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Abstract: The work presented here focuses on identifying and characterizing genetic determinants of antibiotic sensitivity in Mycobacterium abscessus(Mabs), a non-tuberculosis mycobacterial pathogenresponsible for various human infections that are notoriously difficult to treat with standard antibacterial therapy in clinical settings. Usingbacterial genetics and high-throughput DNA sequencing methods, we have identifiedgenes important for growthon amikacin and clarithromycin, two clinically relevantantibiotics used to treat Mabs,and ethionamide(ETH), a second-line drug currently used for treating multidrug-resistant tuberculosis infections.In chapters 1 and 2, I discuss the types of infections caused by Mabs, notable risk factors associated with Mabsinfections, its epidemiology and transmission, and treatment options currently available. In chapter 3, I specifically focus on antibiotic treatment of Mabsinfections and provide an overview on various antibiotic resistance mechanisms that are associated with treatment failure. In chapter 4, I present data that examines the antibacterial activity of ETH alone and in combination with clinically relevant antibiotics in vitro and in vivo. Furthermore, we present sequencing data on MAB_2648c, which encodes for a transcriptional repressor that confers ETH resistance. Wedemonstrate that Mab_2648c-dependent ETH resistance requires the activity of MmpSL5, a putative membrane transporter whosefunction remains unknown. In chapter 5, we attempt to identify the function of MmpSL5 and its potential substrates. In conclusion, we show that ETH is slightly bactericidal against Mabs in vitroand potentially in vivo, does not antagonize with clinicallyrelevant antibiotics used to treat Mabs infections, and identified a biological mechanismthat confers ETH resistance in Mabs. Future work should further examine the repurposing potential of ETH against Mabsand identify compounds that may reverse ETH resistancein vitroand in vivo.