Noncanonical mutations in ribosome nascent peptide exit tunnel confer clarithromycin resistance in Mycobacterium abscessus complex

Mycobacterium abscessus is a non-tuberculous mycobacterial pathogen known to cause pulmonary and skin infections worldwide. Renowned for its multidrug resistance, M. abscessus infections often result in unfavorable clinical outcomes. Clarithromycin plays a pivotal role in treating M. abscessus infections, with resistance commonly leads to treatment failure. While canonical mutations in 23S rRNA residue 2270/2271 are recognized as a major mechanism for acquired clarithromycin resistance, resistant isolates devoid of such mutations have been widely reported. In this study, we conducted a comprehensive investigation into acquired clarithromycin resistance using spontaneous mutants derived from two parental strains characterized by erm(41) T28 and C28 sequevars respectively. A total of 135 resistant mutants were selected from the parental strains. Sequencing of the 78 mutants lacking canonical 2270/2271 mutations identified mutations within the peptidyl-transferase center and in hairpin loops 35, 49, and 74 of the 23S rRNA. Moreover, these noncanonical mutations were identified in 57 out of 1875 genomes of clinical isolates. Thirteen representative mutations were introduced into the bacterial genome via site-directed mutagenesis, and their contribution to macrolide resistance was verified. Mapping these mutations onto the three-dimensional structure of 23S rRNA revealed their localization at the entrance of the nascent peptide exit tunnel, potentially contributing to resistance by disrupting the macrolide binding pocket. The identification of these noncanonical 23S rRNA mutations advances our understanding of macrolide resistance in M. abscessus and underscores their importance as potential markers for detecting clarithromycin resistance.