Synergy testing by Etest, microdilution checkerboard, and time-kill methods for pan-drug-resistant Acinetobacter baumannii

Abstract

Pan-drug-resistant (PDR) Acinetobacter baumannii is an important nosocomial pathogen that poses therapeutic challenges. Tigecycline alone or in combination with agents such as colestimethate, imipenem, and/or amikacin is being used clinically to treat PDR A. baumannii infections. The purpose of this study was to compare in vitro susceptibility testing by epsilometric (Etest) methods and the checkerboard (CB) method with testing by time-kill analysis. PDR A. baumannii clinical strains representing eight unique pulsed-field gel electrophoresis clones selected from a total of 32 isolates were tested in vitro with tigecycline, colestimethate, imipenem, and amikacin in single- and two-drug combinations by using two different methods of Etest (with a fixed ratio method [method 1] and with the incorporation of the active drug in medium [method 2]) and by using CB. The three-drug combination of imipenem, tigecycline, and amikacin was also tested by CB. These results were compared to time-kill results. Synergy was consistently detected with the imipenem plus colestimethate and tigecycline plus imipenem combinations. The Etest method with active drug incorporated into the agar allowed us to detect synergy even in the presence of the active drug and was more comparable to CB and time-kill tests. Synergy was detected with the three-drug combination of imipenem, tigecycline, and amikacin by both CB and time-kill methods among several tested clones. These findings indicate the utility of synergy testing to predict activity of specific antibiotic combinations against PDR A. baumannii.

FIG. 1.

Time-kill analyses of test isolates. Time-kill analysis was performed only for the clones that showed synergy or additiveness with Etest 2 and in more than two wells with CB testing. The log₁₀ decreases in colony counts at 6 and 24 h with the antimicrobial combination compared to the most active single agent are represented for each clonal type. IP, imipenem; TG, tigecycline; CO, colistin; AK, amikacin.