Combination of Amino Acid Substitutions Leading to CTX-M-15-Mediated Resistance to the Ceftazidime-Avibactam Combination

Abstract

Single amino acid substitutions in the Ω loop of KPC β-lactamases are known to lead to resistance to the ceftazidime-avibactam combination. Here, we investigate this mechanism of resistance in CTX-M enzymes, which are the most widely spread extended-spectrum β-lactamases worldwide. Nine single amino acid polymorphisms were identified in the Ω loop of the 172 CTX-M sequences present in the Lahey database of β-lactamases. The corresponding modifications were introduced in CTX-M-15 by site-directed mutagenesis. None of the nine substitutions was associated with ceftazidime-avibactam resistance in Escherichia coli TOP10. However, two substitutions led to 4-fold (P¹⁶⁷S) and 16-fold (L¹⁶⁹Q) increases in the MIC of ceftazidime. We determined whether these substitutions favor the in vitro selection of mutants resistant to ceftazidime-avibactam. The selection provided mutants for the L¹⁶⁹Q substitution but not for the P¹⁶⁷S substitution or for the parental enzyme CTX-M-15. Resistance to the drug combination (MIC of ceftazidime, 16 μg/ml in the presence of 4 μg/ml of avibactam) resulted from the acquisition of the S¹³⁰G substitution by CTX-M-15 L¹⁶⁹Q. Purified CTX-M-15 with the two substitutions, L¹⁶⁹Q and S¹³⁰G, was only partially inhibited by avibactam at concentrations as high as 50,000 μM but retained ceftazidime hydrolysis activity with partially compensatory decreases in k_cat and K_m These results indicate that emergence of resistance to the ceftazidime-avibactam combination requires more than one mutation in most CTX-M-encoding genes. Acquisition of resistance could be restricted to rare variants harboring predisposing polymorphisms such as Q at position 169 detected in a single naturally occurring CTX-M enzyme (CTX-M-93).

Keywords: CTX-M; avibactam; ceftazidime; Ω loop; β-lactamase inhibitor.

FIG 1

Kinetics of CTX-M-15 (A), CTX-M-15 P¹⁶⁷S (B), and CTX-M-15 S¹³⁰G L¹⁶⁹Q (C) inhibition by avibactam. The β-lactamases CTX-M-15 (0.002 μM), CTX-M-15 P¹⁶⁷S (0.002 μM), and CTX-M-15 S¹³⁰G L¹⁶⁹Q (1 μM) were incubated with nitrocefin (100 μM) and avibactam at the indicated concentrations at 20°C in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.4). (D) The efficacy of the carbamoylation reaction was evaluated based on inhibition of nitrocefin hydrolysis (Δε_{486 nm} = 14,600 M⁻¹ cm⁻¹), as previously described (9, 15). Avi, avibactam. (E) Equation 1 was fitted to data to determine the value of the observed rate constant (k_obs) for the various concentrations of avibactam. The carbamoylation efficacy (k₂/K_i) was determined by plotting the values of k_obs as a function of the inhibitor concentration (F) and fitting equation 2 to the data. Abbreviations: [P], concentration of hydrolyzed nitrocefin; v_i, uninhibited enzyme velocity; v_s, fully inhibited enzyme velocity; [S], initial concentration of nitrocefin; [I], avibactam concentration; K_m, Michaelis constant for hydrolysis of nitrocefin by CTX-M β-lactamases.