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Uptake and intracellular activity of ofloxacin isomers in human phagocytic and non-phagocytic cells
International Journal of Antimicrobial Agents 15 (2000) 201 – 205 www.ischemo.org
Original article
Uptake and intracellular activity of ofloxacin isomers in human phagocytic and non-phagocytic cells Isabel Garcı´a, Alvaro Pascual *, Sofı´a Ballesta, Evelio Jose´ Perea Department of Microbiology, School of Medicine, Apdo 914, 41080 Se6ille, Spain Received 14 January 2000; accepted 18 March 2000
Abstract The penetration and intracellular activity of ofloxacin and its isomers (levofloxacin and D-ofloxacin) into human polymorphonuclear leucocytes (PMN), human peritoneal macrophages (PMf) and tissue cultured epithelial cells (McCoy) were evaluated. The cellular to extracellular concentration (C/E) values of the three fluoroquinolones were higher than 3.6 and 2.6 in PMN and PMf, respectively. The C/E ratios in McCoy cells were lower than those in PMN, but still higher than 2.0. The uptake of ofloxacin and its isomers was rapid, non-saturable and reversible. All quinolones (extracellular concentrations: 2, 5 and 10 mg/l) produced a significant reduction of viable intraphagocytic Staphylococcus aureus in phagocytic cells. We concluded that ofloxacin and its isomers reach high intracellular concentrations in phagocytic and non phagocytic cells while remaining active in the former. © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Intracellular penetration; Fluoroquinolones; Phagocytes; Ofloxacin
1. Introduction Fluoroquinolones are broad spectrum antimicrobial agents able to penetrate and remain active within phagocytic cells [1]. Most studies on the intracellular penetration and activity of fluoroquinolones have used phagocytic cells but less information is available on the intracellular pharmacology of these compounds in other types of cell. Bacteria may survive in non-phagocytic cells during several infections; for example, Chlamydia trachomatis and enteroinvasive Escherichia coli are able to invade and multiply within epithelial cells [2,3]. Ofloxacin is a fluoroquinolone able to concentrate in phagocytic cells [4]. Levofloxacin and D-ofloxacin are optically active isomers of racemic ofloxacin. Levofloxacin, considered to be the more active isomer, has an antimicrobial activity twice as high as that of ofloxacin [5,6] and both isomers and the racemic com* Corresponding author. Tel.: +34-5-4557448; fax: 4377413. E-mail address: [email protected] (A. Pascual).
+34-5-
pound have been shown to concentrate several times within human polymorphonuclear leucocytes (PMN) [7,8]. The purpose of this study was to compare the uptake of ofloxacin and its isomers by human PMN, human peritoneal macrophages (PMf) and tissue-cultured epithelial cells. The intracellular activity of the three compounds in phagocytic cells was also evaluated.
2. Material and methods
2.1. Antimicrobial agents Ofloxacin, levofloxacin, and D-ofloxacin were kindly supplied by Hoechst-Marion-Roussel (France) as powders of known potency.
2.2. Isolation of phagocytes PMf were isolated from peritoneal effluents of patients undergoing continuous ambulatory peritoneal di-
0924-8579/00/$20 © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 0 ) 0 0 1 6 1 - 8
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alysis (CAPD) and who were being followed up by the Nephrology Department of the University Hospital of Seville [9]. Donors were clinically uninfected at the time of the study. Cell preparations from the CAPD donors always contained \75% PMf and B15% PMN. PMN were recovered from heparinized venous blood of healthy donors and were purified using methods described previously [10]. PMN preparations were \ 97% pure. Final cell suspensions were adjusted to 2 × 106 PMf/ml or 2×106 PMN/ml in Hanks balanced salt solution containing 0.1% gelatin (GHBSS). Both populations of cells were ]95% viable by trypan blue exclusion criteria.
2.3. Tissue culture cells Tissue culture cells obtained from human synovial fluid (McCoy cells; Flow Laboratories, Irvine, UK) were grown in minimal essential medium (Flow) supplemented with 1 mM HEPES (N-2-hydroxyethylpiperazine-N%-2-ethanesulphonic acid; Flow) and containing 10% foetal calf serum (Flow) without antibiotics. For each experiment, cells were detached from tissue culture bottles with trypsin – EDTA (Flow) and suspended in GHBSS at a concentration of 5×106 cells/ml.
