Minimum Inhibitory Concentration, the Minimum Concentration Inhibiting Colony Formation by 99% (MIC99) and Mutant Prevention Concentration Assays

The MIC values of antibiotics against M. hyopneumoniae was determined using both the standard micro-dilution and agar-dilution methods as described previously (19, 28). The standard method used M. hyopneumoniae cultures in the exponential growth phase period at 10⁶ CFU/mL that was added at 100 μL per well in 96-well cell culture plates containing a series of equal volumes of test drug, growth control (medium only), end point control (blank medium) and sterile control (blank broth). The plate was sealed and cultured as per above and color development was observed every 24 h and when the positive control changed from pink to orange, the minimum drug concentration whose color was consistent with the negative control was taken as the MIC result. The acid produced by M. hyopneumoniae as it grows changes the color of the medium from red to orange, which was used as the basis for interpreting MIC results.

The agar dilution method (29) consisted of agar plates containing a two-fold dilution series of antibiotic concentrations and 10 μL of culture medium (see above) in triplicate were then placed on the surface and the plates were incubated as per above for 7 d. The lowest drug concentration in the drug-containing medium lacking growth was recorded as the MIC as observed using an inverted microscope.

MIC₉₉: MIC₉₉ value of tulathromycin was measured as described previously (30).

The MPC assay was performed as previously described with some modifications (31). Briefly, 1 L logarithmic phase M. hyopneumoniae cultures were centrifuged at 3,000 rpm for 10 min and the supernatant was removed and the pellet was reconstituted with fresh media to obtain ≥10⁹ CFU/mL and 200 μL was spread-plated on tulathromycin agar plates and incubated as per above for 7 d. The lowest drug concentration contained in the plate without colony formation was determined as the primary MPC (MPCpr). Using MPCpr as the baseline, the concentration of 10% drug was linearly decreased to 0.5 MPCpr and the previous steps were repeated. The final drug concentration was taken as the MPC. Each experiment was repeated three times.