Combination of amino acid substitutions leading to CTX-M-15-mediated resistance to the ceftazidime-avibactam combination [PublishAheadOfPrint]

Single amino-acid substitutions in the -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 -loop of the 172 CTX-M sequences present in the Lahey database. 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).

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