2.4. Antimicrobial agents uptake by cells A described previously fluorometric assay to measure quinolone uptake by human PMNs was used [4]. In these experiments PMf, PMN or tissue culture cells were incubated in Hanks balanced salt solution containing different concentrations of the antimicrobial agents (2, 5 and 10 mg/l). After different incubation times (1, 5, 20 and 30 min) at 37°C, cells were separated from extracellular solution by centrifugation through a water-impermeable silicone – oil barrier in a microcentrifuge tube. The entire cell pellet, obtained by cutting off the portion of the microfuge tube containing the pellet, was placed in 2 ml of 0.1 M glycine – HCl buffer (pH 3.0) and agitated vigorously in a vortex shaker. Incubation for 2 h at room temperature was sufficient to release the intracellular antimicrobial agent fully. The samples were centrifuged for 5 min at 5600× g, and the amount of antimicrobial agent determined by fluorescence emission of the supernatants with a fluorescent spectrophotometer. The fluorescence excitation and emission maxima of ofloxacin and its isomers in 0.1 M glycine–HCl (pH 3.0) were 292 and 496 nm, respectively. Controls without antimicrobial agents were always used to determine the background fluorescence. The intracellular water space was measured by using tritiated water and the extracellular marker [14C] polyethylene glycol (1.4 mCi/g; New England Nuclear Corp., Boston, MA). The cells were incubated with
these radiolabeled compounds for 2 min at 37°C and then cells were separated from extracellular fluid by velocity gradient centrifugation, as described above, and counted in a liquid scintillation counter. The total water content of the cell pellet was corrected for trapped extracellular water, i.e. polyethylene glycol space, to obtain the intracellualr water space. From the values obtained by this procedure, cell-associated antimicrobial agent concentrations were calculated and expressed as a ratio of the cellular concentration to extracellular concentration (C/E) [11]. The efflux or reversibility of the binding of cell-associated fluoroquinolones was also studied. PMN, PMf, or McCoy cells were incubated for 10 min at 37°C with these compounds (extracellular concentration, 2 mg/l), collected by centrifugation and rapidly suspended in quinolone-free medium. Cell-associated fluoroquinolone was quantified at various time intervals (5, 10 and 20 min) after the removal of the extracellular antimicrobial agent. All assays were performed in duplicate (n= 5). Organisms and susceptibility testing. Staphylococcus aureus ATCC 25923 was used for the killing assays. Susceptibility studies were determined by dilution assay. The MICs and minimum bactericidal (MBCs) of ofloxacin and its isomers (levofloxacin and D-ofloxacin) for this strain were 0.25, 0.125 and 16 mg/l, respectively.
2.5. Intracellular acti6ity of antimicrobial agents To evaluate the intracellular activity of antimicrobial agents, a previously described method was used [9]. Briefly, 0.1 ml of opsonized bacterial suspension (5× 107 CFU/ml) and 0.1 ml of PMN or PMf (5×106 cells/ml) were combined in a series of polypropylene biovials (Beckman Instruments, Inc., Fullerton, CA) and incubated in a shaker (50 rpm) for 60 min at 37°C. After incubation, extracellular bacteria were removed by differential centrifugation. Cells were then suspended in 0.2 ml of RPMI medium (GIBCO, UK). At this time (designated time zero), the different antimicrobial agents were added and the vials were reincubated in a shaker (50 rpm) at 37°C. Vials were removed at time zero (control) and after 3 h of incubation (control and samples with antimicrobial agents). Cells were lysed in distilled water and samples were diluted and pour plated in agar. Colonies were counted after 24 h of incubation at 37°C. The data were expressed as percentages of surviving staphylococci, compared with controls (without antimicrobial agents) after 3 h of incubation. In addition to determining bacterial survival, morphologic studies were also routinely performed at time 0 and after 3 h of incubation to evaluate the disposition of bacteria (cell associated or extracellular). Samples of 50 ml were removed from biovials and
I. Garcı´a et al. / International Journal of Antimicrobial Agents 15 (2000) 201–205
203
deposited on glass slides. After staining with Wright stain, samples were examined by light microscopy. All assays were performed in duplicate with PMN or PMf from five different donors.
