INTRACELLULAR ACCUMULATION OF NORFLOXACIN IN MYCOBACTERIUM-SMEGMATIS

To evaluate the intracellular accumulation of norfloxacin in mycobacte ria, two methods were used with Mycobacterium smegmatis, A radiometric method (K. V. Cundy, C. E. Fasching, K. E. Willard, and L. R. Peterso n, J. Antimicrob. Chemother. 28:391-497, 1991) was used without great modification, but the fluorometric method (P. G. S. Mortimer and L. J. V. Piddock, J. Antimicrob. Chemother. 28:639-653, 1991) was changed c onsiderably, Indeed, adsorption of the quinolone to the bacterial surf ace was characterized by measuring the level of accumulation at 0 degr ees C. Taking into account the adsorption, the pH of the washing buffe r was increased from 7.0 to 9.0 to improve the desorption of norfloxac in from the cell surface, Both the fluorometric method, with the techn ical improvement, and the radiometric method could be used to estimate the intracellular accumulation of norfloxacin, which resulted from th e difference between the whole uptake measured at 37 degrees C and the adsorption measured at 0 degrees C. A total of 35 ng of norfloxacin p er mg of cells (dry weight) penetrated into the M.smegmatis cell, and the steady state was achieved in 5 min, Use of inhibitors of the proto n motive force revealed that transport of norfloxacin was energy indep endent, Thus, the same mechanisms of quinolone accumulation that occur in eubacteria seem to occur in mycobacteria, at least in M. smegmatis .