title: Reclaiming the efficacy of β-lactam-β-lactamase inhibitor combinations: avibactam restores the susceptibility of CMY-2-producing Escherichia coli to ceftazidime.
abstract: CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and β-lactamase inhibitors in strains carrying blaCMY, novel β-lactam-β-lactamase inhibitor combinations are sought to combat this significant threat to β-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-β-lactam β-lactamase inhibitor in clinical development that reversibly inactivates serine β-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry bla(CMY-2) or bla(CMY-69) and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC(90), ≤ 0.5 mg/liter); in comparison, ceftazidime had a MIC90 of >128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 β-lactamase, as demonstrated by a second-order onset of acylation rate constant (k2/K) of (4.9 ± 0.5) × 10(4) M(-1) s(-1) and the off-rate constant (k(off)) of (3.7 ± 0.4) × 10(-4) s(-1). Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2-avibactam acyl-enzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69.
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Marisa L
authors.initials: ML
authors.lastname: Winkler
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Gatta
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Magdalena A
authors.initials: MA
authors.lastname: Taracila
authors.affiliation: Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Chilakala
authors.affiliation: Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Pathology, University of Maryland, Baltimore, Maryland, USA.
authors.forename: J Kristie
authors.initials: JK
authors.lastname: Johnson
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA robert.bonomo@va.gov.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D002442:Ceftazidime / Q000494:pharmacology*; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006860:Hydrogen Bonding; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D011485:Protein Binding; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; C543519:avibactam; D002442:Ceftazidime; C103022:beta-lactamase CMY-2; D001618:beta-Lactamases