Cls3573

The assay was performed using a BacTiter‐Glo microbial cell viability assay kit (Promega, Madison, WI, USA) as described previously.^[46] Briefly, log‐phased cells were diluted to 10⁶ CFU mL⁻¹ and subsequently incubated with diclofenac or oxacillin. TSB broth without added compounds served as the positive control. After 24 h incubation at 37 °C, bacteria medium was transferred into a black, 384‐well microplate (Corning, CLS3573, NY, USA), and each test sample was mixed with an equal volume of BacTiter‐Glo reagent, which measures the cellular ATP level. The mixed samples were then incubated for 5 min at room temperature, and their luminescence intensities were determined by a Synergy H1 hybrid reader (BioTek, USA) at an integration time of 1 s per well. Relative luminescence units (RLU) values were subtracted from the background control of medium with bacteria.

The integrity of the cell membrane was evaluated as described previously.^[45] Briefly, black 384‐well polystyrene plates (Corning, CLS3573, NY, USA) were filled with 25 µL of PBS per well containing the indicated concentration of compounds. Log phase bacteria were washed three times with PBS and adjusted to 1×10⁸ CFU mL⁻¹ with PBS. Then, PI was added to 10 mL of diluted bacterial suspension to a final concentration of 5 µg mL⁻¹ and incubated at 37 °C for 30 min in the dark. A total 25 µL volume of the bacteria/PI mixture was added to each well of 384‐well plates containing diclofenac. Lastly, fluorescence was measured using a spectrophotometer (Synergy H1, BioTek), with excitation and emission wavelengths of 535 and 620 nm, respectively. Data were corrected by subtraction of fluorescence signal from untreated cells in the presence of PI. All experiments were conducted in triplicate.

To evaluate the ability of the compounds to disrupt the cytoplasmic membrane integrity, the membrane impermeable fluorescent DNA intercalating dye propidium iodide (PI) was used. Early exponential phase cells were pelleted and washed twice in 10 mM HEPES buffer (pH 7.4) containing 50 µg mL⁻¹ of CaCl₂ and 5 mM glucose, and then re-suspended in the same buffer (4 × 10⁸ CFU mL⁻¹). Cell suspension was added to a 384-well black walled polystyrene plate (Corning, CLS3573) containing compounds, giving a final concentration of 16 µg mL⁻¹. After 1 h incubation at 37 °C, 5 µg mL⁻¹ of PI was added and fluorescence was monitored for 90 min using a Tecan Infinite® m1000 Pro Multi-mode reader (excitation/emission 535/620 nm). Data were corrected by subtraction of fluorescence signal arising from untreated cells in the presence of PI. Note that the MIC values for oritavancin and compound 17 increase to 1 µg mL⁻¹, and compound 24 to 2 µg mL⁻¹, in polystyrene plates. Each sample was tested in quadruplicate and independent assays were performed twice showing similar results. Data was analysed and presented using Prism software.