Identification of novel mutations associated with cycloserine resistance in Mycobacterium tuberculosis

Objectives: D-Cycloserine is an important second-line drug used to treat MDR- and XDR-TB. However, the mechanisms of resistance to D-cycloserine are not well understood. Here we investigated the molecular basis of D-cycloserine resistance using in vitro-isolated resistant mutants. Methods: Mycobacterium tuberculosis H37Rv was subjected to mutant selection on 7H11 agar plates containing varying concentrations of D-cycloserine. A total of 18 D-cycloserine-resistant mutants were isolated and subjected to WGS. The identified mutations associated with D-cycloserine resistance were confirmed by PCR and Sanger sequencing. Results: We identified mutations in 16 genes that are associated with D-cycloserine resistance. Interestingly, we found mutations only in alr (rv3423c) encoding alanine racemase, but not in other known D-cycloserine resistance-associated genes such as ddl, cycA or ald. Instead, we identified 13 new genes [rv0059, betP (rv0917), rv0221, rv1403c, rv1683, rv1726, gabD2 (rv1731), rv2749, sugI (rv3331), hisC2 (rv3772), the 50 intergenic region of rv3345c and rv1435c, and the 30 region of rv0759c] that had solo mutations associated with D-cycloserine resistance. Our findings indicate that the mechanisms of D-cycloserine resistance are more complex than previously thought and involve genes participating in different cellular functions such as lipid metabolism, methyltransferase, the stress response and transport systems. Conclusions: New mutations in diverse genes associated with D-cycloserine resistance have been identified that shed new light on the mechanisms of action and resistance of D-cycloserine. Future studies are needed to verify these findings in clinical strains so that molecular detection of D-cycloserine resistance for improved treatment of MDR-TB can be developed.