Antibiotic suceptibility of caprine pathogens

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.