{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T01:23:43Z","timestamp":1790817823568,"version":"4.1.0"},"reference-count":30,"publisher":"American Society for Microbiology","issue":"5","license":[{"start":{"date-parts":[[2017,10,31]],"date-time":"2017-10-31T00:00:00Z","timestamp":1509408000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2017,11,8]],"date-time":"2017-11-08T00:00:00Z","timestamp":1510099200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"Veterans Affairs Merit Review Program Award","award":["1I01BX002872"],"award-info":[{"award-number":["1I01BX002872"]}]},{"name":"Veterans Affairs Merit Review Program Award","award":["1I01BX001974"],"award-info":[{"award-number":["1I01BX001974"]}]},{"name":"Harrington Foundation"},{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R21AI114508"],"award-info":[{"award-number":["R21AI114508"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI100560"],"award-info":[{"award-number":["R01AI100560"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI063517"],"award-info":[{"award-number":["R01AI063517"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"HHS | NIH | 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":[[2017,11,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    The emergence of\n                    <jats:italic>Klebsiella pneumoniae<\/jats:italic>\n                    carbapenemases (KPCs), \u03b2-lactamases that inactivate \u201clast-line\u201d antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent \u03b2-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the \u03a9-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn \u201ctraps\u201d \u03b2-lactams, preferentially binding \u03b2-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the \u03a9-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5\u00a0\u00c5 and rotated more than 90\u00b0, altering the hydrogen bond networks; and (iii) E166 is displaced by 2\u00a0\u00c5 when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel \u03b2-lactams and \u03b2-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>IMPORTANCE<\/jats:bold>\n                    Antibiotic resistance is emerging at unprecedented rates and threatens to reach crisis levels. One key mechanism of resistance is the breakdown of \u03b2-lactam antibiotics by \u03b2-lactamase enzymes. KPC-2 is a \u03b2-lactamase that inactivates carbapenems and \u03b2-lactamase inhibitors (e.g., clavulanate) and is prevalent around the world, including in the United States. Resistance to the new antibiotic ceftazidime-avibactam, which was designed to overcome KPC resistance, had already emerged within a year. Using protein engineering, we uncovered a mechanism by which resistance to this new drug emerges, which could arm scientists with the ability to forestall such resistance to future drugs.\n                  <\/jats:p>","DOI":"10.1128\/mbio.00528-17","type":"journal-article","created":{"date-parts":[[2017,10,31]],"date-time":"2017-10-31T20:10:14Z","timestamp":1509480614000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":133,"title":["<i>Klebsiella pneumoniae<\/i>\n                    Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from \u03b2-Lactamase Protein Engineering"],"prefix":"10.1128","volume":"8","author":[{"given":"Melissa D.","family":"Barnes","sequence":"first","affiliation":[{"name":"Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA"},{"name":"Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marisa L.","family":"Winkler","sequence":"additional","affiliation":[{"name":"Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA"},{"name":"Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdalena A.","family":"Taracila","sequence":"additional","affiliation":[{"name":"Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA"},{"name":"Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Malcolm G.","family":"Page","sequence":"additional","affiliation":[{"name":"Jacobs University, Bremen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric","family":"Desarbre","sequence":"additional","affiliation":[{"name":"Basilea Pharmaceutica International Ltd., Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barry N.","family":"Kreiswirth","sequence":"additional","affiliation":[{"name":"Public Health Research Institute Center, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan K.","family":"Shields","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minh-Hong","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cornelius","family":"Clancy","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brad","family":"Spellberg","sequence":"additional","affiliation":[{"name":"Los Angeles County + University California Medical Center, Los Angeles, California, USA"},{"name":"Division of Infectious Diseases, Keck School of Medicine at USC, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Krisztina M.","family":"Papp-Wallace","sequence":"additional","affiliation":[{"name":"Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA"},{"name":"Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA"},{"name":"Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA"},{"name":"Department of Proteomics, Case Western Reserve University, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert A.","family":"Bonomo","sequence":"additional","affiliation":[{"name":"Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA"},{"name":"Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA"},{"name":"Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA"},{"name":"Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA"},{"name":"Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","volume-title":"High-level meeting on antimicrobial resistance","author":"General Assembly of the United Nations","year":"2016","unstructured":"General Assembly of the United Nations. 21September2016. 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Performance standards for antimicrobial susceptibility testing; 24th informational supplement. CLSI, Wayne, PA. 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