Bacterial ATP level assay

The intracellular ATP levels were determined using a BacTiter-Glo Microbial Cell Viability Assay (Promega) kit. Bacteria were cultured in CA-MHB medium at 37°C until the late-logarithmic phase (OD₆₀₀ = 1). Cells were treated with the indicated concentration of D-11 at 37 °C. 100 μL of cell culture were taken out, mixed with 100 μL BacTiter-Glo reagent every hour and incubated for 5 minutes at room temperature. Luminescence was measured with a luminometer (Varioskan Flash; Thermo Scientific). CCCP (40 μg/mL) was used as a positive control. The ATP concentration was calculated using a standard curve made with a commercial ATP solution. The OD₆₀₀ was determined before ATP levels were measured. Relative ATP levels were calculated by the OD₆₀₀. All the tests were performed in triplicate.