The H33342 bis-benzamide assay was carried out as described by Coldham et al.,26 (link) with the following modifications; strains were grown to an OD at 600 nm of 0.6 and, once resuspended in PBS at room temperature, were adjusted to an OD at 600 nm of 0.1, 0.2, 0.3 or 0.5. Centrifugation steps were carried out at 2200 g. The wells of a black microtitre tray (Corning, Amsterdam, The Netherlands) were inoculated with either 180 μL of cell suspension or 176 μL of cell suspension with 4 μL of the EI carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) or phenylalanine-arginine-β-naphthylamide (PAβN) at the required concentration (see the Results section). Fluorescence was measured and data were analysed as previously described.26 (link) The level at which maximum fluorescence was reached and remained unchanged within the time period of the assay was taken as the steady-state accumulation level. In order to quantitatively compare the efflux rate of the strains, the time needed for a 4-fold increase in dye fluorescence after H33342 injection was calculated. Each assay was repeated three times with three biological replicates. Differences in accumulation between clinical isolates and AYE were analysed for statistical significance using Student's t-test; a P value ≤0.05 was considered significant.
Ethidium bromide assays were carried out essentially as the H33342 accumulation assays described above, except that cultures were resuspended in 1 M sodium phosphate buffer with 5% glucose. A 1 mM ethidium bromide stock solution was prepared and 20 μL was injected to give a final concentration of 0.1 mM in the assay. Fluorescence was measured over 117 min at excitation and emission wavelengths of 530 nm and 600 nm, respectively, in a FLUOstar Optima. Norfloxacin assays were carried out as previously by Mortimer and Piddock.31 (link)