Ceftazidime-Avibactam Resistance Mediated by the NY Substitution in Various AmpC β-Lactamases.

Chromosomal and plasmid-borne AmpC cephalosporinases are a major resistance mechanism to β-lactams in and The new β-lactamase inhibitor avibactam effectively inhibits class C enzymes and can fully restore ceftazidime susceptibility. The conserved amino acid residue Asn of AmpC cephalosporinases directly interacts with the avibactam sulfonate. Disruption of this interaction caused by the NY amino acid substitution in AmpC was previously shown to confer resistance to the ceftazidime-avibactam combination (CAZ-AVI). The aim of this study was to phenotypically and biochemically characterize the consequences of the NY substitution in various AmpC backgrounds. Introduction of NY into AmpC (AmpC), plasmid-mediated DHA-1, and PDC-5 led to 270-, 12,000-, and 79-fold decreases in the inhibitory efficacy (/ ) of avibactam, respectively. The kinetic parameters of AmpC and DHA-1 for ceftazidime hydrolysis were moderately affected by the substitution. Accordingly, AmpC and DHA-1 harboring NY conferred CAZ-AVI resistance (MIC of ceftazidime of 16 μg/ml in the presence of 4 μg/ml of avibactam). In contrast, production of PDC-5 NY was associated with a lower MIC (4 μg/ml) since this β-lactamase retained a higher inactivation efficacy by avibactam in comparison to AmpC NY. For FOX-3, the IY substitution did not reduce the inactivation efficacy of avibactam and the substitution was highly deleterious for β-lactam hydrolysis, including ceftazidime, preventing CAZ-AVI resistance. Since AmpC and DHA-1 display substantial sequence diversity, our results suggest that loss of hydrogen interaction between Asn and avibactam could be a common mechanism of acquisition of CAZ-AVI resistance.