Departments of 1 Microbiology and Immunology and 2 Otorhinolaryngology, Shimane Medical University, Izumo, Shimane 693-8501; 3 Shimane Institute of Health Science, Izumo, Shimane 693-0021, Japan
Received 17 July 2002; returned 17 August 2002; revised 2 January 2003; accepted 29 May 2003
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Abstract |
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Keywords: gatifloxacin, sitafloxacin, macrophages, type II alveolar epithelial cells, Mycobacterium tuberculosis, fluoroquinolones, tuberculosis
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Introduction |
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In pulmonary M. tuberculosis infections, the cells, which initially encounter the pathogens, are alveolar Ms and alveolar epithelial cells.11 By an electron microscopic study on the M. tuberculosis infection in mice, we have demonstrated that the pathogens invaded type II lung pneumocytes during the early phases of the infection.12 It has also been found that M. tuberculosis internalized and vigorously multiplied within not only M
s but also type II lung epithelial cells.11,13 These findings strongly suggest that the pneumocytes act as sites of bacterial growth in lung infections due to mycobacterial pathogens. We thus compared the antimicrobial activities of new fluoroquinolones, including a C-8-methoxy quinolone gatifloxacin14 and a C-8-chloro quinolone sitafloxacin,15 against M. tuberculosis replicating within human M
s and type II alveolar cells.
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Materials and methods |
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M. tuberculosis Kurono strain isolated from a tuberculosis patient was used. The organisms were grown in Middlebrook 7H9 broth and bacterial suspension in PBS containing 1% (v/v) BSA was frozen at 80°C until use. Staphylococcus aureus 209P strain (standard strain for drug susceptibility testing in Japan) was cultured in tryptosoy (TS) broth.
Antimicrobial agents
Gatifloxacin (Kyorin Pharmaceutical Co., Tokyo, Japan), sitafloxacin (Daiichi Pharmaceutical Co., Tokyo, Japan), and levofloxacin (Daiichi Pharmaceutical Co.) were used. The drugs were dissolved in 0.1 N NaOH at 3 mg/mL, and diluted in appropriate culture media before use. The MICs of gatifloxacin, sitafloxacin and levofloxacin measured by the broth microdilution method using 7HSF medium16 were 0.125, 0.06 and 0.25 mg/L, respectively. The Cmax values in human blood after oral administration of clinical dosages were as follows: 1.7 mg/L for gatifloxacin (4 mg/kg),17 1.0 mg/L for sitafloxacin (2 mg/L),18 and 2.0 mg/L for levofloxacin (4 mg/kg) (personal communication from Dr K. Namba, Daiichi Pharmaceutical Co.).
Cell lines
Mono Mac 6 human monocytic cell line (MM6-Ms) and A-549 human type II alveolar epithelial cell line (A-549 cells) were obtained from the German Collection of Microorganisms and Cell Cultures (Mascheroder, Braunschweig, Germany) and the American Type Culture Collection (Rockville, MD, USA), respectively. MM6-M
s were subcultured in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS; Bio Whittaker Co., Walkersville, MD, USA), 1% (v/v) nonessential amino acids (Bio Whittaker), 1 mM sodium pyruvate (Bio Whittaker) and 9 mg/L bovine insulin (Wako Pure Chemical Industries, Osaka, Japan). A-549 cells were subcultured in Hams F-12K medium (Dainippon Pharmaceutical Co., Osaka, Japan) supplemented with 10% FBS.
Antimicrobial activity against intracellular organisms
The antimicrobial activities of test drugs against organisms replicating within MM6-Ms and A-549 cells were measured as follows.
MM6-Ms: MM6-M
s (4 x 106 cells) were suspended in 5 mL of 5% FBS-RPMI 1640 medium in 75 cm2 tissue culture flask, mixed with an equal volume of M. tuberculosis suspension (1.2 x 108 cfu: MOI = 30) in the same medium, and incubated at 37°C in a CO2 incubator for 3 h. The cells infected with the organisms were collected by centrifugation (150g, 5 min) and washed five times with Hanks balanced salt solution (HBSS) containing 2% FBS by centrifugation (150g, 5 min), since the MM6-M
s were non-adherent to plastic wells. The resultant MM6-M
cells (4 x 104 cells) resuspended in 200 µL of 1% FBS-RPMI 1640 medium were seeded in microculture wells (96-well tissue culture plate: U-bottom: Becton Dickinson & Company, Lincoln Park, NJ, USA) in the presence or absence of test drugs, and cultivated for up to 7 days. At intervals, the cells were lysed with 0.07% (w/v) SDS followed by subsequent neutralization with 6% (w/v) BSAPBS, and were then washed twice with 0.05% (v/v) Tween 80 by centrifugation (2000g, 30 min). The recovered organisms were resuspended in 250 µL of 0.05% (v/v) Tween 80 and counted for cfu on 7H11 agar plates.
