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Study Highlights Genomic Contributions to Amerithrax Investigation

By Andrea Anderson

NEW YORK (GenomeWeb News) – In a study appearing online this week in the Proceedings of the National Academy of Sciences, an American research team describes how they used genomics to help track down the source of Bacillus anthracis spores used in the 2001 Amerithrax attacks.

The study marks the first time that bacterial whole-genome sequencing was used as a microbial forensics tool, lead author David Rasko, a microbiology and immunology researcher with the University of Maryland School of Medicine's Institute for Genome Sciences, told GenomeWeb Daily News.

Five people died and more than a dozen other individuals contracted anthrax in 2001, when B. anthracis-tainted letters were delivered to the New York Post, NBC television, and the offices of two American senators in Washington, DC.

As part of the investigation into the attacks, researchers from the University of Maryland, the US Federal Bureau of Investigation, and elsewhere used whole-genome sequencing, comparative genomics, and high-throughput PCR screening to characterize B. anthracis spores belonging to the Ames strain that were found in the letters. The analyses uncovered sub-populations of spores with specific mutations and morphological features distinct from those detected in Ames ancestor isolates or environmental anthrax samples.

By using this genetic information to screen through a repository of Ames isolates from labs around the world, researchers helped narrow down the number of suspects in the case. The information, along with other lines of evidence, linked the attacks to prime suspect Bruce Ivins, a US Army biodefense scientist who had access to B. anthracis with genetic features matching the Amerithrax morphotypes.

"This study highlights the resolving power of whole-genome sequencing and careful comparative genomic analyses," Rasko and his co-authors wrote. "When applied to microbial forensics, bacterial whole-genome sequence data appear to be the ultimate evidence; much like a human genetic fingerprint, it could link microbial evidence to its source."

Traditionally, forensics investigations involving microbes have relied on strategies such as targeted sequencing and genotyping, Rasko and his co-authors noted. In the Amerithrax case, though, investigators were tasked with trying to eke out clues from a collection of genetically homogenous B. anthracis spores.

Through multiple-locus variable-number tandem repeat (MLVA) and multi-locus sequence typing, for instance, the team found that the anthrax spores in all of the letters belonged to the Ames strain. But that's as far as they could get using such approaches.

"Using those traditional methods we could narrow it down: it was an Ames strain and it was of a specific genotype," Rasko said. "But once we got past that there was really no way to do this other than genome sequencing — complete genome sequencing."

Over the course of the investigation, researchers not only sequenced the 5.2 million base pair genomes of wild type and mutant morphotype spores collected during the Amerithrax investigation, they also generated a reference genome sequence for the so-called Ames Ancestor — an isolate collected in Texas in the early 1980s that's thought to be ancestral to all Ames isolates currently found in labs worldwide.

Generating this reference sequence was a key first step, Rasko noted, providing a framework for the team's subsequent analyses. All of the whole-genome sequencing for the study was performed using the Sanger approach.

During their subsequent genome comparisons, researchers found that wild type spores collected during the Amerithrax investigation all matched the Ames reference genome.

But sub-populations of spores with distinct morphological features — including specific sporulation features — were also present in the Amerithrax letters. And spores from each of these morphotypes contained SNPs, insertions, and deletions not present in the wild type spores or the Ames Ancestor.

From these genetic differences, the team came up with high-throughput PCR assays that they subsequently used to screen a collection of B. anthracis Ames samples assembled by the FBI.

Although some samples in the repository carried one or two of these genetic changes, Rasko explained, only a small handful harbored isolates with all of the genetic markers.

And, he added, all of these could be traced back to a parent sample at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) lab where Ivins worked. Ivins committed suicide in 2008 before the case went to trial.

"This paper and the Amerithrax investigation really marked the beginning of a new approach for the science we call forensic genomics," senior author Jacques Ravel, a University of Maryland microbiology and immunology researcher and associate director for genomics at the Institute for Genome Sciences, said in a statement. "Without genomics, it would have been extremely difficult to narrow the pool of potential suspects."

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