Accumulation of Norfloxacin by Bacteroides fragilis

The accumulation of norfloxacin by Bacteroides fragilis NCTC 9343 was determined by the modified fluorescence method. The time required to achieve a steady-state concentration (SSC) after allowing B. fragilis to accumulate norfloxacin in an aerobic or an anaerobic environment was approximately 2 min; the SSC achieved in air was 90.28 +/- 9.32 ng of norfloxacin/mg (dry weight) of cells, and that achieved anaerobically was 98.45 +/- 3.7 ng of norfloxacin/mg (dry weight) of cells. Initial rates of accumulation were determined with a range of external concentrations, as up to 8 microg/ml the concentration of norfloxacin accumulated increased proportionally to the external concentration, 12.13 ng/mg (dry weight) of cells per microg of exogenous norfloxacin per ml. At concentrations above 10 microg/ml no increase in the rate of norfloxacin accumulation was observed. From the kinetic data, a Lineweaver-Burk plot calculated a K(m) of 5.03 microg/ml and a V(max) of 25.1 ng of norfloxacin/s. With an increase in temperature of between 0 and 30 degrees C, the concentration of norfloxacin accumulated also increased proportionally at 4.722 ng of norfloxacin/mg (dry weight) of cells/ degrees C. At low concentrations of glucose (<0.2%; 11 mM), the concentration of norfloxacin accumulated was decreased. With the addition of 100 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) the mean SSC of norfloxacin was increased to 116 +/- 7.01 ng of norfloxacin/mg (dry weight) of cells; glucose had no significant effect in the presence of CCCP. Magnesium chloride (20 mM) decreased the SSC of norfloxacin to 40.5 +/- 3.76 ng of norfloxacin per mg (dry weight) of cells. These data suggest that the mechanism of accumulation of norfloxacin by B. fragilis is similar to that of aerobic bacteria and that the fluoresence procedure is suitable for use with an anaerobic bacterium.