TGen-NAU study generates Soviet anthrax pathogen genome from autopsy specimens

Next generation genomic analysis used to probe the former USSR’s biological weapons program

FLAGSTAFF, Ariz. — Sept. 7, 2016 — A new study by the Translational Genomics Research Institute (TGen) and Northern Arizona University (NAU) used deep DNA sequencing methods to generate the anthrax genome sequence from the victims of the 1979 anthrax outbreak in Sverdlovsk, Russia, when it was part of the USSR.

The Soviet Union produced anthrax spores on an industrial scale but repeatedly denied the existence of their biological weapons program. This study, to be published in the September issue of the journal mBio, represents a precise and detailed examination of the anthrax strain used in their weapons development, and includes an anthrax genetic database that puts the weapons strain into a global context.

“I have been studying this anthrax outbreak and these specimens for more than 20 years. Finally, using genomic technology, we could comprehensively characterize this pathogen genome,” said Dr. Paul Keim, a Regents Professor of Biology and the Cowden Endowed Chair of Microbiology at NAU, Director of TGen’s Pathogen Genomics Division, and the study’s lead author.

“This is the signature agent of the world’s largest biological weapons program and now we have it in our genetic databases. Anywhere this strain shows up again, we will be able to identify it and track it back to its source. This is now an essential part of our forensic arsenal,” said Dr. Keim, who also is Director of NAU’s Microbial Genetics & Genomics Center (MGGen).

The anthrax bacterium produces small capsules, or spores, that can lie dormant for decades. After settling inside the human lung, for example, it can cause a severe disease that, if not treated with antibiotics, kills 90 percent of those it infects.

Anthrax is found in many parts of the globe and dispersed through the human movement of animal parts contaminated with spores. Wool and hair from goats and sheep are moved globally as textiles or their precursors. When these originate in anthrax endemic regions, they can carry the spores, which are long-lived. While this bacterium has little variation from strain to strain, whole genome sequencing has identified DNA fingerprints that enable molecular epidemiology, tracing it to its source. When anthrax outbreaks occur, their whole genome profile are now routinely compared to the genetic database to identify possible sources and exclude others. This type of analysis was used by the FBI to track the spores in the 2001 anthrax letter attacks, which infected 22 people and killed five.

The Soviet Union had signed the Biological Weapons convention that prohibited the use of biological agents, including anthrax, as weapons. The United States’ biological weapons program was eliminated in a decree by President Richard Nixon in 1969, but the Soviet program was maintained and expanded in a covert fashion for decades.

In 1992, an investigative team from the United States led by noted Harvard biologist Dr. Matt Meselson characterized the 1979 Sverdlovsk outbreak by interviewing local physicians, visiting cemeteries and examining autopsy specimens. This investigation, along with accounts by Ken Alibek, a former Soviet scientist, revealed that the Sverdlovsk anthrax outbreak was due to an industrial accident.  A faulty filter at a Soviet spore production facility allowed anthrax spores, in a silent plume, to drift with the wind over the city and into the nearby countryside. Nearly 70 Sverdlovsk inhabitants died as far as three miles downwind from the facility, but more anthrax-susceptible farm animals died over 25 miles away. It remains the world’s deadliest human outbreak of inhalation anthrax.

The bacterial genomes were generated from autopsy tissue specimens of two Sverdlovsk anthrax victims. These tissues were moved to the United States with permission of Sverdlovsk pathologists to continue the investigation into the disease outbreak. From these, it was established that the anthrax pathogen was detected within their tissues and the victims died from inhaling the spores.

The Sverdlovsk anthrax genome was compared to the global genome database maintained by NAU to identify its close relatives and to look for evidence of genetic engineering. The Flagstaff research team found that this strain was closely related to other Asian isolates with very few differences to naturally occurring anthrax. There were no signs of genetic engineering.

Dr. Keim notes that the Soviets had to be very meticulous to avoid mutant variants from dominating their production stock. Invariably when wild anthrax strains are grown extensively in the laboratory, they adapt to those conditions and lose the killing power.

“The Sverdlovsk strain’s genome looked very much like those of wild strains we see across Asia,” Dr. Keim said.

Dr. Meselson, who was not part of the current paper, notes: “If this strain had been grown repeated in the laboratory, it would have mutated to a form that had less virulence and less capacity to cause anthrax. The Soviet scientists must been very meticulous in their maintenance of the natural form.”