2.6. Statistical analysis of data Data were expressed as the mean9 standard deviations. Differences among groups were compared by variance analysis; statistical significance was taken to be P 50.05.
3. Results Fig. 1. Ofloxacin, levofloxacin and D-ofloxacin uptake by human PMN and efflux of human PMN-associated fluoroquinolone (dotted line) after removal of the extracellualr drug (n= 5). Extracellular concentration 2 mg/l.
Fig. 2. Ofloxacin, levofloxacin and D-ofloxacin uptake by human PMf and efflux of human PMf-associated fluoroquinolone (dotted line) after removal of the extracellular drug (n= 5). Extracellular concentration 2 mg/l.
3.1. Uptake of ofloxacin, le6ofloxacin and by phagocytic and non phagocytic cells
D -ofloxacin
The uptake and efflux kinetics of these fluoroquinolones in human PMN, PMf and McCoy cells are shown in Figs. 1–3, respectively. The three compounds penetrate into three types of cells, rapidly reaching intracellular concentrations three to five times greater than the extracellular ones in phagocytic cells and slightly lower in epithelial cells (37°C, 20 min). The intracellular accumulation of the three fluoroquinolones in both PMf and McCoy cells continued increasing after 60 min incubation. The efflux of ofloxacin and its isomer from PMN and McCoy cells was rapid, with 90–95% of the cell-associated drug being lost within 5 min. The efflux of levofloxacin and ofloxacin from PMf, was slower. After 5 min incubation in quinolone-free medium, the percentages of PMf-associated levofloxacin and ofloxacin were 30 and 35% respectively. No significant differences were observed between fluoroquinolones and cells after 20 min incubation (compare values at 20 and 25 min in Figs. 1–3). The effect of different extracellular concentrations on the uptake of the three quinolones in PMf and McCoy cells is shown in Fig. 4. The higher extracellular concentrations, the greater the intracellular accumulation in both type of cells with no significant differences between ofloxacin and its isomers. Similar results were observed in PMN (data not shown).
3.2. Intracellular acti6ity of ofloxacin, le6ofloxacin and D -ofloxacin
Fig. 3. Ofloxacin, levofloxacin and D-ofloxacin uptake by McCoy cells and efflux of McCoy cells-associated fluroquinolone (dotted line) after removal of the extracellular drug (n=5). Extracellualr concentration 2 mg/l.
The intracellular activity of these agents against S. aureus was evaluated by a 3-h assay (Fig. 5). At each of the extracellular concentrations evaluated, ofloxacin and levofloxacin showed significant intracellular activity compared with the control without antimicrobial agent in both phagocytic cells. In PMf this activity was higher than that observed in PMN. How-
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Fig. 4. Effect of the extracellular concentration in the intracellular penetration of ofloxacin, levofloxacin and D-ofloxacin in human PMf (A) and McCoy cells (B). Incubation time: 20 min (n= 5).
Fig. 5. Intracellular activity against S. aureus ATCC 25923 of ofloxacin, levofloxacin and D-ofloxacin in human PMN (A) and PMf (B). Data are expressed as percentages of surviving staphylococci after 3 h incubation (control 100%) (*P50.05 compared to control) (n =5)
ever, D-ofloxacin only showed significant intracellular activity at an extracellular concentration of 5 and 10 mg/l.
