National Reference Centre of Hygiene and Institute of Environmental Medicine and Hospital Epidemiology, Freiburg University Hospital, Hugstetter Strasse 55, D-79106 Freiburg, Germany
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Abstract |
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Introduction |
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The aim of this study was to compare the in vitro activity of the novel quinolone moxifloxacin with trovafloxacin, clinafloxacin and levofloxacin and to determine its effect on intracellular L. pneumophila.
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Materials and methods |
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Trovafloxacin (Pfizer, Karlsruhe, Germany), moxifloxacin (Bayer Vital, Wuppertal, Germany), levofloxacin (Aventis Pharma, Bad Soden, Germany), clinafloxacin (Gödecke Parke-Davis, Freiburg, Germany) and gentamicin (Merck, Darmstadt, Germany) were laboratory reference standards. All agents were dissolved and stored according to the manufacturers' recommendations. Biological activities were assured by microdilution susceptibility testing of Staphylococcus aureus ATCC 29213 in MuellerHinton broth (MHB) as described elsewhere.14
Bacterial strains
Six clinical isolates of L. pneumophila serogroup 1 (Nos 1, 3, 6, 26, 5 and 16) were obtained from bronchoalveolar lavage of patients suffering from legionnaires' disease. The isolates were identified by standard microbiological methods and PCR.15 These strains were determined as representing five different genotypes by means of arbitrarily primed PCR (data not shown).16 After five passages on buffered charcoal yeast extract -ketoglutarate (BCYE
) agar (Heipha, Heidelberg, Germany), the isolates were stored in aliquots of BYE
brothglycerol (1:1) at 70°C. BYE
broth contained 0.9% (w/v) yeast extract (Difco, Augsburg, Germany) and 10% (v/v) LegionellaBCYE Supplement SR 110A (Oxoid Unipath, Wesel, Germany) adjusted to pH 6.9 at a final volume of 100 mL. For each experiment an aliquot was thawed and grown on BCYE
agar in 5% CO2 at 37°C for 4 days.
MICs
Antimicrobial susceptibilities were determined by microdilution susceptibility testing in BYE broth as described previously.17,18 Each experiment was performed in duplicate and repeated several times. Bacteria were added at a final concentration of 5 x 104 cfu/100 µL to a geometric dilution series of the antimicrobial agent. Ciprofloxacin was compared with the four newer quinolones. After incubation of the dilution series for 48 h at 37°C, MICs for bacterial growth were read visually. Geometric mean value and modal value were calculated from repeated MIC determinations.
Mono Mac 6 infection model
The monocytic cell line Mono Mac 6 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and was kept at 106 cells/mL in RPMI medium without antibiotics. HEPES-buffered RPMI-1640 with 2 g/L NaHCO3 (Gibco-BRL, Eggenstein, Germany) was mixed with 10% heat inactivated fetal calf serum (FCS) (Biochrom, Berlin, Germany), 1% OPI media supplement (Sigma), 2 mM l-glutamine and 1% MEM non-essential amino acids (both Gibco-BRL). The cells were grown at 37°C in a humidified atmosphere with 5% CO2 and were checked regularly for Mycoplasma infection by means of cultural microbiology methods.
Infection of the cells with L. pneumophila was performed as described previously.19,20 Briefly, 3.2 x 109 L. pneumophila and 3.2 x 107 Mono Mac 6 cells were re-suspended in 6 mL of tissue culture medium (pH 7.4) without FCS and incubated at 37°C for 2 h. Gentamicin was added to the suspension to kill extracellular bacteria; the end concentration was 100 mg/L. After 1 h the cells were centrifuged at 400g for 5 min at 37°C and washed three times, and then re-suspended in 21 mL of medium. The cell suspension containing intracellular bacteria was incubated for 20 h and distributed in 1 mL aliquots into the wells of a 24-well tissue culture plate (Nunc, Wiesbaden, Germany). At this point (day 0) the test antibiotic was added to duplicate wells and the antimicrobial effect was checked after 48 h, when the largest increase in intracellular cfu was observed in the controls without antibiotics.20
Intracellular Legionella were counted in duplicate from samples removed from the wells. The cells were pelleted and washed, and lysed in 1 mL of ice-cold distilled water by vortexing with sterile beads. One hundred microlitres of different lysate dilutions were spread on to BCYE agar plates. Colony-forming units were counted after 1 week of incubation. Changes in viable intracellular L. pneumophila were expressed as the ratio cfu day 2:cfu day 0. The mean change in growth was calculated from five independent experiments with moxifloxacin and four experiments with the other quinolones. The calculated mean values and standard errors of the ratios for the individual antimicrobials were depicted on a logarithmic scale.