A-549 cells: A-549 cells (4 x 104 cells) suspended in 200 µL of 5% FBS-Hams F-12K medium were seeded in tissue culture plates (96 wells: flat-bottom: Becton Dickinson) and incubated for 18 h in order to allow attachment to the culture wells. The resultant monolayer culture of A-549 cells was rinsed with 2% FBS-HBSS and 100 µL of the fresh medium of 5% FBS-Hams F-12K was added. Then the A-549 cells were infected with M. tuberculosis (4 x 105 cfu: MOI = 100) in a 100 µL portion of the same medium for 2 h. This condition yielded the same levels of mycobacterial entry into the A-549 cells as those achieved in the case of MM6-Ms. After washing with 2% FBS-HBSS, the infected cells were cultured in 200 µL of 1% FBS-Hams F-12K medium in the presence or absence of test drugs for up to 7 days. At intervals, viable count analysis was carried out as mentioned above.
Uptake of quinolones by MM6-Ms and A-549 cells
MM6-Ms and A-549 cells (2 x 106 cells) were cultured in 10 mL of culture medium (MM6-M
s: 5% FBS-RPMI 1640 medium; A-549 cells: 5% FBS-Hams F-12K medium) in plastic culture plates (90 mm diameter) in the presence of 100 mg/L of test quinolones at 37°C for 24 h. After rinsing the cultured cells with PBS, cells were collected by centrifugation (800g, 5 min), suspended in 100 µL of distilled water, and gently sonicated in a sonicator bath at 37°C for 2 h. The resultant cell lysate was mixed with 5 volumes of TS broth and the resultant solution (50 µL each) was then subjected to serial two-fold dilution with TS broth in microculture wells (96 wells: U-bottom: Becton Dickinson). To each well containing drug dilutions was added 10 µL of TS broth containing 5 x 103 cfu of S. aureus, thereafter cultured at 37°C for 24 h, and observed for the maximal dilution that caused growth inhibition of S. aureus (growth inhibition titre). For calibration of the concentrations of quinolones in test cell lysates, standard serial two-fold dilutions of test quinolones were also determined for their growth inhibition titre.
Statistical analysis
Statistical analysis was carried out by Students t-test.
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Results |
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We examined the antimicrobial activities of gatifloxacin, sitafloxacin and levofloxacin at the MIC and Cmax against M. tuberculosis residing within MM6-Ms. As shown in Figure 1(a), sitafloxacin and gatifloxacin added at the MIC caused growth inhibition of intramacrophage M. tuberculosis, and levofloxacin caused growth inhibition of the organisms at day 3. The efficacy was in the order of sitafloxacin > gatifloxacin > levofloxacin. Notably, complete inhibition of bacterial growth was noted for sitafloxacin. On the other hand, the test quinolones exhibited bactericidal activity against intramacrophage M. tuberculosis when added at the Cmax [Figure 1(b)]. The efficacy was in the order of gatifloxacin > levofloxacin
sitafloxacin, in terms of the rate of bacterial elimination during 3- and 5-day cultivation of infected M
s due to drugs. As shown in Table 1, when the test drugs were added at lower concentrations (1/8Cmax to 1/2Cmax) and the bactericidal activities of drugs were determined in terms of bacterial elimination during 7-day cultivation, the efficacy was in the order of sitafloxacin > gatifloxacin > levofloxacin. In this experiment, sitafloxacin added at the Cmax exerted the same level of bactericidal activity as gatifloxacin.
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Next, we examined the antimicrobial activities of test quinolones against M. tuberculosis residing inside A-549 cells. As shown in Figure 2(a), when the test quinolones were added at the MIC, sitafloxacin and gatifloxacin but not levofloxacin caused growth inhibition of M. tuberculosis inside the A-549 cells. The efficacy was in the order of sitafloxacin gatifloxacin > levofloxacin. On the other hand, the test quinolones more or less exhibited bactericidal activity against the organisms residing in A-549 cells, when added at the Cmax [Figure 2(b)]. The efficacy was in the order of gatifloxacin > sitafloxacin > levofloxacin, in terms of the rate of bacterial elimination during 3- and 5-day cultivation of infected M
s due to drugs. As shown in Table 2, when the test drugs were added at lower concentrations (1/8Cmax to 1/2Cmax) and the bactericidal activities of drugs were determined in terms of bacterial elimination during 7-day cultivation, the efficacy was in the order of sitafloxacin > gatifloxacin > levofloxacin. In this experiment, both sitafloxacin and gatifloxacin when added at Cmax caused complete elimination of intracellular M. tuberculosis.
