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 Mycobacterium abscessus. We recently identified two aminoglycoside-acetylating genes [aac(2=) and eis2] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. M. abscessus BLASTP analysis identified MAB-2385 as a close homologue of the 3″-O-phosphotransferase [APH(3″)] from the opportunistic pathogen Mycobacterium fortuitum as a putative streptomycin resistance determinant. Heterologous expression of MAB-2385 in Mycobacterium smegmatis increased the streptomycin MIC, while the gene deletion mutant M. abscessus ΔMAB-2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in M. abscessus ΔMAB-2385. This demonstrates that MAB-2385 encodes a specific and prime innate streptomycin resistance determinant in M. abscessus. We further explored the feasibility of applying rpsL-based streptomycin counterselection to generate gene deletion mutants in M. abscessus. Spontaneous streptomycin-resistant mutants of M. abscessus ΔMAB-2385 were selected, and we demonstrated that the wild-type rpsL is dominant over the mutated rpsL K43R in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an rpsL-based counterselection method in M. abscessus.