Evaluating TIG-R EV Transfer Activity

To evaluate the transfer activity of TIG-R EVs to AB, bacterial growth (optical density, at 600 nm, OD₆₀₀) of TIG-S AB treated with or without TIG-R EVs (5 μg; BCA quantified) under different concentrations of TIG (0–4 µg·mL⁻¹) was monitored every 30 min for 24 h at 37 °C with 500 rpm using a SPECTROStar^® Nano (BMG LABTECH GmbH, Ortenberg, Germany). Aliquots (5 μL) from cells grown for 24 h were spotted onto LB-agar plates to determine MIC values for bactericidal activity. The transfer activity of TIG-R EVs from AB to other bacteria was evaluated using a 96-well MIC assay [18 (link)]. LB-agar plates or 96-well plates were imaged by ChemiDoc^TM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab Software (ver.5.2.1, Bio-Rad, Hercules, CA, USA). One of representative of the three is shown.