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Therapeutic options remain limited due to the scarcity of effective \u03b2-lactam\/\u03b2-lactamase inhibitor combinations. Pilabactam (formerly ANT3310) is a novel diazabicyclooctane (DBO) \u03b2-lactamase inhibitor featuring a fluorine substituent that extends its activity spectrum, relative to approved DBOs like avibactam and relebactam, to include CHDLs. Pilabactam is currently in phase I clinical trials in combination with meropenem, and its activity and mechanism against CRAB remain incompletely defined. Using engineered\n                      <jats:italic toggle=\"yes\">A. baumannii<\/jats:italic>\n                      strains producing individual \u03b2-lactamases, we show that pilabactam restores meropenem activity against serine \u03b2-lactamase producers, including difficult-to-inhibit CHDLs. This was corroborated in 68 whole-genome-sequenced meropenem-resistant clinical isolates, yielding MIC\u2085\u2080 and MIC\u2089\u2080 values for meropenem\/pilabactam of 1 and 2 mg\/L, respectively. Frequency of resistance studies in representative CHDL producers demonstrated suppression of resistance selection at 4\u00d7 MIC. Kinetic analyses revealed that pilabactam inhibits OXA-23 via a two-step tight binding mechanism, with slightly higher inactivation rates (1.7 \u00d7 10\u2074 M\u207b\u00b9s\u207b\u00b9) than that of durlobactam (3.5 \u00d7 10\u00b3 M\u207b\u00b9s\u207b\u00b9). Pilabactam also yielded a low dissociation constant (\n                      <jats:italic toggle=\"yes\">\n                        K\n                        <jats:sub>d<\/jats:sub>\n                      <\/jats:italic>\n                      \u2248 4 nM) and slow off-rate, indicating durable inhibition. Molecular dynamics simulations revealed the critical role of the fluorine substituent in forming stabilizing hydrogen-bonding and CH\u2013F interactions within the tunnel-like OXA-23 active site. These findings identify pilabactam as a potent novel DBO supporting its development with meropenem for treating CRAB infections.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aac.01691-25","type":"journal-article","created":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T15:06:45Z","timestamp":1771513605000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Disarming carbapenemase-producing\n                    <i>Acinetobacter baumannii<\/i>\n                    : high potency of the novel therapeutic combination of meropenem and the innovative diazabicyclooctane \u03b2-lactamase inhibitor pilabactam (formerly 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