Break Up Anthrax Attacks, Sequence the Pieces

Irradiation, a genome-fragmenting defense against weaponized anthrax and other bioterror agents, needn’t frustrate forensic investigations that rely on whole-genome sequencing. [Janice Haney Carr, CDC]

A strong dose of gamma-irradiation—the standing defense against weaponized anthrax—is so good at breaking apart anthrax DNA that it could complicate forensic analysis, the next step in responding to a bioterror attack. Forensic analysis has the difficult job of reading the subtle genomic signatures of weaponized anthrax and other bioterror agents, which are hard to distinguish from those of common laboratory strains. Yet these signatures, however shredded, may still be readable by means of whole-genome sequencing.

Although forensic science has found that gamma-irradiation of microbial DNA won’t necessarily complicate investigations based on the real-time polymerase chain reaction (PCR), relatively little attention has been devoted to establishing the usefulness of whole-genome sequencing, an alternative technique that is coming into wider use. Unlike real-time PCR, which requires small DNA fragments for amplification, some whole-genome sequencing approaches require larger fragments and potentially several enzymatic processing steps to construct the libraries used in sequencing instruments.

To resolve the uncertainty over the usefulness of whole-genome sequencing in tracing genomic variants to their sources, microbiologists based at the U.S. Army’s Edgewood Chemical Biological Center at the Aberdeen Proving Grounds studied an anthrax surrogate, Bacillus atrophaeus. Like anthrax, B. atrophaeus makes spores, but it is nonpathogenic. The investigators also used a vaccine strain of Yersinia pestis, a non-spore-forming bacterium that causes plague.

In their work, the microbiologists used strains that had a number of genetic mutations, as compared to the reference strains. The microbiologists hoped that these mutations would be detectable by means of whole-genome sequencing even after the genomes harboring the mutations had been irradiated to fragments.

After the investigators sequenced the fragments, they documented the sequences. Then the investigators entered the fragment sequences into a computer, which they programmed to determine the full sequences of the strains. “The full sequences were almost indistinguishable from control materials,” said the Chemical Biological Center’s Henry S. Gibbons, Ph.D. “All known mutations were found in the irradiated materials.”

This encouraging result appeared November 13 in the journal Applied and Environmental Microbiology, in an article entitled, “Whole Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials.”

“[We] determine the effects of high-dose gamma-irradiation on forensic markers of bacterial biothreat agent surrogate organisms with particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis,” wrote the article’s authors. “While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we find that irradiated material could be utilized to generate accurate whole-genome sequence data on Illumina and Roche 454 sequencing platforms.”

This work research was motivated by the need for quick recovery of microbial signatures following deliberate releases such as the 2001 anthrax attacks, said Dr. Gibbons. These attacks, which began a week after 9-11, and were aimed partly at Congressional offices, killed a photo editor, two postal workers, and two others, the first anthrax deaths in the United States in 25 years. Since then, irradiation of mail has become routine at sensitive government facilities.

“Rapid sequencing of irradiated materials from a biocrime would allow quick characterization of the material,” explained Dr. Gibbons. That could answer important questions. For example, the genome might contain a drug-resistance cassette, important information for knowing how to treat any infections.

More generally, rapid sequencing could also reveal whether a strain had been genetically engineered. “This was one of the first questions asked during the anthrax investigation,” Dr. Gibbons noted. “It was motivated by the fact that the Soviet program had developed some engineered pathogens.” Dr. Gibbons added that rapid sequencing could also help determine the sophistication of the microbial agents, which would be important in tracking down the perpetrators of biocrimes and bioterror attacks.