Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays.

Background: Fluoroquinolones (FQs) are being used as second-line agents in the treatment of tuberculosis caused by multidrug-resistant strains. Ofloxacin (OFX) is being tried as a part of modified multidrug therapy regimens for leprosy. A preliminary study was carried out to evaluate the accumulation of FQs - OFX, levofloxacin (LFX), norfloxacin (NFX), and ciprofloxacin (CIF) in Mycobacterium smegmatis.

Methods: M. smegmatis were grown in Sauton's medium till log phase, harvested and resuspended in phosphate buffer (0.1 M, pH 7.2, Optical Density (OD) of 0.4-0.5) The suspensions were incubated with OFX, LFX, NFX, and CIF (10 μg/ml) at 37°C. The drugs were estimated in the supernatants using spectrofluorimeteric methods. The experiments were also conducted with the addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a proton motive force inhibitor, at 100 μM, 10 min before and/or immediately after the addition of the drugs.

Results: The time taken to achieve a Steady State Concentration (SSC) of OFX in M. smegmatis was 3 min and the level of accumulation was 102 ng/mg dry weight of the bacilli; with LFX the time for SSC was 5 min and the level of accumulation was 90 ng/mg; in case of NFX the accumulation to SSC was 87 ng/mg in 3 min. CIF accumulation attained a steady state (SSC level of 79 ng/mg) in 4 min. The accumulation kinetics for NFX in M. smegmatis using the spectrofluorimetric method is comparable with radioactive assays. Dose-related accumulation was observed with 10 μg/ml exposure concentrations. The addition of CCCP failed to influence the accumulation of each of these quinolones.

Conclusion: The findings of dose-related accumulation of OFX, LFX NFX, and CIF suggest simple diffusion as the possible mechanism of transport of these drugs.