Abstract
Mutations in KPC-2 and KPC-3 β-lactamase can confer resistance to the β-lactam/β-lactamase inhibitor antibacterial intravenous drug combination ceftazidime-avibactam, introduced in 2015. Avibactam was the first of the diazabicyclooctane class of non-β-lactam β-lactamase inhibitors to be approved for clinical use. The orally bioavailable prodrug ETX0282 of the diazabicyclooctane β-lactamase inhibitor ETX1317 is in clinical development in combination with the oral β-lactam prodrug cefpodoxime proxetil for use against complicated urinary tract infections. We investigated the effects of 3 ceftazidime-avibactam resistance mutations in KPC-3 (V240G, D179Y, and D179Y/T243M) on the ability of ETX1317 to overcome KPC-3-induced cefpodoxime resistance. Isogenic Escherichia coli strains, each expressing the wild-type or a mutant KPC-3 at similar levels, retained susceptibility to cefpodoxime-ETX1317 (1:2) with essentially identical minimal inhibitory concentrations of 0.125-0.25 μg/mL cefpodoxime. The KPC-3 mutations had little or no effect on the kinact/Ki values for inhibition by each of 3 diazabicyclooctanes: avibactam, durlobactam (ETX2514), and ETX1317. The KM values for hydrolysis of cefpodoxime were similar for all 4 variants, but the kcat values of the D179Y and D179Y/T243M variants were much lower than those of the wild-type and V240G mutant enzymes. All 4 KPC-3 variants formed stable, reversibly covalent complexes with ETX1317, but dissociation of ETX1317 was much slower from the D179Y and D179Y/T243M mutants than from the wild-type and V240G mutant enzymes. Thus, the KPC-3 variants examined here that cause resistance to ceftazidime-avibactam do not cause resistance to cefpodoxime-ETX1317.
Highlights
Mutations in KPC-2 and KPC-3 β-lactamase can confer resistance to the β-lactam/β-lactamase inhibitor antibacterial intravenous drug combination ceftazidime−avibactam, introduced in 2015
The cephalosporin nitrocefin is frequently used as a β-lactamase substrate in inhibition measurements because of its large absorbance increase upon hydrolysis
For ceftazidime−avibactam resistance, it is clear from the results presented here that the considerable ceftazidime KM decrease in the D179Y and D179Y/T243M mutants of KPC-3 is responsible
Summary
Mutations in KPC-2 and KPC-3 β-lactamase can confer resistance to the β-lactam/β-lactamase inhibitor antibacterial intravenous drug combination ceftazidime−avibactam, introduced in 2015. The orally bioavailable prodrug ETX0282 of the diazabicyclooctane β-lactamase inhibitor ETX1317 is in clinical development in combination with the oral β-lactam prodrug cefpodoxime proxetil for use against complicated urinary tract infections. Isogenic Escherichia coli strains, each expressing the wild-type or a mutant KPC-3 at similar levels, retained susceptibility to cefpodoxime−ETX1317 (1:2) with essentially identical minimal inhibitory concentrations of 0.125−0.25 μg/mL cefpodoxime. ETX0282 is an orally bioavailable prodrug of the diazabicyclooctane BLI ETX1317 (Figure 1D).[12] In combination with the cephalosporin prodrug cefpodoxime proxetil, ETX0282 is intended for a cUTI indication and would be used to treat infections with Enterobacteriales, including Escherichia coli and Klebsiella pneumoniae, which would include strains expressing KPC β-lactamases.[27] It is of interest to determine whether the ceftazidime− avibactam resistance mutations in KPC enzymes confer resistance to cefpodoxime−ETX1317. This work reports the results of our investigation of the effects of certain previously reported KPC-3 mutations: V240G (KPC-8), D179Y (KPC31), and D179Y/T243M (KPC-32)[25] that confer ceftazidime−
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