4. Discussion Levofloxacin and D-ofloxacin are optically active isomers of racemic ofloxacin. Levofloxacin is considered the most active isomer, showing antibacterial activity as least twice as high as that of ofloxacin [5,6]. All three compounds have shown a high intracellular accumulation in human PMN [7]. The high intracellular accumulation of ofloxacin and levofloxacin, suggested they could be useful in infections caused by intracellular pathogens. Many intracellular pathogens multiply within cells ther than PMN. For example, C. trachomatis invade and multiply within epithelial cells, and ofloxacin is a drug of choice for the treatment such infections [12]. There is not much information about the activity of ofloxacin and its isomers within this type of cell [8]. In this study we have observed that ofloxacin, levofloxacin and D-ofloxacin reach similar intracellular levels in two types of phagocytic cell and tissue-cultured epithelial cells. The C/E ratio values of the three quinolones for McCoy cells were lower than those in PMN but still higher than two. The uptake of ofloxacin and its isomer by both phagocytic and non-phagocytic
cells was rapid, non saturable and reversible. This high intracellular accumulation of ofloxacin and levofloxacin in McCoy cells could partially explain the excellent activity of these compounds against C. trachomatis both in vitro and in vivo [13]. The results are similar to those observed for other fluoroquinolones [14–16]. Only sparfloxacin has shown a higher intracellular concentration than ofloxacin and its isomers in epithelial cells. This might be because sparfloxacin is more hydrophobic than ofloxacin and theoretically could pass more easily through the cell membrane [15]. Ofloxacin and levofloxacin showed high intracellular activity againts S. aureus in both PMN and PMf, this activity being markedly lower for D-ofloxacin at low extracellular concentrations. The differences in the intracellular activity among these compoundsmight be related to differences in the intrinsic activity against S. aureus. In summary, levofloxacin and D-ofloxacin reach intracellular concentrations in phagocytic and epithelial cells similar to those of racemate ofloxacin and remain active intracellularly.
Acknowledgements We gratefully acknowledge Janet Dawson and Patricia Hidalgo for manuscript preparation.
I. Garcı´a et al. / International Journal of Antimicrobial Agents 15 (2000) 201–205
References [1] Pascual A. Uptake and intracellular activity of antimicrobial agents in phagocytic cells. Rev Med Microbiol 1995;64:228 – 35. [2] Noumi T, Nishida N, Minami S, Watnabe Y, Yasuda T. Intracellular activity of tosufloxacin (T-3262) against Salmonella enteritidis and ability to penetrate into tissue culture cells of human origin. Antimicrob Agents Chemother 1990;34:949–53. [3] Daronide RO, Hamill RJ. Antibiotic penetration and bactericidal activity within endothelial cells. Antimicrob Agents Chemother 1994;38:1059–64. [4] Pascual A, Garcı´a I, Perea EJ. Fluorometric measurement of ofloxacin uptake by human polymorphonuclear leukocytes. Antimicrob Agents Chemother 1989;33:653–6. [5] Hayakawa I, Atarashi S, Yokohama S, Imamura M, Sakano K, Furukawa M. Synthesis and antibacterial activities of optically active ofloxacin. Antimicrob Agents Chemother 1986;29:163 – 4. [6] Imamura M, Shibamura I, Hayakawa I, Osada Y. Inhibition of DNA gyrase by optically active ofloxacin. Antimicrob Agents Chemother 1987;31:325–7. [7] Pascual A, Garcı´a I, Perea EJ. Uptake and intracellular activity of an optically active ofloxacin isomer in human neutrophils and tissue culture cells. Antimicrob Agents Chemother 1990;34:277 – 80. [8] Taira K, Koya H, Kohno S. Accumulation of a newly developed fluoroquinolone OPC-17116, by human polymorphonuclear leukocytes. Antimicrob Agents Chemother 1993;37:1877– 81.
