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

<b>Background</b>: Infections caused by the multidrug-resistant pathogen <i>Mycobacterium abscessus</i> (<i>Mab</i>) are notoriously difficult to treat. The novel β-lactamase inhibitor durlobactam, in combination with β-lactams, shows potent bactericidal activity against <i>Mab</i>, but the potential for acquired resistance remains a clinical concern. <b>Objectives</b>: To identify and characterize mechanisms of acquired resistance to durlobactam in <i>Mab</i>. <b>Methods</b>: In vitro single-step resistance selection was performed by plating wild-type <i>Mab</i> ATCC 19977 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. <b>Results</b>: Whole-genome sequencing of durlobactam-resistant mutants identified loss-of-function mutations in <i>ponA1</i>, a gene encoding a class A penicillin-binding protein involved in cell wall synthesis. Targeted deletion of <i>ponA1</i> (Δ<i>ponA1</i>) and CRISPRi-mediated knockdown of <i>ponA1</i> 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. <b>Conclusions</b>: Inactivation of the peptidoglycan synthase PonA1 is a novel mechanism of resistance to durlobactam in <i>Mab</i> 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.