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A method for measuring fluoroquinolone accumulation by Streptococcus pneumoniae was rigorously examined. The accumulation of ciprofloxacin, clinafloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, sitafloxacin, and trovafloxacin in the presence and absence of either carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or reserpine was determined for two wild-type fluoroquinolone-susceptible capsulated S. pneumoniae strains (M3 and M4) and the noncapsulated strain R6. Two efflux mutants, R6N (which overexpresses PmrA) and a mutant of M4, M22 (no expression of PmrA), were also examined. Essentially, the fluoroquinolones fell into two groups. (i) One group consisting of ciprofloxacin, grepafloxacin, and norfloxacin accumulated to 72 to 92 ng/mg (dry weight) of cells in all strains. (ii) The remainder of the agents accumulated to 3 to 30 ng/mg (dry weight) of cells. With a decrease in hydrophobicity, there was a decrease in the concentration accumulated. With an increase in the molecular weight of the free form of each agent, there was also a decrease in the concentration accumulated. The strains differed in their responses to reserpine and CCCP. For the three fluoroquinolone-susceptible strains, only reserpine had a significant effect upon accumulation of moxifloxacin and clinafloxacin by M3 and showed no effect for the other agents and strains. For M3 and M4, CCCP enhanced the concentration of ciprofloxacin and norfloxacin accumulated, whereas for R6, the effect was only statistically significant for ofloxacin. Efflux mutant M22 accumulated less ciprofloxacin, gatifloxacin, and ofloxacin than M4 did. M22 accumulated more norfloxacin than M4 did. Reserpine and CCCP had variable effects as for the other strains. Differences in the accumulation of fluoroquinolones by R6 and R6N were highly dependent upon growth phase, and only for norfloxacin was there a significant difference between two strains.

Accumulation of 10 Fluoroquinolones by Wild-Type or Efflux Mutant Streptococcus pneumoniae