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Ceftazidime-Avibactam Resistance Mediated by the N346Y Substitution in Various AmpC β-Lactamases
Fabrice Compain1,2, Agathe Debray1, Pauline Adjadj1
1INSERM, Sorbonne Université, Université de Paris, Centre de Recherche des Cordeliers, Paris, France.
Abstract:
Chromosomal and plasmid-borne AmpC cephalosporinases are a major resistance mechanism to β-lactams in Enterobacteriaceae and Pseudomonas aeruginosa The new β-lactamase inhibitor avibactam effectively inhibits class C enzymes and can fully restore ceftazidime susceptibility. The conserved amino acid residue Asn346 of AmpC cephalosporinases directly interacts with the avibactam sulfonate. Disruption of this interaction caused by the N346Y amino acid substitution in Citrobacter freundii 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 N346Y substitution in various AmpC backgrounds. Introduction of N346Y into Enterobacter cloacae AmpC (AmpCcloacae), plasmid-mediated DHA-1, and P. aeruginosa PDC-5 led to 270-, 12,000-, and 79-fold decreases in the inhibitory efficacy (k2/K ) of avibactam, respectively. The kinetic parameters of AmpCcloacae and DHA-1 for ceftazidime hydrolysis were moderately affected by the substitution. Accordingly, AmpCcloacae and DHA-1 harboring N346Y 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 N346Y was associated with a lower MIC (4 μg/ml) since this β-lactamase retained a higher inactivation efficacy by avibactam in comparison to AmpCcloacae N346Y. For FOX-3, the I346Y 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 AmpCcloacae and DHA-1 display substantial sequence diversity, our results suggest that loss of hydrogen interaction between Asn346 and avibactam could be a common mechanism of acquisition of CAZ-AVI resistance.
Insights
A specific amino acid change (N346Y) in AmpC cephalosporinases can cause resistance to ceftazidime-avibactam (CAZ-AVI). This disruption of interaction with avibactam highlights a common mechanism for CAZ-AVI resistance in bacteria.
Area of Science:
- Molecular biology
- Microbiology
- Biochemistry
Background:
- AmpC cephalosporinases confer resistance to beta-lactams in Enterobacteriaceae and Pseudomonas aeruginosa.
- Avibactam is a novel beta-lactamase inhibitor that restores susceptibility to ceftazidime by inhibiting class C enzymes.
- The Asn346 residue is crucial for avibactam's inhibitory activity, and its substitution can lead to resistance.
Purpose of the Study:
- To investigate the phenotypic and biochemical effects of the Asn346Tyr (N346Y) substitution in various AmpC cephalosporinase backgrounds.
- To understand the mechanism by which the N346Y substitution confers resistance to the ceftazidime-avibactam combination (CAZ-AVI).
Main Methods:
- Introduction of the N346Y substitution into AmpC cephalosporinases from Enterobacter cloacae (AmpCcloacae), plasmid-mediated DHA-1, and Pseudomonas aeruginosa PDC-5.
- Phenotypic characterization of CAZ-AVI resistance through minimum inhibitory concentration (MIC) determination.
- Biochemical assessment of avibactam's inhibitory efficacy (k2/K) and kinetic parameters for ceftazidime hydrolysis.
Main Results:
- The N346Y substitution significantly decreased avibactam's inhibitory efficacy against AmpCcloacae (270-fold), DHA-1 (12,000-fold), and PDC-5 (79-fold).
- AmpCcloacae and DHA-1 with the N346Y substitution conferred resistance to CAZ-AVI, with ceftazidime MICs of 16 μg/ml.
- PDC-5 N346Y showed lower resistance (MIC of 4 μg/ml) due to retained avibactam inactivation, while FOX-3 I346Y was deleterious for hydrolysis, preventing resistance.
Conclusions:
- The N346Y substitution disrupts the critical interaction between AmpC cephalosporinases and avibactam, leading to ceftazidime-avibactam resistance.
- Loss of the hydrogen bond between Asn346 and avibactam represents a potential common mechanism for acquiring CAZ-AVI resistance.
- Understanding these resistance mechanisms is crucial for developing effective strategies against carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa.
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