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[9] Pascual A, Tsakayama J, Kovarick J, Gekker G, Peterson PK. Uptake and activity of rifapentine in human peritoneal macrophages and polymorphonuclear leukocytes. Eur J Clin Microbiol 1987;6:152 – 7. [10] Peterson PK, Vehoef J, Schmeling D, Quie PG. Kinetics of phagocytosis and bacterial killing by human polymorphonuclear leukocytes and monocytes. J Infect Dis 1977;136:502 – 9. [11] Klempner MS, Styrt B. Clindamycin uptake by human neutrophils. J Infect Dis 1981;144:472 – 5. [12] Perea EJ, Aznar J, Herrera A, Mazuecos J, Rodrı´guez-Pichardo A. Clinical efficacy of new quinolones for therapy of non-gonococcal urethritis. Sex Trans Dis 1989;16:7 – 10. [13] Perea EJ. Treatment of genital chlamydial infections with ciprofloxacin and ofloxacin. Quinolones Bull 1989;5:1 –3. [14] Pascual A, Garcı´a I, Perea EJ. Entry of lomefloxacin and temafloxacin into human neutrophils, peritoneal macrophages and tissue culture cells. Diagn Microbiol Infect Dis 1992;15:393– 8. [15] Garcı´a I, Pascual A, Guzman MC, Perea EJ. Uptake and intracellular activity of sparfloxacin in human polymorphonuclear leukocytes and tissue culture cells. Antimicrob Agents Chemother 1992;36:1053 – 6. [16] Pascual A, Garcı´a I, Ballesta S, Perea EJ. Uptake and intracellular activity of trovafloxacin in human phagocytes and tissue-cultured epithelial cells. Antimicrob Agents Chemother 1997; 41:274 – 7.
Original article
Uptake and intracellular activity of ofloxacin isomers in human phagocytic and non-phagocytic cells Isabel Garcı´a, Alvaro Pascual *, Sofı´a Ballesta, Evelio Jose´ Perea Department of Microbiology, School of Medicine, Apdo 914, 41080 Se6ille, Spain Received 14 January 2000; accepted 18 March 2000
Abstract The penetration and intracellular activity of ofloxacin and its isomers (levofloxacin and D-ofloxacin) into human polymorphonuclear leucocytes (PMN), human peritoneal macrophages (PMf) and tissue cultured epithelial cells (McCoy) were evaluated. The cellular to extracellular concentration (C/E) values of the three fluoroquinolones were higher than 3.6 and 2.6 in PMN and PMf, respectively. The C/E ratios in McCoy cells were lower than those in PMN, but still higher than 2.0. The uptake of ofloxacin and its isomers was rapid, non-saturable and reversible. All quinolones (extracellular concentrations: 2, 5 and 10 mg/l) produced a significant reduction of viable intraphagocytic Staphylococcus aureus in phagocytic cells. We concluded that ofloxacin and its isomers reach high intracellular concentrations in phagocytic and non phagocytic cells while remaining active in the former. © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Intracellular penetration; Fluoroquinolones; Phagocytes; Ofloxacin
1. Introduction Fluoroquinolones are broad spectrum antimicrobial agents able to penetrate and remain active within phagocytic cells [1]. Most studies on the intracellular penetration and activity of fluoroquinolones have used phagocytic cells but less information is available on the intracellular pharmacology of these compounds in other types of cell. Bacteria may survive in non-phagocytic cells during several infections; for example, Chlamydia trachomatis and enteroinvasive Escherichia coli are able to invade and multiply within epithelial cells [2,3]. Ofloxacin is a fluoroquinolone able to concentrate in phagocytic cells [4]. Levofloxacin and D-ofloxacin are optically active isomers of racemic ofloxacin. Levofloxacin, considered to be the more active isomer, has an antimicrobial activity twice as high as that of ofloxacin [5,6] and both isomers and the racemic com* Corresponding author. Tel.: +34-5-4557448; fax: 4377413. E-mail address: [email protected] (A. Pascual).
+34-5-
pound have been shown to concentrate several times within human polymorphonuclear leucocytes (PMN) [7,8]. The purpose of this study was to compare the uptake of ofloxacin and its isomers by human PMN, human peritoneal macrophages (PMf) and tissue-cultured epithelial cells. The intracellular activity of the three compounds in phagocytic cells was also evaluated.
2. Material and methods
2.1. Antimicrobial agents Ofloxacin, levofloxacin, and D-ofloxacin were kindly supplied by Hoechst-Marion-Roussel (France) as powders of known potency.
