Unité de pharmacologie cellulaire et moléculaire, Université catholique de Louvain, Brussels, Belgium
Received 16 September 2004; returned 23 October 2004; revised 27 December 2004; accepted 14 January 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: We assessed the activities of ciprofloxacin, levofloxacin, moxifloxacin and garenoxacin against the extracellular (broth) and intracellular (infected J774 macrophages) forms of Listeria monocytogenes (cytosolic infection) and Staphylococcus aureus (phagolysosomal infection) using a range of clinically meaningful extracellular concentrations (0.064 mg/L).
Results: All four quinolones displayed concentration-dependent bactericidal activity against extracellular and intracellular L. monocytogenes and S. aureus for extracellular concentrations in the range 14-fold their MIC. Compared at equipotent extracellular concentrations, intracellular activities against L. monocytogenes were roughly equal to those that were extracellular, but were 50100 times lower against S. aureus. Because quinolones accumulate in cells (ciprofloxacin, 3 times; levofloxacin,
5 times; garenoxacin,
10 times, moxifloxacin,
13 times), these data show that, intracellularly, quinolones are 510 times less potent against L. monocytogenes (P=0.065 [ANCOVA]), and at least 100 times less potent (P < 0.0001) against S. aureus. Because of their lower MICs and higher accumulation levels, garenoxacin and moxifloxacin were, however, more active than ciprofloxacin and levofloxacin when compared at similar extracellular concentrations.
Conclusions: Quinolone activity is reduced intracellulary. This suggests that either only a fraction of cell-associated quinolones exert an antibacterial effect, or that intracellular activity is defeated by the local environment, or that intracellular bacteria only poorly respond to the action of quinolones.
Keywords: cellular pharmacodynamics , cellular pharmacokinetics , bactericidal activity , accumulation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We used a haemolysin-producing strain (EGD serotype 1/2a) for L. monocytogenes (obtained from P. Berche, Laboratoire de Microbiologie, Faculté de Médecine, Necker, Paris, France), and a methicillin-susceptible strain of S. aureus (ATCC 25923) obtained from the American Tissue Cell Collection (Manassas, VA, USA). MICs and MBCs were determined in tryptic soy broth (L. monocytogenes) or MuellerHinton broth (S. aureus) as in our previous publications.8,13 MICs for S. aureus were also determined in the same medium adjusted to pH 5. For killing-curve experiments, bacteria in logarithmic growth were resuspended at a density of 106 cfu/mL in broth. The number of viable bacteria was determined after incubation at 37 °C with antibiotics for suitable periods of time (up to 24 h) by plate assay with appropriately diluted samples.
Cell infection and measurement of intracellular activity
All experiments were conducted with J774 macrophages, a continuous reticulosarcoma cell line of murine origin,14 following exactly the procedures described earlier.13,15 In brief, infection was achieved by incubating macrophages with bacteria for 1 h [5 cfu/cell for L. monocytogenes and 0.5 cfu/cell for S. aureus (human serum-opsonized)]. Extracellular bacteria were eliminated by washing in PBS (for S. aureus, a first washing was made by bathing the cells for 1 h in a medium supplemented by 50 mg/L gentamicin). For experiments in which infected cells were maintained in culture 24 h post-phagocytosis, gentamicin was added at its MIC (1 mg/L for L. monocytogenes and 0.5 mg/L for S. aureus) during the whole incubation period to prevent the extracellular growth of released bacteria, and the ensuing fast acidification of the medium and subsequent loss of cell viability.13
Cellular accumulation of quinolones
Infected and uninfected cells were collected and analysed following the general procedure described earlier.16
In brief, cell sheets were washed three times with ice-cold PBS and collected by scraping in 0.1 M glycine-NaOH pH 3 buffer for fluorimetric determinations, or in distilled water for radiochemical assays. Cell-associated ciprofloxacin, moxifloxacin and levofloxacin were assayed by fluorimetry (exc=275, 298 and 298 nm;
em=450, 504 and 500 nm respectively), as previously described.8
Garenoxacin was assayed by scintillation counting using 14C-labelled drug. These methods have been fully validated with respect to specificity and reproducibility, and linearity under our conditions of assay. All drug contents in cell samples were expressed by reference to the cell protein (assayed by the Folin-ciocalteu/biuret method),17
and the apparent cellular-to-extracellular concentration ratio calculated using a conversion factor of 3.08 µL of cellular volume per mg cell protein, as determined for J774 macrophages by the sucrose/urea partition method.16
Data analyses
Curve-fitting and statistical analyses [one way analysis of variance (ANOVA), analysis of covariance (ANCOVA) and Tukey's Honestly Significant Difference (HSD) tests (for differences between groups with a confidence interval of 95%)] were made using Prism version 4.02 and InStat version 3.00 (GraphPad Software, San Diego, CA, USA), or XLSTAT version 6.0 (Addinsoft SARL, Paris, France).
