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Metagenomic Analysis Finds Ancient TB Victims Harbored Multiple Bacterial Strains

NEW YORK (GenomeWeb) – Metagenomic analysis of samples from 18th century tuberculosis victims indicates that infection with multiple bacterial strains was common, according to researchers from the University of Warwick in the UK and elsewhere. The researchers also uncovered a direct link between such historic strains and ones circulating in Europe today.

As they reported in Nature Communications today, Warwick's Mark Pallen and his colleagues collected DNA samples from more than two dozen people buried in a Hungarian churchyard in the 1700s. After aligning sequences obtained from TB victims against the Mycobacterium tuberculosis genome and constructing a phylogenetic tree, the researchers found that the most common ancestor of this lineage could be traced back to the late Roman period.

"We have shown that metagenomic approaches can document past infections," Pallen said in a statement. "However, we have also recently shown that metagenomics can identify and characterize pathogens in contemporary samples, so such approaches might soon also inform current and future infectious disease diagnosis and control."

The researchers collected DNA samples from 26 bodies found in a crypt in Vác, Hungary and mapped the reads generated by sequencing those samples against the M. tuberculosis reference genome. From this, they generated draft tuberculosis sequences from eight of the bodies, five of which harbored multiple M. tuberculosis sequences, to yield a total of 14 historical M. tuberculosis sequences. Of those, four were sequenced to more than 10X coverage.

These 14 historical tuberculosis sequences all harbored a seven-basepair deletion that's found in the Euro-American M. tuberculosis lineage, also known as Lineage 4.

Using more than 1,580 contemporary Lineage 4 genomes, along with a Beijing lineage genome as an outgroup, Pallen and his colleagues assembled a phylogenetic tree based on SNVs located in non-repetitive regions of the M. tuberculosis genome.

Through this, the researchers found that the four historical strains with high-coverage genomes belong to phylogenetically distinct genotypes.

With a new algorithm called MGplacer, the researchers then added the low-coverage historical genomes onto the phylogenetic tree. Their results, they noted, indicated that there were at least 12 different strains of tuberculosis circulating in 18th century Hungary. Further, as these strains were nested deep within contemporary subdivisions of Lineage 4, the researchers said that likely indicated a continuity of strain lineages over the past two centuries.

Pallen and his colleagues also used the recorded dates of death for the victims who harbored the four high-coverage historical TB strains along with 161 modern Lineage 4 genomes that were isolated between 1992 and 2012 to calculate the date of the most recent common ancestor of the lineage. From this, they traced the emergence of the lineage back to about 396 AD, or about the late Roman period.

Additionally, the effective population size of the lineage grew continuously, according to the researchers' calculations, from its origin until the 20th century when it underwent a precipitous drop in size.

The researchers also found that five of the eight bodies from which they were able to isolate M. tuberculosis DNA harbored more than one genotype — one person in particular had three distinct M. tuberculosis genotypes. This, they noted, contrasts with many microbial analyses of contemporary patients, though mixed infections have been observed in modern patients living in high-prevalence areas.

Based on this, the researchers hypothesized that multi-strain infection was common during peak TB in Europe.

"[A]s culture-based TB microbiology appears to be poorly suited to the detection of mixed infections, the approaches we describe here might deliver improvements in [the] diagnosis and management of contemporary infections," Pallen and his colleagues said in their paper.