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To compare the activity of cloxacillin and vancomycin against methicillin-susceptible Staphylococcus aureus and to determine how rapidly their bactericidal activity occurs in cardiac vegetations.In vitro and in vivo studies using an experimental model of endocarditis in rabbits. Animals were treated for 1, 2 or 3 days with cloxacillin 200 mg/kg intramuscularly three times a day or vancomycin 25 mg/kg intravenously twice a day.Cloxacillin and vancomycin at concentrations 4- and 16-fold the MIC produced a modest decrease in the number of microorganisms at 4 h. After 24 h, cloxacillin produced a decrease in the counts of staphylococci from 2.19 to 4.84 log10 cfu/mL of inoculum. Only concentrations of vancomycin from 16- to 32-fold the MIC resulted in equivalent decreases. After 24 h of treatment, both antibiotics were equally effective in preventing mortality of rabbits. Cloxacillin produced a greater decrease in the number of staphylococci than vancomycin (3.50 ± 2.18 log10 cfu/g vegetation and 6.25 ± 1.28 log10 cfu/g vegetation, respectively; P < 0.05) and 41% of rabbits had sterile vegetations in comparison with none with vancomycin (P=0.035). After 48 and 72 h of treatment, both antimicrobials exhibited equivalent activity.Vancomycin was less rapidly bactericidal than cloxacillin in vivo.