M. DAL MOLIN¹, M. GUT¹, K. HALDIMANN¹, P. SANDER<sup>1

1</sup>Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

Mycobacterium abscessus is an emerging pathogen which exhibits a broad range of intrinsic drug resistance mechanisms. The high level of drug resistance not only limits treatment options for patients infected with M. abscessus but also hampers genetic experiments with M. abscessus. Amongst others, M. abscessus shows high levels of resistance towards first- and second-line TB drugs, including resistance towards Streptomycin (STR), the first effective TB drug. Clinically acquired STR resistance in Mycobacterium tuberculosis is either due to alterations in specific codons of rpsL (encoding for small ribosomal subunit protein S12) or due to mutations in specific regions of rrs (encoding for the small ribosomal RNA; 16S rRNA). Sequence alignments between M. tuberculosis and M. abscessus did not indicate any evidence for resistance associated polymorphism in rpsL or rrs of M. abscessus. Instead, an Open Reading Frame (MAB_2385) encoding a putative antibiotic modifying enzyme, a potential 3’’-Streptomycin phosphotransferase, is present in the genome of M. abscessus, while a corresponding gene is missing in the genome of M. tuberculosis and Mycobacterium smegmatis, respectively. Heterologous expression of MAB_2385 in M. smegmatis significantly increased STR Minimal Inhibitory Concentration (MIC) of the recombinant strain as compared to the parental M. smegmatis thus indicating that MAB_2385 encodes a functional 3’’-Streptomycin phosphotransferase. Accordingly, deletion of MAB_2385 from the genome of M. abscessus decreased the STR MIC, while MIC towards other aminoglycosides or unrelated drugs remained unaltered. Wild-type like STR resistance was restored upon transformation of the deletion mutant with a single copy complementation vector. Taken together, our experiments indicate that MAB_2385 is the major STR resistance determinant in M. abscessus.