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This warrants the identification of novel targets for aiding the discovery of new drugs or drug combinations to treat\n              <jats:italic>M. abscessus<\/jats:italic>\n              infections. This study is inspired by the drug-hypersensitive profile of a mutant\n              <jats:italic>M. abscessus<\/jats:italic>\n              (U14) with transposon insertion in\n              <jats:italic>MAB_1915<\/jats:italic>\n              . We validated the role of\n              <jats:italic>MAB_1915<\/jats:italic>\n              in intrinsic drug resistance in\n              <jats:italic>M. abscessus<\/jats:italic>\n              by constructing a selectable marker-free in-frame deletion in\n              <jats:italic>MAB_1915<\/jats:italic>\n              and complementing the mutant with the same or extended version of the gene and then followed by drug susceptibility testing. Judging by the putative function of MAB_1915, cell envelope permeability was studied by ethidium bromide accumulation assay and susceptibility testing against dyes and detergents. In this study, we established genetic evidence of the role of\n              <jats:italic>MAB_1915<\/jats:italic>\n              in intrinsic resistance to rifampicin, rifabutin, linezolid, clarithromycin, vancomycin, and bedaquiline. Disruption of\n              <jats:italic>MAB_1915<\/jats:italic>\n              has also been observed to cause a significant increase in cell envelope permeability in\n              <jats:italic>M. abscessus<\/jats:italic>\n              . Restoration of resistance is observed to depend on at least 27 base pairs upstream of the coding DNA sequence of\n              <jats:italic>MAB_1915<\/jats:italic>\n              . MAB_1915 could therefore be associated with cell envelope permeability, and hence its role in intrinsic resistance to multiple drugs in\n              <jats:italic>M. abscessus<\/jats:italic>\n              , which presents it as a novel target for future development of effective antimicrobials to overcome intrinsic drug resistance in\n              <jats:italic>M. abscessus<\/jats:italic>\n              .\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              This study reports the role of a putative fadD (MAB_1915) in innate resistance to multiple drugs by\n              <jats:italic>M. abscessus<\/jats:italic>\n              , hence identifying MAB_1915 as a valuable target and providing a baseline for further mechanistic studies and development of effective antimicrobials to check the high level of intrinsic resistance in this pathogen.\n            <\/jats:p>\n          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