| الوصف: |
A 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 D29A mutation, 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. abscessusantibiotic susceptible strains: ATCC19977, 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, demonstrating that the presence of the genetic barcode is linked to the targeted incorporation of the desired mutation in its chromosomal location. All recombinants displayed the same minimal inhibitory concentration to bedaquiline than 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, performed in independentM. abscessusstrains and by independent researchers. |
