The Activity of a Combination of Penicillin and Novobiocin Against Bovine Mastitis Pathogens: Development of a Disk Diffusion Test

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Abstract
The combination of penicillin and novobiocin is currently available for the treatment of bovine mastitis, but methods are not available for susceptibility testing of the combination by veterinary diagnostic laboratories. The minimum inhibitory concentration (MIC) and disk diffusion data were determined for penicillin, novobiocin, and a combination of the two in a 1:2 ratio for 225 staphylococcal, streptococcal, and Gram-negative isolates from bovine intramammary infections. Based on the drug concentrations in milk following infusion, linear regression analysis, and error rate bounding, the interpretive zone diameters selected were < or = 16 mm for resistant isolates and > or 17 mm for susceptible isolates with a disk containing 10 U of penicillin and 30 micrograms of novobiocin. Additionally, MIC breakpoints of < or = 2 micrograms/ml of penicillin and 4 micrograms/ml of novobiocin were selected to categorize isolates as susceptible and > or = 4 micrograms/ml of penicillin and 8 micrograms/ml of novobiocin were selected to categorize isolates as resistant. The MIC and disk diffusion results, as well as studies to monitor bacterial killing by antimicrobial agents over time, indicated that the combination of penicillin and novobiocin in a 1:2 ratio was more active than were the individual drugs. Kinetics of the kill curves with the penicillin and novobiocin combination (1:2 ratio) showed that the combination was bactericidal for Staphylococcus aureus and Staphylococcus xylosus.