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International Team Tracks Plague Pathogen Patterns Using Sequencing and Phylogenetics

This article has been updated to clarify a quote regarding the potential utility of the Y. pestis phylogenetic tree.

By Andrea Anderson

NEW YORK (GenomeWeb News) – A genetic study appearing online yesterday in Nature Genetics has uncovered new details about the evolution and spread of the bacterial species behind Black Death and other plague outbreaks.

Researchers from the US, Europe, and China used a combination of sequencing, genotyping, and phylogenetic analyses to characterize Yersinia pestis isolates from around the world, exploring the pathogen's genetic diversity and relationships between the isolates.

"It's a very high resolution phylogenomic framework that we established," co-lead author Mark Eppinger, a microbiology and immunology researcher at the University of Maryland School of Medicine's Institute for Genome Sciences, told GenomeWeb Daily News, explaining that the study uncovered new biomarkers that can be used to explore the plague bacterium's evolutionary history.

Their results indicate that Y. pestis originated in and around China and was carried by traders and explorers to southeast Asia, Europe, South America, and Africa in a series of separate events. With the phylogenetic tree in hand, researchers are also learning more about the sources of historical plague outbreaks. And, they say, similar genetic analyses may also be useful for tracking and treating modern-day plague cases.

In an effort to learn more about plague strains from various parts of the world and their historical relationships, the team sequenced 11 Y. pestis isolates from China, Madagascar, Uganda, Turkey, Angola, and the US, comparing these with another six previously sequenced isolates.

Given the hurdles involved in transporting the pathogen between and within countries, Eppinger explained, the team opted to decentralize the sequencing stage of the project, with sequencing being performed at a handful of centers in the US and abroad using the Sanger and/or Roche 454 sequencing.

The researchers then analyzed patterns in the non-repetitive, core regions of new and previously sequenced genomes, using bioinformatics to help find as many informative SNPs as possible.

In so doing, the researchers were able to dig up 1,364 SNPs in coding regions of the Y. pestis genome. They also found additional changes by sifting through data on another 370 isolates.

"The tricky part of the research is actually to find these minor differences in the genomes and make sense out of them," Eppinger noted.

The team then applied this knowledge of genetic variation for their phylogenetic analyses of the bug, using the Sequenom MassArray SNP array to assess 933 SNP sites in another 286 isolates.

Overall, they noted, Y. pestis strains clustered based on geography, with isolates from specific countries or regions sharing SNPs that could be used to distinguish them from isolates found in other parts of the world.

In general, their results indicate that the plague pathogen is more genetically diverse in a geographic region in and around China, where it appears to have originated more than 2,600 years ago before spreading around the world through several distinct events.

"Plague clearly evolved from — or in the vicinity of — China," Eppiner said. "All the Chinese isolates we tested are pretty much scattered all over the four major phylogenetic branches."

Researchers were able to combine phylogenetic clues to the bacteria's migration with historical information to examine its spread, Eppinger explained, and to learn more about the sources of strains involved in the three plague outbreaks that have occurred during recorded human history.

In general, he said, genetic patterns coincided quite well with known historical events. For instance, the researchers found evidence that some plague pathogens moved from China to western Asia and beyond along the so-called Silk Road that facilitated trade between the regions.

And consistent with historical records, their findings indicate that plague isolates currently found in the US are linked to those introduced by a ship carrying Y. pestis infected rats that stopped in multiple American port cities in the late 1800s.

"All the US isolates that we tested reflect one radiation from a single import," Eppinger said. "If we try to link our phylogenetic data to historical records we see that plague was most likely imported to the United States in 1899 by a plague ship that departed in Hong Kong and docked on its way in Hawaii and then actually arrived in San Francisco."

Meanwhile, isolates collected from Madagascar — a region known to harbor plague pathogens that can be resistant to the antibiotics typically used to treat plague infections — belonged to a lineage apparently introduced to Madagascar from India in 1898.

Such findings suggest it may ultimately be possible to apply information from the Y. pestis phylogenetic tree to further investigate plague cases, Eppinger noted, by genotyping patient-isolated strains at SNPs that are known to correspond to specific countries or lineages. For instance, he explained, plague infections caused by Y. pestis strains resembling those in the Madagascar lineage are more likely to be multi-drug resistant than those belonging to other lineages.

In the future, the researchers hope to get their hands on additional isolates from Asia so that they can get an even more refined view of Y. pestis phylogeny and history, Eppinger noted. They also plan to genotype lineage informative SNPs in Y. pestis from ancient plague cases, such as victims of the medieval plague epidemic, to look at how these patterns relate to those in their phylogenomic framework.

"This extensive SNP-based framework will facilitate future investigations of under-sampled regions, such as Africa and the [former Soviet Union], for which details are still lacking," the team concluded. "It will also help to elucidate the basis of historical pandemics such as Justinian's plague and the Black Death through ancient DNA studies."