3.3. Time-Kill Assays

Infection of planktonic bacterial cells with a starting amount of approximately 5 × 10⁸ cfu/mL was done at the exponential phase of bacterial growth in Lysogeny broth (LB) (NaCl 1% w/v, tryptone 1% w/v, yeast extract 0.5% w/v). The bacteria were challenged with phages at four different multiplicities of infection (MOI 10, 10⁻¹, 10⁻³, and 10⁻⁵), either alone or in combination with oxacillin. Five different oxacillin concentrations in the range below and above the MICs of the propagation strain SA19 (100 µg/mL, 50 µg/mL, 20 µg/mL, 10 µg/mL, and 5 µg/mL) were used, which largely reflected achievable serum levels in human anti-infective therapy [30,31]. The dynamics of phage- or antibiotic-based challenges on bacterial populations were determined via measurements of the changes in optical densities (OD₅₉₀), which is particularly suited for high throughput analysis [32,33,34]. According to our previous studies, the presence of a β-lactam antibiotic does not interfere with the optical density values, which correlated with the reduction of bacterial cells rather than with altered cell morphology [14,15].

For the infection assays, 10 mL of 2× LB were inoculated with 100 µL of the host strain and incubated overnight at 37 °C and 200 rpm. Subsequently, 5 mL of this culture was again mixed with 5 mL 2× LB and shaken at room temperature for another hour to reach a bacterial concentration of approximately 5 × 10⁸ CFU/mL. The phage-lysates were diluted by tenfold serial dilution, and 100 µL of phage-lysates were inoculated with 98 µL of a bacterial strain and 2 µL of the selected antibiotic on a 96-well microtiter plate. For the controls, 98 µL of the host strain or LB medium were mixed with 2 µL of RNase-free water and 100 µL PBS. After sealing the microtiter plate with adhesive tape, a hole was made in every well to supply the bacteria with oxygen throughout the experiment. The time-kill assays were run for 16 h at 37 °C, and the OD₅₉₀ was measured every 20 min using the SpectraMax i3 microplate reader (Molecular Devices, Sunnyvale, CA, USA).