Exploring the Inactivation Mechanism by Avibactam (AVI) of an Inhibitor-Resistant Carbapenemase, PenA from Burkholderia multivorans (Bm)　　Session:	Structure-Function of Beta-Lactamases　Monday, Sep 21, 2015, 8:30 AM -11:00 AM

Interscience Conference of Antimicrobial Agents and Chemotherapy 2015,San Diego, CA　San Diego Convention Center

K. M. Papp-Wallace, J. A. Gatta, N. Ohuchi, M. L. Winkler, S. A. Becka, J. J. LiPuma, R. A. Bonomo, M. Nukaga
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Background: Bm is a significant health threat to people with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. A major resistance determinant expressed in Bm, the class A β-lactamase, PenA possesses a very broad substrate profile including carbapenems and β-lactamase inhibitors. As a result, β-lactams and β-lactamase inhibitors currently on the market do not inhibit this β-lactamase. A panel of Bm clinical isolates from CF patients were studied and a novel β-lactam-β-lactamase inhibitor combination was found to be effective. Methods: Susceptibility testing was performed on 50 Bm clinical isolates. PenA was purified for steady-state kinetics, mass spectrometry, and X-ray crystallographic determination with AVI. Results:The antimicrobials currently used to treat Bm were not able to inhibit the growth of all isolates (Table 1). However, the isolates were found to be susceptible to ceftazidime(CAZ)-AVI (Table 1). PenA was potently inhibited by AVI with a k2/K of 2 ± 1 X 106 M-1s-1 and a koff of 2 ± 1 x 10-3 s-1. Mass spectrometry revealed that AVI formed a stable complex with PenA for up to 24 hours. Crystallographic analysis of AVI carbamylated in the PenA active site reveals several interactions that stabilize the acyl-enzyme complex. The deacylation water molecule possesses decreased nucleophicility preventing decarbamylation and Lys-73 is unlikely to abstract a proton from Ser-130 to initiate recyclization. Conclusions: Highly drug-resistant pathogens, such as Bm severely limit treatment options in patients. Here, we found that when AVI is combined with CAZ susceptibility to CAZ of Bm is restored in vitro. AVI forms a stable complex with PenA. Decarbamylation and intramolecular AVI recyclization are blocked by distinctive mechanisms.
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