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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.

The intracellular location of certain strains of Staphylococcus aureus serves as a reservoir of bacteria which is thought to be important in therapy of recurrent infections in humans and in chronic staphylococcal mastitis in dairy cows. This overview summarizes data pertaining to the intracellular survival of Staphylococcus aureus within polymorphonuclear neutrophils (PMNs) both in vitro and in vivo in the face of antibiotic treatment. While compounds such as rifampin, clindamycin, erythromycin, and ciprofloxacin have been shown to be rapidly taken up by PMNs, the ability of antibiotics to concentrate within PMNs did not strictly correlate with their ability to kill intracellular Staphylococcus aureus. Rifampin and ciprofloxacin have been shown to be the most effective intraphagocytic killing agents, while clindamycin and erythromycin were inactive in these in vitro assays. In vivo, in therapy of Staphylococcus aureus arthritis and peritonitis in humans and in certain mouse models rifampin has generally been shown to be more effective than comparator antibiotics. In a staphylococcal subcutaneous abscess model, however, clindamycin was very effective in curing the Staphylococcus aureus abscesses in this system where PMNs were the primary inflammatory cells involved. The intracellular bacterial counts decreased as rapidly as the extracellular bacteria. Rifampin was also effective in the abscess model but ciprofloxacin was ineffective at the highest doses tested. The relevance of in vitro and in vivo models and the importance of PMNs as a reservoir of infection in staphylococcal diseases in humans and the dairy cow are discussed.

A bovine polymorphonuclear leukocyte (PMN) monolayer system was used to determine the ability of different antibiotics to kill surviving intracellular Staphylococcus aureus. The following classes of antimicrobial agents were tested in this high-volume assay procedure: aminocyclitol, beta-lactam, coumarin, lincosaminide, macrolide, naphthalenic ansamycin, paulomycin, peptide, quinolone, and tetracycline. The activities of these compounds were compared with those of positive (rifampin), negative (cloxacillin), and non-antibiotic-treated controls. Only oxytetracycline, the ansamycins (rifampin, rifamycin SV, streptovaricin A, C, and D), paulomycin, and paldimycin caused a significant reduction in the viable count of intracellular S. aureus. Of these, however, the intracellular killing by the streptovaricins was directly related to the cytotoxicity (as determined by trypan blue exclusion) of these compounds for the PMNs. Although the paulomycins were cytotoxic for the PMNs, the cytotoxic and the intracellular killing activity of these new compounds could be distinguished. The relevance of these results to the therapeutic effectiveness of these antibiotics in the treatment of bovine staphylococcal mastitis is discussed.

Bovine mastitis due to Staphylococcus aureus may become chronic and refractory to antibiotic therapy because of the organism's ability to survive within the mammary gland macrophages and/or polymorphonuclear neutrophils (PMNs). Therefore, phagocytosis and killing of S. aureus by bovine udder macrophages, udder and blood neutrophils and blood monocytes were studied. Gland and blood PMNs were about equally effective at phagocytosing (2.5 log reduction in supernatant) and killing the bacteria (92% reduction of viable bacteria by two hours). Gland macrophages phagocytosed at a lower rate (1.5 log reduction) and were less effective at killing the bacteria (73% reduction by two hours). Blood monocytes phagocytosed and killed S. aureus at the lowest rate. An udder macrophage monolayer system was developed and used to evaluate the ability of antibiotics to kill surviving intracellular S. aureus. This assay was similar to our previously described system with blood PMNs. Several classes of antibiotics were investigated. These included naphthalenic ansamycin, lincosaminide, tetracycline, coumarin, peptide, paulomycin, quinolone, macrolide, cephalosporin, and penicillin-class antibiotics. The activity of these compounds was compared to positive (rifampicin), negative (cloxacillin), and nonantibiotic treated controls. Only naphthalenic ansamycin class antibiotics, paulomycin, paldimycin and ciprofloxacin caused significant reduction in viable intracellular bacteria in the macrophage system. These results were similar to those obtained in the blood PMN monolayer system. Because a low intraphagolysosomal pH could affect an antibiotic's ability to kill intracellular bacteria by affecting the drug itself or inhibiting bacterial growth, the effect of low pH on the minimum inhibitory concentration and the minimum lethal concentration of clindamycin and rifampicin against three strains of S. aureus was also tested. While the activity of clindamycin at pH 5.0 compared to pH 7.0 was not affected greatly, the activity of rifampicin was greatly enhanced at acidic pH. These results suggest that at least some of the excellent activity of rifampicin for intracellular S. aureus is due to potentiation of its activity in the intracellular acidic compartment of the phagolysosome.