{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T12:50:21Z","timestamp":1766580621385,"version":"3.48.0"},"reference-count":29,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,20]],"date-time":"2025-12-20T00:00:00Z","timestamp":1766188800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH-NIAID","award":["R01-AI132374"],"award-info":[{"award-number":["R01-AI132374"]}]},{"name":"NIH-NIAID","award":["R01-AI184502"],"award-info":[{"award-number":["R01-AI184502"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background: Infections caused by the multidrug-resistant pathogen Mycobacterium abscessus (Mab) are notoriously difficult to treat. The novel \u03b2-lactamase inhibitor durlobactam, in combination with \u03b2-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 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 (\u0394ponA1) 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.<\/jats:p>","DOI":"10.3390\/antibiotics15010007","type":"journal-article","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T12:22:27Z","timestamp":1766578947000},"page":"7","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Loss-of-Function Mutations in the Penicillin-Binding Protein PonA1 Confer Agar-Dependent Resistance to Durlobactam in Mycobacterium abscessus"],"prefix":"10.3390","volume":"15","author":[{"given":"Dereje Abate","family":"Negatu","sequence":"first","affiliation":[{"name":"Center for Discovery and Innovation, Hackensack Meridian Health, 111 Ideation Way, Nutley, NJ 07110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4199-2224","authenticated-orcid":false,"given":"Wassihun Wedajo","family":"Aragaw","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation, Hackensack Meridian Health, 111 Ideation Way, Nutley, NJ 07110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min","family":"Xie","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation, Hackensack Meridian Health, 111 Ideation Way, Nutley, NJ 07110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9470-5009","authenticated-orcid":false,"given":"V\u00e9ronique","family":"Dartois","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation, Hackensack Meridian Health, 111 Ideation Way, Nutley, NJ 07110, USA"},{"name":"Department of Medical Sciences, Hackensack Meridian School of Medicine, 123 Metro Boulevard, Nutley, NJ 07110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9604-9452","authenticated-orcid":false,"given":"Thomas","family":"Dick","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation, Hackensack Meridian Health, 111 Ideation Way, Nutley, NJ 07110, USA"},{"name":"Department of Medical Sciences, Hackensack Meridian School of Medicine, 123 Metro Boulevard, Nutley, NJ 07110, USA"},{"name":"Department of Microbiology and Immunology, Georgetown University, Washington, DC 20057, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1016\/j.chest.2021.10.003","article-title":"Nontuberculous Mycobacterial Pulmonary Disease: Clinical Epidemiologic Features, Risk Factors, and Diagnosis: The Nontuberculous Mycobacterial Series","volume":"161","author":"Kumar","year":"2022","journal-title":"Chest"},{"key":"ref_2","first-page":"712","article-title":"Epidemiology of Mycobacterium abscessus","volume":"30","author":"Varley","year":"2023","journal-title":"Clin. 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