The emergence of clarithromycin resistance is a challenge in treating Mycobacterium abscessus infections. Known mechanisms that contribute to intrinsic clarithromycin resistance focus on rrl gene-related mutations, but resistant clinical isolates often exhibit an inconsistent rrl genotype. Patients and Methods: In this study, 194 clinical Mycobacterium abscessus isolates were collected from patients with lung infections and the whole genome of each isolate was sequenced. A comprehensive examination of the molecular mechanisms underlying intrinsic clarithromycin resistance was performed, combining MIC determination, comparative genome sequence analysis and qRT-PCR. Results: Of the 194 isolates, 13 (6.7%) were clarithromycin resistant; only seven of these harbored a rrl 2270/2271 mutation. The remaining six resistant isolates did not exhibit a specific resistance-associated mutation in the clarithromycin target-site genes, rrl, rplC, rplD and rplV, or in the rrl modification gene erm(41). qRT-PCR analysis showed that the increased expression of the efflux pump genes, MAB_2355c, MAB_1409c and MAB_1846, as well as their positive regulatory gene whiB7, consistently correlated with increased clarithromycin resistance. The presence of efflux pump inhibitors significantly decreased the MIC of clarithromycin for nonsusceptible isolates, especially the intrinsic resistant isolates that exhibited no rrl 2270/2271 mutation. Conclusion: These findings indicate that efflux pumps play a prominent role in the intrinsic resistance of M. abscessus to clarithromycin, complementing other known resistance mechanisms.

Chemotherapeutic options against Mycobacterium abscessus infections are very limited. Bedaquiline, a new antituberculosis (anti-TB) drug, is effective for the treatment of multidrug-resistant TB. However, few data are available on bedaquiline for treatment of M. abscessus infections. In this study, we determined the profile for in vitro susceptibility of M. abscessus clinical isolates to bedaquiline and investigated the potential molecular mechanisms of decreased susceptibility. A total of 197 M. abscessus clinical isolates were collected from sputum and bronchoalveolar fluid of patients with lung infections. Standard broth microdilution test revealed that bedaquiline exhibited high in vitro killing activity against M. abscessus isolates, with a MIC50 of 0.062 and a MIC90 of 0.125 mg/liter. Whole-genome sequencing data showed that no nonsynonymous mutation occurred in atpE, the gene encoding the bedaquiline-targeted protein. However, of 6 strains with decreased susceptibility of bedaquiline (MIC = 0.5 to 1 mg/liter), 3 strains had nonsynonymous mutations in mab_4384, the gene encoding the repressor of efflux pump MmpS5/MmpL5. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the expression of MmpS5/MmpL5 in the group with decreased susceptibility to bedaquiline was significantly higher than in those with medium MICs (MIC = 0.125 to 0.5 mg/liter) or in the low-MIC group (MIC ≤ 0.062 mg/liter). Two isolates with increased MICs did not show overexpression of MmpS5/MmpL5, which could not be explained by known molecular mechanisms. This is the first report showing the association of MmpS5/MmpL5 with decreased bedaquiline susceptibility in M. abscessus clinical isolates and suggesting the presence of other, yet-to-be identified mechanisms for decreased bedaquiline susceptibility in M. abscessus.

Mycobacterium abscessus accounts for a large proportion of lung disease cases caused by rapidly growing mycobacteria. The association between clarithromycin sensitivity and treatment outcome is clear. However, M. abscessus culture and antibiotic susceptibility testing are time-consuming. Clarithromycin susceptibility genotyping offers an alternate, rapid approach to predicting the efficacy of clarithromycin-based antibiotic therapy. M. abscessus lung disease patients were divided into two groups based upon the clarithromycin susceptibility genotype of the organism isolated. A retrospective analysis was conducted to compare the clinical features, microbiological characteristics, and treatment outcomes of the two groups. Several other potential predictors of the response to treatment were also assessed. Sixty-nine patients were enrolled in the clarithromycin-resistant genotype group, which included 5 infected with rrl 2058-2059 mutants and 64 infected with erm(41)T28-type M. abscessus; 31 were in the clarithromycin-sensitive group, i.e., 6 and 25 patients infected with genotypes erm(41)C28 and erm(41) M type, respectively. The results showed that lung disease patients infected with clarithromycin-sensitive and -resistant M. abscessus genotypes differed significantly in clarithromycin-based combination treatment outcomes. Patients infected with the clarithromycin-sensitive genotype exhibited higher initial and final sputum-negative conversion and radiological improvement rates and better therapeutic outcomes. Multivariate analysis demonstrated that genotyping was a reliable and, more importantly, rapid means of predicting the efficacy of clarithromycin-based antibiotic treatment for M. abscessus lung disease.