In Vitro Antibiotic Synergy in Extensively Drug-Resistant Acinetobacter baumannii : the Effect of Testing by Time-Kill, Checkerboard, and Etest Methods

Abstract

Treatment of extensively drug-resistant (XDR) Acinetobacter baumannii infections is challenging because of both the limited choice of antibiotic and the tendency of such infections to occur in critically ill hosts with limited physiologic reserves (17). Polymyxins demonstrate in vitro activity against A. baumannii, but resistance is also reported (8,17). In the absence of feasible alternatives, unconventional antibiotics, such as minocycline, rifampin, and tigecycline, have been used, both singly and in combination (19). In vitro combination susceptibility testing poses a significant challenge for clinical microbiology laboratories, with a lack of standardization and a variety of in vitro testing methods. This study evaluated the effects of various antibiotic combinations against a panel of XDR-Acinetobacter baumannii and compared in vitro synergy testing results obtained from time-kill, checkerboard, and Etest methods.(Some elements of this study were presented at the 20th European Congress of Clinical Microbiology and Infectious Diseases, 2010.) Clinical isolates of antibiotic-resistant A. baumannii were collected from four hospitals in Singapore over a 2-year period. Species identification was confirmed by a multiplex PCR assay (2). MICs to ampicillin-sulbactam, ciprofloxacin, gentamicin, imipenem, meropenem, aztreonam, piperacillin-tazobactam, polymyxin B, tigecycline, ceftazidime, amikacin, and cefepime were obtained by broth microdilution (Sensititre, Trek Diagnostics, United Kingdom), while MICs for rifampin were obtained by broth macrodilution (3). Categorical susceptibility was based on Clinical and Laboratory Standards Institute breakpoints (4). XDR A. baumannii isolates were defined as isolates that were resistant to all the tested antimicrobials except for tigecycline, rifampin, and polymyxin B (5, 6), while pandrug-resistant (PDR) A. baumannii was resistant to all tested antimicrobial agents (5, 6). Clonal relatedness of study isolates was determined by multiplex PCR strain typing (11), with cluster analysis of banding data performed using the unweighted pair group method with arithmetic mean.All strains were tested for the presence of in vitro synergy to polymyxin B-rifampin, polymyxin B-tigecycline, and tigecycline-rifampin combinations by three methods. These combinations were selected based on previously published data, which demonstrated a high likelihood of achieving in vitro synergy (10). Time-kill assays were performed following methods published by the Clinical and Laboratory Standards Institute (13), with colony enumeration performed at 0-and 24-h time points. The lower limit of detection was 400 CFU/ml. Antibiotic concentrations used for the time-kill assay were 2 mg/liter for each antibiotic, representing achievable serum concentrations for polymyxin B (9) and rifampin (7) and achievable tissue levels for tigecycline (18).Etest and checkerboard synergy testing were performed according to published methods (12,20). Fractional inhibitory concentrations (FIC) were calculated as follows: ...

Evaluation of the best method to assess antibiotic potentiation by phytochemicals against Staphylococcus aureus

“…Time-kill assay is very laborious if a high number of combinations are required, and the results are greatly influenced by differences on the inoculum size and on the interpretation of the results (because relatively few antibiotic concentrations are examined) (White et al, 1996). Also, methods for the interpretation of kill-kinetic studies vary, and synergy has been defined by some authors as at least a 100-fold increase in killing at 24 h (Bonapace et al, 2000;Jung et al, 2004;Kiraz et al, 2009;Mayer and Nagy, 1999), while other authors consider a 200-fold increase (Guo et al, 2008;Leonard, 2012;Tan et al, 2011). Relatively to checkerboard, the endpoint (i.e., the complete inhibition of growth) is a qualitative measure (Tariq et al, 1995), and the occurrence of synergy appears to be highly dependent on the method of interpretation (Bonapace et al, 2002).…”

In Vitro Pharmacodynamics of Polymyxin B and Tigecycline Alone and in Combination against Carbapenem-Resistant Acinetobacter baumannii

“…Most synergy studies against A. baumannii, however, are conducted in vitro by applying time-kill or checkerboard methodology, where only specific fixed concentrations of antibiotics are studied. Using this methodology, numerous studies have observed synergistic interactions between polymyxins and various other antibiotic classes, including carbapenems, tigecycline, rifampin, and most recently, vancomycin (15)(16)(17)(18)(19)(20)(21)(22). These time-kill and checkerboard experiments, however, ignore the contribution of human pharmacokinetics and the dynamic effects of varied drug concentrations over the duration of the experiment.…”

Antibiotic Resistance Profiles and Genotypes of <i>Acinetobacter baumannii</i> Isolates and <i>In Vitro</i> Interactions of Various Antibiotics in Combination with Tigecycline and Colistin

“…The highest synergy was seen in beta-lactam-fluoroquinolone and fluoroquinolone-AK combinations. 24 While in a study conducted in 2011, Tan et al 8 detected rate of synergy by CB method between polymyxin B-RF and TGC-RF as 19%, those between polymyxin B-TGC was 12%. The same rates obtained by TK methods were 56%, 19% and 44% respectively.…”

“…1 Of these alternative classes, polymyxin and tigecycline (TGC) remain the most active treatments in vitro against MDR A. baumannii but resistance against these antibiotics is also reported. 4,6,7,8 Combination therapy is often used in the treatment of MDR A. baumannii infections to prevent the emergence of resistance and obtain a synergistic effect. 1,3,8 The aim of this study was to determine the antimicrobial resistance profile, clonal relation and efficacy of antimicrobial combinations on nosocomial MDR A. baumannii.…”