Synergy h1 hybrid multi mode fluorescence spectrophotometer

The tetracycline uptake assay was performed according to the previously described method^{8 (link)}. The tetracycline uptake assay was monitored by fluorescence enhancement of tetracycline after cellular uptake. Cultures of A. baumannii AYE, A. baumannii SDF, E. coli MG1655, and E. coli RPTU54 were grown to OD_600nm ~ 0.6. Cells were pelleted down at 1200 × g for 10 min and resuspended in 10 mM HEPES buffer (pH 7.2) to a final OD_600nm ~ 0.5. Tetracycline was added to the cells at a final concentration of 128 mg/L at this stage. Then, nalidixic acid was added at different concentrations (for A. baumannii AYE 1×, 2×, 4×, and 8× MIC represents 512, 1024, 2048, and 4096 mg/L, respectively; concentrations of nalidixic acid used for A. baumannii SDF, E. coli MG1655, and E. coli RPTU54 is described in Supplementary Fig. 3) in cell suspension and finally pipetted into the black polystyrene plates (Corning®, USA) at 100 µl/well. The fluorescence reading was monitored (at room temperature) using a Synergy^TM H1 Hybrid Multi-Mode fluorescence spectrophotometer (BioTek, USA) at an excitation wavelength of 405 nm and an emission wavelength of 535 nm for 60 min at every 5 min. The fluorescence readings were normalized with respect to tetracycline fluorescence at time zero and plotted against time.

Antibiotics can disturb PMF directly, which may be responsible for cellular lethality^{17 (link)}. We also directly measured the role of nalidixic acid, tetracycline, or their combination on PMF in A. baumannii AYE. PMF was monitored using a potential-sensitive probe, DiBAC₄(3) (Invitrogen™, USA). The fluorescence of DiBAC₄(3) changes in response to fluctuation in membrane potential. A. baumannii AYE cells were grown till OD_600nm ~ 0.2 and washed twice in 1× PBS containing 0.4% glucose (wt./vol). Cells were pre-incubated with 10 µM of DiBAC₄(3) for 10 min^{42 (link)}. After 10 min, cells were treated with either nalidixic acid (at 512 mg/L), tetracycline (at 64 mg/L) alone, or in combination (nalidixic acid and tetracycline at 64 and 8 mg/L, respectively). Immediately after the addition of antibiotics, fluorescence reading was monitored (at 37 °C) using a Synergy^TM H1 Hybrid Multi-Mode fluorescence spectrophotometer (BioTek, USA) at an excitation wavelength of 490 nm and an emission wavelength of 516 nm for 60 min at every minute. Triton X-100 (0.01% vol/vol) acts as positive control for this experiment.