Fig. (1).

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method. MDR strains were swabbed in TSA plates con-taining different concentrations of EtBr and incubated for 16 hours at 37ºC. Controls and clinical isolates were swabbed on EtBr-containing TSA plates, according to the diagram. Each EtBr-TSA plate can accommodate as many as twelve bacterial strains. The distribution of the strains in the TSA plate can be altered according to the desired experiment. After this, fluorescence was recorded and strains that showed lower fluorescence than the control (indicative of efflux activity) were selected. The efflux activity was further confirmed by determining the MIC of antibiotics in the presence of efflux inhibitors. Two control strains can be inserted in this study. For example, Control 1 should pre-sent the highest fluorescence (evidence of no efflux activity or physiological activity) and Control 2 should be a strain showing no fluores-cence or very low levels of fluorescence (indicative of an active over-expressed efflux system). These strains should be previously well char-acterized for their efflux systems.