Ceftazidime-Avibactam Resistance Mediated by the N346Y Substitution in Various AmpC β-Lactamases

Chromosomal and plasmid-borne AmpC cephalosporinases are a major resistance mechanism to beta-lactams in Enterobacteriaceae and Pseudomonas aeruginosa. The new beta-lactamase inhibitor avibactam effectively inhibits class C enzymes and can fully restore ceftazidime susceptibility. The conserved amino acid residue Asn(346) of AmpC cephalosporinases directly interacts with the avibactam sulfonate. Disruption of this interaction caused by the (NY)-Y-346 amino acid substitution in Citrobacter freundii AmpC was previously shown to confer resistance to the ceftazidimeavibactam combination (CAZ-AVI). The aim of this study was to phenotypically and biochemically characterize the consequences of the (NY)-Y-346 substitution in various AmpC backgrounds. Introduction of (NY)-Y-346 into Enterobacter cloacae AmpC (AmpC(cloacae)), plasmid-mediated DHA-1, and P. aeruginosa PDC-5 led to 270-, 12,000-, and 79-fold decreases in the inhibitory efficacy (k(2)/K-i) of avibactam, respectively. The kinetic parameters of AmpC(cloacae) and DHA-1 for ceftazidime hydrolysis were moderately affected by the substitution. Accordingly, AmpC(cloacae) and DHA-1 harboring (NY)-Y-346 conferred CAZ-AVI resistance (MIC of ceftazidime of 16 mu g/ml in the presence of 4 mu g/ml of avibactam). In contrast, production of PDC-5 (NY)-Y-346 was associated with a lower MIC (4 mu g/ml) since this beta-lactamase retained a higher inactivation efficacy by avibactam in comparison to AmpC(cloacae) (NY)-Y-346. For FOX-3, the (IY)-Y-346 substitution did not reduce the inactivation efficacy of avibactam and the substitution was highly deleterious for beta-lactam hydrolysis, including ceftazidime, preventing CAZ-AVI resistance. Since AmpC(cloacae) and DHA-1 display substantial sequence diversity, our results suggest that loss of hydrogen interaction between Asn(346) and avibactam could be a common mechanism of acquisition of CAZ-AVI resistance.