How the Smoking-Gun "Anthrax" Flask at USAMRIID Became a Smoking Gun

In a more complete account of last week's "anthrax" science briefing by the FBI, USA Today describes how the incriminating flask of Bacillus anthracis at USAMRIID, RMR-1029, became so genetically distinctive.

RMR-1029 started out as spores from an original Ames strain isolate, which was obtained from a dead Texas calf in 1981. At the US Army's Dugway Proving Ground, 13 production runs were initially conducted with this Ames isolate. Then USAMRIID scientist Bruce Ivins ran another 22 runs, to produce 164 liters of spores in 1997. Later Ivins concentrated the spore collection, called at this time RMR-1029, to 2 flasks in 2001 and then one 1-liter flask in 2004.

Because RMR-1029 had therefore been produced from so many generations of B. anthracis (Paul Keim of Northern Arizona University estimated that one spore colony might represent up to a trillion generations, wrote the paper), subpopulations of spores in the collection harbored distinctive mutations.* Four of these mutations were used by investigators to trace the letter spores back to RMR-1029.

*Strains of B. anthracis are usually highly genetically conserved, because spores in the wild typically remain dormant in the soil for such a long period of time before growth in an infected animal.

Scanning electron micrograph of spores of Ames strain of B. anthracis from CDC/Janice Haney Carr.

Addendum: USA Today, unlike other media outlets, also provided the FBI's list of scientific publications that relate directly to the anthrax investigation.

• Read TD et al. Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science. 2002;296:2028-2033.
• Cummings CA, Relman DA. Genomics and microbiology: microbial forensics—"cross-examing pathogens." Science. 2002;296:1976-1979.
• Read TD et al. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature. 2003;423:81-86.
• Whiteaker JR et al. Quantitative determination of heme for forensic characterization of Bacillus spores using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem. 2004;76:2836-2841.
• Easterday WR et al. Specific detection of Bacillus anthracis using the TaqMan mismatch amplification mutation assay. Biotechniques. 2005;38:731-735.
• Beecher DJ. Forensic application of microbiological culture analysis to identify mail intentionally contaminated with Bacillus anthracis spores. Appl Environ Microbiol. 2006;72:5304-5310.
• Van Ert MN et al. Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain. J Clin Microbiol. 2007;45:47-53. (Discussed in a previous post, here.)
• Brewer LN et al. Forensic analysis of bioagents by X-ray and TOF-SIMS hyperspectral imaging. Forensic Sci Int. 2008;179:98-106.