Materials
Ciprofloxacin (potency, 85.0%) and moxifloxacin (potency, 91%) were obtained as laboratory samples for microbiological evaluation from Bayer AG (Leverkusen, Germany), and garenoxacin (purity 99.8%) from Bristol Myers Squibb, New Brunswick, CT, USA. Levofloxacin and gentamicin were procured as Tavanic and Geomycin, the registered commercial products available for intravenous administration in Belgium. 14C-labelled garenoxacin (0.8 MBq/mg; 98.3% of radiochemical purity) was obtained from the Bristol Myers Squibb Research Institute, Princeton, NJ, USA. Cell culture media and fetal calf serum (FCS) were purchased from Gibco Biocult (Paisley, Scotland, UK). Human serum for opsonization of S. aureus was obtained from healthy volunteers as pooled samples stored as aliquots at 80 °C until use. Unless stated otherwise, all other reagents were of analytic grade and purchased form E. Merck AG (Darmstadt, Germany) or from Sigma-Aldrich (St Louis, MO, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Table 1 shows the MIC and MBC values of the four quinolones under investigation against the two bacterial strains used in this study. Bearing in mind the results obtained at pH 7, the four drugs were less active against L. monocytogenes than S. aureus, and levofloxacin or ciprofloxacin were in all cases less active than moxifloxacin and garenoxacin. All MICs, however, were still lower than the maximal serum concentrations observed in healthy volunteers (Cmax, see Table 1). On investigating the extracellular activities of quinolones, we observed that the MIC/MBC ratios were between 2 and 8, demonstrating the bactericidal activity of these drugs. MICs and MBCs measured at acid pH against S. aureus (to mimic the conditions prevailing in lysosomes)18 were 2 (ciprofloxacin) to 33 (garenoxacin) times higher than at pH 7 but still lower than Cmax.
|
The activity of the four quinolones was then examined against extracellular and intracellular forms of L. monocytogenes and S. aureus. In the first series of experiments (Figure 1), all drugs were compared at a fixed post-phagocytosis time point (5 h for L. monocytogenes, 24 h for S. aureus; these times were selected based on previous observations showing that intracellular L. monocytogenes grows after a lag period of about 1 h only, whereas this lag period extends for up to 8 h for S. aureus)8,13 and at the same extracellular concentration (4 mg/L). We observed that the growth of L. monocytogenes was similar in broth and in infected macrophages, whereas that of S. aureus was significantly lower intracellularly as compared with broth. On examining the extracellular activities of quinolones, we see that these were only slightly bactericidal towards L. monocytogenes (achieving an inoculum reduction from about 1.2 log for ciprofloxacin, 1.5 log for levofloxacin and garenoxacin, and about 2 log for moxifloxacin (these differences were statistically significant). In contrast, they were highly bactericidal against S. aureus, achieving an inoculum reduction of about 4 log for ciprofloxacin and levofloxacin, and of 4.5 log for moxifloxacin and garenoxacin (these differences were statistically significant). However, when considering activities against intracellular bacteria, a global analysis of the results showed that these were always significantly lower than what was seen against extracellular bacteria, the difference being, however, smaller for L. monocytogenes (especially for moxifloxacin) than for S. aureus. Comparison between drugs showed a ranking (with statistically significant differences) of ciprofloxacin < levofloxacin < garenoxacin < moxifloxacin for L. monocytogenes, and of ciprofloxacin = levofloxacin < moxifloxacin = garenoxacin for S. aureus.
