National Reference Centre of Hygiene and Institute of Environmental Medicine and Hospital Epidemiology, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany
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Abstract |
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Introduction |
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The effect of high concentrations of azithromycin on L. pneumophila has already been shown in different infection models,35 but it was not taken into consideration that the maximum serum concentrations of azithromycin are as low as 0.45 mg/L or that this compound is enriched in macrophages by a factor of at least 40.6,7 Some antibiotics, such as macrolides, become greatly enriched in lysosomes, but their concentration within the endosomes, where the bacteria reside, is unknown.8
We chose the mature monocytic cell line Mono Mac 6 to investigate the effects of antimicrobial agents on intracellular L. pneumophila.9 The Mono Mac 6 infection model has recently been employed in various studies with L. pneumophila.1012
The aims of this study were to investigate whether the Mono Mac 6 cell line is suitable for identifying antibiotics with intracellular activity against legionellae and whether azithromycin is effective in vitro against L. pneumophila at the low concentrations attainable in vivo.
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Materials and methods |
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Azithromycin (Pfizer, Karlsruhe, Germany), ciprofloxacin (Bayer, Wuppertal, Germany), imipenem/cilastatin sodium (Merck Sharp & Dohme, Munich, Germany), amoxycillin, clavulanic acid (both from SmithKline Beecham, Munich, Germany), ceftizoxime (Boehringer Mannheim, Mannheim, Germany) and gentamicin (Merck, Darmstadt, Germany) were laboratory reference standards; they 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.13
Bacterial strains
Two clinical isolates of L. pneumophila serogroup 1 were obtained from bronchoalveolar lavages of patients suffering from legionnaires' disease; they were identified by standard microbiological methods and PCR as described elsewhere.14 These strains were stored after five passages in aliquots in BYE broth and glycerol (1:1) at 70°C. For each experiment a fresh aliquot was thawed and grown on BCYE
agar (Heipha, Heidelberg, Germany) in 5% CO2 at 37°C for 4 days.
MICs
Antimicrobial susceptibilities were determined in duplicate by microdilution susceptibility testing as described previously.15,16 Briefly, freshly grown bacteria were adjusted to an optical density at 415 nm of 108 cfu/mL in water. Fifty microlitres of the suspension was added to 5 mL of BYE broth, which was prepared by dissolving 0.9 g of yeast extract (Difco, Augsburg, Germany) in 90 mL distilled water, adding 10 mL of Legionella BCYE Supplement SR 110A (Oxoid Unipath, Wesel, Germany) and adjusting the pH to 6.9. Testing was carried out in BYE
medium without charcoal, to prevent any interaction with the antibiotic.
Bacteria were added at a final concentration of 5 x 105 cfu/mL to a geometric dilution series of the respective antibiotics. The MIC was read after incubation for 48 h at 37°C. MICs were calculated as the geometric mean value of different, independent experiments.
Mono Mac 6 infection model
The monocytic cell line Mono Mac 6 was obtained from the DSMZ German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were kept at 106 cells/mL in RPMI medium in the absence of 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% oxaloacetate, pyruvate and bovine insulin medium supplement (Sigma, Munich, Germany), 2 mM l-glutamine and 1% Minimum Essential Medium non-essential amino acids (both from Gibco BRL). The cells were grown at 37°C in a humidified atmosphere with 5% CO2. The tissue culture medium was changed twice a week. The cells were checked regularly for mycoplasma infection.
Cells were infected with L. pneumophila as described previously.10 Briefly, 3.2 x 109 legionellae and 3.2 x 107 Mono Mac 6 cells were resuspended in 6 mL of tissue culture medium (pH 7.4) without FCS and incubated for 2 h in a 50 mL tube. Extracellular bacteria were killed by addition of gentamicin to a final concentration of 100 mg/L, which reduced viable counts by more than six orders of magnitude (not shown). After another hour, the cells were washed three times by centrifugation at 400g for 5 min at 37°C. Cells were resuspended in 21 mL of medium as before, and incubated for another 20 h before addition of the antibiotics to intracellular multiplying bacteria. Finally, the cell suspension was distributed in 1 mL aliquots into the wells of a 24-well tissue culture plate (Nunc, Wiesbaden, Germany). At this time (defined as day 0), the test antibiotic was added to duplicate wells. The effects of the antimicrobial agents were studied on the next two days.
Intracellular legionellae were counted in duplicate from aliquots. The cells were pelleted, washed and lysed with 1 mL of ice-cold distilled water and by vortexing with sterile glass beads. Complete lysis was assured by checking microscopically. A serial 10-fold dilution of the lysate was prepared and 100 µL of different dilution steps were spread on to BCYE agar plates. Colonies were counted after 1 week of incubation, because these released bacteria grew more slowly than after a normal passage on agar.
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Results |
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L. pneumophila strains 6 and 26 were tested for their susceptibility to azithromycin, ciprofloxacin, ceftizoxime, imipenemcilastatin and amoxycillinclavulanic acid (Table). For both L. pneumophila strains, ciprofloxacin and imipenem were more active than azithromycin or amoxycillinclavulanic acid.
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The effect of different antibiotics on bacterial growth in Mono Mac 6 cells
L. pneumophila ingested by Mono Mac 6 were incubated for 2 days in the presence of different antimicrobial agents. A representative experiment is shown in Figure 1. After only 1 day of incubation with either azithromycin or ciprofloxacin, the number of viable legionellae decreased. Ceftizoxime, imipenem and amoxycillinclavulanic acid did not affect intracellular replication when compared with untreated cells.
