The resistance mechanism in three strains of Pseudomonas aeruginosa, DeltaABM (devoid of MexAB-OprM), WT, and nalB-1 (overexpression of MexAB-OprM), was investigated using real-time single live cell imaging and fluorescence spectroscopy. Time courses of fluorescence intensity of these three strains in ethidium bromide (EtBr) showed that accumulation kinetics and extrusion machinery were highly dependent upon pump substrate (Etl3r) concentration. At high substrate concentration (100 muM), the accumulation kinetic profiles in the cells at earlier incubation times were similar to those observed in low concentration. As EtBr accumulated in the cells reached a critical concentration, the fluorescence intensity of DeltaABM decreased below the fluorescence intensity of EtBr in buffer solution. This result suggested an inductive mechanism in the development of substrate resistance in P. aeruginosa. Substrates appeared to trigger the degradation of EtBr in DeltaABM. Unlike bulk measurements, single live cell imaging overcame the ensemble measurement of bulk analysis and showed that efflux machinery and resistance mechanism in individual cells were not synchronized. (C) 2003 Elsevier Science (USA). All rights reserved.