Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes

Abstract

Epidemics of drug-resistant bacteria emerge worldwide, even as resistant strains frequently have reduced fitness compared to their drug-susceptible counterparts. Data from model systems suggest that the fitness cost of antimicrobial resistance can be reduced by compensatory mutations; however, there is limited evidence that compensatory evolution has any significant role in the success of drug-resistant bacteria in human populations. Here we describe a set of compensatory mutations in the RNA polymerase genes of rifampicin-resistant M. tuberculosis, the etiologic agent of human tuberculosis (TB). M. tuberculosis strains harboring these compensatory mutations showed a high competitive fitness in vitro. Moreover, these mutations were associated with high fitness in vivo, as determined by examining their relative clinical frequency across patient populations. Of note, in countries with the world's highest incidence of multidrug-resistant (MDR) TB, more than 30% of MDR clinical isolates had this form of mutation. Our findings support a role for compensatory evolution in the global epidemics of MDR TB.

Figure 1. Putative compensatory mutations in a) rpoA and b) rpoC of Mycobacterium tuberculosis

Mutations identified after genome sequencing of experimentally evolved strains or paired clinical isolates are indicated above the gene by a circle and a triangle, respectively. Mutations identified by screening a global and a high-burden collection of MDR strains are indicated by stars below the gene. Colours indicate the respective strain lineage (blue - lineage 2, red - lineage 4, brown - lineage 5, pink - lineage 1). Some of these mutations occurred in multiple lineages and/or affect the same codon position.

Figure 2. Putative compensatory mutations in rpoA and rpoC fall at the interface of RNA polymerase subunits

Mutations identified in rifampicin-resistant experimentally evolved isolates and paired clinical isolates were mapped onto the RNA polymerase of Escherichia coli. The mutations fall in predicted interacting residues of RpoA (light blue) and RpoC (orange) of the E. coli RNA polymerase. Residue numbers are indicated according to M. tuberculosis coordinates. Colour code: RpoA (α-subunit) - blue, RpoB (β-subunit) - red, RpoC (β'-subunit) - yellow, RpoD (σ-subunit) - green.

Figure 3. Experimental and clinical relevance of putative compensatory mutations

a) Experimental competitive fitness of 10 clinical isolates that acquired rifampicin resistance during treatment compared to their susceptible counterparts. High-probability compensatory mutations (HCM) are indicated in the pair in which they were identified. Bar colours indicate strain lineage (blue - lineage 2, red - lineage 4). b) Difference in relative fitness between 10 rifampicin-resistant paired clinical isolates compared to laboratory-generated mutants carrying the same rifampicin resistance-conferring mutation and genetic background as defined by strain lineage. Data are shown for clinical strains harbouring (or not) an HCM. c) Time in months between the first and the second isolate of each clinical pair. The horizontal line indicates the median time interval. d) Percentage of MDR strains carrying putative compensatory mutations in rpoA or rpoC. Grey bars refer to the percentage of strains carrying HCMs and black bars refer to strains carrying any putative compensatory mutation. Data for a global collection of strains and for the high MDR-TB burden regions of Abkhazia/Georgia, Uzbekistan and Kazakhstan are shown.