Bacitracin

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.

Minimum inhibition concentrations were determined using a previously described microdilution method (Kawada‐Matsuo, Yoshida, et al., 2013) for nisin A (Sigma‐Aldrich, St. Louis, MO, USA), gallidermin (Santa Cruz Biotechnology, TX, USA), and bacitracin (WAKO Chemicals, Osaka, Japan).

The antibacterial properties of the products were assayed against S. mutans, A. naeslundii, and P. gingivalis. Each bacterium was procured from the American Type Culture Collection (Manassas, VA, USA) and frozen until further analysis. First, the frozen bacterial stocks were thawed and grown on brain heart infusion (BHI) medium (Pearlcore^®, Eiken Chemical, Co., Ltd., Tokyo, Japan) supplemented with 0.1% antibiotic (0.05% gramicidin D and 0.05% bacitracin, FUJIFILM Wako Pure Chemical Corporation) and 1% sucrose (FUJIFILM Wako Pure Chemical Corporation).
In a 96-well plate, S. mutans (1.1 × 10⁶ CFU/200 µL/well), A. naeslundii (1.0 × 10⁶ CFU/200 µL/well), and P. gingivalis (2.8 × 10⁹ CFU/200 µL/well) were incubated for 24 h at 37 °C under anaerobic conditions in the BHI medium supplemented with the product at various doses: 0 (ctrl), 0.001, 0.01, and 0.1 w/v%. After incubation, the metabolic activity of the bacteria (proportional to the number of living bacteria) was assayed using a microbial viability assay kit-WST (DOJINDO Laboratories, Mashiki, Japan) according to the manufacturer’s instructions, and the absorbance was measured at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA). The pH of each bacterial suspension was measured before and after incubation using a portable pH meter (LAQUA-PH-SE, HORIBA, Ltd., Kyoto, Japan).