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The antimicrobial effect of low concentrations of azithromycin, which would be attainable in vivo, on intracellular L. pneumophila was determined. Changes in viable intracellular L. pneumophila from day 0 to day 2 were determined, in analogy to the incubation period used in broth dilution susceptibility experiments. The mean and s.e. changes in the numbers of viable legionellae of both strains are shown in Figure 2. In both cases, there was a similar doseresponse after day 2. Compared with untreated cells, azithromycin inhibited the intracellular growth of both strains at a concentration as low as 0.125 mg/L on day 2. After 2 days of exposure to azithromycin 0.1250.25 mg/L, the L. pneumophila counts were the same as before addition of the antibiotic. The concentration of 0.125 mg/L is significantly below the MIC determined by microdilution. After 2 days of incubation, a maximum antimicrobial effect was reached at a concentration of 0.25 mg/L azithromycin. Doses above 0.25 mg/L did not further decrease viable counts significantly.
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For comparison, the effect of ciprofloxacin was determined on phagocytosed legionellae at concentrations below, equal to and above the MIC. Since the MICs of both strains tested were almost identical, only experimental data with strain 6 are presented here. The means and s.e. of three independent experiments are shown in Figure 3. Compared with untreated cells, ciprofloxacin significantly inhibited intracellular growth at a concentration of 0.0040.008 mg/L. After exposure to ciprofloxacin concentrations between 0.008 and 0.015 mg/L, the L. pneumophila count remained unchanged for 2 days. The concentration of 0.015 mg/L is approximately the same as the MIC determined by microdilution. Maximum antimicrobial effect was reached at a ciprofloxacin concentration of 0.03 mg/L.
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Discussion |
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The use of cell lines has been questioned because of the marked effects seen with ß-lactamase inhibitors such as sulbactam, clavulanic acid and imipenem, which have no effect in vivo.18,19,23 Our study shows that the Mono Mac 6 infection model is also suitable for testing the intracellular activity of antimicrobial agents on L. pneumophila. The addition of ciprofloxacin or azithromycin at concentrations near to their respective MICs strongly reduced viable, intracellular counts, but ceftizoxime, amoxycillin/clavulanic acid or imipenem had hardly any effect.
In contrast to the fluoroquinolones, azithromycin has comparatively high modal MICs for legionellae (0.1250.5 mg/L) and a low maximum serum concentration (0.45 mg/L), while intracellularly it distributes mainly into the lysosomes.6,8,24
The in vitro efficacy of azithromycin against phagocytosed L. pneumophila has already been reported, but the concentrations used in the infection models corresponded to or were higher than the MIC.35 Moreover, the dosages used in most of these experiments exceeded the maximum serum concentrations achieved in clinical practice, so their relevance is questionable. We therefore examined the effect of attainable serum concentrations. Intracellular growth was inhibited by a concentration as low as 0.125 mg/L. A significant reduction of intracellular L. pneumophila occurred at the clinically meaningful azithromycin concentration of 0.25 mg/L.
Data on Legionella micdadei phagocytosed by human monocytes suggested that azithromycin was potent at a concentration as low as 0.02 mg/L.25 The MIC of this particular strain, determined by agar dilution on BCYE agar, is 1 mg/L,25 but this may be erroneously high, since the charcoal in BCYE
agar inhibits antibiotics such as fluoroquinolones and macrolides.3,15,26 Therefore, in our experiments the MIC was determined by means of microdilution susceptibility testing in BYE
broth.16 Since macrolides appear to lose potency at low pH,13 the azithromycin MICs of an S. aureus reference strain were determined in BYE
broth and in tissue culture medium and compared with standard MHB. As expected, there was a marked loss of azithromycin potency when testing the S. aureus ATCC 29213 reference strain at pH 6.9, which is the pH of BYE
broth and within the cell.
The MICs of ciprofloxacin and azithromycin for L. pneumophila strains 6 and 26 were similar to those previously determined using media without charcoal.5,17,24,27,28 Even in this rigorous experimental setting, intraphagocytic growth of legionellae was clearly inhibited at concentrations of azithromycin below the MIC, i.e. the concentration inhibiting extracellular growth.
Similar experiments were performed with ciprofloxacin. This fluoroquinolone does not accumulate within cells to such a great extent as azithromycin (it is enriched <10-fold); it is found mainly in the cytosol.29,30 Ciprofloxacin inhibited intracellular growth when used at a concentration between 0.008 and 0.015 mg/L. This ciprofloxacin concentration is below the MIC of 0.02 mg/L determined as the geometric mean value. These results are in accordance with reported sub-MIC effects of fluoroquinolones on phagocytosed L. pneumophila.20
In conclusion, the Mono Mac 6 infection model was proven to be suitable for investigating the intracellular activity of antimicrobial agents. Using this model we have shown that, at clinically relevant concentrations, azithromycin reduces the number of intracellular legionellae in vitro. The Mono Mac 6 infection model may prove to be a valuable tool for in vitro investigation of new compounds in the treatment of legionnaires' disease.
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Acknowledgments |
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Notes |
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References |
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Received 5 January 2000; returned 20 March 2000; revised 2 May 2000; accepted 10 May 2000