Accumulation of enoxacin by Escherichia coli and Bacillus subtilis

Abstract

Several methods were used to determine enoxacin uptake in Escherichia coli strains because washing of cells removed all or most cell-associated enoxacin whereas no washing was associated with large amounts of cell-bound enoxacin. Washing after up to 40 to 45 min of exposure to enoxacin followed by suspension in drug-free medium prevented a significant effect of enoxacin on cell growth. Cell uptakes obtained with different methods showed no difference in the shape of the timed uptake curves but did show significant quantitative differences. These results are consistent with cell-associated enoxacin comprising a freely exchangeable pool of drug. Lineweaver-Burk plots of uptake were consistent with uptake of enoxacin by simple diffusion. No saturability and no competition with ciprofloxacin were observed. Low temperature (4 degrees C) was associated with decreased uptake. Arsenate, carbonyl cyanide m-chlorophenylhydrazone, sodium fluoride, sodium azide, and 2,4-dinitrophenol had no effect on uptake. We conclude that the mechanism of transport of enoxacin into cells is by simple diffusion. Mutants of E. coli with deficiency of outer membrane proteins F and C and an enoxacin-resistant mutant selected by serial passage with increasing enoxacin concentrations demonstrated that F porins play a significant role in enoxacin uptake and influence susceptibility to enoxacin. Uptake was shown to be similar in a strain of Bacillus subtilis.