Avibactam inactivation of PER-2 beta-lactamase: a new understanding of beta-lactamase inhibition.

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

Rosario, Sanata Fe

Congreso; XXIII Congreso Latinoamericano de Microbiología, XIV Congreso Argentino de Microbiología; 2016

Asociación Argentina de Microbiología

Avibactam inactivation of PER-2 β-lactamase: a new understanding ofβ-lactamase inhibition. Ruggiero M1, Papp-Wallace K M2, Gutkind G1, Bonomo R A2,Power P1 1 Laboratoriode Resistencia Bacteriana, Facultad de Farmacia y Bioquímica, Universidad deBuenos Aires, Junin 956 (1113), Argentina.2 Cleveland VAMC, Cleveland, OH, USA. Introduction: Avibactam (AVI) is a diazabicyclooctane (DBO)derivative acting as a bridged reversible β-lactamase inhibitor thatinactivates Ambler class A and class C β-lactamases. PERβ-lactamases are prevalent in South America and the Middle East and readily hydrolyzeoxyimino-cephalosporins. This enhanced hydrolytic activity may result from thepresence of a unique fold in the Ω loop (A164-N179) and in the β3-β4 loop(S238-A243, which includes a 4-amino acid insertion compared to other class Aβ-lactamases). These structural differences enlarge the active site entrance by2-fold compared to other class A β-lactamases. PER-2 possesses higher catalyticefficiency towards ceftazidime (CAZ) compared to PER-1 (~22-fold higher). Examinationof the apo-structure reveals that the H-bonding (HB) network between S70-Q69-HOH-T237-R220may also play a role in their different catalytic profiles. Objectives: We aimed to assess the ability of AVI to inhibit PER-2, restoresusceptibility to PER-2-producing clinical strains when combined with CAZ, ceftaroline(TAR), and aztreonam (ATM), and to probe the mechanism of inhibition.Methods: MIC testing was conducted using clinicalisolates of Enterobacteriaceae (Ent) producingPER-2, using ampicillin (AMP), AMP-AVI, CAZ, CAZ-AVI, TAR, TAR-AVI, ATM, and/orATM-AVI. AVI inhibition constants (k2/K)were determined using nitrocefin as a reporter substrate. Modelling of PER-2with AVI was conducted with Autodock Vina for evaluating the Michaelis complex(non-covalent binding), and Yasara for assessing the covalent association withthe active site serine.Results: Ent possessing PER-2 demonstrated high MICsto CAZ (≥ 512 mg/ml),TAR (16-512 mg/ml),and ATM (≥ 128 mg/ml).When CAZ, TAR, and ATM were combined with AVI, only the TAR-AVI combination restoredsusceptibility (MICs ≤1 mg/ml) for all isolates. The k2/Kvalue for AVI was 2,000 M-1s-1, similar to AmpC inactivationconstants (103 M-1s-1 range), but 100-fold lessthan that for other class A β-lactamases (e.g., TEM-1 and CTX-M-15), and10-fold less than KPC-2. As observed in other class A β-lactamases, HBinteractions between AVI and S130, E166, T235, and T237 are postulated. The Q69,H170, N132 and T104 residues are predicted to form hydrophobic interactionswith AVI. Conclusion: Compared to CAZ and ATM, AVI combinedwith TAR effectively lowers MICs vs. Ent. The unique structural features of thePER active site and new interactions formed impact AVI inhibition of PER-2 and likelyeffect the deacylation of the AVI-PER-2 complex.