Dr. Meselson, who is the Thomas Dudley Cabot Professor of the Natural Sciences at Harvard, is known for his 1961 discovery of messenger RNA.

This study was supported by a grant from the U.S. Department of Homeland Security.

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Study validates TGen developed test for healthcare-acquired infections

Molecular-based KlebSeq assay could save lives and lower healthcare costs

FLAGSTAFF, Ariz. — Sept. 2, 2016 — A new study by the Translational Genomics Research Institute (TGen) details the design and validation of a low-cost, rapid and highly accurate screening tool — known as KlebSeq  — for potentially deadly healthcare-acquired infections (HAIs), such as Klebsiella pneumoniae. HAIs affect hundreds of thousands of patients annually and add nearly $10 billion in associated healthcare costs.

The findings, to be published in the October issue of the Journal of Clinical Microbiology, detail the workings of the KlebSeq test at detecting HAIs earlier, in particular Klebsiella, which has multiple strains, such as ST258, which are increasingly resistant to treatment by antibiotics.

Unlike traditional assays that require growing a live culture in a laboratory setting, which adds days to the testing process and layers on cost, KlebSeq employs a technique called amplicon sequencing that identifies the presence of Klebsiella and stratifies its characteristics, such as strain type and whether it may be antibiotic resistant.

“KlebSeq is able to accurately and consistently identify and characterize Klebsiella from many different types of specimen samples, including blood, urine, nasal swabs, and respiratory fluids,” said Dr. Jolene Bowers, a Post-Doctoral fellow in TGen’s Pathogen Genomics Division, TGen North, and the paper’s first author.

In 2015, Bowers co-led a study published in PLOS One, in collaboration with the U.S. Centers for Disease Control and Prevention, which documented the rapid global spread of ST258.

According to the CDC, nearly 2 million Americans annually contract bacterial infections that are resistant to at least one antibiotic, and 23,000 die each year from such infections, nearly twice as many who die of AIDS.

“Improved testing technology holds great potential for the rapid detection of HAIs and more quickly identifying antibiotic-resistant infections, such as K. pneumoniae, which have become an urgent public health crisis,” said Bowers. “KlebSeq is a perfect example of the power of genomic-based analytical tools that deliver results faster, more accurately and at a lower cost.”

According to Dr. David Engelthaler, Director of Programs and Operations for TGen North, and one of the authors of the study, transmission of multidrug-resistant strains of K. pneumoniae is rapid and without initial symptoms, leading to outbreaks in the healthcare system and the community that often go undetected.

“Early detection of K. pneumoniae in healthcare patients, especially those with multidrug-resistant strains, is critical to infection control,” said Dr. Engelthaler, who also is a former epidemiologist for the state of Arizona. “Perhaps most concerning is that Kleb acts like a shuttle for critical resistance genes, often transmitting them to other HAI species. It is important for us to detect both the bacteria and these critical genes.”

KlebSeq can be used for routine screening and surveillance, enabling healthcare staff to make more informed patient decisions, and curb outbreak situations by rapidly identifying transmissions prior patients showing signs of infection. Classifying the type of infection in each patient would help enable an institution to decide when and which intervention procedures to enact.

Study results suggest that KlebSeq would be especially helpful for high-risk patients — those in intensive-care units, centers specializing in bone marrow transplantation or chronically immunosuppressed patients, long-term care facilities, and travelers returning from endemic regions.

“The sensitivity of KlebSeq is superior to culture-based methods,” said Dr. Paul Keim, Director of TGen North and the senior author of the study.

“KlebSeq is an important step toward a comprehensive, yet accessible, tool for all pathogen identification and characterization,” said Dr. Keim, who also is the Cowden Endowed Chair of Microbiology at Northern Arizona University, and Director of NAU’s Center for Microbial Genetics and Genomics (MGGen).

The results also suggest that KlebSeq could be easily modified to detect other healthcare-acquired infectious agents, and identify those with antimicrobial resistance. It could also be used for outbreak detection, transmission mapping and tracing the source of infections by being able to screen hundreds of patient samples simultaneously, at a cost of tens of dollars per patient.

KlebSeq: A Diagnostic Tool for Surveillance, Detection, and Monitoring of Klebsiella pneumoniae, will be published in the October 2016 issue of the Journal of Clinical Microbiology.