ALT Experimental design and workflow. (A) Serial passaging of planktonic and biofilms evolved independently by a daily aliquot/bead passage over 10 days. (B) Intermittent colistin experimental treatment conditions during evolution: 0 mg/L (no treatment, gray), 0.5 mg/L, (static, light blue), 1 mg/L, (static, blue), and 0.5–8 mg/L, (increasing, dark blue) at days 1, 3, 5, 7, and 9. (C) Daily population analysis profiling (PAP) tests were performed to determine the survival frequency in each evolved population. (D) Whole-genome sequencing analysis was performed to the ancestral PAO1 strain as well as representative resistant clones from the last day of evolution.

ALT Sertaconazole and oxiconazole inhibit S. aureus efflux pumps in vitro. Using ethidium bromide (EtBr) accumulation and the EtBr efflux assay, the inhibition of the efflux pump was studied. Panels A and C show bar graphs that depict the concentration-dependent increase in relative fluorescence of EtBr in the presence of sertaconazole (Sert) and oxiconazole (Oxi), respectively. Thioridazine (TZ) is a known efflux pump inhibitor, used as a positive control at 16 µg/mL, while glucose (Glu), known to enhance active efflux, was used as a negative control at 0.4%. Panels B and D illustrate the decrease in EtBr efflux in the presence of sertaconazole and oxiconazole. Chlorpromazine (CPZ) and TZ, known EPIs, were used as positive controls at 16 µg/mL.