A3. Resistance mechanisms
Oral Abstract Submission
Melissa D. Barnes, Ph.D.
Senior Research Associate
Case Western Reserve University
Cleveland, OH
Magdalena A. Taracilla, MS
Researcher
Research Service, Louis Stokes Veterans Affairs Medical Center
Cleveland, OH
Disclosure: Nothing to disclose
Joseph D. Rutter, BS
Research Assistant
Louis Stokes Cleveland VA Medical Center
Cleveland, OH
Disclosure: Nothing to disclose
Minh-Hong Nguyen, MD
Professor of Medicine
University of Pittsburgh
Pittsburgh, PA
Disclosure: Astellas: Consultant, Grant/Research Support
Cidara: Consultant, Grant/Research Support
Melinta: Grant/Research Support
Merck: Consultant, Grant/Research Support
Scynexis: Consultant
Ryan K. Shields, PharmD, MS
Associate Professor of Medicine
University of Pittsburgh
Pittsburgh, PA
Disclosure: Allergan: Consultant, Grant/Research Support
Melinta Therapeutics: Grant/Research Support
Merck, Sharpe, and Dohme: Consultant, Grant/Research Support
Pfizer, Inc: Consultant
Shionogi: Consultant, Grant/Research Support
Tetraphase Pharmaceuticals: Grant/Research Support
VenatoRx: Grant/Research Support
Cornelius J. Clancy, MD
Associate Professor of Medicine
University of Pittsburgh
Pittsburgh, PA
Disclosure: Astellas: Consultant, Grant/Research Support
Cidara: Consultant, Research Grant or Support
Melinta: Grant/Research Support
Merck: Consultant, Grant/Research Support
Needham Associates: Consultant
Qpex: Consultant
Scynexis: Consultant
Shionogi: Consultant
Robert A. Bonomo, MD
Chief of Medicine
Louis Stokes Cleveland VA Medical Center
Cleveland, Ohio
Disclosure: Nothing to disclose
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 Quadropole 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 (Fig. 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.
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