Quantitative analysis of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) activities against intracellular Staphylococcus aureus in mouse J774 macrophages

Abstract

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 0.5 mg of gentamicin/liter (1x MIC) in controls. When tested at extracellular concentrations equivalent to their maximum concentrations in human serum, all antibiotics except azithromycin caused a significant reduction of the postphagocytosis inoculum within 24 h, albeit to markedly different extents (telithromycin [2 mg/liter], 0.60 log; ciprofloxacin [4.3 mg/liter], 0.81 log; gentamicin [18 mg/liter], 1.21 log; moxifloxacin [4 mg/liter], 1.51 log; oritavancin [25 mg/liter], 3.49 log). Intracellular activities were not systematically related to drug accumulation (apparent cellular-to-extracellular concentration ratios in infected cells: ciprofloxacin, 3.2; gentamicin, 6.8; telithromycin, 8.7; moxifloxacin, 13.4; azithromycin, 50; oritavancin, 348). 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 (>or=32 x) and fairly extensively reduced that of ciprofloxacin and moxifloxacin (>or=4 x) 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.

FIG. 1.

Confocal microscopy of J774 macrophages observed 5 h after phagocytosis of opsonized S. aureus. Cell actin (located mostly on the inner face of the pericellular membrane) was labeled with rhodamine-phalloidin (red signal), and bacteria were labeled with FITC (green signal). (Left panel) Incubation was performed in the absence of any antibiotic (in the inset, a vertical section of the cell shown in the main panel is depicted; actin surrounds the bacteria, demonstrating its intracellular localization). (Right panel) Incubation performed in the presence of 0.5 mg of gentamicin/liter. No difference was seen with cells incubated without gentamicin.

FIG. 2.

Electron microscopy of J774 macrophages fixed 1 h (A and B) or 24 h (C and D) after phagocytosis of opsonized S. aureus. In both cases, incubation was carried out in the presence of 0.5 mg of gentamicin/liter. Bacteria appeared isolated (A) or sometimes in clusters (B) at 1 h after phagocytosis, without evidence of damage but with no sign of division. In contrast, most bacteria observed at 24 h were in the active process of division. In both cases, all bacteria were seen in membrane-bounded structures with no evidence of transfer to cytosol. Bars are 0.3 μm (A, B, and D) and 1 μm (C).

FIG. 3.

Variations in the number of CFU per milligram of protein (± SD; n = 3) collected from J774 macrophages after 24 h of incubation postphagocytosis compared to that seen with the original postphagocytosis inoculum. ctr, control cells incubated with 0.5 mg of gentamicin/liter; RIF, cells incubated with rifampin (4 mg/liter); OXA, cells incubated with oxacillin (6 mg/liter); VAN, cells incubated with vancomycin (50 mg/liter). These concentrations correspond to peak concentrations in serum observed for patients after administration of conventional doses of oxacillin and vancomycin (14, 22) and to the low side of the range of peak concentrations in serum observed for rifampin (21). They also represent values 250 fold (rifampin) and 50 fold (oxacillin and vancomycin) larger than the MIC (in broth) of the corresponding antibiotic for the strain of S. aureus used in the present study.

FIG. 4.

Variations in the number of CFU upon incubation for up to 24 h with increasing concentrations of gentamicin. (Left panel) Results for bacteria in broth (initial pH, 7.3). (Right panel) Results for bacteria collected from J774 macrophages (after phagocytosis). The lowest drug concentration (0.5 mg/liter) corresponds to the MIC (in broth) of gentamicin for the strain of S. aureus used in this study; the highest concentration (18 mg/liter) corresponds to the C_max observed for patients after the administration of conventional doses of gentamicin (26). Each point corresponds to the mean value for three independent determinations. SD values were calculated, but the corresponding bars are smaller than the symbols in many cases. For the results obtained with broth, symbols placed outside of the bottom of the graph indicate samples in which no or only a few colonies were observed (i.e., numbers were too close to the lowest limit of detection [10² CFU/ml] to be fully reliable). Cells incubated with 0.5 mg of gentamicin/liter served as controls for all subsequent experiments.

FIG. 5.

Variations in the number of CFU upon incubation for up to 24 h with increasing concentrations of azithromycin (top two panels) or telithromycin (bottom two panels). (Left two panels) Results for bacteria in broth (initial pH, 7.3). (Right two panels) Results for bacteria collected from J774 macrophages (after phagocytosis). The lowest drug concentrations investigated (0.5 mg/liter for azithromycin, 0.06 mg/liter for telithromycin) correspond to the MICs (in broth) of the drugs for the strain of S. aureus used in this study; note that for azithromycin, the peak concentration observed for patients receiving a conventional dose (500 mg) did not exceed 0.4 mg/liter (24); for telithromycin, the highest concentration investigated (2 mg/liter) corresponds to the C_max observed for patients after administration of a conventional dose (800 mg) (42). Each point corresponds to the mean value for three independent determinations. SD values were calculated, but the corresponding bars are smaller than the symbols in many cases. Note that cells incubated in the absence of azithromycin or telithromycin were maintained in the presence of 0.5 mg of gentamicin/liter to prevent extracellular growth of S. aureus.

FIG. 6.

Variations in the number of CFU upon incubation for up to 24 h with increasing concentrations of ciprofloxacin and moxifloxacin. (Left two panels) Results for bacteria in broth (initial pH, 7.3). (Right two panels) Results for bacteria collected from J774 macrophages (after phagocytosis). The lowest drug concentrations investigated (0.125 mg/liter for ciprofloxacin, 0.06 mg/liter for moxifloxacin) correspond to the MICs (in broth) of the drugs for the strain of S. aureus used in this study; the highest concentrations (4.3 mg/liter for ciprofloxacin, 4 mg/liter for moxifloxacin) correspond to the C_max observed for patients after administration of the currently recommended doses of each of these two fluoroquinolones (reference and U.S. ciprofloxacin package insert, Bayer Corporation, West Haven, Conn.). Each point corresponds to the mean value for three independent determinations. SD values were calculated, but the corresponding bars are smaller than the symbols in many cases. Symbols placed outside of the bottom of the lower left graph indicate samples in which no or only a few colonies were observed (i.e., numbers were too close to the lowest limit of detection [10² CFU/ml] to be fully reliable). Note that cells incubated in the absence of ciprofloxacin or moxifloxacin were maintained in the presence of 0.5 mg of gentamicin/liter to prevent extracellular growth of S. aureus.

FIG. 7.

Variations in the number of CFU upon incubation for up to 24 h with increasing concentrations of oritavancin. (Left two panels) Results for bacteria in broth (initial pH, 7.3). (Right two panels) Results for bacteria collected from J774 macrophages after phagocytosis. The lowest drug concentration investigated (0.25 mg/liter) corresponds to the MIC (in broth) of oritavancin for the strain of S. aureus used in this study, and the highest concentration investigated (25 mg/liter) corresponds to the C_max observed for patients after administration of the dose currently used in the clinical development program of this antibiotic (D. K. Braun et al., Abstracts of the 11th European Congress of Clinical Microbiology and Infectious Diseases, Clin. Microbiol. Infect., 7[Suppl. 1]:P434, 2001). Each point corresponds to the mean value of three independent determinations. SD values were calculated, but the corresponding bars are smaller than the symbols in many cases. Symbols placed outside of the bottom of the lower left graph indicate samples in which no or only a few colonies were observed (i.e., numbers were too close to the lowest limit of detection [10² CFU/ml] to be fully reliable). Note that cells incubated in the absence of oritavancin were maintained in the presence of 0.5 mg of gentamicin/liter to prevent extracellular growth of S. aureus.