Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa

Abstract

The purpose of this study was to determine if synergism was maintained for various combinations of beta-lactams with an aminoglycoside against four clinical strains and one laboratory strain of Pseudomonas aeruginosa which were resistant, according to the MICs, to the beta-lactams and/or aminoglycoside. The results from both the checkerboard and killing curve methodologies were compared. The laboratory strain (ATCC 27853) was manipulated in vitro by serial passage onto agar containing increasing concentrations of each antibiotic to select for resistance. One clinical isolate (R61) was also serially passed to raise the MIC of piperacillin from 128 to 1,024 micrograms/ml. The fractional inhibitory concentration indices for all isolates indicated indifference for all combination therapies, with values ranging from 0.6 to 3. In contrast, killing curve results for all isolates demonstrated synergism with drug concentrations at either one-fourth or one-half the MIC for each organism. The MIC of piperacillin for the laboratory-manipulated clinical isolate R61 was 1,024 micrograms/ml, and synergism was still observed with concentrations of one-half the MIC of piperacillin and amikacin. For clinical isolate R166, which was beta-lactam and tobramycin resistant, synergism continued to be demonstrated with concentrations of tobramycin (1/16 MIC) in combination with piperacillin and cefepime at 1/2 the MIC. The results of this study indicate that against P. aeruginosa, synergism is observed in spite of resistance to beta-lactams and/or aminoglycosides. Synergism appears to be maintained even at very high MICs (piperacillin, 1,024 micrograms/ml; tobramycin, 128 micrograms/ml) with drug concentrations within achievable therapeutic ranges. With current definitions of synergism there was a complete lack of correlation between the results obtained by the checkerboard and killing curve methodologies, with the fractional inhibitory concentration indices showing indifference and killing curves resulting in synergism. The methodologies and definitions of synergism or antagonism are variable and not standardized and should be reevaluated.