ABSTRACTA bedaquiline-resistantMycobacterium abscessusisolate was sequenced, and a candidate mutation in theatpEgene was identified as responsible for the antibiotic resistance phenotype. To establish a direct genotype-phenotype relationship of this mutation which results in a Asp-to-Ala change at position 29 (D29A), we developed a recombineering-based method consisting of the specific replacement of the desired mutation in the bacterial chromosome. As surrogate bacteria, we used twoM. abscessusbedaquiline-susceptible strains: ATCC 19977 and the SL541 clinical isolate. The allelic exchange substrates used in recombineering carried either the sole D29A mutation or a genetic barcode of silent mutations in codons flanking the D29A mutation. After selection of bedaquiline-resistantM. abscessuscolonies transformed with both substrates, we obtained equivalent numbers of recombinants. These resistant colonies were analyzed by allele-specific PCR and Sanger sequencing, and we demonstrated that the presence of the genetic barcode was linked to the targeted incorporation of the desired mutation in its chromosomal location. All recombinants displayed the same MIC to bedaquiline as the original isolate, from which the D29A mutation was identified. Finally, to demonstrate the broad applicability of this method, we confirmed the association of bedaquiline resistance with theatpEA64P mutation in analysis performed in independentM. abscessusstrains and by independent researchers.IMPORTANCEAntimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections caused by microorganisms. On the other hand, infections caused byMycobacterium abscessusaffect people with chronic lung diseases, and their incidence has grown alarmingly in recent years. Further, these bacteria are known to easily develop AMR to the few therapeutic options available, making their treatment long-lasting and challenging. The recent introduction of new antibiotics againstM. abscessus, such as bedaquiline, makes us anticipate a future when a plethora of antibiotic-resistant strains will be isolated and sequenced. However, in the era of whole-genome sequencing, one of the challenges is to unequivocally assign a biological function to each identified polymorphism. Thus, in this study, we developed a fast, robust, and reliable method to assign genotype-phenotype associations for putative antibiotic-resistant polymorphisms inM. abscessus.