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Results |
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In order to prevent any interaction with the antibiotic, testing was carried out in BYE media without charcoal. Susceptibilities of strain No. 6, which was used in subsequent Mono Mac 6 infection experiments, were determined in duplicate in five independent experiments, thus ensuring the accuracy of this non-standardized susceptibility testing method. The MICs for the other five strains were tested in duplicate in three independent experiments. In repeated testing the MIC for each strain did not show a variation greater than one dilution step. The results are shown in the Table
. To facilitate comparison of dose-dependent effects of the quinolones tested on intracellular legionellae, geometric mean and modal values are used. The MICs of moxifloxacin, levofloxacin and ciprofloxacin against tested strains were similar; geometric mean values ranged from 0.015 to 0.03 mg/L. The activities of clinafloxacin and trovafloxacin were almost identical, but were substantially higher than those of moxifloxacin, levofloxacin and ciprofloxacin; geometric mean values ranged from 0.003 to 0.004 mg/L.
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Dose-dependent effect of fluoroquinolones on intracellular growth of L. pneumophila
Mono Mac 6 cells were infected with L. pneumophila strain No. 6, incubated for 20 h and exposed to the antibiotic. The antimicrobial concentrations were chosen from preliminary experiments according to the biggest impact on intracellular counts in a dose-dependent manner (data not shown) and ranged from 0.125 to 2 x MIC. The mean change in growth for the individual antimicrobials is shown in the Figure.
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Compared with untreated cells, all antibiotics impaired intracellular growth at 0.25 x MIC. After 2 days' exposure, at a concentration corresponding to 0.25 x MIC of moxifloxacin, levofloxacin or clinafloxacin, L. pneumophila counts showed practically no change compared with the value before addition of the antibiotics. Similar effects on intracellular growth were achieved by trovafloxacin at higher concentrations of between 0.5 and 1 x MIC. After 2 days' incubation, a maximal antimicrobial effect was reached at a concentration of 1 x MIC. Doses above 2 x MIC did not lead to a further significant decrease in viable count (data not shown).
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Discussion |
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It has been shown that charcoal in BCYE agar inhibits various antibiotics, including fluoroquinolones and macrolides.17,22,23 Therefore, in our experiments the MIC was determined by means of microdilution susceptibility testing in BYE
broth.18 The MICs of trovafloxacin and clinafloxacin were approximately one order of magnitude lower than the corresponding values for levofloxacin and moxifloxacin. These results were found to be in keeping with published data comparing expanded-spectrum quinolones with either levofloxacin or ciprofloxacin.58 In our study, the MICs of each individual quinolone showed almost no variation for six different clinical isolates. For this reason, intracellular activities were determined and compared with strain No. 6 only.
The mature monocytic cell line Mono Mac 6 was used for the investigation of the intracellular activities of the quinolones.24 This in vitro infection model is a well-established method for the investigation of phagocytosed legionellae as well as for studying the efficacy of antimicrobials acting intracellularly.19,20,25,26
The intracellular activities of the quinolones of differing potencies were compared with each other on the basis of their respective MICs. As clinafloxacin had the lowest MIC it showed the highest efficacy against intracellular legionella; trovafloxacin exhibited less intracellular activity at dosages corresponding to the MIC. A lower intracellular concentration does not explain this since trovafloxacin is highly concentrated within macrophages.27 Quinolones have been noted to lose their potency in the presence of increased divalent cation concentrations.14 In contrast to other quinolones, the in vitro activity of trovafloxacin is only marginally affected by magnesium concentrations raised to 9 mM within MHB test medium.28 Trovafloxacin is highly bound to human serum protein; its binding rate of 70% is considerably higher than that of other quinolones.29,30 This fact might explain why its intracellular activity against legionellae is low in relation to the MIC. Unfortunately, trovafloxacin and clinafloxacin will not be available for clinical use because of serious adverse reactions.
Although moxifloxacin and levofloxacin were less effective in vitro, they displayed similar dose-dependent intracellular activities, inhibiting a net growth of L. pneumophila at dosages that corresponded to just 0.25 x MIC. This might be explained by the intracellular enrichment of these agents.31,32 Others have described similar inhibition of intracellular net growth at dosages below the respective MIC.12
Pharmacokinetic studies in humans have shown moxifloxacin or levofloxacin to reach much higher intracellular concentrations in alveolar macrophages than clinafloxacin or trovafloxacin.33 In vivo this might outweigh the lower potency of moxifloxacin against legionellae measured in vitro.
Our data on the intracellular activity of novel quinolones correspond with results presented by other investigators.9,10,12,13 Our findings, however, add valuable new data, especially on the intracellular potency of moxifloxacin against L. pneumophila. In conclusion, our results suggest that newer quinolones, and above all moxifloxacin, could be of value in the empirical treatment of lower respiratory tract infections including those caused by L. pneumophila.
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Acknowledgments |
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Notes |
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References |
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Received 30 March 2000; returned 1 August 2000; revised 18 September 2000; accepted 9 October 2000