title: Avibactam and inhibitor-resistant SHV β-lactamases.
abstract: β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.
authors.affiliation: Louis Stokes Veterans Affairs Medical Center, 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: Louis Stokes Veterans Affairs Medical Center, 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: Louis Stokes Veterans Affairs Medical Center, 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: Louis Stokes Veterans Affairs Medical Center, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, 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 Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA robert.bonomo@va.gov.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000215:Acylation; D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D020134:Catalytic Domain / Q000187:drug effects; D002442:Ceftazidime / Q000494:pharmacology; D004338:Drug Combinations; D024881:Drug Resistance, Bacterial / Q000502:physiology*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D065093:beta-Lactamase Inhibitors / Q000494:pharmacology*; D001618:beta-Lactamases / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure
chemical_list: D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; D004338:Drug Combinations; D065093:beta-Lactamase Inhibitors; C543519:avibactam; D000667:Ampicillin; D002442:Ceftazidime; C473109:beta-lactamase CTX-M-15; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases