The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran

Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates.

Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP).

All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ. Conclusions: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.