1385. Mechanism-Based, In Vitro Inhibition of Mycobacterium abscessus: Assessing β-Lactam Therapy

Background M. abscessus (Mab) is an emerging pathogen, a highly drug-resistant rapidly-growing nontuberculous mycobacteria. Mab L, D transpeptidases (LdtMab 1–5), D,D carboxypeptidase and BlaMab β-lactamase are important targets. Herein, we tested the susceptibility of ceftaroline (TAR) and imipenem (IMI) alone and in combinations with two diazabicyclooctanone β-lactamase inhibitors (BLI), relebactam (REL) and avibactam (AVI), against representative clinical isolates belonging to the Mab complex and assessed the mechanism of inhibition using mass spectrometry (QTOF-MS) Methods Minimum inhibitory concentrations (MICs) of TAR and IMI with or without AVI and REL and a TAR-IMI combination with and without REL were determined using microdilution. Approximately 5 x 105 colony-forming units (CFU) per milliliter were inoculated into Middlebrook 7H9 broth supplemented with 10% (vol/vol) oleic albumin dextrose catalase and 0.05% (vol/vol) Tween 80. AVI or REL were added at fixed concentration of 4 µg/mL to serial dilutions of TAR or IMI. For the TAR-IMI combinations, IMI at 1 µg/mL, and serial dilutions of TAR were used. Mab isolates were incubated with test agents at 30°C for 48 h, and MIC was defined as lowest antibiotic concentration that prevented visible bacterial growth. (QTOF-MS) was used to assess intermediates of BlaMab, LdtMab1 and LdtMab2 with TAR, IMI, AVI, and REL Results In-vitro susceptibility testing on representative clinical Mab strains (table). MIC90 was > 128 μg/mL for TAR and 8 μg/mL for IMI. Combination of TAR and IMI lowered MIC’s of all clinical isolates to <0.06 μg/mL. Addition of REL or AVI lowered TAR MICs but had minimal or no impact on IMI or TAR-IMI MICs. Mass spectrometry analyses of BlaMab, LdtMab (1–2) alone and incubated with IMI, TAR, REL and AVI (figure). BlaMab β-lactamase bound the AVI and REL, but acyl complexes with TAR or IMI were not detected. LdtMab (1–2) form stable acyl complexes with AVI, REL, TAR, and IMI. Conclusion Addition of IMI to TAR lowers MICs of TAR against Mab to therapeutically achievable concentrations. It would be welcome news for clinicians who are treating patients with highly resistant Mab infection that the combination of TAR and IMI is commercially available and thus might be considered as part of a rescue regimen. Disclosures All authors: No reported disclosures.

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

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.