{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:02:49Z","timestamp":1785402169813,"version":"3.56.0"},"reference-count":49,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T00:00:00Z","timestamp":1749600000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T00:00:00Z","timestamp":1749600000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI072219"],"award-info":[{"award-number":["R01AI072219"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2025,6,11]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Antimicrobial resistance in\n                      <jats:italic toggle=\"yes\">Acinetobacter baumannii<\/jats:italic>\n                      is partly mediated by chromosomal class C \u03b2-lactamases, the\n                      <jats:italic toggle=\"yes\">Acinetobacter<\/jats:italic>\n                      -derived cephalosporinases (ADCs). Recently, a growing number of emerging variants were described, expanding this threat. Consistent with other \u03b2-lactamases, one of the main areas of variance exists in the \u03a9-loop region near the site of cephalosporin binding. Interestingly, a common alanine duplication (Adup) is found in this region. Herein, we studied specific Adup variants expressed in a uniform\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      genetic background that demonstrated high-level resistance to multiple oxyimino-cephalosporins. For ceftolozane and ceftazidime, the Adup ADCs significantly increased levels of resistance (minimum inhibitory concentration [MIC] \u2265 512 \u00b5g\/mL and MIC \u2265 1,024 \u00b5g\/mL, respectively). These observations were consistent with the increased\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>\n                        <jats:italic toggle=\"yes\">M<\/jats:italic>\n                      <\/jats:sub>\n                      for ceftazidime. For cefiderocol, three Adup variants exhibited increased MICs and increased\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>\n                        <jats:italic toggle=\"yes\">M<\/jats:italic>\n                      <\/jats:sub>\n                      for this compound. Timed electrospray ionization mass spectrometry demonstrated stable cephalosporin:ADC adducts with ADC-30 (non-Adup), but not with ADC-33 (Adup), consistent with turnover. The X-ray crystal structure of Adup variant ADC-33 in complex with ceftazidime was determined (1.57 \u00c5 resolution) and suggests that increased turnover is facilitated by conformational changes (shift in Tyr221 and orientation of the oxyimino portion of the R1 side chain) and repositioning of water in the active site. These changes appear to favor substrate-assisted catalysis as an alternative mechanism to base-assisted catalysis. These studies also provide unprecedented insight into the mechanism underlying oxyimino-cephalosporin hydrolysis by expanded-spectrum ADC \u03b2-lactamases and possibly other class C \u03b2-lactamases, which is of critical importance to future drug design.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        The characterization of emerging\n                        <jats:italic toggle=\"yes\">Acinetobacter<\/jats:italic>\n                        -derived cephalosporinase (ADC) variants is necessary to understand the increasing resistance to \u03b2-lactam antibiotics in\n                        <jats:italic toggle=\"yes\">Acinetobacter<\/jats:italic>\n                        spp. In this study, cefiderocol retains effectiveness against ADC variants with and without an \u03a9-loop alanine duplication (Adup). However, the presence of the Adup appears to introduce loop flexibility and structural alterations resulting in increased resistance and steady-state turnover of larger cephalosporins. Further characterization provides unprecedented insight into the mechanism of cephalosporin hydrolysis by ADC \u03b2-lactamases and supports a concomitant increase in ADC structural flexibility and cephalosporin affinity that leads to more efficient hydrolysis. In addition, the crystal structure of ADC-33 in complex with ceftazidime is consistent with a substrate-assisted catalysis mechanism. The structural differences in the ADC-33 active site leading to ceftazidime catalysis provide a better understanding of \u03b2-lactamase Adup variants and open important opportunities for future drug design and development.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00287-25","type":"journal-article","created":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T09:00:47Z","timestamp":1747386047000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Resistance to oxyimino-cephalosporins conferred by an alternative mechanism of hydrolysis by the\n                    <i>Acinetobacter<\/i>\n                    -derived cephalosporinase-33 (ADC-33), a class C \u03b2-lactamase present in carbapenem-resistant\n                    <i>Acinetobacter baumannii<\/i>\n                    (CR\n                    <i>Ab<\/i>\n                    )"],"prefix":"10.1128","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9968-8284","authenticated-orcid":false,"given":"Rachel A.","family":"Powers","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/001m1hv61","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemistry, Grand Valley State University","place":["Allendale, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1466-4794","authenticated-orcid":false,"given":"Bradley J.","family":"Wallar","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/001m1hv61","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemistry, Grand Valley State University","place":["Allendale, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah R.","family":"Jarvis","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/001m1hv61","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemistry, Grand Valley State University","place":["Allendale, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zoe X.","family":"Ziegler","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/001m1hv61","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemistry, Grand Valley State University","place":["Allendale, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia M.","family":"June","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/001m1hv61","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemistry, Grand Valley State University","place":["Allendale, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher 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