|
|
|
Table 2 compares the apparent cellular accumulations of the four quinolones in both uninfected and infected cells, under the conditions used for the experiments described in Figure 1. Marked differences were observed among drugs, with the following ranking: ciprofloxacin < levofloxacin < garenoxacin <moxifloxacin. Differences between data obtained at 5 and 24 h in uninfected cells were either not significant (ciprofloxacin, moxifloxacin) or small (levofloxacin, garenoxacin), indicating that an apparent steady state had been reached or was close to being obtained. Differences between uninfected and infected cells were also not significant (ciprofloxacin, garenoxacin) or small (levofloxacin, moxifloxacin).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two main conclusions emerge from the present data. First, whereas quinolones appear to be concentration-dependent drugs for L. monocytogenes (whether extra- or intracellularly), this seems not to be the case for S. aureus. However, closer examination of the data suggests that concentration-dependency is observed for both organisms but may be limited to a range of concentrations that span from the MIC up to a maximum of 4 times the MIC. Future studies will need to explore these limits in more detail, perhaps by using strains of S. aureus with higher MICs.
Second, and surprisingly, our data show that the intracellular activity of quinolones against both L. monocytogenes and S. aureus is only a fraction of what could be anticipated if their apparent accumulation in cells is taken into account. Thus, whereas all quinolones used in the present study show higher concentrations in cells compared with medium, all are also characterized by somewhat weaker activity against intracellular L. monocytogenes and drastically reduced activity against intracellular S. aureus in comparison with broth. To substantiate this conclusion further, we used all data generated in this studypooling them with a series of data obtained previously with the same models13,15 to compare activities in broth and in cells after normalization of concentrations for differences in MIC (Figure 4). Thus, the activity of quinolones against intracellular L. monocytogenes was lower (five- to 20-fold depending on the concentration) than against the extracellular forms, but the global difference was at the limit of statistical significance (P=0.065 by ANCOVA). For S. aureus, the difference is at least 100-fold and was highly significant. Several factors could account for such a loss of activity. For instance, we know that intracellular L. monocytogenes is surrounded by a thick layer of actin that confers motility to the bacteria,22,23 but could also partially protect it from antibiotics. For S. aureus, the slow intracellular growth of this organism may make it poorly sensitive to quinolones, as suggested from studies in broth with slowly growing bacteria.2426 (This could result from the decreased expression of quinolone targets as recently found for topoisomerase IV).27 Part of the drastic reduction in activity against intracellular S. aureus could also be due to the acidic environment of phagolysosomes, which is unfavourable to their activity (see Table 1). Moreover, we have to consider that we do not know with certainty where the drugs are located intracellularly. Whereas in fractionation studies the bulk of cell-associated ciprofloxacin is recovered in the cytosol,15 we cannot exclude a binding to proteins28 or lipids,29 or simply the formation of complexes with ions.30 These various, non-mutually exclusive hypotheses are now open to experimental evaluation.
|
![]() |
Footnotes |
---|
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Wright, D. H., Brown, G. H., Peterson, M. L. et al. (2000). Application of fluoroquinolone pharmacodynamics. Journal of Antimicrobial Chemotherapy 46, 66983.
