2.5. Determination of Efflux Pump Activity

Accumulation of ethidium bromide was assayed as described previously [20]. Strain PAO1 was grown in TSB overnight and washed with 50 mM sodium phosphate buffer (pH 7.0). In order to measure the presence of active efflux, ethidium bromide (10 µg/mL) was added to control (TSB only) and antibiotic-containing wells (6.4 µg/mL clindamycin and 0.8 µg/mL rifampicin) and fluorescence was read every 5 min at excitation 500 nm and emission 590 nm for 60 min in a fluorescence microplate reader using SoftMax Pro software (Molecular Devices, San Jose, CA, USA). All experiments were performed in triplicate. Error bars indicate standard deviation from the mean.