Reclaiming the Efficacy of β-Lactam-β-Lactamase Inhibitor Combinations: Avibactam Restores the Susceptibility of CMY-2-Producing Escherichia coli to Ceftazidime

CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and beta-lactamase inhibitors in strains carrying bla(CMY), novel beta-lactam-beta-lactamase inhibitor combinations are sought to combat this significant threat to beta-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1] octanone non-beta-lactam beta-lactamase inhibitor in clinical development that reversibly inactivates serine beta-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry bla(CMY-2) or bla(CMY-69) and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC90, <= 0.5 mg/liter); in comparison, ceftazidime had a MIC90 of > 128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 beta-lactamase, as demonstrated by a second-order onset of acylation rate constant (k(2)/K) of (4.9 +/- 0.5) x 10(4) M-1 s(-1) and the off-rate constant (k(off)) of (3.7 +/- 0.4) x 10(-4) s(-1). Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2-avibactam acylenzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69.