Molecular Mechanisms of Intrinsic Streptomycin Resistance in Mycobacterium abscessus

Abstract

Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen <i>Mycobacterium abscessus</i> We recently identified two aminoglycoside-acetylating genes [<i>aac(2')</i> and <i>eis2</i>] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. <i>M. abscessus</i> BLASTP analysis identified MAB_2385 as a close homologue of the 3″-<i>O</i>-phosphotransferase [APH(3″)] from the opportunistic pathogen <i>Mycobacterium fortuitum</i> as a putative streptomycin resistance determinant. Heterologous expression of <i>MAB_2385</i> in <i>Mycobacterium smegmatis</i> increased the streptomycin MIC, while the gene deletion mutant <i>M. abscessus</i> ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in <i>M. abscessus</i> ΔMAB_2385. This demonstrates that <i>MAB_2385</i> encodes a specific and prime innate streptomycin resistance determinant in <i>M. abscessus</i> We further explored the feasibility of applying <i>rpsL</i>-based streptomycin counterselection to generate gene deletion mutants in <i>M. abscessus</i> Spontaneous streptomycin-resistant mutants of <i>M. abscessus</i> ΔMAB_2385 were selected, and we demonstrated that the wild-type <i>rpsL</i> is dominant over the mutated <i>rpsL^K43R</i> in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an <i>rpsL</i>-based counterselection method in <i>M. abscessus</i>.