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Table 3 shows the profiles of the intracellular accumulation of test quinolones in MM6-Ms and A-549 cells. First, the efficacies of the drug uptake by these cells were in the order of sitafloxacin > gatifloxacin > levofloxacin. Next, there was no significant difference in the uptake of individual quinolones between MM6-M
s and A-549 cells, although a tendency was noted that MM6-M
s showed a somewhat greater accumulation of sitafloxacin and levofloxacin than did the A-549 cells. In this context, it appears that the profiles of intracellular drug accumulation may be dependent on the media used for cultivation of individual cells (RPMI 1640 and Hams F-12K medium for MM6-M
s and A-549 cells, respectively), since variation in composition of culture media can influence the antibiotic intracellular equilibrium in these cells. Since this experiment was carried out using drugs at a non-physiological concentration (100 mg/L), it is possible that such a high concentration of test drugs might reduce the viability of test cells. Our separate experiments, using nigrosin exclusion testing, indicated that the percentage of viable cells did not reduce even after 24-h cultivation of MM6-M
s and A-549 cells in the medium containing 100 mg/L of test drugs, as follows: (1) MM6-M
s: control (drug), 98.5%; +gatifloxacin, 98.0%; +sitafloxacin, 98.5%; +levofloxacin, 98.5%: (2) A-549 cells: control (drug), 99.9%; +gatifloxacin, 99.9%;+sitafloxacin, 99.9%; +levofloxacin, 99.9%. Therefore, it is thought that the concentrations of drugs used in this experiment (100 mg/L) were not cytotoxic to these cells.
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Discussion |
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Second, when these quinolones were added to the culture medium at the Cmax, such a relationship was not observed. Although both quinolones exerted bactericidal effects against the intracellular organisms, gatifloxacin exhibited much more potent activity against M. tuberculosis residing inside MM6-Ms or A-549 cells compared to sitafloxacin, when the activity of drugs was determined in terms of the rate of drug-mediated bacterial elimination during 3- and 5-day cultivation of infected cells. Notably, when drugs were added to the culture medium of test cells at the 1/8Cmax to 1/2Cmax and the bactericidal activities of drugs were determined in terms of bacterial elimination during 7-day cultivation, the efficacy was in the order of sitafloxacin > gatifloxacin > levofloxacin.
Third, the efficacies of intracellular uptake of these quinolones by MM6-Ms and A-549 cells were both markedly different from quinolone to quinolone, in the order of sitafloxacin > gatifloxacin > levofloxacin. This indicates that sitafloxacin has superior pharmacological characteristics in terms of intracellular uptake and accumulation by lung cells including M
s and alveolar epithelial cells. However, the possibility cannot be excluded that the observed profiles of M
uptake of quinolone are limited to the case of MM6-M
s, since it has been observed that the efficacies of the uptake of levofloxacin and sitafloxacin by THP-1 human M
cell line did not markedly differ from each other (personal communication from Dr K. Namba, Daiichi Pharmaceutical Co.).
It is enigmatic to note that profiles of the antimicrobial activity expression of the test quinolones, especially sitafloxacin, against intracellular M. tuberculosis significantly changed depending on the concentration of the drugs which were added to the culture medium. For instance, although sitafloxacin exhibited the most potent activity against M. tuberculosis inside MM6-Ms and A-549 cells when added at the MIC or 1/8Cmax to 1/2 Cmax concentrations, its activity was significantly less than gatifloxacin when added at higher concentration such as Cmax. This situation may be simply explained by the recent finding of Paillard et al.19 that the ratio of the cellular to extracellular concentration of a certain quinolone (moxifloxacin) is different in infected and uninfected cells (THP-1 M
s). Indeed, in this study, we examined profiles of the drug accumulation using uninfected cells. Alternatively, it is also possible that the mode of intracellular accumulation of the test quinolones may differ depending on the concentration at which the drugs were added to the culture medium of MM6-M
s or A-549 cells infected with M. tuberculosis.
In any case, the present findings may indicate that the MIC values of fluoroquinolones are not always predictive of their antimicrobial activity against intracellular M. tuberculosis replicating within Ms and type II pneumocytes. Since, in the early phases of M. tuberculosis infection in the lungs, the majority of infected tubercle bacilli are replicating within alveolar M
s, interstitial M
s, or type II alveolar epithelial cells,12,20 this result means that the MIC values of quinolones are not sufficiently predictive of their therapeutic effects against tuberculosis especially in the early stage. This may also be the case in disseminated tuberculosis in which most M. tuberculosis organisms are residing within M
s. In addition, this study indicated that a C-8-methoxy quinolone, gatifloxacin, exhibited strong microbicidal activity against intracellular M. tuberculosis regardless of the host cells, such as M
s and type II pneumocytes, when added at the Cmax in the blood. It is reported that the concentration of gatifloxacin in the lungs of rats given an oral administration of the drug was higher than its serum concentration (4.58 mg/L in the lungs versus 2.60 mg/L in the blood 1 h after a single oral administration of gatifloxacin at a dose of 10 mg/kg).21 It thus appears that this quinolone may exert good therapeutic effects in treating patients during the early phases of pulmonary tuberculosis.
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Acknowledgements |
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Footnotes |
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