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With Genomic Analysis, Researchers Characterize Tuberculosis Lineage, History, and Spread

NEW YORK (GenomeWeb) – Through genetic analysis, an international team of researchers was able to reconstruct the evolutionary history of a set of Mycobacterium tuberculosis strains dubbed the Beijing lineage.

As it reported in Nature Genetics today, the team analyzed the genomes of nearly 5,000 M. tuberculosis isolates and performed whole-genome sequencing on a subset of 110 representative isolates. Based on this, the team reported that this Beijing lineage of M. tuberculosis originated in the Far East some 6,600 years ago and spread globally in a number of waves, many of which coincided with major events in human history such as the Industrial Revolution, World War I, and the HIV epidemic.

"[O]ur results show for the first time, to our knowledge, the important dynamic changes that have occurred in the worldwide population of a major M. tuberculosis lineage," the team led by Thierry Wirth from Ecole Pratique des Hautes Etudes wrote in their paper. "Although the exact timing remains dependent upon uncertainties in mutation rates, especially over the long term, the conjunction of the most recent changes in the bacterial population with specific chief events in human history … is intriguing."

Tuberculosis causes some 1.3 million deaths a year, the researchers noted, and multi-drug resistant strains — strains that are resistance to the first-line drugs isoniazid and rifampicin — are spreading, particularly in southern Africa and in the former Soviet states. The drug-resistant strains circulating in Eurasia mostly belong to the Beijing lineage, and strains of this lineage have been suggested to harbor some selective advantages over other lineages, such as an increased ability to become drug resistant or increased transmissibility, the researchers said.

Wirth and his colleagues genotyped 4,987 clinical isolates from 99 countries linked to drug resistance and used that 24-locus mycobacterial interspersed repetitive unit-variable-number tandem repeats (MIRU-VNTR) genotyping data to construct a minimum-spanning tree.

This tree classified the isolates into six major clonal complexes (CC) and three distant branches that together made up basal sub-lineage 7 (BL7). The clonal complexes they dubbed CC1 through CC5 contained typical or modern Beijing strains, while CC6 and BL7 contained atypical, ancestral Beijing variants.

The CC1 and CC2 populations, the researchers noted, appeared to be expanding, as did, though to a lesser degree, CC5. However, CC6 and BL7 did not appear to be undergoing such a population expansion, underscoring their likely ancient origins.

The spatial distribution of these clonal complexes indicated that the Beijing lineage likely originated in East Asia and the Far East, as the strains there were the most diverse, Wirth and his colleagues said. CC5 became more prominent closer to the Pacific Ocean, while CC1 and CC2 dominated the Central Asia and Eastern Europe isolates.

The researchers further estimated the time to the most recent common ancestor for the Beijing lineage to be some 6,600 years, or about the time that agriculture was adopted in the region. This analysis also pegged CC6 and BL7 as the oldest sub-lineages, dating back 6,000 and 5,000 years ago. CC5, meanwhile, was the youngest sub-lineage at 1,500 years.

At the same time, Wirth and his colleagues sequenced the genomes of 110 isolates that represented the main clonal complexes. After excluding genes associated with drug resistance as well as repetitive and mobile elements, the researchers uncovered some 6,000 SNPs.

A phylogenetic tree based on sequencing data from these isolates largely matched with the MIRU-VNTR-based tree, they reported.

Using a previously determined short-term mutation rate, the researchers traced the timing of Beijing lineage population growth phases to around the time of the Industrial Revolution and around World War I. A decrease in effective population size, meanwhile, could be linked to the time of widespread use of anti-tuberculosis drugs.

Additionally, the researchers noticed a temporary reversal of this decline in population size that coincided with the start of the HIV epidemic and outbreaks of drug-resistant strains in the former Soviet Union and in the US.

CC2, Wirth and his colleagues reported, harbored the greatest proportion of drug-resistant strains, about 75 percent, while CC4, CC3, and CC5, had 17.2 percent, 12.1 percent, and 3.5 percent resistance, respectively. Further, nearly a third of CC1 isolates were drug-resistant, and nearly all of them shared a MIRU-VNTR haplotype.

These CC1 and CC2 strains, which are linked to the central Asian outbreak and a European-Russian outbreak, respectively, had high clustering rates and low pairwise distance, indicating to the researchers that there was recent expansion of these MDR clones in central Asia and Russia.

Wirth and his colleagues also scoured the M. tuberculosis genomes for targets that could be linked to the expansion of the Beijing lineage that occurred some 200 years to 700 years ago. They examined 81 SNPs characteristic of the modern strains, finding SNPs in the mammalian cell entry and virulence-associated protein gene families. SNPs in coding regions of those gene families were fixed in both the modern and ancestral subgroups of the Beijing lineages, suggesting that they are under positive selection.

They also uncovered SNPs that were branch-specific. For instance, a frameshift mutation in kdpD, which is involved in the kdpDE operon and signal transduction system, was specific to all European-Russian outbreak strains.

A partial deletion of this operon, the researchers said, had been linked to greater virulence in a mouse model.

"[W]e propose that the expansion of modern Beijing strains has been favored by mutations in a number of gene targets under positive selection," Wirth and his colleagues said. "The data obtained here thus suggest further experiments to investigate which of these candidate genes were involved. Such work may ultimately contribute to the detection of new targets for combating tuberculosis."