Analysis of the mutant selection window and killing of Mycoplasma hyopneumoniae for doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin

Abstract

Mycoplasma hyopneumoniae is the major pathogenic microorganism causing enzootic pneumonia in pigs. With increasing resistance of M. hyopneumoniae to conventional antibiotics, treatment is becoming complicated. Herein, we investigated the mutant selection window (MSW) of doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin for treating the M. hyopneumoniae type strain (ATCC 25934) to determine the likelihood of promoting resistance with continued use of these antibiotics. Minimum inhibitory concentration (MIC) values against M. hyopneumoniae were determined for each antimicrobial agent based on microdilution broth and agar dilution methods (bacterial numbers ranged from 105 colony-forming units (CFU)/mL to 109 CFU/mL). The minimal concentration inhibiting colony formation by 99% (MIC99) and the mutant prevention concentration (MPC) were determined by the agar dilution method with three inoculum sizes. Antimicrobial killing was determined based on MIC99 and MPC values for all five agents. MIC values ranged from 0.001 to 0.25 μg/mL based on the microdilution broth method, and from 0.008 to 1.0 μg/mL based on the agar dilution method. MPC values ranged from 0.0016 to 10.24 μg/mL. MPC/MIC99 values were ordered tylosin > doxycycline > danofloxacin > tiamulin > valnemulin. MPC achieved better bactericidal action than MIC99. Based on pharmacodynamic analyses, danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.

Fig 1. Minimum inhibitory concentration (MIC) determination for danofloxacin, tiamulin, tylosin, doxycycline, and valnemulin against M. hyopneumoniae in artificial medium using liquid and solid agar methods with inoculum sizes of 10⁵, 10⁶, and 10⁷ CFU/mL.

Fig 2. M. hyopneumoniae killing curves at the minimal concentration inhibiting colony formation by 99% (MIC₉₉) and at the mutant prevention concentration (MPC) with an inoculum of 10⁵ CFU/mL.

Fig 3. M. hyopneumoniae killing curves at the minimal concentration inhibiting colony formation by 99% (MIC₉₉) and at the mutant prevention concentration (MPC) with an inoculum of 10⁷ CFU/mL.

Fig 4. M. hyopneumoniae killing curves at the minimal concentration inhibiting colony formation by 99% (MIC₉₉) and at the mutant prevention concentration (MPC) with an inoculum of 10⁹ CFU/mL.