Bioterrorism Agent Case
Studies
Beth Schweitzer MT(ASCP), SM
Microbiology Specialist
Website: www.nphl.org
NPHL
Nebraska Public
Health Lab
Case Format
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Lack of pertinent patient information
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Travel history
Occupation
Symptoms
Duration of Illness
Case Format
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Cultures are set according to NPHL
Operating Procedures
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BAP
Chocolate
MacConkey
Thioglycolate Broth
Zoonotic Diseases
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Naturally occurring

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Cases in NE
Safety concerns for laboratorian
Manuel’s Mystery


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Blood Cultures drawn on 16 y/o Hispanic
male
Instrument flags bottles positive at day 4
 Gram stain: NOS
 Bottles blind subbed and placed back on
machine
Bottles discarded on day 5 (standard
operating procedure)
Con’t

BAP and Choc show a haze of growth at 48
hours ( 6th day of original Blood culture)

Gram stain: tiny gram- negative coccobacilli

What organisms should you be
thinking!!!
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Haemophilus
Pasturella
Oligella
Bordetella
Brucella
Francisella

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Acinetobacter
Psychrobacter
Think Biosafety
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In a biosafety cabinet you perform
following:
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Identification for Haemophilus
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Rapid NH
Quad/Tri Plate
Staph streak
Think Biosafety

Reactions do not fit the pattern for
Haemophilus!!

What now?
Think Biosafety

Do not perform automated commercial
identification on slow growing-GNCB
that do not grow on MAC/EMB.

The risk of aerosols is too high!
Think Biosafety

In a biosafety cabinet you perform
following:


Urease slant (Pos in 2 hours)
Tube motility (Neg at 48 hours)
TEST
Oxidase Motility Urease Nitrate BAP X/V
Brucella
Bordetella
Acinetobacter
Psychrobacter
Oligella
Haemophilus
+ 95%
+
+
+
V
+
V
-
+
+
V
+
+
V
+
+
V
+
+
+
+
+
+
+
-
+
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What organism can’t we rule out?
Brucella spp.
Brucella spp. - Biosafety Alert:


Brucellosis was 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 (e.g. culture manipulation)
Brucellosis:

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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.
NO Person to person transmission
Mortality: <5%
Persistence of organism: very stable
Brucella spp.-Specimen Selection:

Serum
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Blood or bone marrow
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The diagnosis of brucellosis is frequently achieved by
serology. An acute & convalescent phase specimen
should be collected (21 days apart)
Most often isolated from these
Tissue (spleen, liver)

Occasionally isolated
Brucella spp. – Technical hints

Oxidase positive, rapid urease
positive, non-motile, slow growing
gram negative-coccobacillus
Naturally occurring

Patient was a visitor from Mexico and
had ingested unpasteurized goat cheese
Loretta’s Lymph

Lymph node aspirate from a 35 y/o
female
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Gram stain: Moderate WBC’s
Few Gram- negative rods
Growth at 24 hours
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Tiny (1-2 mm) grey colonies on BAP
and Choc
Non-lactose fermenter on Mac
Biochemical tests
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Oxidase (-)
Indole (-)
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Use commercial ID system
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What organism could it be?
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Fits Enterobacteriaceae pattern
Without patient history hard to create a
differential
Y. pestis – commercial ID systems
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Included in the data base of MicroScan,
Vitek, and API 20E.. etc
True accuracy not yet determined
60% of California’s Y. pestis ID’s are from
commercial systems
Y. pestis – commercial ID systems


If ID as Y. pestis – send to NPHL
May ID as Shigella ssp, H2S- negative
Salmonella, Acinetobacter, or Y.
pseudotuberculosis
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Biochemically inert nature of organism
Y. pestis –commercial ID systems
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How do we know when to question ID
results??
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Source
Colony morphology
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H2S neg Salmonella
Shigella characteristics
Inpatient/outpatient status-nosocomial
infection.
Plague Epidemiology:
 During 1988-2002, 112 human cases were reported
from 11 western states
 30% of cases are in Native Americans in the
Southwest.
 15% case fatality rate
 80% of cases occur in summer and near the patient’s
residence- FLEAS
Y. pestis - Specimen Selection:
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Specimen selection is important
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Bubonic
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Bubo - lymph node aspirate
Septicemic - blood - organisms may be
intermittent. Take three specimens 10-30
minutes apart
Pneumonic
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Sputum/throat
Bronchial washings
Y. pestis - Gram stain from sputum
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Small, gram-negative rods
Y. pestis - Direct Culture
Growth on Agar Plate:
Y. pestis - Growth in Broth:
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Thioglycolate Broth (may see from original
specimen set-up)
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Do not shake tubes
Observe suspended flocculent clumps on side and
bottom of tube. Broth remains clear
May also happen with Y. pseudotuberculosis or S.
pneumoniae
Y. pestis – Growth in Broth culture:
Y. pestis
Y. pseudotuberculosis
Y. pestis - Technical Hints:
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Small gram-negative, poorly staining rods from
blood, lymph node aspirate, or respiratory
specimens, BLOODY SPUTUM
Naturally Occurring
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Couple from New York City infected by
flea bites on trip to New Mexico
Returned home to NYC before
becoming ill
Bob’s Booboo
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Aerobic culture is collected from wound
on the hand of a 45 y/o male
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Gram stain: NOS
Mod WBC
Con’t
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At 48 hours there is slight haze growth
on the BAP and Choc

