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β-Lactamases can accumulate stepwise mutations that increase their resistance profiles to the latest β-lactam agents. CMY-185 is a CMY-2-like β-lactamase and was identified in an Escherichia coli clinical strain isolated from a patient who underwent treatment with ceftazidime-avibactam. CMY-185, possessing four amino acid substitutions of A114E, Q120K, V211S, and N346Y relative to CMY-2, confers high-level ceftazidime-avibactam resistance, and accumulation of the substitutions incrementally enhances the level of resistance to this agent. However, the functional role of each substitution and their interplay in enabling ceftazidime-avibactam resistance remains unknown. Through biochemical and structural analysis, we present the molecular basis for the enhanced ceftazidime hydrolysis and impaired avibactam inhibition conferred by CMY-185. The substituted Y346 residue is a major driver of the functional evolution as it rejects primary avibactam binding due to the steric hindrance and augments oxyimino-cephalosporin hydrolysis through a drastic structural change, rotating the side chain of Y346 and then disrupting the H-10 helix structure. The other substituted residues E114 and K120 incrementally contribute to rejection of avibactam inhibition, while S211 stimulates the turnover rate of the oxyimino-cephalosporin hydrolysis. These findings indicate that the N346Y substitution is capable of simultaneously expanding the spectrum of activity against some of the latest β-lactam agents with altered bulky side chains and rejecting the binding of β-lactamase inhibitors. However, substitution of additional residues may be required for CMY enzymes to achieve enhanced affinity or turnover rate of the β-lactam agents leading to clinically relevant levels of resistance.IMPORTANCECeftazidime-avibactam has a broad spectrum of activity against multidrug-resistant Gram-negative bacteria including carbapenem-resistant Enterobacterales including strains with or without production of serine carbapenemases. After its launch, emergence of ceftazidime-avibactam-resistant strains that produce mutated β-lactamases capable of efficiently hydrolyzing ceftazidime or impairing avibactam inhibition are increasingly reported. Furthermore, cross-resistance towards cefiderocol, the latest cephalosporin in clinical use, has been observed in some instances. Here, we clearly demonstrate the functional role of the substituted residues in CMY-185, a four amino-acid variant of CMY-2 identified in a patient treated with ceftazidime-avibactam, for high-level resistance to this agent and low-level resistance to cefiderocol. These findings provide structural insights into how β-lactamases may incrementally alter their structures to escape multiple advanced β-lactam agents., Competing Interests: Y.D. has received research support from Entasis and Shionogi; consulting fees from Gilead, Moderna, GSK, MeijiSeika Pharma, Shionogi, and bioMérieux; and honoraria from Gilead, MSD, and Shionogi.

Objectives: To characterize a blaCMY variant associated with ceftazidime/avibactam resistance from a serially collected Escherichia coli isolate., Methods: A patient with an intra-abdominal infection due to recurrent E. coli was treated with ceftazidime/avibactam. On Day 48 of ceftazidime/avibactam therapy, E. coli with a ceftazidime/avibactam MIC of >256 mg/L was identified from abdominal drainage. Illumina and Oxford Nanopore Technologies WGS was performed on serial isolates to identify potential resistance mechanisms. Site-directed mutants of CMY β-lactamase were constructed to identify amino acid residues responsible for ceftazidime/avibactam resistance., Results: WGS revealed that all three isolates were E. coli ST410. The ceftazidime/avibactam-resistant strain uniquely acquired a novel CMY β-lactamase gene, herein called blaCMY-185, harboured on an IncI-γ/K1 conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2, including A114E, Q120K, V211S and N346Y, and conferred high-level ceftazidime/avibactam resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced ceftazidime/avibactam susceptibility. However, double and triple mutants containing N346Y previously associated with ceftazidime/avibactam resistance in other AmpC enzymes, conferred ceftazidime/avibactam MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to steric hindrance between the side chain of Y346 and the sulphate group of avibactam., Conclusions: We identified ceftazidime/avibactam resistance in E. coli associated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer ceftazidime/avibactam resistance., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)