safety pin look
Wayson stain alone is not diagnostic
Y.pestis
48 h culture on SBA
72 h culture on SBA
Yersinia pestis
Technical Hints
• Small Gram-negative, poorly staining rods
from blood, lymph node aspirate, or
respiratory specimens
• Safety pin appearance in Gram, Wright,
Giemsa, or Wayson stain
• More than one patient in a short, specified
period with fever, lymphadenopathy
• Refer to state lab
Francisella tularensis
Tularemia
Tularemia: Overview
• Disease of Northern Hemisphere
• In U.S., most cases associated with rabbits/hares
and ticks
• About 200 cases/year in U.S.
– most in South central and Western states
– majority of cases in summer, some in winter
Reported Cases of Tularemia 1990-1998
Tularemia: Overview (cont’d)
• Several forms of human tularemia exist:
- Ulceroglandular, glandular, oculoglandular,
oropharyngeal, intestinal, pneumonic, and
typhoidal
• Low infectious dose
–1 to 10 organisms by aerosol or intradermal
route
• No person-to-person transmission
Tularemia: Specimen Selection
• Serum - acute and convalescent
• Blood cultures
• Sputum
• Swab – ulcer or eye
Sentinel Lab Procedures
Francisella tularensis
• This is a dangerous, highly virulent organism
and it should not be manipulated at the
bench. Laboratory-acquired infections can
occur easily.
• Gram stain
• Growth characteristics in broth
• Growth characteristics in agar
Francisella tularensis
• Poorly staining, tiny Gram-negative coccobacilli
Francisella tularensis
Growth Characteristics
• Fastidious, requires cysteine for robust growth:
Cysteine Heart Agar (CHA) is ideal
– Enriched chocolate agar + 9% sheep blood + cysteine
– Not part of Sentinel Lab routine procedures
– BCYE (for Legionella) also works
• Will grow initially on sheep and chocolate blood agar
and Thayer-Martin agar, but poorly or not at all on
passage
• Grows slowly at 35oC, poorly at 28oC
Francisella tularensis
Growth Characteristics
• 24 hours
– gray-white, translucent colonies
– usually too small to be seen individually
• 48 hours
– Sheep Blood Agar - <1 mm, gray-white, opaque, no
hemolysis
Francisella tularensis
Sheep blood agar
Chocolate agar
Cysteine heart agar
Francisella tularensis
Technical Hints
If you see:
• Tiny, Gram-negative coccobacilli from
blood, lymph node aspirate, or respiratory
specimens
• Blood isolates that will grow slowly on
chocolate agar but poorly or not at all on
blood agar in 24 hours
• Faint growth in thio; requires cysteine in
other broth
• Refer to state lab
Brucella spp.
Brucellosis
BRUCELLOSIS
• A zoonotic disease caused by any of 4
Brucella sp.: abortus, melitensis, suis,
and canis
• A systemic infection characterized by
an undulant fever pattern
• But relatively rare in the U.S. with
approximately 100 cases/yr
BRUCELLOSIS:
TRANSMISSION
• Unpasteurized dairy products
– The most common mode of transmission
• Direct skin contact
– Occupational hazard for farmers,
butchers, veterinarians, and laboratory
personnel
• Aerosols
– Highly infectious
BRUCELLOSIS
•Infective dose = 10 -100 organisms
•Incubation period = 5 days - > 6 months
•Duration of illness = weeks to months
•Fever, profuse sweating, malaise, headache
and muscle/back pain.
•Person to person transmission = no
•Mortality = <5%
•Persistence of organism = very stable
Brucella spp.
Specimen Selection
• Serum
– The diagnosis of brucellosis is frequently
achieved by serology. An acute &
convalescent phase specimen should be
collected (21d apart)
• Blood or bone marrow
– Sources from which Brucellae are most
often isolated
• Tissue (spleen, liver)
– Brucellae occasionally isolated
Brucella spp.
Biosafety Alert
• Brucellosis is THE most commonly
reported laboratory-associated
bacterial infection.
• Cases have occurred in clinical
laboratory settings by “sniffing”
cultures, direct skin contact with
cultures, and aerosol generating
procedures
Sentinel Lab Tests
Brucella spp.
• Colonial morphology on SBA
• Gram stain morphology
• Oxidase positive
• Urea hydrolysis positive
Brucella spp.
Key Sentinel Lab Tests
Colonial morphology on SBA
–Fastidious
–Visible growth may take 48 - 72 hrs
–Small (0.5-1.0mm), convex, glistening
– Non-hemolytic and non-pigmented
B. melitensis on sheep blood agar
Brucella spp.
Key Sentinel Lab Tests
Gram Stain Morphology
–Tiny (very)
–Faintly staining
–Gram-negative coccobacilli
–0.5 - 0.7 x 0.6 - 1.5
Brucella spp.
Review of Key Tests
• Tiny, faintly staining, gram-negative
coccobacilli from blood or bone marrow
• Slow growth on Sheep Blood Agar, 2-3 days
for colony appearance
• Oxidase +
• Urease +
• Handle plates with care
• Refer to state lab
Clostridium botulinum
Botulism
Botulism: Overview
• Caused by toxin from Clostridium botulinum
– toxin types A, B, E, most commonly associated with
human disease
– most potent lethal substance known to man (lethal
dose 1ng/kg)
• C. botulinum spores found in soil worldwide
• Approximately 100 reported cases/year in the U.S.
– infant most common (72%)
– food borne not common
• No person-to-person transmission
FOODBORNE BOTULISM
• Infective dose: 0.001 g/kg
• Incubation period: 18 - 36 hr (6hr to 10 d)
• Dry mouth, double vision, droopy eyelids,
dilated pupils
• Generalized, progressive descending bilateral
muscle weakness & paralysis
• Respiratory failure and death
• Mortality usually 5 – 10%
FOODBORNE
BOTULISM
• Among 309 persons with clinically diagnosed
botulism reported to CDC from 1975 to 1988:
– Stool cultures for C. botulinum: 51% +
– Serum botulinum toxin testing: 37% +
– Stool botulinum toxin testing: 23% +
• Overall, at least one of the above tests was
positive for 65% of all patients
• Diagnosis is primarily clinical
Sentinel Lab Procedures
for Botulism Event
• Properly collected specimens are to be referred
to designated testing laboratories
• Prior to the shipment of any botulismassociated specimen, testing must be arranged
with MDCH laboratory
Sentinel Lab Procedures
for Botulism Event
Clinical specimens to be collected:
1. Serum
2. Feces
3. Food samples
Autopsy specimens:
1. Serum
2. Gastric and intestinal contents
Botulism
Biosafety Alert
Materials suspected of containing
botulism toxin must be handled:
–Biological Safety Cabinet (Class II)
–Laboratory Coats
–Disposable surgical gloves
–Face shield (as needed)
BOTULISM
• The diagnosis of botulism is made
clinically, i.e., based on the patient’s case
history and physical findings
• Health care providers suspecting
botulism should contact the Michigan
Department of Community Health
Botulism
Referral Lab Procedures
• Mouse bioassay
• Isolation of C. botulinum