Loss-of-Function Mutations in the Penicillin-Binding Protein PonA1 Confer Agar-Dependent Resistance to Durlobactam in <em>Mycobacterium abscessus</em>

Background: Infections caused by the multidrug-resistant pathogen Mycobacterium abscessus (Mab) are notoriously difficult to treat. The novel β-lactamase inhibitor durlobactam, in combination with β-lactams, shows potent bactericidal activity against Mab, but the potential for acquired resistance remains a clinical concern. Objectives: To identify and characterize mechanisms of acquired resistance to durlobactam in Mab. Methods: In vitro single-step resistance selection was performed by plating wild-type Mab ATCC 19977 on agar containing durlobactam. Resistant mutants were isolated, and their genomes were sequenced. The resistance phenotype was confirmed by constructing a targeted gene deletion mutant and by transcriptional silencing using a CRISPR interference (CRISPRi) system. Minimum inhibitory concentrations (MICs) were determined by both, an agar-based method and broth microdilution. Results: Whole-genome sequencing of durlobactam-resistant mutants identified loss-of-function mutations in ponA1, a gene encoding a class A penicillin-binding protein involved in cell wall synthesis. Targeted deletion of ponA1 (ΔponA1) and CRISPRi-mediated knockdown of ponA1 expression both recapitulated the resistance phenotype, resulting in a significant increase in the durlobactam MIC on solid agar media. Strikingly, broth microdilution MICs remained largely unaffected. Conclusions: Inactivation of the peptidoglycan synthase PonA1 is a novel mechanism of resistance to durlobactam in Mab that is phenotypically expressed only during growth on solid surfaces. This finding identifies a specific genetic pathway for resistance and highlights that standard broth-based susceptibility testing could miss clinically relevant resistance mechanisms.