Avibactam (AVI) inactivation of PER-2 beta-lactamase: Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition

M. RUGGIERO; K. M. PAPP-WALLACE; G. GUTKIND; R. A. BONOMO; P. POWER

Boston

Congreso; ASM Microbe 2016; 2016

Background: PER β-lactamases are prevalent in South America and the Middle East and readily hydrolyze oxyimino-cephalosporins. This enhanced hydrolytic activity may result from the presence of a unique fold in the Ω loop and in the β3 strand. These structural differences enlarge the active site entrance by 2-fold compared to other class A β-lactamases. PER-2 possesses higher catalytic efficiency towards ceftazidime (CAZ) compared to PER-1 (~22-fold higher). Examination of the apo-structure reveals that the H-bonding (HB) network between S70-Q69-HOH-T237-R220 may also play a role in their different catalytic profiles. AVI is a reversible DBO β-lactamase inhibitor (BLI) that inactivates Ambler class A and C β-lactamases. Our goal was to assess the ability of AVI to inhibit PER-2, restore susceptibility to clinical strains when combined with CAZ, ceftaroline (TAR), and aztreonam (ATM) and to probe the mechanism of inhibition. Methods: MIC testing was conducted using clinical isolates of Enterobacteriaceae (Ent) possessing PER-2 using ampicillin (AMP), AMP-AVI, CAZ, CAZ-AVI, TAR, TAR-AVI, ATM, and/or ATM-AVI. AVI inhibition constants (k2/K) were determined using nitrocefin as a reporter substrate. Modeling of PER-2 with AVI was conducted with Autodock Vina and Yasara. Results: Ent possessing PER-2 demonstrated high MICs to CAZ (≥ 512 g/ml), TAR (16-512 g/ml), and ATM (≥ 128 g/ml). When CAZ, TAR, and ATM were combined with AVI, only the TAR-AVI combination restored susceptibility (MICs ≤1 g/ml) for all isolates. The k2/K value for AVI was 2,000 M-1s-1, similar to AmpC inactivation constants (103 M-1s-1 range), but 100-fold less than that for other class A β-lactamases (e.g., TEM-1 and CTX-M-15), and 10-fold less than KPC-2. As observed in other class A β-lactamases, HB interactions between AVI and S130, E166, T235, and T237 are postulated. The Q69, H170, N132 and T104 residues are predicted to form hydrophobic interactions with AVI. Conclusion: Compared to CAZ and ATM, AVI combined with TAR effectively lowers MICs vs. Ent. The unique structural features of the PER active site and new interactions formed impact AVI inhibition of PER-2 and likely effect the deacylation of the AVI-PER-2 complex.