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.
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.
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