1829. The Paradox of KPC Bearing Strains of Klebsiella pneumoniae with the D179Y Substitution: Resistance to Ceftazidine/Avibactam (CZA) and Susceptibility to Meropenem (MEM)

Abstract Background Resistance to CZA is a serious limitation of treatment for KPC bearing Enterobacteriaceae infections. Recently, a single amino acid substitution (D179Y) was described in KPC-2 and KPC-3 bearing CZA-resistant K. pneumoniae recovered from patients failing treatment. In class A β-lactamases the D179 residue is located at the neck of the omega loop and is critical for KPC catalytic activity. In attempts to understand the evolution of substrate specificity in KPC-2, the D179Y variant of KPC-2 was shown to be resistant to CZA (ceftazidime forms a long-lived acyl enzyme with in KPC-2), but susceptible to MEM. A similar observation was made in clinical and laboratory-generated K. pneumoniae and E. coli strains bearing D179Y KPC-3. We were compelled to explore the catalytic mechanisms of susceptibility to MEM of the D179Y variants in KPC-2 vs. KPC-3. Methods KPC-2, KPC-3, and D179Y in the respective KPC were cloned into an expression vector and the β-lactamase proteins were purified. 5 mg of each β-lactamase with and without MEM (1:1 molar ratio) was incubated for the time indicated and analyzed using the Quadrupole Time-of-Flight (QTOF) timed mass spectrometry for the reaction intermediates. To assess thermal stability, denaturation melting curves were run for 2 hours using 12 µM β-lactamase. Results The D179Y variant forms prolonged acyl-complexes with meropenem in KPC-3 and KPC-2, which can be detected up to 24 hours (Figure 1). This prolonged trapping of meropenem by D179Y variants is not evident with the respective KPCs. Further, the tyrosine substitution at the D179 position (Tm = 48–52°C) destabilizes the KPC β-lactamases (TmKPC-2/3 = 52–56°C). Conclusion These data suggest that MEM acts as a covalent β-lactamase inhibitor more than as a substrate for KPC-2 and -3. The mechanistic basis of paradoxical susceptibility to carbapenems provides an impetus to develop better therapeutic approaches to the increasing threat of carbapenem resistance and highlights how the rational design of novel β-lactam/β-lactamase inhibitors must consider mechanistic bases of resistance. Disclosures All Authors: No reported Disclosures.