Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution.

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global tuberculosis (TB) control, yet the mechanisms enabling rapid evolution of resistance in Mtb remain poorly understood. Here, we show that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INH), challenging the paradigm that resistance mutations must precede compensatory adaptation. Using Mycobacterium smegmatis mc155 (Msm) as a model, we demonstrate that brief exposure to sublethal isoniazid (INH) enriches for "low-level resistance and tolerance" (LLRT) mutants in a single step. LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (>500× IC) at ~6-fold higher rates than wildtype, primarily through otherwise deleterious mycothiol biosynthesis mutations that become tolerable in an oxidative stress-buffered background. Crucially, sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INHevolution. Analysis of 1578 clinical Mtb isolates revealed significant enrichment of oxidative stress-related loci among those associated with INH. Reanalysis of genome-wide CRISPRi data further linked oxidative stress response pathways to survival under multiple antibiotics. Together, these findings suggest that host-imposed oxidative stress and suboptimal drug exposure may prime Mtb populations for rapid resistance evolution, highlighting oxidative stress defenses as potential targets to limit resistance emergence.