2.2. Isolation of phagocytes PMf were isolated from peritoneal effluents of patients undergoing continuous ambulatory peritoneal di-
0924-8579/00/$20 © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 0 ) 0 0 1 6 1 - 8
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I. Garcı´a et al. / International Journal of Antimicrobial Agents 15 (2000) 201–205
alysis (CAPD) and who were being followed up by the Nephrology Department of the University Hospital of Seville [9]. Donors were clinically uninfected at the time of the study. Cell preparations from the CAPD donors always contained \75% PMf and B15% PMN. PMN were recovered from heparinized venous blood of healthy donors and were purified using methods described previously [10]. PMN preparations were \ 97% pure. Final cell suspensions were adjusted to 2 × 106 PMf/ml or 2×106 PMN/ml in Hanks balanced salt solution containing 0.1% gelatin (GHBSS). Both populations of cells were ]95% viable by trypan blue exclusion criteria.
2.3. Tissue culture cells Tissue culture cells obtained from human synovial fluid (McCoy cells; Flow Laboratories, Irvine, UK) were grown in minimal essential medium (Flow) supplemented with 1 mM HEPES (N-2-hydroxyethylpiperazine-N%-2-ethanesulphonic acid; Flow) and containing 10% foetal calf serum (Flow) without antibiotics. For each experiment, cells were detached from tissue culture bottles with trypsin – EDTA (Flow) and suspended in GHBSS at a concentration of 5×106 cells/ml.
2.4. Antimicrobial agents uptake by cells A described previously fluorometric assay to measure quinolone uptake by human PMNs was used [4]. In these experiments PMf, PMN or tissue culture cells were incubated in Hanks balanced salt solution containing different concentrations of the antimicrobial agents (2, 5 and 10 mg/l). After different incubation times (1, 5, 20 and 30 min) at 37°C, cells were separated from extracellular solution by centrifugation through a water-impermeable silicone – oil barrier in a microcentrifuge tube. The entire cell pellet, obtained by cutting off the portion of the microfuge tube containing the pellet, was placed in 2 ml of 0.1 M glycine – HCl buffer (pH 3.0) and agitated vigorously in a vortex shaker. Incubation for 2 h at room temperature was sufficient to release the intracellular antimicrobial agent fully. The samples were centrifuged for 5 min at 5600× g, and the amount of antimicrobial agent determined by fluorescence emission of the supernatants with a fluorescent spectrophotometer. The fluorescence excitation and emission maxima of ofloxacin and its isomers in 0.1 M glycine–HCl (pH 3.0) were 292 and 496 nm, respectively. Controls without antimicrobial agents were always used to determine the background fluorescence. The intracellular water space was measured by using tritiated water and the extracellular marker [14C] polyethylene glycol (1.4 mCi/g; New England Nuclear Corp., Boston, MA). The cells were incubated with
these radiolabeled compounds for 2 min at 37°C and then cells were separated from extracellular fluid by velocity gradient centrifugation, as described above, and counted in a liquid scintillation counter. The total water content of the cell pellet was corrected for trapped extracellular water, i.e. polyethylene glycol space, to obtain the intracellualr water space. From the values obtained by this procedure, cell-associated antimicrobial agent concentrations were calculated and expressed as a ratio of the cellular concentration to extracellular concentration (C/E) [11]. The efflux or reversibility of the binding of cell-associated fluoroquinolones was also studied. PMN, PMf, or McCoy cells were incubated for 10 min at 37°C with these compounds (extracellular concentration, 2 mg/l), collected by centrifugation and rapidly suspended in quinolone-free medium. Cell-associated fluoroquinolone was quantified at various time intervals (5, 10 and 20 min) after the removal of the extracellular antimicrobial agent. All assays were performed in duplicate (n= 5). Organisms and susceptibility testing. Staphylococcus aureus ATCC 25923 was used for the killing assays. Susceptibility studies were determined by dilution assay. The MICs and minimum bactericidal (MBCs) of ofloxacin and its isomers (levofloxacin and D-ofloxacin) for this strain were 0.25, 0.125 and 16 mg/l, respectively.
