Clofazimine has been repurposed for the treatment of tuberculosis, especially for multidrug-resistant tuberculosis (MDR-TB). To test the susceptibility to clofazimine of Mycobacterium tuberculosis clinical isolates, MICs of clofazimine were determined using the microplate alamarBlue assay (MABA) method for 80 drugresistant isolates and 10 drug-susceptible isolates for comparison. For five clofazimineresistant strains isolated from previously treated pre-extensively drug-resistant TB (pre-XDR-TB) and XDR-TB patients without prior exposure to clofazimine or bedaquiline, clofazimine MICs were Ն1.2 g/ml. Four isolates with cross-resistance to bedaquiline had Rv0678 mutations. The other isolate with no resistance to bedaquiline had an Rv1979c mutation. This study adds to a recent study showing that 6.3% of MDR-TB patients without prior clofazimine or bedaquiline exposure harbored isolates with Rv0678 mutations, which raises concern that preexisting resistance to these drugs may be associated with prior TB treatment. Furthermore, we propose a tentative breakpoint of 1.2 g/ml for clofazimine resistance using the MABA method. Morewidespread surveillance and individualized testing for clofazimine and bedaquiline resistance, together with assessment of their clinical usage, especially among previously treated and MDR-TB patients, are warranted.

Purpose: Linezolid (LZD) and pretomanid (PA-824) are promising candidates in regimens for the treatment of drug-resistant tuberculosis. However, research on LZD and PA-824 dual drug-resistant (LPDR) strains is rarely reported. This study aimed to investigate the genotypic and virulence characteristics of LPDR strains. Methods: To obtain the LPDR strains (marked as LP or PL strains), we used a two-way induction method, namely, we first induced LZD-or PA-824-resistant mutants from the parental Mycobacterium tuberculosis (MTB) strain H37Rv in vitro, then we obtained the LPDR strains from induction of LZD-or PA-824-resistant mutants. Mutations in rplC, rrl, or ddn and fgd1 were identified in all mutants. To investigate the virulence of these strains, six strains were selected as representative strains, including LZD-resistant strains, PA-824resistant strains and LPDR strains. We performed the animal survival study as virulence of MTB can be measured as survival time of an animal after being infected. Results: We induced 38 mutant strains of LZD and PA-824 mono or dual drug resistance from H37Rv in vitro. The mutation frequency of rplC (C154R) gene in LPDR strains was 100% and 86%, respectively. In the animal survival study, animals infected with different drug-resistant strains survived significantly longer than those infected with H37Rv; animals infected with LPDR strains and PA-824-resistant strains survived similarly and both of which survived significantly shorter than those infected with LZD-resistant strains. Conclusion: Our study showed that rplC gene had a high mutation frequency in LPDR strains. The virulence of LPDR strains was similar to PA-824-resistant strains, and the virulence of the LZD-resistant strains was weaker than PA-824-resistant strains.