3 . Tulkens, P. M. (1991). Intracellular distribution and activity of antibiotics. European Journal of Clinical Microbiology and Infectious Diseases 10, 1006.[ISI][Medline]
4 . Carlier, M. B., Scorneaux, B., Zenebergh, A. et al. (1990). Cellular uptake, localization and activity of fluoroquinolones in uninfected and infected macrophages. Journal of Antimicrobial Chemotherapy 26, Suppl. B, 2739.[ISI][Medline]
5 . Facinelli, B., Magi, G., Prenna, M. et al. (1997). In vitro extracellular and intracellular activity of two newer and two earlier fluoroquinolones against Listeria monocytogenes. European Journal of Clinical Microbiology and Infectious Diseases 16, 82733.[ISI][Medline]
6 . Michelet, C., Avril, J. L., Arvieux, C. et al. (1997). Comparative activities of new fluoroquinolones, alone or in combination with amoxicillin, trimethoprim-sulfamethoxazole, or rifampin, against intracellular Listeria monocytogenes. Antimicrobial Agents and Chemotherapy 41, 605.[Abstract]
7
.
Ouadrhiri, Y., Scorneaux, B., Sibille, Y. et al. (1999). Mechanism of the intracellular killing and modulation of antibiotic susceptibility of Listeria monocytogenes in THP-1 macrophages activated by gamma interferon. Antimicrobial Agents and Chemotherapy 43, 124251.
8
.
Carryn, S., Van Bambeke, F., Mingeot-Leclercq, M. P. et al. (2002). Comparative intracellular (THP-1 macrophage) and extracellular activities of beta-lactams, azithromycin, gentamicin, and fluoroquinolones against Listeria monocytogenes at clinically relevant concentrations. Antimicrobial Agents and Chemotherapy 46, 2095103.
9
.
Jonas, D., Engels, I., Friedhoff, C. et al. (2001). Efficacy of moxifloxacin, trovafloxacin, clinafloxacin and levofloxacin against intracellular Legionella pneumophila. Journal of Antimicrobial Chemotherapy 47, 14752.
10 . Yamamoto, T., Kusajima, H., Hosaka, M. et al. (1995). Uptake and intracellular activity of fleroxacin in phagocytic cells. Chemotherapy 41, 3539.[ISI][Medline]
11 . Sanchez, M. S., Ford, C. W. & Yancey, R. J. Jr. (1988). Evaluation of antibiotic effectiveness against Staphylococcus aureus surviving within the bovine mammary gland macrophage. Journal of Antimicrobial Chemotherapy 21, 77386.[Abstract]
12 . Pascual, A., Garcia, I., Ballesta, S. et al. (1997). Uptake and intracellular activity of trovafloxacin in human phagocytes and tissue-cultured epithelial cells. Antimicrobial Agents and Chemotherapy 41, 2747.[Abstract]
13
.
Seral, C., Van Bambeke, F. & Tulkens, P. M. (2003). Quantitative analysis of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) activities against intracellular Staphylococcus aureus in mouse J774 macrophages. Antimicrobial Agents and Chemotherapy 47, 228392.
14 . Snyderman, R., Pike, M. C., Fischer, D. G. et al. (1977). Biologic and biochemical activities of continuous macrophage cell lines P388D1 and J774.1. Journal of Immunology 119, 20606.[ISI][Medline]
15
.
Seral, C., Carryn, S., Tulkens, P. M. et al. (2003). Influence of P-glycoprotein and MRP efflux pump inhibitors on the intracellular activity of azithromycin and ciprofloxacin in macrophages infected by Listeria monocytogenes or Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 51, 116773.
16
.
Michot, J. M., Van Bambeke, F., Mingeot-Leclercq, M. P. et al. (2004). Active efflux of the fluoroquinolone antibiotic ciprofloxacin from J774 macrophages through MRP-like transporter. Antimicrobial Agents and Chemotherapy 48, 267382.
17
.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. et al. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 26575.
18 . Ohkuma, S. & Poole, B. (1978). Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proceedings of the National Academy of Sciences, USA 75, 332731.[Abstract]
19
.
