Whole Genome Sequencing of Mycobacterium tuberculosis Reveals Slow Growth and Low Mutation Rates during Latent Infections in Humans

Abstract

Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mutation analysis of M. tuberculosis isolates from a multi-decade tuberculosis outbreak of the New Zealand Rangipo strain. We used epidemiological and phylogenetic analysis to identify four cases of tuberculosis acquired from the same index case. Two of the tuberculosis cases occurred within two years of exposure and were classified as recently transmitted tuberculosis. Two other cases occurred more than 20 years after exposure and were classified as reactivation of latent M. tuberculosis infections. Mutation rates were compared between the two recently transmitted pairs versus the two latent pairs. Mean mutation rates assuming 20 hour generation times were 5.5X10−10 mutations/bp/generation for recently transmitted tuberculosis and 7.3X10−11 mutations/bp/generation for latent tuberculosis. Generation time versus mutation rate curves were also significantly higher for recently transmitted tuberculosis across all replication rates (p = 0.006). Assuming identical replication and mutation rates among all isolates in the final two years before disease reactivation, the u20hr mutation rate attributable to the remaining latent period was 1.6×10−11 mutations/bp/generation, or approximately 30 fold less than that calculated during the two years immediately before disease. Mutations attributable to oxidative stress as might be caused by bacterial exposure to the host immune system were not increased in latent infections. In conclusion, we did not find any evidence to suggest elevated mutation rates during tuberculosis latency in humans, unlike the situation in rhesus macaques.

Substantial molecular evolution and mutation rates in prolonged latent Mycobacterium tuberculosis infection in humans

“…We therefore found no real evidence supporting that oxidative stress is the dominant contributing factor during latent infection in our data. A similar observation was also made by Colangeli et al (Colangeli et al, 2014).…”

“…Furthermore, Colangeli et al (Colangeli et al, 2014) have provided contrasting evidence by showing mutation rates ten times lower during latency than active disease by examining four strains, two of them latent for more than 20 years, derived from the same index case.…”

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

“…Furthermore, two studies by Lillebaek et al provide compelling evidence from human LTBI that bacillary replication is minimal, as restriction fragment length polymorphism and insertion sequence patterns do not change over decades of latent infection (2,25). Similarly, although the number of isolates studied was very small, Colangeli et al used whole-genome sequencing to show that the mutation rates were not elevated in cases occurring more than 20 years after a TB outbreak relative to those representing recent transmission (361).…”

A Microbiological Revolution Meets an Ancient Disease: Improving the Management of Tuberculosis with Genomics

“…They provided evidence that the mutations that arise during latency are a result of oxidative damage, which is consistent with the exposure of the bacterium to oxidative stress during adaptation to intracellular survival within macrophages (87). In order to further characterize the mutation rate of M. tuberculosis during latency in humans, Colangeli et al used epidemiological and phylogenetic analyses of two recently acquired M. tuberculosis isolates and two cases of reactivated latent infection (20 years after initial exposure) from a multidecade outbreak in New Zealand (93). They reported that during latent infection, the mutation rate was approximately 30-fold lower than that during active disease, in contrast to the macaque study results, and they did not report any evidence to suggest that oxidative damage increases the mutation rate of M. tuberculosis during latency (87,93).…”

“…Two studies, one in macaques and the other in humans, estimated the mutation rate of M. tuberculosis during latency, with conflicting results (87,93). Ford et al showed that in M. tuberculosis infections of macaques, the mutation rates of M. tuberculosis were identical during active disease, latent infection, and in vitro growth (87).…”