2.2. Determination of the Minimum Inhibitory Concentration (MIC)

The MIC of tilmicosin against M. hyopneumoniae was determined by a modified version of the MIC assay, as described by Tanner and Wu [19]. Briefly, dilutions of exponential-phase cells at 10⁵, 10⁶ and 10⁷ CFU/mL (100 μL) were added to an equal volume of drug-containing medium in each well in a 96-well plate. The tilmicosin concentration in the 96-well plate was 0.05–12.8 μg/mL. A growth control (lacking antibiotic), sterility control (sterile broth at pH 7.7) and endpoint control (blank medium at pH 6.5) were included. Plates were incubated at 37 °C in a humidified atmosphere of 5% CO₂ until the growth group and endpoint control were the same color. The MIC was defined as the minimal concentration of antibacterial agent that resulted in no color change. All experiments were carried out in triplicate.

According to the method described by Hannan et al. [20], plates containing a series of tilmicosin concentrations (1.6–25.6 μg/mL) were prepared. Samples (10 μL) of cultures with an inoculum of 10⁵, 10⁶ and 10⁷ CFU/mL were also applied to the drug plates. A blank-growth control group was also used and comprised cells spread on plates lacking the drug. Plates were incubated for ≥7 days. The lowest concentration without M. hyopneumoniae growth was determined as the MIC. All experiments were carried out in triplicate.