Mutation accumulation assay under rifampicin selective pressure of Mycobacterium smegmatis mc2 155 wild type and nucS-deficient strains

In this study, we performed a mutation accumulation (MA) assay in presence of increasing concentrations of rifampicin to explore the effect of this antibiotic on mutation rate and study the evolutionary trajectory to drug resistance in mycobacteria. We generated 20 independent lines from the wild type strain and the same number from its hypermutator nucS-null derivative. The experimental evolution was carried out for 40 weeks, starting with a rifampicin concentration of 0.25 ug/ml and doubling the concentration every 5 weeks. Considering the low survival rate at the end of the evolution, genomic DNA was extracted from the MA lines evolved for 35 weeks (up to 16 ug/ml of rifampicin), 18 wild type lines and 20 nucS-deficient lines.