Natural product-based putative efflux inhibitors restore bedaquiline susceptibility in a drug-resistant Mycobacterium tuberculosis mutant

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a potent threat to global public health. Moreover, the alarming surge in the number of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb strains will continue to imperil TB control efforts. Thus, the discovery of new TB agents with novel modes of action or resistance-reversing therapeutics is a pressing priority. In this study, we report the generation of spontaneous Mtb mutants exhibiting bedaquiline (BDQ) resistance and the subsequent evaluation of natural product-derived efflux inhibitors (EIs) that restored the antimicrobial efficacy of BDQ against the mutants. BDQ-resistant mutants were successfully isolated, and colonies were observed on agar plates with concentrations up to 100x the minimum inhibitory concentration (MIC). Upon screening against BDQ, the resistant strains exhibited MIC values ranging from 0.098 M to 3.136 M, corresponding to 1-32-fold increases relative to the wild type, with higher resistance observed on 50x- and 100x-selection plates. Genetic analysis identified point mutations and frameshifts in key resistance-related genes, including Rv0678, pepQ, and atpE. Notably, combining BDQ with EIs such as berberine (BER), reserpine (RES), piperine (PIP), and lyoniresinol (LYO) remarkably lowered the MIC in the selected mutant strain. Synergistic effects were observed for BDQ+BER (FICI = 0.188; 16-fold MIC reduction) and BDQ+RES (FICI = 0.37; 8-fold MIC reduction). For BDQ+LYO, the FICI could not be calculated because LYO did not have an MIC at the highest concentration tested; however, this combination produced the strongest effect, restoring susceptibility with a 64-fold MIC reduction and exhibiting bactericidal activity. These results highlight the role of efflux pumps in BDQ resistance and support the use of natural product-derived EIs as potential supplementary therapies against drug-resistant Mtb.