Anthrax investigation still yielding findings: Chemical composition of spores doesn't match suspect flask/Nature

This is an important short piece from Nature (online February 25, 2009) that I missed last February. It explains why the anthrax letter spores did not come directly from Ivins' flask: they incorporate metals not found in Ivins' spores, and were therefore grown under different conditions. Their progenitors probably did come from the flask, but dozens and possibly hundreds of scientists had access to samples from that flask.

Furthermore, the Bacillus subtilis contaminant present in the letters has not been matched to the strain found in Ivins' lab. The FBI speculates it might have come from another lab at Fort Detrick, but has not yet identified any lab that has it. The case against Ivins just gets weaker.

The deadly bacterial spores mailed to victims in the US anthrax attacks, scientists say, share a chemical 'fingerprint' that is not found in bacteria from the flask linked to Bruce Ivins, the biodefence researcher implicated in the crime.
The Federal Bureau of Investigation (FBI) alleges that Ivins, who committed suicide last July, was the person responsible for mailing letters laden with Bacillus anthracis to news media and congressional offices in 2001, killing five people and sickening 17. The FBI used genetic analyses to trace the mailed spores back to a flask called RMR-1029, which Ivins could access in his laboratory at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) in Fort Detrick, Maryland.

At a biodefence meeting on 24 February, Joseph Michael, a materials scientist at Sandia National Laboratories in Albuquerque, New Mexico, presented analyses of three letters sent to the New York Post and to the offices of Senators Tom Daschle and Patrick Leahy. Spores from two of those show a distinct chemical signature that includes silicon, oxygen, iron, and tin; the third letter had silicon, oxygen, iron and possibly also tin, says Michael. Bacteria from Ivins' RMR-1029 flask did not contain any of those four elements.

Two cultures of the same anthrax strain grown using similar processes — one from Ivins' lab, the other from a US Army facility in Utah — showed the silicon-oxygen signature but did not contain tin or iron. Michael presented the analyses at the American Society for Microbiology's Biodefense and Emerging Diseases Research Meeting in Baltimore, Maryland.

The chemical mismatch doesn't necessarily mean that deadly spores used in the attacks did not originate from Ivins' RMR-1029 flask, says Jason Bannan, a microbiologist and forensic examiner at the FBI's Chemical Biological Sciences Unit in Quantico, Virginia. The RMR-1029 culture was created in 1997, and the mailed spores could have been taken out of that flask and grown under different conditions, resulting in varying chemical contents. "It doesn't surprise me that it would be different," he says.

The data suggest that spores for the three letters were grown using the same process, says Michael. It is not clear how tin and iron made their way into the culture, he says. Bannan suggests that the growth medium may have contained iron and tin may have come from a water source.

Hard to tell apart

The meeting offered scientists who collaborated with the FBI during the investigation an opportunity to share detailed data. The analyses will eventually be published in peer-reviewed journals, the FBI has said.

Jacques Ravel, a genomics scientist at the University of Maryland School of Medicine in Baltimore, described his team's efforts to find genetic differences between various cultures of the Ames strain, theB. anthracis strain identified in the anthrax letters. At first, the team was surprised to find that the DNA sequences of a reference Ames strain and Ames samples from the investigation, such as bacteria isolated from the spinal fluid of the first victim, were exactly the same. "It was kind of a shock," says Ravel.

For help, the researchers turned to variants found by a team at USAMRIID. Patricia Worsham and her colleagues had noticed differences in shape, colour and rate of spore formation even within a single anthrax culture. Ravel's team identified the genetic mutations associated with four variants and developed an assay for one of them, called Morph E. Researchers at Commonwealth Biotechnologies in Richmond, Virginia, and the Midwest Research Institute's Florida Division in Palm Bay created assays for three other variants.

The FBI then used that arsenal of tests to pin down the origins of the anthrax letters, matching the mix of genetic variants in the mailed spores to Ivins' RMR-1029 flask. "It has the genetic signatures that identify it as the most likely source of the growth," says Bannan.

Ravel also sequenced the genome of a Bacillus subtilis strain that was found in one of the letters. That sample did not match a B. subtilis strain found in Ivins' lab, says Bannan, but the bacterial contamination still could have come from somewhere else in Ivins' institution.

The FBI has asked the National Academy of Sciences (NAS) to convene an independent panel of experts to review the anthrax investigation data. The academy is still in the process of drawing up a contract with the FBI that lays out an agreement to perform the study, says NAS spokeswoman Christine Stencel.