Tetracycline

S. aureus treated with plasma or plasma-activated saline was diluted six times with MH medium containing antibiotics. The final antibiotic concentrations, corresponding to 10 × the minimum inhibitory concentration (MIC), were as follows: tetracycline, 500 μg/ml (MP Biomedicals); gentamycin, 200 μg/ml (Sigma); clindamycin, 5,000 μg/ml (TCI); chloramphenicol, 500 μg/ml (MP Biomedicals); ciprofloxacin, 50 μg/ml (Sigma); rifampicin, 4 μg/ml (Sigma); and vancomycin, 50 μg/ml (Sigma). The numbers of surviving bacteria were determined at the time points indicated by harvesting 100 μl of bacterial culture, which was centrifuged at 10,000 × g for 1 min. The resulting pellet was resuspended in 100 μl 0.9% NaCl. Samples were serially diluted, and 10 μl of each dilution was spotted onto MH agar plates and incubated overnight at 37°C and the numbers of surviving bacteria were determined by counting the resulting CFUs.

The MICs of streptomycin (MP Biomedicals LLC, Solon, OH), tetracycline (MP Biomedicals LLC, Solon, OH), and ciprofloxacin (Acros Organics, Fair Lawn, NJ) for Pph were determined by evaluation of turbidity using a previously described method (58 (link)), with some modifications. An overnight culture of Pph was diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 in King’s B medium, and 20 μl of the cell suspension was added to 180 μl of King’s B medium amended with antibiotics to achieve final antibiotic concentrations of 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, and 0 μg ml⁻¹ in a 200-μl volume. Growth was assessed by measuring the OD₆₀₀ over 20 h using an absorbance plate reader (Bio-Tek). The MIC of each antibiotic was the lowest concentration at which no increase in turbidity was measured across at least three independent cultures. The MIC of tailocin was similarly determined in activity units (AU), starting with nine 1:2 serial dilutions of the initial tailocin preparation.