Carryn, S., Van Bambeke, F., Mingeot-Leclercq, M. P. et al. (2003). Activity of beta-lactams (ampicillin, meropenem), gentamicin, azithromycin and moxifloxacin against intracellular Listeria monocytogenes in a 24 h THP-1 human macrophage model. Journal of Antimicrobial Chemotherapy 51, 10512.
20 . Hirota, M., Totsu, T., Adachi, F. et al. (2001). Comparison of antimycobacterial activity of grepafloxacin against Mycobacterium avium with that of levofloxacin: accumulation of grepafloxacin in human macrophages. Journal of Infection and Chemotherapy 7, 1621.[CrossRef][Medline]
21 . Edelstein, P. H., Edelstein, M. A., Ren, J. et al. (1996). Activity of trovafloxacin (CP-99,219) against Legionella isolates: in vitro activity, intracellular accumulation and killing in macrophages, and pharmacokinetics and treatment of guinea pigs with L. pneumophila pneumonia. Antimicrobial Agents and Chemotherapy 40, 3149.[Abstract]
22 . Mounier, J., Ryter, A., Coquis-Rondon, M. et al. (1990). Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2. Infection and Immunity 58, 104858.[ISI][Medline]
23 . Dramsi, S. & Cossart, P. (1998). Intracellular pathogens and the actin cytoskeleton. Annual Review of Cell and Developmental Biology 14, 13766.[CrossRef][ISI][Medline]
24 . Lewin, C. S. & Smith, J. T. (1988). Bactericidal mechanisms of ofloxacin. Journal of Antimicrobial Chemotherapy 22, 18.[ISI][Medline]
25 . Eng, R. H., Padberg, F. T., Smith, S. M. et al. (1991). Bactericidal effects of antibiotics on slowly growing and nongrowing bacteria. Antimicrobial Agents and Chemotherapy 35, 18248.[ISI][Medline]
26 . Dalhoff, A., Matutat, S. & Ullmann, U. (1995). Effect of quinolones against slowly growing bacteria. Chemotherapy 41, 929.[ISI][Medline]
27
.
Ince, D. & Hooper, D. C. (2003). Quinolone resistance due to reduced target enzyme expression. Journal of Bacteriology 185, 688392.
28 . Bergogne-Berezin, E. (2002). Clinical role of protein binding of quinolones. Clinical Pharmacokinetics 41, 74150.[ISI][Medline]
29 . Vazquez, J., Montero, M., Merino, S. et al. (2001). Location and nature of the surface membrane binding site of ciprofloxacin: a fluorescence study. Langmuir 17, 100914.[CrossRef][ISI]
30 . Turel, I. (2002). The interactions of metal ions with quinolones antibacterial agents. Coordination Chemistry Reviews 232, 2747.[CrossRef][ISI]
31 . Shah, A., Liu, M. C., Vaughan, D. et al. (1999). Oral bioequivalence of three ciprofloxacin formulations following single-dose administration: 500 mg tablet compared with 500 mg/10 mL or 500 mg/5 mL suspension and the effect of food on the absorption of ciprofloxacin oral suspension. Journal of Antimicrobial Chemotherapy 43, 4954.[CrossRef][ISI][Medline]
32 . Fish, D. N. & Chow, A. T. (1997). The clinical pharmacokinetics of levofloxacin. Clinical Pharmacokinetics 32, 10119.[ISI][Medline]
33
.
Sullivan, J. T., Woodruff, M., Lettieri, J. et al. (1999). Pharmacokinetics of a once-daily oral dose of moxifloxacin (Bay 12-8039), a new enantiomerically pure 8-methoxy quinolone. Antimicrobial Agents and Chemotherapy 43, 27937.
34
.
Gajjar, D. A., Bello, A., Ge, Z. et al. (2003). Multiple-dose safety and pharmacokinetics of oral garenoxacin in healthy subjects. Antimicrobial Agents and Chemotherapy 47, 225663.