Gentamicin

CHO cells were cultured at 37° C with 5% CO₂ in FreeStyle CHO expression medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 4 mM L-glutamate (Thermo Fisher Scientific). COLO 205 cells were cultured at 37° C with 5% CO₂ in RPMI1640 (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum and 50 μg/mL gentamicin (Nacalai, Kyoto, JAPAN) or penicillin/streptomycin (final concentrations 100 U/mL and 100 μg/mL, respectively; Nacalai Tesque, Kyoto, Japan).

HeLa cells and HaCaT cells were cultured in DMEM containing 10% fetal bovine serum (Gibco) and 50 μg/mL gentamicin (Nacalai Tesque).

Two methods were used for the evaluation of antibacterial activity. To measure the MIC value of nisin A (Sigma–Aldrich, St. Louis, USA), the microdilution method was used as described elsewhere [23 (link)]. Bacitracin (Fujifilm Wako chemicals, Osaka, Japan), vancomycin (Sigma–Aldrich), Metronidazole (Fujifilm Wako chemicals), ampicillin (Nacalai Tesque, Kyoto, Japan), chloramphenicol (Wako chemicals), gentamicin (Nacalai Tesque), ofloxacin (Sigma–Aldrich) and imipenem (Fujifilm Wako chemicals) were also used for MIC evaluations.
To assess the antibacterial activity of the bacteriocins, a direct assay was performed by a method described elsewhere [23 (link)]. An overnight culture (3 μl) of the bacteriocin-producing strain, as indicator bacteria, was spotted on a TSA plate and cultured at 37°C for 24 h. Then, 3.5 ml of prewarmed BHI soft agar (0.75%) containing C. difficile cells (10⁸ cells/ml) was poured over the TSA plate. The plates were incubated anaerobically at 37°C for 24 h. Then, the diameters of the growth inhibitory zones were measured in three directions. Three independent experiments were performed, and the average diameters were calculated.

HeLa cells were purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (Gibco) and 50 μg/mL gentamicin (Nacalai Tesque) in a 5% CO₂ incubator at 37°C. Plasmid transfections were performed using polyethylenimine (Polysciences, Inc.), Lipofectamine 3000 (Invitrogen), or Lipofectamine RNAiMAX (Invitrogen), according to the manufacturers' protocols.

Bacterial association with and invasion into hBMECs or the human alveolar cell line A549 were quantified as previously described, with minor modifications [12,19,26,52]. hBMECs or A549 cells were seeded at 2 × 10⁵ cells into 24-well plates 1 day prior to bacterial infection. In each well, ~ 2.0 × 10⁶ CFU of bacteria from exponential phase bacterial cultures (OD₆₀₀ = ~ 0.5) was added to infect hBMECs, while bacteria at ~ 1.0 × 10⁷ CFU was added to infect A549 cells. For assays using heparin, S. pneumoniae organisms were pre-incubated with 10, 50, or 100 U of heparin or buffer for 30 minutes prior to infection. To determine bacterial association, hBMECs and A549 cells were infected with the bacteria for 1 and 2 hours, respectively, then the cells were washed and harvested with PBS containing 0.05% trypsin and 0.025% Triton X-100. To examine bacterial invasion, hBMECs and A549 cells were washed following incubation for 1 and 2 hours, respectively, then medium containing 100 μg/mL of gentamicin (Nacalai) was added, after which the both types of cells were incubated for an additional 1 hour and harvested. The number of bacteria in each sample was quantified by serial dilution plating.