Study of in vitro selected resistance of Pseudomonas aeruginosa to cefoperazone, ceftazidime, azthreonam and imipenem

Abstract

Ten strains of Pseudomonas aeruginosa were plated on agar containing cefoperazone, ceftazidime, azthreonam or imipenem alone and in combination with ampicillin or cefoxitin as inducers, and/or amikacin (MIC/4), according to Szybalsky. Regrowing colonies were randomly selected for MIC and MBC determinations. The numbers of regrowing colonies were occasionally increased by a beta-lactamase inducer, although amikacin significantly reduced it. Some 243 colonies were further studied. The most frequent phenotypes of resistance (195 mutants) were: resistance to cefoperazone, ceftazidime, azthreonam (99 regrowing colonies); resistance to cefoperazone, azthreonam (36); resistance to cefoperazone (17); resistance to cefoperazone, ceftazidime, azthreonam, imipenem (17). A total of 28/31 R mutants retained their phenotype after 15 subcultures in antibiotic-free medium. The mutants R cefoperazone, ceftazidime, azthreonam were studied by quantitative beta-lactamase evaluation in agar with substrate and non-substrate antibiotics, substrate profile study spectrophotometrically, crypticity study by comparing beta-lactamase activity of intact and sonicated cells spectrophotometrically, isoelectric focusing of sonicate extracts and dialysis experiments for trapping evaluation. These mutants were characterized by a 240-fold increase in beta-lactamase production (Pi = 8.7) and unchanged permeability. beta-lactamase trapping was demonstrated but could not explain alone the phenotype of resistance. These studies suggest that mutants constitutive for beta-lactamase production were easily selected in vitro. Amikacin was able significantly to prevent their emergence.