2.5. Intracellular acti6ity of antimicrobial agents To evaluate the intracellular activity of antimicrobial agents, a previously described method was used [9]. Briefly, 0.1 ml of opsonized bacterial suspension (5× 107 CFU/ml) and 0.1 ml of PMN or PMf (5×106 cells/ml) were combined in a series of polypropylene biovials (Beckman Instruments, Inc., Fullerton, CA) and incubated in a shaker (50 rpm) for 60 min at 37°C. After incubation, extracellular bacteria were removed by differential centrifugation. Cells were then suspended in 0.2 ml of RPMI medium (GIBCO, UK). At this time (designated time zero), the different antimicrobial agents were added and the vials were reincubated in a shaker (50 rpm) at 37°C. Vials were removed at time zero (control) and after 3 h of incubation (control and samples with antimicrobial agents). Cells were lysed in distilled water and samples were diluted and pour plated in agar. Colonies were counted after 24 h of incubation at 37°C. The data were expressed as percentages of surviving staphylococci, compared with controls (without antimicrobial agents) after 3 h of incubation. In addition to determining bacterial survival, morphologic studies were also routinely performed at time 0 and after 3 h of incubation to evaluate the disposition of bacteria (cell associated or extracellular). Samples of 50 ml were removed from biovials and
I. Garcı´a et al. / International Journal of Antimicrobial Agents 15 (2000) 201–205
203
deposited on glass slides. After staining with Wright stain, samples were examined by light microscopy. All assays were performed in duplicate with PMN or PMf from five different donors.
2.6. Statistical analysis of data Data were expressed as the mean9 standard deviations. Differences among groups were compared by variance analysis; statistical significance was taken to be P 50.05.
3. Results Fig. 1. Ofloxacin, levofloxacin and D-ofloxacin uptake by human PMN and efflux of human PMN-associated fluoroquinolone (dotted line) after removal of the extracellualr drug (n= 5). Extracellular concentration 2 mg/l.
Fig. 2. Ofloxacin, levofloxacin and D-ofloxacin uptake by human PMf and efflux of human PMf-associated fluoroquinolone (dotted line) after removal of the extracellular drug (n= 5). Extracellular concentration 2 mg/l.
3.1. Uptake of ofloxacin, le6ofloxacin and by phagocytic and non phagocytic cells
D -ofloxacin
The uptake and efflux kinetics of these fluoroquinolones in human PMN, PMf and McCoy cells are shown in Figs. 1–3, respectively. The three compounds penetrate into three types of cells, rapidly reaching intracellular concentrations three to five times greater than the extracellular ones in phagocytic cells and slightly lower in epithelial cells (37°C, 20 min). The intracellular accumulation of the three fluoroquinolones in both PMf and McCoy cells continued increasing after 60 min incubation. The efflux of ofloxacin and its isomer from PMN and McCoy cells was rapid, with 90–95% of the cell-associated drug being lost within 5 min. The efflux of levofloxacin and ofloxacin from PMf, was slower. After 5 min incubation in quinolone-free medium, the percentages of PMf-associated levofloxacin and ofloxacin were 30 and 35% respectively. No significant differences were observed between fluoroquinolones and cells after 20 min incubation (compare values at 20 and 25 min in Figs. 1–3). The effect of different extracellular concentrations on the uptake of the three quinolones in PMf and McCoy cells is shown in Fig. 4. The higher extracellular concentrations, the greater the intracellular accumulation in both type of cells with no significant differences between ofloxacin and its isomers. Similar results were observed in PMN (data not shown).
3.2. Intracellular acti6ity of ofloxacin, le6ofloxacin and D -ofloxacin
Fig. 3. Ofloxacin, levofloxacin and D-ofloxacin uptake by McCoy cells and efflux of McCoy cells-associated fluroquinolone (dotted line) after removal of the extracellular drug (n=5). Extracellualr concentration 2 mg/l.
The intracellular activity of these agents against S. aureus was evaluated by a 3-h assay (Fig. 5). At each of the extracellular concentrations evaluated, ofloxacin and levofloxacin showed significant intracellular activity compared with the control without antimicrobial agent in both phagocytic cells. In PMf this activity was higher than that observed in PMN. How-
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Fig. 4. Effect of the extracellular concentration in the intracellular penetration of ofloxacin, levofloxacin and D-ofloxacin in human PMf (A) and McCoy cells (B). Incubation time: 20 min (n= 5).
