Selective Pressure by Rifampicin Modulates Mutation Rates and Evolutionary Trajectories of Mycobacterial Genomes.

Resistance to the frontline antibiotic rifampicin constitutes a challenge to the treatment and control of tuberculosis. Here, we analyzed the mutational landscape of Mycobacterium smegmatis during long-term evolution with increasing concentrations of rifampicin, using a mutation accumulation assay combined with whole-genome sequencing. Antibiotic treatment enhanced the acquisition of mutations, doubling the genome-wide mutation rate of the wild-type cells. While antibiotic exposure led to extinction of almost all wild-type lines, the hypermutable phenotype of the ΔnucS mutant strain (noncanonical mismatch repair deficient) provided an efficient response to the antibiotic, leading to high rates of survival. This adaptative advantage resulted in the emergence of higher levels of rifampicin resistance, an accelerated acquisition of drug resistance mutations in rpoB (ß RNA polymerase), and a wider diversity of evolutionary pathways that led to drug resistance. Finally, this approach revealed a subset of adaptive genes under positive selection with rifampicin that could be associated with the development of antibiotic resistance. IMPORTANCE Rifampicin is the most important first-line antibiotic against mycobacterial infections, including tuberculosis, one of the top causes of death worldwide. Acquisition of rifampicin resistance constitutes a major global public health problem that makes the control of the disease challenging. Here, we performed an experimental evolution assay under antibiotic selection to analyze the response and adaptation of mycobacteria, leading to the acquisition of rifampicin resistance. This approach explored the total number of mutations that arose in the mycobacterial genomes under long-term rifampicin exposure, using whole-genome sequencing. Our results revealed the effect of rifampicin at a genomic level, identifying different mechanisms and multiple pathways leading to rifampicin resistance in mycobacteria. Moreover, this study detected that an increase in the rate of mutations led to enhanced levels of drug resistance and survival. In summary, all of these results could be useful to understand and prevent the emergence of drug-resistant isolates in mycobacterial infections.

Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies.

The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.