Important information on anthrax for clinical microbiologists

Clinical microbiology laboratories should take care not to regard all isolates of Bacillus species as contaminants, especially if isolated from sterile sites (blood, cerebrospinal fluid) and/or multiple cultures are positive from the same patient. Public Health recommends that all sterile site Bacillus isolates be further evaluated. If non-motile or non-hemolytic, and/or if the clinical syndrome is suggestive of anthrax, contact Public Health immediately at 206-296-4774 (24 hours) and refer the isolates immediately to the WA State Public Health Laboratory (PHL) for further testing (Public Health can facilitate transport of isolates to the PHL).

Laboratory issues with respect to diagnosing Bacillus anthracis include:

Bacillus anthracis can be isolated primarily from blood, sputum, CSF, vesicular fluid, a swab of exudate from the eschar, a tissue biopsy and stool (if gastrointestinal anthrax).
Clinical laboratory specimens should be handled in Biosafety Level 2 facilities.
Confirmatory diagnostic testing is available through the WA State PHL; positive specimens would be sent to the CDC for additional testing.

Presumptive identification key for Bacillus anthracis:

Non-hemolytic
Non-motile
Encapsulated (requires India ink to visualize the capsule)
Gram-positive, spore-forming rod

Gram stain morphology of B. anthracis:

Broad, gram-positive rod: 1-1.5 x 3-5 µ occurring singly or in short chains, often with squared off ends
Oval, central to subterminal spores: 1 x 1.5 µ with no significant swelling of cell
Spores usually NOT present in clinical specimens unless exposed to atmospheric O₂
In advanced disease, a gram stain of unspun blood may be positive.

Colonial and isolate characteristics of B. anthracis:

After incubation on a blood agar plate for 15-24 hours at 35-37^o C, well isolated colonies are 2-5 mm in diameter; heavily inoculated areas may show growth in 6-8 hours
Gray-white, flat or slightly convex colonies are irregularly round, with edges that slightly undulate, and have "ground glass" appearance
Often have comma-shaped protrusions from colony edge ("Medusa head" colonies)
Tenacious consistency (when teased with a loop, stands up like a beaten egg white)
Non-hemolytic (weak hemolysis may be observed under areas of confluent growth in aging cultures and should NOT be confused with real ß-hemolysis)
Non-motile
Susceptible to gamma phage lysis

Detailed guidelines for testing for Bacillus anthracis are available on the CDC website at: www.bt.cdc.gov/Agent/Anthrax/LevelAProtocol/
Anthracis20010417.pdf or www.bt.cdc.gov

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