Luciferase reporter mycobacteriophage (TM4:: <i>GeNL</i> ) enables rapid assessment of drug susceptibilities and inducible macrolide resistance in <i>Mycobacterium abscessus</i> complex

<i>Mycobacterium abscessus</i> (MAB) infections are challenging to treat due to high-level resistance to anti-tuberculosis drugs, carbapenems, fluoroquinolones, and tetracyclines. Clarithromycin and amikacin are considered cornerstone drugs for MAB treatment due to their superior efficacy; however, assessing clarithromycin susceptibility typically takes 7 to 14 days because of inducible macrolide resistance. We developed a 48 h luciferase reporter mycobacteriophage drug susceptibility testing (LRM-DST) assay using TM4::<i>GeNL</i> to evaluate MAB drug susceptibility. For this proof-of-principle study, we performed DST on 26 MAB clinical isolates using TM4::<i>GeNL</i> and Sensititre RAPMYCO2 plates. Genome sequencing was performed to identify the MAB sub-species and for understanding intrinsic and acquired drug resistance mechanisms. We detected clarithromycin resistance in 12/26 (46%) by both Sensititre and LRM-DST. All clarithromycin-resistant isolates carried full-length inducible erythromycin ribosomal methylase-41 [<i>erm(41</i>)] gene, and none had <i>rrl</i> (23S rRNA) mutations. Amikacin resistance or intermediate amikacin resistance was detected in 6/26 (23%) isolates. Mutations in the <i>rrs</i> (16S rRNA) gene at positions 1375A > G (<i>E. coli</i> 1408A > G) conferred amikacin resistance in four isolates; however, no mutations were identified in two of the amikacin intermediate-resistant isolates. LRM-DST results were in 100% and 92.3% concordance with Sensititre DST results of clarithromycin (kappa coefficient, 1.0; 95% confidence interval [CI], 0.84 to 1.0) and amikacin (kappa coefficient, 0.752; 95% confidence interval [CI], 0.43 to 1.0). In addition, LRM-DST results for bedaquiline, moxifloxacin, imipenem, linezolid, and cefoxitin correlated well with Sensititre DST. Our findings suggest that LRM-DST can provide reliable phenotypic MAB-DST information in 48 h for prompt clinical management.IMPORTANCE<i>Mycobacterium abscessus</i> (MAB) is a notorious human pathogen causing severe infections in individuals with cystic fibrosis and immunocompromised patients. Treatment options for MAB are very limited as they are resistant to multiple drugs through intrinsic and acquired resistance mechanisms. While macrolides and amikacin are the key drugs in the fight against MAB infections, emerging drug resistance is compromising their efficacy. Current growth-based drug susceptibility testing (DST) method takes 7 to 14 days to identify clarithromycin susceptibility due to the presence of inducible macrolide resistance and 3 to 5 days for other drugs. We developed a novel luciferase reporter mycobacteriophage (LRM) based phenotypic DST method using TM4::<i>GeNL</i> to assess MAB drug susceptibility based on metabolic inhibition rather than growth. LRM-DST significantly reduces the DST turnaround time to 48 h and provides rapid assessment of MAB susceptibility to drugs, including inducible macrolide resistance, thereby accelerating the treatment process and improving patient outcomes.