title: Variants of β-lactamase KPC-2 that are resistant to inhibition by avibactam.
abstract: KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M(-1) s(-1) to 1.2 M(-1) s(-1)). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants.
authors: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors: Krisztina M
authors: KM
authors: Papp-Wallace
authors: 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: Marisa L
authors: ML
authors: Winkler
authors: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors: Magdalena A
authors: MA
authors: Taracila
authors: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA robert.bonomo@va.gov.
authors: Robert A
authors: RA
authors: Bonomo
mesh_terms: D000215:Acylation; D019943:Amino Acid Substitution / Q000235:genetics; D000667:Ampicillin / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D002442:Ceftazidime / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004338:Drug Combinations; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D018440:beta-Lactam Resistance / Q000187:drug effects; D065093:beta-Lactamase Inhibitors / Q000494:pharmacology; D001618:beta-Lactamases / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure
chemical_list: D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; D004338:Drug Combinations; C000595613:avibactam, ceftazidime drug combination; D065093:beta-Lactamase Inhibitors; C543519:avibactam; D000667:Ampicillin; D002442:Ceftazidime; C491478:beta-lactamase KPC-2; D001618:beta-Lactamases