A. ROMINSKI¹, P. SELCHOW¹, J. K. BRÜLLE¹, K. BECKER¹, A. RODITSCHEFF¹, E. C. BÖTTGER¹, P. SANDER<sup>1

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

Mycobacterium abscessus is one of the most pathogenic and chemotherapy-resistant organisms among rapidly growing mycobacteria. The M. abscessus genome encodes various enzymes that are able to modify crucial residues of aminoglycoside (AG) drugs leading to their inactivation. The presence of these drug resistance genes hampers treatment options. M. abscessus is predicted to possess a single AG 2´-N-acetyltransferase and two putative multi-acetylating AG acetyltransferases of the Eis (Enhanced intracellular survival) family. However, the exact function, their role in drug resistance and the interplay of these genes remain under cover. Elucidation of drug resistance heavily relies on the ability for genetic manipulation of M. abscessus, particularly on generation of mutants by targeted gene inactivation. By applying established and novel genetic tools, the aim of this study is to elucidate the detailed resistance mechanisms of M. abscessus towards AGs. Based on drug susceptibility patterns of clinical isolates, we exploited our newly developed genetic tools to generate targeted deletion mutants of M. abscessus aac(2´), eis1 and eis2 and characterized their drug resistance profile. Compared to the parental strain, the Δaac(2´) and Δeis2 mutants proved to be highly sensitive towards specific AGs. Our MIC data clearly demonstrate that antibacterial activity of AGs characterized by an NH2 group at the 2´-position against M. abscessus WT is low, as compared to AGs carrying a corresponding OH group. Genetic inactivation of aac(2´) in M. abscessus confers susceptibility to AGs carrying a 2´-NH2 group while not affecting susceptibility towards AGs with a 2´-OH group. Modification of AGs with an L-HABA side chain prevents Aac(2´) mediated drug inactivation. Genetic inactivation of eis2 increased susceptibility towards a distinct set of AGs, while targeted deletion of eis1 did not alter susceptibility against any of the AGs tested. Characterization of the M. abscessus Aac(2´) and Eis2 mediated AG resistance mechanisms at the molecular level may have significant implementation for the development of new intervention strategies against this highly resistant pathogen.