CC BY 4.0Ghodousi, ArashArashGhodousiIannucci, I.I.IannucciSaluzzo, FrancescaFrancescaSaluzzoDreisbach, JuliaJuliaDreisbachMirold-Mei, S.S.Mirold-MeiHoelscher- von Lovenberg, MichaelMichaelHoelscher- von LovenbergCirillo, D. M.D. M.Cirillo2025-12-182025-12-182025https://publica.fraunhofer.de/handle/publica/502179https://doi.org/10.24406/publica-687610.5588/ijtldopen.25.030110.24406/publica-68762-s2.0-105022238470Multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis complex (MTBC) remain a significant global health challenge. This study investigates resistance mechanisms to BTZ-043, a novel decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1) inhibitor, and its potential cross-resistance with bedaquiline (BDQ) and clofazimine (CFZ). BTZ-043-resistant mutants were generated in M. tuberculosis H37Rv by serial exposure to escalating drug concentrations. Minimum inhibitory concentrations (MICs) for BTZ-043 were determined for 130 wild-type strains, including 60 H37Rv independent cultures and 70 diverse clinical isolates, plus 33 non–wild-type clinical strains with known BDQ susceptibility. MICs were correlated with whole-genome sequencing (WGS) data to identify genetic factors underlying resistance. The MIC distribution for clinical MTBC strains was similar to the reference strain, with a mode of 0.002 μg/mL. WGS of resistant mutants revealed mutations in dprE1 and Rv0678. Rv0678 and dprE1 mutations resulted in 4- to 8-fold and >1,000-fold increase in MIC compared with the reference mode, respectively. Sequential clinical strains from BDQ-treated patients showed increased MICs and Rv0678 mutations, indicating low-level cross-resistance. However, Rv0678 mutations in BDQ-susceptible strains did not affect BTZ-043 MICs. Rv0678 mutations confer low-level cross-resistance to BTZ-043, BDQ, and CFZ, with variable effects on susceptibility. These findings highlight the complexity of resistance mechanisms and the need for ongoing surveillance and early resistance assessments in drug development.entrueextensively drug-resistantMDR-TBMICmultidrug-drug resistanttuberculosisWGSjournal article