Antibiotic suceptibility of caprine pathogens

Standard conditions or modification from CLSI methods by 25% goat serum supplementation were performed to determine the MIC (mg/L) of tildipirosin, gamithromycin, oxytetracycline, and danofloxacin. Minimum inhibitory concentration tests were performed by microdilution broth technique (Clinical and Laboratory Standards Institute 2009) using U-bottom 96-well microtitre plates. Serial two-fold dilutions of the antimicrobial agents were prepared starting from the stock solution. Broth dilutions were made using Mueller-Hinton broth (MHB) (Merck, Madrid, Spain) for Coagulase-negative staphylococci, Staphylococcus aureus, and Escherichia coli. For investigating Streptococcus spp., cation-adjusted Mueller-Hinton broth (Merck, Madrid, Spain) with 5% of defibrinated horse blood (Thermo Fisher Scientific, Massachusetts, USA) was used. Concentrations of all antibiotics ranging from 0.03 to 128 mg/l were used. Inocula were prepared by diluting an overnight MHB culture in buffered saline solution to a density of 0.5 on McFarland Turbidity Scale and finally diluting again 40-fold before testing. The U-bottomed microtiter plates were incubated at 37°C and observed 24 h after. The MIC was defined as the lowest concentration of antibiotic at which the bacterial growth was completely inhibited. The reference strains S. aureus (ATCC 29213) and E. coli (ATCC 25922) were used as controls. Minimal inhibitory concentration was determined according to the recommendations of Hannan (Hannan 2000). Briefly, a stationary-phase culture of each isolate was carried out in mycoplasma medium without antimicrobials, supplemented with phenol red (0.005%) in 96-well round-bottomed plates. Each antibiotic was added to achieve each of the pre-established final concentrations (from 32 μg/ml to 0.006 μg/ml) and a final concentration of the mycoplasma cultures of 105 to 103 colour-changing units/ml. Positive (lacking antibiotic) and negative (lacking mycoplasmas) controls were also added. The plates were then sealed and incubated at 37 °C. After 48 h, plates were examined for colour change. MIC was defined as the lowest concentration of each antibiotic at which no M. agalactiae growth (no colour change) was observed.