Targeted Chromosomal Barcoding Establishes Direct Genotype-Phenotype Associations for Antibiotic Resistance in...

Area of Science:

• Microbiology and Infectious Diseases
• Genetics and Genomics
• Antimicrobial Resistance Research

Background:

• Antimicrobial resistance (AMR) is a growing global health threat, particularly for infections caused by *Mycobacterium abscessus*.
• *M. abscessus* infections, often affecting individuals with chronic lung diseases, are increasingly difficult to treat due to rapid development of AMR.
• New antibiotics like bedaquiline are vital, but their use anticipates the emergence of resistant strains, necessitating methods to understand resistance mechanisms.

Purpose of the Study:

• To establish a direct genotype-phenotype relationship for a specific mutation (Asp-to-Ala at position 29, D29A) in the *atpE* gene conferring bedaquiline resistance in *M. abscessus*.
• To develop and validate a robust, fast, and reliable recombineering-based method for assigning biological function to identified genetic polymorphisms in *M. abscessus*.

Main Methods:

• A recombineering-based allelic exchange strategy was employed to introduce the specific D29A mutation into the *atpE* gene of bedaquiline-susceptible *M. abscessus* strains (ATCC 19977 and SL541).
• Allelic exchange substrates included either the D29A mutation alone or with a linked genetic barcode of silent mutations for tracking.
• Allele-specific PCR and Sanger sequencing were used to confirm targeted mutation incorporation, and minimum inhibitory concentrations (MICs) were determined to assess bedaquiline resistance.

Main Results:

• The recombineering method successfully and specifically incorporated the D29A mutation into the chromosomal *atpE* gene of *M. abscessus*.
• Recombinant *M. abscessus* strains harboring the D29A mutation exhibited bedaquiline resistance comparable to the original resistant isolate.
• The method's applicability was confirmed by associating another mutation (*atpE* A64P) with bedaquiline resistance in independent strains.

Conclusions:

• The developed recombineering method provides a fast, robust, and reliable means to establish genotype-phenotype associations for antibiotic resistance in *M. abscessus*.
• This approach is crucial for unequivocally assigning function to genetic variations identified through whole-genome sequencing in the context of AMR.
• The validated method will accelerate the understanding and characterization of emerging antibiotic resistance mechanisms in *M. abscessus*, aiding in the development of effective treatment strategies.