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The effect of penicillin G on Staphylococcus aureus after phagocytosis by human monocytes was compared with the effect on nonphagocytosed bacteria. Intracellular S. aureus was obtained by incubation of monocytes and preopsonized S. aureus for 3 min at 37 C; phagocytosis was stopped by cooling the test tubes in crushed ice. Nonphagocytosed bacteria were removed by differential centrifugation followed by two washes of the cells. Morphological studies with lysostaphin confirmed the intracellular localization of approximately 70% of the bacteria after this procedure. The antibacterial activity of penicillin G was assessed by incubation of monocytes containing ingested S. aureus with the drug at 37 C, and determination of the number of viable cell-associated bacteria was assessed microbiologically at various intervals. Inactivation of the intracellular killing mechanism of the monocytes by omission of serum during incubation allowed measurement of the effect of penicillin G apart from the bactericidal activity of the cells. The effect of penicillin G on nonphagocytosed bacteria was assessed after incubation of preopsonized S. aureus in medium with the drug. The results show that the effect of penicillin G on intracellular S. aureus is two to seven times stronger than the effect on nonphagocytosed S. aureus.
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