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Gram stain: faintly staining gram- negative
coccobacilli
What organisms should you be
thinking!!!






Haemophilus
Pasturella
Oligella
Bordetella
Brucella
Francisella



Acinetobacter
Psychrobacter
Actinobacillus
Think Biosafety

Do not perform automated commercial
identification on slow growing-GNCB
that do not grow on MAC/EMB.

The risk of aerosols is too high!
Think Biosafety

In a biosafety cabinet growth you
perform the following:

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Oxidase (Negative)
Manual commercial identification system
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No identification
Reisolate to BAP and Choc
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No growth on BAP after reisolation
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What to do now???!?!?!
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Haemophilus is a likely organism
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Haemophilus quad plate is set for
identification
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No growth is seen
Its NOT Haemophilus… What Now?
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In a biosafety cabinet the following
tests are performed:
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Motility (Negative at 24 hours)
Urease (Negative at 24 hours)
B-lactamase (+)
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What organism can’t we rule out?
Francisella
F. tularensis – Gram stain:
Poorly staining, tiny Gram-negative coccobacilli
Francisella/E. coli
F. tularensis – growth
characteristics:
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Will grow initially on sheep blood agar,
chocolate agar and Thayer-Martin agar, but
poorly or not at all on sub
Grows slowly at 35oC, poorly at 28oC
24 hours on BAP and Choc

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gray-white, translucent colonies
usually too small to be seen individually
F. tularensis – growth
characteristics:

Thayer Martin is a good selective media
from a mixed culture
F. tularensis - Colony morphology
F. tularensis SCHU strain on cysteine heart agar with 9% sheep blood
(left) and chocolate agar (right), 72 hours
F. tularensis - Colony Morphology
F. tularensis NV972342 isolate on CHAB agar. Opalescent purplish
sheen under oblique light
F. tularensis:
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Infective dose: 10-50 organisms Very Low !
Incubation period: 1-21 days (average=3-5 days)
Duration of illness: ~2 weeks
NOT Contagious person to person
Mortality: treated: low
untreated: moderate
Persistence of organism: months in moist soil
Vaccine efficacy: good, ~80%
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Not presently available
Francisella – specimen collection
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Serum- like Brucella most often diagnosed by
serology testing, not by culture
Ulceroglandualar
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Pneumonic
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Wound culture
Sputum
Lymph node aspirate
Typhoidal
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Blood
CSF
sputum
F. tularensis - Technical Hints:
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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
Naturally Occurring
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Patient is a veterinarian
Gordo’s Glass Jaw
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Anaerobic culture received from jaw of
67 y/o male
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Gram stain: large chaining Gram- positive
rods
What could you do now?
Visualize capsule
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Perform India Ink directly from sample
to visualize capsule
Will only see capsule directly from
specimen
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It will not be visible upon sub-culture
B. anthracis – Colony Morphology:
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Colonial morphology of 18-24hr @ 35 C:
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Well isolated colonies are 2-5 mm in
diameter
Flat or slightly convex, irregularly round
Edges: slightly undulate, often curly tailing
edges- “Medusa Head”
Ground glass appearance
“Sticky” or “Tenacious” consistency….stands
up like beaten egg whites
Presence of Spores

Spores will only be seen after
incubation in ambient conditions
Capsule
B. anthracis - colony on SBA
Sticky colonies
Bacillus anthracis – India Ink
Anthrax: Inhalational

Infective dose
 Naturally occurring= 8,000 - 15,000 spores
 Weaponized = ??? maybe 10 to 100 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
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NO Person to person transmission
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Anthrax- Specimen Selection
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Inhalational: Sputum and Blood
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Cutaneous: Vesicles and Eschar
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Gastrointestinal: Stool and Blood
B. anthracis- Technical Hints:

Catalase positive, non-motile, spore forming
Gram- positive rods
Naturally Occurring

Patient was a rancher from North
Dakota that recently had 5 cows die of
anthrax