Using J774 macrophages, the intracellular activities of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) against Staphylococcus aureus (strain ATCC 25923) have been quantitatively assessed in a 24-h model. S. aureus was positively localized in phagolysosomes by confocal and electron microscopy, and extracellular growth was prevented with 0. . Intracellular activity was not directly correlated to extracellular activity as measured in broth. Conditions of pH 5 (i.e., mimicking that of phagolysosomes) markedly reduced the activity of gentamicin, azithromycin, and telithromycin (>32؋) and fairly extensively reduced that of ciprofloxacin and moxifloxacin (>4؋) but did not affect oritavancin activity. We conclude that the cellular accumulation of antibiotics is not the only parameter to take into account for intracellular activity but that local environmental conditions (such as pH) and other factors can also prove critical.Staphylococcus aureus, a ubiquitous pyogenic bacterium, is causing severe infections in humans as well as in animals (63). S. aureus adheres to and easily invades nonprofessional as well as professional phagocytes (1, 33). In the latter cells, S. aureus tends to be restricted to the phagolysosomal compartment, where it largely escapes destruction and survives in a semiquiescent state for prolonged periods (29, 52). These intraphagocytic forms are considered responsible for the well-known recurrent character of staphylococcal infections as well as for the many failures of apparently appropriate antibiotic treatments (16,27,36). The main and most current treatment option for S. aureus infections is the administration of a -lactam resistant to -lactamases (often combined with an aminoglycoside and/or rifampin) (61). Clindamycin and fusidic acid are second-line antibiotics for treatment of these infections. Glycopeptides, oxazolidinones, or synergistins are recommended for multiresistant strains only (47). Yet it is usually recognized that -lactams are poorly active against the intracellular organisms because of their lack of cellular accumulation (56, 59). A number of models have therefore been developed to assess the potential of other antibiotics, such as rifampin, clindamycin, or glycopeptides (2, 12, 34), against intracellular S. aureus. Antibiotics with known cellular accumulation such as macrolides (23,38,48,60), synergistins (17), or fluoroquinolones (3, 4, 10, 46, 50) have also been studied. While providing useful information, these models have so far been used to examine only short periods of incubation and have not fully taken into account (i) the slow rate of intracellular accumulation of some antibiotics (see reference 57 for comments) and (ii) the reduced growth rate of intracellular S. aureus in comparison with that of bacteria in broth or other more favorable media (29). Moreover, contamination by bacteria growing extracellularly has often proven difficult to control (36).We present here data obtained with a model of S. aureusinfected J...