Fig. 5. Intracellular activity against S. aureus ATCC 25923 of ofloxacin, levofloxacin and D-ofloxacin in human PMN (A) and PMf (B). Data are expressed as percentages of surviving staphylococci after 3 h incubation (control 100%) (*P50.05 compared to control) (n =5)
ever, D-ofloxacin only showed significant intracellular activity at an extracellular concentration of 5 and 10 mg/l.
4. Discussion Levofloxacin and D-ofloxacin are optically active isomers of racemic ofloxacin. Levofloxacin is considered the most active isomer, showing antibacterial activity as least twice as high as that of ofloxacin [5,6]. All three compounds have shown a high intracellular accumulation in human PMN [7]. The high intracellular accumulation of ofloxacin and levofloxacin, suggested they could be useful in infections caused by intracellular pathogens. Many intracellular pathogens multiply within cells ther than PMN. For example, C. trachomatis invade and multiply within epithelial cells, and ofloxacin is a drug of choice for the treatment such infections [12]. There is not much information about the activity of ofloxacin and its isomers within this type of cell [8]. In this study we have observed that ofloxacin, levofloxacin and D-ofloxacin reach similar intracellular levels in two types of phagocytic cell and tissue-cultured epithelial cells. The C/E ratio values of the three quinolones for McCoy cells were lower than those in PMN but still higher than two. The uptake of ofloxacin and its isomer by both phagocytic and non-phagocytic
cells was rapid, non saturable and reversible. This high intracellular accumulation of ofloxacin and levofloxacin in McCoy cells could partially explain the excellent activity of these compounds against C. trachomatis both in vitro and in vivo [13]. The results are similar to those observed for other fluoroquinolones [14–16]. Only sparfloxacin has shown a higher intracellular concentration than ofloxacin and its isomers in epithelial cells. This might be because sparfloxacin is more hydrophobic than ofloxacin and theoretically could pass more easily through the cell membrane [15]. Ofloxacin and levofloxacin showed high intracellular activity againts S. aureus in both PMN and PMf, this activity being markedly lower for D-ofloxacin at low extracellular concentrations. The differences in the intracellular activity among these compoundsmight be related to differences in the intrinsic activity against S. aureus. In summary, levofloxacin and D-ofloxacin reach intracellular concentrations in phagocytic and epithelial cells similar to those of racemate ofloxacin and remain active intracellularly.
Acknowledgements We gratefully acknowledge Janet Dawson and Patricia Hidalgo for manuscript preparation.
I. Garcı´a et al. / International Journal of Antimicrobial Agents 15 (2000) 201–205
References [1] Pascual A. Uptake and intracellular activity of antimicrobial agents in phagocytic cells. Rev Med Microbiol 1995;64:228 – 35. [2] Noumi T, Nishida N, Minami S, Watnabe Y, Yasuda T. Intracellular activity of tosufloxacin (T-3262) against Salmonella enteritidis and ability to penetrate into tissue culture cells of human origin. Antimicrob Agents Chemother 1990;34:949–53. [3] Daronide RO, Hamill RJ. Antibiotic penetration and bactericidal activity within endothelial cells. Antimicrob Agents Chemother 1994;38:1059–64. [4] Pascual A, Garcı´a I, Perea EJ. Fluorometric measurement of ofloxacin uptake by human polymorphonuclear leukocytes. Antimicrob Agents Chemother 1989;33:653–6. [5] Hayakawa I, Atarashi S, Yokohama S, Imamura M, Sakano K, Furukawa M. Synthesis and antibacterial activities of optically active ofloxacin. Antimicrob Agents Chemother 1986;29:163 – 4. [6] Imamura M, Shibamura I, Hayakawa I, Osada Y. Inhibition of DNA gyrase by optically active ofloxacin. Antimicrob Agents Chemother 1987;31:325–7. [7] Pascual A, Garcı´a I, Perea EJ. Uptake and intracellular activity of an optically active ofloxacin isomer in human neutrophils and tissue culture cells. Antimicrob Agents Chemother 1990;34:277 – 80. [8] Taira K, Koya H, Kohno S. Accumulation of a newly developed fluoroquinolone OPC-17116, by human polymorphonuclear leukocytes. Antimicrob Agents Chemother 1993;37:1877– 81.
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