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Cefepime\/enmetazobactam is a novel combination with demonstrated activity against extended-spectrum \u03b2-lactamase-producing Enterobacterales, but its activity against\n                      <jats:italic toggle=\"yes\">Acinetobacter<\/jats:italic>\n                      has not yet been thoroughly explored. We aimed to assess its activity against\n                      <jats:italic toggle=\"yes\">Acinetobacter<\/jats:italic>\n                      spp., including multidrug-resistant strains producing carbapenem-hydrolyzing class D \u03b2-lactamases (CHDLs). We analyzed 208 clinical isolates of\n                      <jats:italic toggle=\"yes\">Acinetobacter<\/jats:italic>\n                      spp., including 67 carbapenem-resistant\n                      <jats:italic toggle=\"yes\">Acinetobacter baumannii<\/jats:italic>\n                      (CRAB). Antibiotic susceptibility testing was conducted with cefepime, sulbactam, and imipenem, alone and in combination with enmetazobactam; the latter was also tested individually. Additionally, MICs of enmetazobactam\/durlobactam and sulbactam\/durlobactam were determined for CRAB and CHDL-producing\n                      <jats:italic toggle=\"yes\">A. baumannii<\/jats:italic>\n                      ATCC 17978 transformants. PBP binding assays (IC\u2085\u2080), molecular docking, simulation studies with the enmetazobactam\/OXA-23 adduct, hydrolysis kinetics (\n                      <jats:italic toggle=\"yes\">k<\/jats:italic>\n                      <jats:sub>cat<\/jats:sub>\n                      ,\n                      <jats:italic toggle=\"yes\">K<\/jats:italic>\n                      <jats:sub>m<\/jats:sub>\n                      ), and OXA-23 inhibition assays (IC\u2085\u2080,\n                      <jats:italic toggle=\"yes\">k<\/jats:italic>\n                      <jats:sub>off<\/jats:sub>\n                      ,\n                      <jats:italic toggle=\"yes\">t<\/jats:italic>\n                      <jats:sub>\u2081\/\u2082<\/jats:sub>\n                      ) were performed to elucidate the mechanism of enmetazobactam and detect reduced susceptibility. Enmetazobactam showed high intrinsic activity against\n                      <jats:italic toggle=\"yes\">Acinetobacter<\/jats:italic>\n                      spp., displaying reduced MICs against carbapenem-susceptible isolates. MIC\n                      <jats:sub>50\/90<\/jats:sub>\n                      of the enmetazobactam\/durlobactam combination was 2\/2 mg\/L for CHDL-producing\n                      <jats:italic toggle=\"yes\">A. baumannii<\/jats:italic>\n                      . Enmetazobactam exhibited bactericidal activity comparable to sulbactam. Binding assays revealed that the antimicrobial activity is driven by selective affinity for PBP2 (IC\u2085\u2080 3.6 mg\/L) and PBP3 (IC\u2085\u2080 4.2 mg\/L). OXA-23 readily inactivated enmetazobactam, confirming the major role of CHDLs in resistance to enmetazobactam, via substrate-assisted de-acylation. This study evidences the potent antimicrobial activity of enmetazobactam against\n                      <jats:italic toggle=\"yes\">A. baumannii<\/jats:italic>\n                      via inhibition of PBP2 and PBP3. Its combination with new OXA-type inhibitors (e.g., durlobactam) represents a potential therapeutic alternative for multidrug-resistant\n                      <jats:italic toggle=\"yes\">A. baumannii<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aac.01206-25","type":"journal-article","created":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T14:00:27Z","timestamp":1766412027000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Antibacterial activity of enmetazobactam against\n                    <i>Acinetobacter<\/i>\n                    spp.: a molecular dissection of mechanism of action and resistance determinants"],"prefix":"10.1128","volume":"70","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-1042-8174","authenticated-orcid":false,"given":"Gabriela-Alejandra","family":"B\u00e1ez-Barroso","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/044knj408","id-type":"ROR","asserted-by":"publisher"}],"name":"Servicio de Microbiolog\u00eda and Instituto de Investigaci\u00f3n Biom\u00e9dica A Coru\u00f1a (INIBIC), Complexo Hospitalario Universitario A Coru\u00f1a (CHUAC)","place":["A Coru\u00f1a, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arianna","family":"Rodr\u00edguez-Coello","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/044knj408","id-type":"ROR","asserted-by":"publisher"}],"name":"Servicio de Microbiolog\u00eda and Instituto de Investigaci\u00f3n Biom\u00e9dica A Coru\u00f1a (INIBIC), Complexo Hospitalario Universitario A Coru\u00f1a (CHUAC)","place":["A Coru\u00f1a, 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