Antimicrobial susceptibility testing was performed by using a customized 96-well Sensititre plate (Trek Diagnostics, Oakwood, GA, USA) with the following antimicrobials: enrofloxacin (ENRO), gamithromycin (GAM), tulathromycin (TUL), tildipirosin (TIP), tilmicosin (TIL), tylosin tartrate (TYL), florfenicol (FFN), oxytetracycline (OXY), chlortetracycline (CTET), and penicillin (PEN). Serial two-fold dilutions were prepared as follows: ENRO, 0.12–128 µg/mL; TIP, 0.12–128 µg/mL; GAM, 0.25–256 µg/mL; TUL, 0.25–256 µg/mL; TIL, 1–256 µg/mL; TYL, 1–128 µg/mL; FFN, 0.25–256 µg/mL; OXY, 0.5–256 µg/mL; and CTET, 1–256 µg/mL. PEN (2–8 µg/mL) served as a control. Growth was assessed by using a color redox indicator, alamarBlue (Invitrogen, Fisher Scientific), based on a blue-to-pink color change.
For AST, the reserve culture was inoculated into the PPLO broth with 0.5% sodium pyruvate and incubated for 72 h. Following incubation, the actively growing broth cultures of isolates were subcultured into a neat PPLO broth and incubated for 24 h. Next, the optical density (OD) at 450 nm was determined by using a NanoDrop One Spectrophotometer (Fisher Scientific) and cultures were normalized to an OD450 = 0.1. Cultures were further diluted up to 10× in neat PPLO media prior to preparation of the inoculum. This inoculum (culture: media, 1:50) was prepared in a 20% alamarBlue solution in neat PPLO media and added to each well according to the manufacturer’s procedure (Trek Diagnostics, Thermo Fischer Scientific, Oakwood, OH, USA). This provided a final concentration of 103 to 5 × 105 CFU/mL in a 10% alamarBlue solution (as per manufacturer’s instructions). The plates were sealed with a CO2 permeable film and incubated for 48–72 h. Minimum inhibitory concentrations (MIC) were visually determined at 48 and 72 h and reported as per the Clinical and Laboratory Standards Institute (CLSI) guidelines [24 ].
If growth was observed in the positive control wells (no antibiotics), then the MIC values for that isolate were accepted. The MIC was defined as the lowest concentration of antimicrobial that prevented visible growth of the inoculated M. bovis culture. A M. bovis reference strain (Mycoplasma bovis ATCC® 25523™) was tested five times for quality control.
For AST, the reserve culture was inoculated into the PPLO broth with 0.5% sodium pyruvate and incubated for 72 h. Following incubation, the actively growing broth cultures of isolates were subcultured into a neat PPLO broth and incubated for 24 h. Next, the optical density (OD) at 450 nm was determined by using a NanoDrop One Spectrophotometer (Fisher Scientific) and cultures were normalized to an OD450 = 0.1. Cultures were further diluted up to 10× in neat PPLO media prior to preparation of the inoculum. This inoculum (culture: media, 1:50) was prepared in a 20% alamarBlue solution in neat PPLO media and added to each well according to the manufacturer’s procedure (Trek Diagnostics, Thermo Fischer Scientific, Oakwood, OH, USA). This provided a final concentration of 103 to 5 × 105 CFU/mL in a 10% alamarBlue solution (as per manufacturer’s instructions). The plates were sealed with a CO2 permeable film and incubated for 48–72 h. Minimum inhibitory concentrations (MIC) were visually determined at 48 and 72 h and reported as per the Clinical and Laboratory Standards Institute (CLSI) guidelines [24 ].
If growth was observed in the positive control wells (no antibiotics), then the MIC values for that isolate were accepted. The MIC was defined as the lowest concentration of antimicrobial that prevented visible growth of the inoculated M. bovis culture. A M. bovis reference strain (Mycoplasma bovis ATCC® 25523™) was tested five times for quality control.
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