(T1130-04-28) Periplasmic Target Site Penetration Rates of Four β-Lactamase Inhibitors in Klebsiella Pneumoniae Carbapenemase-2 (KPC-2) Producing Klebsiella pneumoniae

Purpose: Carbapenem-resistant K. pneumoniae isolates are posing significant clinical challenges due to their resistance towards many β-lactams. Combining available β-lactams with a novel β-lactamase inhibitor (BLI) has yielded new and effective combinations. However, there are substantial gaps regarding the outer membrane penetration and periplasmic target site concentrations of BLIs. This study aims to characterize the rate and extent of penetration for four clinically-relevant BLIs into periplasm in a carbapenem-resistant K. pneumoniae via UPLC-MS/MS.

Methods: A KPC-2 producing clinical KP isolate, KP3800, was grown to high inocula (10^7.7 or 10^7.85 CFU/mL). Half of the bacteria were lysed to evaluate the periplasmic β-lactamase inhibition activity by the BLIs (avibactam [AVI], relebactam [REL], vaborbactam [VAB] and taniborbactam [TAN]). The other half served as intact bacteria arms to characterize the outer membrane penetration of BLIs. Imipenem (IPM; 10 mg/L) was added at 0 min as a rapidly penetrating victim β-lactam. At 4 min, BLIs were added at 0, 0.0315, 0.125, 0.5, 2, 8, 16, 32 mg/L to the intact and lysed bacteria arms. The time-course of IPM degradation was determined over 60 min by UPLC-MS/MS.

Results: For near-complete inhibition of IPM degradation, AVI required ~0.125 mg/L in lysed and ~2 mg/L in intact cells (Figure 1). REL required ~0.5 mg/L in lysed and ~8 mg/L in intact cells. VAB required ~0.5 mg/L in lysed and ~8 mg/L in intact cells. TAN required ~0.125 to 0.5 mg/L in lysed and ~2 mg/L in intact cells. In lysed cells, β-lactamase inhibition occurred within < 1-2 min. In intact cells, near-complete inhibition started at 6-10min (i.e. at 2-6 min after BLI addition), depending on BLI concentration. This suggested a similar rate of penetration for the four BLIs (full time course modeling is ongoing).

Conclusion: The four BLIs required ~16x higher concentrations in intact compared to lysed cells for β-lactamase inhibition and their rate of penetration into periplasm was roughly comparable. This novel UPLC-MS/MS assay allowed us to compare the rate and extent of BLI penetration into periplasm. These target site penetration data provide important insights to design efficacious β-lactam/BLI combination dosing strategies of current and future BLIs.

Acknowledgements: Support: This work was supported by NIH/NIAID R01 AI136803 and R01 AI148560

Figure 1. Rapid OM penetration of four BLIs in a KPC-producing CRKP. A: AVI, B: REL, C: VAB and D: TAN. The BLI was added at 4 min. In intact cells (top) imipenem hydrolysis was stopped within 2 to 6 min, whereas hydrolysis stopped within < 1 to 2 min in lysed cells (bottom).