Lefamulin harbors promising anti-tuberculosis activity against multidrug-resistant <i>Mycobacterium tuberculosis</i> isolates

Multidrug-resistant tuberculosis (MDR-TB) is often associated with poor clinical outcomes. This study evaluated the <i>in vitro</i> activity of lefamulin (LEF) and intracellular activities against <i>Mycobacterium tuberculosis</i>. In this study, we evaluated the potential of LEF as a new drug candidate for treating <i>M. tuberculosis</i> infections, including MDR-TB. The antimicrobial susceptibility testing was performed to determine the minimum inhibitory concentrations (MICs) of LEF against 132 clinical isolates of <i>M. tuberculosis</i>. The intracellular activity of LEF and its interaction with other anti-tuberculosis drugs were also evaluated using <i>M. tuberculosis</i> H37Rv. From the 132 <i>M</i>. <i>tuberculosis</i> clinical isolates<i>,</i> the MIC₅₀ and MIC₉₀ were 0.5 µg/mL and 1 µg/mL, respectively. The tentative epidemiological cut-off (ECOFF) against LEF was defined at 1 µg/mL. After 5 days of incubation, LEF at 2 µg/mL inhibited 89.88% ± 1.73% of intracellular bacterial growth, which was comparable with the inhibitory rate of 94.29% ± 1.32% achieved by INH at 2 µg/mL. In addition, a synergy between LEF and bedaquiline (BDQ) was observed with a fractional inhibitory concentration index = 0.5. Furthermore, LEF showed no correlation with resistance to 10 anti-tuberculosis drugs. The minimum bactericidal concentration/MIC of LEF values suggested that it is a bacteriostatic drug against <i>M. tuberculosis</i>, and the bactericidal activity is mainly characterized by a concentration-dependent pattern. LEF has potent inhibitory activities against <i>M. tuberculosis in vitro</i> as well as in macrophages. Furthermore, the synergistic effect with BDQ also favors LEF as a promising drug candidate for tuberculosis treatment, especially for MDR-TB.IMPORTANCELefamulin (LEF), the first systemic pleuromutilin antibiotic approved for human use, exhibits broad-spectrum activity against Gram-positive bacteria. However, its <i>in vitro</i> activity against <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) remains unexplored. This study evaluated the potential of LEF for treating <i>Mtb</i> infections, including multidrug-resistant tuberculosis. Our findings demonstrate that LEF possesses potent bacteriostatic activity against <i>Mtb in vitro</i> and exhibits synergistic effects when combined with bedaquiline. These results suggest LEF as a promising therapeutic candidate for tuberculosis treatment.