Chacha, R. | bioRxiv Science

bioRxiv2026

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

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.

microbiology↗

bioRxiv2025

Identification of mycobacterial efflux pump inhibiting compounds from Berberis holstii Engl. and determination of their mechanism of action

Mycobacterium tuberculosis (Mtb), the causative agent for tuberculosis (TB), is the leading infectious killer of humankind. In 2023, an estimated 10.8 million new cases of TB and 1.25 million deaths were reported globally. Sub-Saharan Africa faces a heavy TB burden, worsened by high HIV-AIDS prevalence and rising drug resistance, making novel anti-TB therapies a pressing priority. This study investigated the efflux inhibition (EI) activity of compounds characterised from Berberis holstii Engl. extracts. Following in vitro evaluation of the antimycobacterial activity of aqueous and organic extracts against the non-pathogenic mycobacterial model, Mycobacterium smegmatis (Msm), and Mtb. Thereafter, molecular docking of several compounds identified in chromatographic fractions onto Rv1258c, MmpS5-MmpL5 and Rv2333c Mtb efflux pumps (EPs) was undertaken to infer their binding modes and affinities. Subsequently, fractions containing potentially active compounds were tested in combination with spectinomycin (SPEC) and fractional inhibitory concentration index (FICI) values determined. Validation of Mtb efflux pump inhibition was performed using CRISPRi knockdown strains of Rv1258c and Rv2333c. MeOH extracts of the root and the stem bark, and aqueous extracts of the roots, exhibited minimum inhibitory concentration (MIC99) values of 906.25g/ml, 4500 g/ml and 1875 g/ml, respectively, against wild-type Msm. On the other hand, MeOH extract of the leaves and aqueous extract of the stem bark had MIC99 values of 60.43 g/ml and 1.74 g/ml respectively, against wild-type Mtb. Molecular docking of chillanamine, isoboldine, berberrubine, reticuline, N-methylcoclaurine, thalifoline, apoglaziovine, and orientine onto Mtb Rv1258c, MmpS5-MmpL5 and Rv2333c revealed string binding affinities of <-5 kcal/mol. Of the seven fractions containing the bioactive compounds evaluated in checkerboard assays, six were synergistic with SPEC (FICI <0.5), with two fractions lowering the MIC99 of SPEC by 8-fold against wild-type Mtb. This study identified phytochemicals with probable Mtb efflux pump inhibition, warranting further investigation for potential use in TB combination therapy.

microbiology↗