Efflux pump regulation and metabolic rewiring define the ceiling of efflux-mediated drug resistance in Mycobacteria

Multidrug-resistant efflux pumps play a critical role in antimicrobial resistance, yet the physiological limits of their overexpression remain poorly understood. While gene amplifications and regulatory mutations commonly drive this overexpression, these pumps remain transcriptionally inactive under normal cellular conditions due to their high energetic cost, making it challenging to assess their full impact on resistance evolution and the maximum attainable minimum inhibitory concentration (MIC). To delineate the extent and spectrum of efflux-based resistance, we subjected Mycobacterium smegmatis mc{superscript 2}155(Msm) wild-type and efflux pump-deleted strains ({Delta}lfrA and {Delta}efpA) to adaptive laboratory evolution in the presence of a broad-spectrum efflux substrate ethidium bromide (EtBr). Despite prolonged selection, resistance plateaued at 8-16x MIC, revealing an intrinsic ceiling to efflux-based adaptation. Evolved populations integrated multiple layers of adaptation: enhanced efflux kinetics, reduced intracellular EtBr accumulation, and mutations spanning global regulators, ribosomal genes, and membrane components. Notably, deletion of specific pumps ({Delta}lfrA) triggered compensatory upregulation of alternative efflux systems, highlighting functional redundancy. Beyond efflux activation, evolved strains exhibited metabolic reprogramming, including downregulation of the tricarboxylic acid cycle and upregulation of lipid biosynthesis and {beta}-oxidation, which altered NAD/NADH homeostasis and membrane potential. Extending this adaptive evolution approach to Mycobacterium tuberculosis H37Ra (Mtb) demonstrated that these adaptive constraints and metabolic alterations are conserved across mycobacterial species. Together, these results uncover fundamental limits to efflux evolution and reveal how bacteria integrate regulatory and metabolic plasticity to maximize drug resistance. Targeting these interconnected networks may offer a powerful strategy to contain the rise of multidrug resistance. ImportanceEfflux pumps are widely recognized as major contributors to antimicrobial resistance; however, the extent to which resistance can be driven solely by efflux remains unclear. Here, we define the limits of efflux-mediated resistance using adaptive laboratory evolution to ethidium bromide in Mycobacterium smegmatis and Mycobacterium tuberculosis. We show that resistance reproducibly plateaus despite continued selection and the presence of multiple efflux pumps in the genome, indicating a physiological ceiling to efflux-based adaptation. This limit is achieved through distinct evolutionary trajectories involving regulatory mutations that converge on enhanced efflux activity. Importantly, upregulation of efflux pumps is coupled to extensive cellular remodelling, including metabolic rewiring, and changes in membrane energetics, revealing system-wide trade-offs associated with resistance. Together, our findings demonstrate that efflux-driven resistance is constrained by cellular physiology and shaped by global adaptive costs, thereby refining current models of antimicrobial resistance and identifying vulnerabilities that may be exploited to limit the evolution of resistance.