Accumulation of norfloxacin by Bacteroides fragilis

The accumulation of norfloxacin by Bacteroides fragilis NCTC 9343 was deter mined by the modified fluorescence method. The time required to achieve a s teady-state concentration (SSC) after allowing B. fragilis to accumulate no rfloxacin in an aerobic or an anaerobic environment was similar to 2 min; t he 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 norfloxa cin/mg (dry weight) of tells. Initial rates of accumulation were determined with a range of external concentrations, as up to 8 mu g/ml the concentrat ion of norfloxacin accumulated increased proportionally to the external con centration, 12.13 ng/mg (dry might) of cells per pg of exogenous norfloxaci n per mi. At concentrations above 10 mu g/ml no increase in the rate of nor floxacin accumulation was observed. From the kinetic data, a Lineweaver-Bur k plot calculated a K-m of 5.03 mu g/ml and a V-max of 25.1 ng of norfloxac in/s. With an increase in temperature of between 0 and 30 degrees C, the co ncentration of norfloxacin accumulated also increased proportionally at 4.7 22 ng of norfloxacin/mg (dry weight) of cells/degrees C. At low concentrati ons of glucose (<0.2%; 11 mM), the concentration of norfloxacin accumulated was decreased. With the addition of 100 mu M carbonyl cyanide m-chlorophen ylhydrazone (CCCP) the mean SSC of norfloxacin was increased to 116 +/- 7.0 1 ng of norfloxacin/mg (dry weight) of cells; glucose had no significant ef fect 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 ce lls, These data suggest that the mechanism of accumulation of norfloxacin b y B. fragilis is similar to that of aerobic bacteria and that the fluoresen ce procedure is suitable for use with an anaerobic bacterium.