Caffeine (1,3,7-trimethylxanthine) and antibiotics novobiocin sodium salt, cefepime hydrochloride, gentamycin sulfate salt hydrate, azithromycin dihydrate, and ticarcillin disodium salt, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Refer to Figure 5 for the chemical structures of the compounds. For UV-Vis spectroscopy and isothermal titration calorimetry (ITC) measurements, 100 mM sodium phosphate buffer (pH 6.8) was used. The buffer was filtered through a 0.2 μm pore Millex Millipore filter and degassed before experiments. Caffeine stock solutions were prepared in the buffer or deionized water at concentrations of approximately 10−1 M, and stored at 4 °C in darkness. Antibiotic stock solutions were prepared by dissolving their weight amounts in the buffer or deionized water immediately before the experiments.
Solid media for bacterial cultivation and testing included tryptone soya agar (TSA, Oxoid Ltd., Basingstone, UK) and Mueller-Hinton agar (BioMaxima, Lublin, Poland). Cation-adjusted Mueller-Hinton broth (CA-MHB) for antimicrobial susceptibility testing using the broth microdilution method was purchased from Beckton Dickinson (BD Difco™ BBL™; Franklin Lakes, NJ, USA).
Solid media for bacterial cultivation and testing included tryptone soya agar (TSA, Oxoid Ltd., Basingstone, UK) and Mueller-Hinton agar (BioMaxima, Lublin, Poland). Cation-adjusted Mueller-Hinton broth (CA-MHB) for antimicrobial susceptibility testing using the broth microdilution method was purchased from Beckton Dickinson (BD Difco™ BBL™; Franklin Lakes, NJ, USA).