Wildtype and laboratory evolved isoniazid resistant strains of Mycobacterium smegmatis mc2155

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global TB control, yet the mechanisms enabling rapid evolution of drug resistance in Mtb remain poorly understood. Here we reveal that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INHR) without fitness costs, challenging the paradigm that resistance mutations always precede their fitness compensatory adaptations. Using M. smegmatis mc2155 (Msm) as a model, we show that brief exposure to sublethal INH (2x IC50) enriches for "low-level resistance and tolerance" (LLRT) mutants in a single step. These LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (>500x IC50) at 6-fold higher rates than wildtype, primarily through otherwise-deleterious mycothiol biosynthesis mutations that become tolerable in the oxidative stress-buffered background.