Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli

Abstract

We have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity in Escherichia coli and the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action in E. coli However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump.

FIG 1

Growth kinetics of JW0452, JW0451, and JW5503 compared to those of the wild-type parent strain BW25113. The growth curves were generated by incubating 1% overnight inocula of strains BW25113, JW0452, JW0451, and JW5503 in LB medium (control) (A), LB medium supplemented with 10 mM GSH (B), LB medium with 4 ng/ml ciprofloxacin (subinhibitory ciprofloxacin concentration for BW25113) (C), LB medium with 10 mM GSH and 16 ng/ml ciprofloxacin, and LB medium with 16 ng/ml ciprofloxacin (inhibitory concentration for BW25113). The growth kinetics experiment was performed for two biological replicates. Generation times of the strains were calculated from the data of a single representative set.

FIG 2

Total efflux activities of wild-type E. coli BW25113 and efflux mutants JW0452, BW0451, and JW5503 (carrying acrA, acrB, and tolC deletions, respectively) determined in presence and absence of GSH. (A) Representative flow cytometric histograms depicting the extent of intracellular fluorescence due to Hoechst 33342 retention in different bacterial strains in the presence and absence of GSH (n = 3 per group). In each of the histograms, the x axis represents the signal acquired from the FL4 channel, and the y axis represents the count. (B) Bar chart showing the amount of Hoechst 33342 retained inside the bacterial cell (n = 3 per group). Data points show means ± SEM. One-way Analysis of variance (ANOVA) followed by Tukey's posttest was used to determine the statistical significance. *, compared with control (P < 0.01); #, compared with the absence of GSH for a given bacterial strain (P < 0.05).

FIG 3

Ciprofloxacin accumulation inside wild-type E. coli BW25113 (A) and efflux mutants JW0451 (B), BW452 (C), and JW5503 (D) in the presence and absence of 10 mM GSH. Separate bars depict the extents of fluorescence due to ciprofloxacin accumulation in the presence and absence of GSH (n = 3 per group). Error bars represent ± SEM. *, significantly different compared to the corresponding absence of GSH treatment (P < 0.05).