Bacterial agents ofbioterroism

Laboratory network forbiological terrorism

Bacillus anthracis

Anthrax

• 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

Anthrax: Overview

CDC

ANTHRAX

• Three forms of human anthrax occur:1. Cutaneous2. Gastrointestinal

•Oropharyngeal•Abdominal

3. Inhalation (Woolsorter’s Disease)

Vesicle development, day 2Eschar formation, day 4

Cutaneous anthrax

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 anthracisKey Sentinel Lab Tests

• Gram stain• Growth characteristics on

agar• Sporulation, in air• Motility• Capsule by India Ink

• 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

Bacillus anthracisBacillus anthracisGram Stain MorphologyGram Stain Morphology

B. anthracis, Gram staindemonstrating spores

• 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. B. anthracisanthracisColonial MorphologyColonial Morphology

B. anthracis, colony on SBA

““STICKY” consistency of STICKY” consistency of B. anthracis’B. anthracis’ colony on colony on SBASBA

• Gram-positive, broad rod, catalase-positive, 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 anthracisBacillus anthracisPresumptive IdentificationPresumptive Identification

• Nonmotile: B anthracis and B cereus var mycoides (and B. megaterium)

• Nonhemolytic, forms capsule: Presumptive B. anthracis

• Refer to state lab for testing

Bacillus anthracisBacillus anthracisPresumptive Identification, con’tPresumptive Identification, con’t

Yersinia pestis

Plague

• Natural vector - Rodent flea • Mammalian hosts

– rats, squirrels, chipmunks, rabbits, and carnivores

• Enzootic or Epizootic

Plague: OverviewPlague: Overview

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 pestisSpecimen 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 pestisGram stain

• Small, gram-negative coccobacilli

Yersinia pestisWayson 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 pestisWayson Stain

Wayson stain alone is not diagnostic

• Pink-blue cells with a closed safety pin look

Y.pestis

48 h culture on SBA

72 h culture on SBA

• 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

Yersinia pestisYersinia pestisTechnical HintsTechnical Hints

Francisella tularensisFrancisella 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 Reported Cases of Tularemia - 1990-1998- 1990-1998

• Low infectious dose

–1 to 10 organisms by aerosol or intradermal route

• No person-to-person transmission

Tularemia: Overview (cont’d)

• Several forms of human tularemia exist:

- Ulceroglandular, glandular, oculoglandular,

oropharyngeal, intestinal, pneumonic, and

typhoidal

Tularemia: Specimen Selection

• Serum - acute and convalescent

• Blood cultures

• Sputum

• Swab – ulcer or eye

Sentinel Lab ProceduresFrancisella 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 tularensisGrowth 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 tularensisGrowth 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 tularensisTechnical Hints

• 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

If you see:

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

•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

BRUCELLOSISBRUCELLOSIS

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

• 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

Brucella Brucella spp.spp.Biosafety AlertBiosafety Alert

• Colonial morphology on SBA

• Gram stain morphology

• Oxidase positive

• Urea hydrolysis positive

Sentinel Lab TestsSentinel Lab TestsBrucellaBrucella spp. spp.

Colonial morphology on SBA

–Fastidious

–Visible growth may take 48 - 72 hrs

–Small (0.5-1.0mm), convex, glistening

– Non-hemolytic and non-pigmented

BrucellaBrucella spp. spp.Key Sentinel Lab TestsKey Sentinel Lab Tests

B. melitensis on sheep blood agar

Gram Stain Morphology

–Tiny (very)

–Faintly staining

–Gram-negative coccobacilli

–0.5 - 0.7 x 0.6 - 1.5

BrucellaBrucella spp. spp.Key Sentinel Lab TestsKey Sentinel Lab 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

Brucella spp.Brucella spp.Review of Key TestsReview of Key Tests

Clostridium botulinum

Botulism

• 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

Botulism: OverviewBotulism: Overview

• 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 BOTULISMFOODBORNE BOTULISM

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 botulism-

associated specimen, testing must be arranged with MDCH laboratory

Clinical specimens to be collected:

1. Serum

2. Feces

3. Food samples

Autopsy specimens:

1. Serum

2. Gastric and intestinal contents

Sentinel Lab ProceduresSentinel Lab Proceduresfor Botulism Eventfor Botulism Event

Materials suspected of containing botulism toxin must be handled:

–Biological Safety Cabinet (Class II)

–Laboratory Coats

–Disposable surgical gloves

–Face shield (as needed)

BotulismBotulismBiosafety AlertBiosafety Alert

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