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The activity of novobiocin against Escherichia coli ATCC 25922 and three E. coli strains that were isolated from cases of bovine mastitis was determined in timekill studies in the presence of bovine lactoferrin. Lactoferrin alone did not affect the growth of any of the strains of E. coli. A combination of 1.0 mg/ml of lactoferrin and novobiocin at 1/16x minimum inhibitory concentration (MIC) was bactericidal for E. coli ATCC 25922. When the concentration was increased to 3.0 mg/ml of lactoferrin, novobiocin was bactericidal at 1/64x MIC. Among the mastitis strains tested, 6789 and 6806 were more susceptible to killing by novobiocin than was strain 6800. Strains 6789 and 6806 were killed when treated with novobiocin concentrations of 2, 1/2, and 1/4x MIC. When these strains were also treated with lactoferrin at 3.0 mg/ml, there was a bacteriostatic effect at novobiocin concentrations of 1/8 and 1/16x MIC for strains 6789 and 6800. Strain 6806 appeared to be more susceptible to the combination of lactoferrin and novobiocin as was evidenced by a bactericidal effect over the 24-h testing period. The combination treatment with cephapirin and lactoferrin showed that there was a synergistic bactericidal effect against all of the E. coli strains tested. These studies indicate that lactoferrin can potentiate the activity of antibiotics against Gram-negative bacteria.

The mouse model was used to determine the efficacy of the cytokine, tumor necrosis factor-alpha, and antibiotic in treatment of experimentally induced staphylococcal mastitis. Recombinant human tumor necrosis factor-alpha alone administered to the mammary glands of lactating mice recruited significantly more polymorphonuclear neutrophils into the gland by 4 h posttreatment than did the untreated control. One hundred times less recombinant mouse tumor necrosis factor-alpha than human tumor necrosis factor-alpha was required to enhance the killing of Staphylococcus aureus within the gland. Human tumor necrosis factor-alpha effectively enhanced the killing of the bacteria when it was administered 4 to 0 h prior to infection, but not 4 h after infection. When mice were first pretreated with tumor necrosis factor-alpha, infected, and then treated with antibiotics (ciprofloxacin and pirlimycin, but not cloxacillin), the combination of antibiotic and cytokine significantly reduced the number of bacteria within the gland compared with that for mice treated with antibiotic alone, cytokine alone, or placebo. Recombinant tumor necrosis factor-alpha may be an effective adjunct to antimicrobial therapy in treatment of staphylococcal mastitis in the bovine.

A bovine blood polymorphonuclear neutrophil system was used to determine the ability of cytokines, alone or in combination with various antibiotics, to enhance the intracellular killing of Staphylococcus aureus sequestered within these phagocytes. Pretreatment of blood polymorphonuclear neutrophils with human recombinant tumor necrosis factor-alpha, bovine macrophage supernatant tumor necrosis factor-alpha, or human recombinant interleukin-1 beta enhanced the killing of S. aureus by the phagocytes compared with that of untreated polymorphonuclear neutrophils. This enhancement was dose-dependent and required 30 min of contact between the cytokine and the blood polymorphonuclear neutrophils. However, mammary gland polymorphonuclear neutrophils, macrophages, and blood monocytes showed no enhanced killing activity following pretreatment with tumor necrosis factor. Pretreatment of polymorphonuclear neutrophils with tumor necrosis factor and subsequent exposure to various antibiotics after infection with S. aureus enhanced intracellular bacterial killing by certain antibiotics, including paldimycin, rifampin, and ciprofloxacin (antibiotics that normally kill intracellular S. aureus). However, no enhancement occurred with antibiotics that do not normally kill intracellular S. aureus. Cytokine therapy may be a useful adjunct to antibiotic therapy in the treatment of bovine staphylococcal infection and mastitis.