Phenotypic Conversion of Amikacin Resistant Acinetobacter baumannii to Susceptible by Zinc

LIN D.; TRAN T.; HERRON S.; RAMIREZ M.S.; TOLMASKY M.E.

Denver, Colorado

Congreso; 53rd ICAAC 2013; 2013

American Society for Microbiology

Introduction: Aminoglycosides are an important component of the armamentarium against serious infections. However, bacteria are becoming resistant to all of them reducing their effectiveness. A large number of resistance isolates owe this property to the presence of aminoglycoside N-acetyltransferases. One way to deal with the problem of rising aminoglycoside resistance levels is to find inhibitors that interfere with the enzymatic activity and restore susceptibility. A clinically relevant enzyme that confers resistance to several aminoglycosides such as amikacin and netilmicin is the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6?)-Ib]. Here we report that zinc is a potent inhibitor of this enzyme that shows an effect in vivo in Acinetobacter baumannii harboring the aac(6?)-Ib gene. Methods: In vitro acetylation activity was measured using a 5,5′- dithiobis(2-nitrobenzoic acid)-based colorimetric assay. MICs were determined using E-tests and growth curves were carried out measuring OD600 in a microplate reader in the presence of varying concentrations of ZnCl2 and antibiotic. Results: A systematic study of the effects of cations on the acetylation reaction showed that Zn+2 is a potent inhibitor of AAC(6?)-Ib. Inhibition was competitive with acetyl CoA as determined by Lineweaver-Burk plots and the IC50 was 1.5 microM. MICs determined on agar plates did not show inhibition when testing Escherichia coli harboring aac(6?)-Ib. However, consistent reduction in MIC for amikacin was detected when testing A. baumannii harboring aac(6?)-Ib and the assays in liquid medium did not show any growth at 6 or 4 microg/ml amikacin in the presence of 50 or 100 microM ZnCl2, respectively. Conclusions: Zn+2 is a potent inhibitor of the acetylation reaction in vitro. This activity leads to phenotypic conversion to amikacin susceptibility in A. baumannii but not E. coli, probably due to permeability and/or presence of efflux pumps. Further studies to better understand these mechanisms and their impact in the action of Zn+2 may provide ideas for improvement of the in vivo effect and a utilization of this cation to overcome the action of AAC(6?)-Ib.