The effect of azithromycin on intracellular Legionella pneumophila in the Mono Mac 6 cell line at serum concentrations attainable in vivo

Daniel Jonas*, Inge Engels, Franz D. Daschner and Uwe Frank

National Reference Centre of Hygiene and Institute of Environmental Medicine and Hospital Epidemiology, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We investigated the antimicrobial efficacy of clinically meaningful, low concentrations of azithromycin against intracellular growth of two clinical isolates of Legionella pneumophila. The mature monocytic cell line Mono Mac 6 was used as a model to investigate the effects of antimicrobial agents on L. pneumophila. Extracellular susceptibility was determined by microdilution susceptibility testing in BYE{alpha} broth after 48 h of incubation. Mono Mac 6 cells infected with L. pneumophila were incubated with various concentrations of azithromycin. After 2 days of incubation, intracellular bacteria were released from the phagocytes and plated on to BCYE{alpha} agar. Addition of the intracellular-acting antibiotics azithromycin or ciprofloxacin at their MICs (0.5 and 0.015 mg/L, respectively) resulted in a significant decrease in cfu, of up to approximately 1 log10 after 48 h of incubation. In contrast, incubation of intraphagocytic L. pneumophila in the presence of antibiotics without intracellular activity (ceftizoxime, imipenem or amoxycillin–clavulanic acid) did not have any effect. Azithromycin inhibited intracellular replication at concentrations as low as 0.125 mg/L, approximately one-quarter of the extracellular MIC. The Mono Mac 6 cell line is a useful infection model for investigating the intracellular activity of antimicrobial agents in vitro. In accordance with clinical data and animal experiments, azithromycin and ciprofloxacin inhibited the intraphagocytic replication of L. pneumophila. In particular, azithromycin killed ingested legionellae in vitro at concentrations below the peak serum concentrations and below the MIC.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although Legionella pneumophila is susceptible to many antibiotics, such as aminoglycosides and ß-lactam antibiotics and their derivatives, in vitro, these drugs are often ineffective in treating legionnaires' disease, because these bacteria grow within cells, which may be penetrated poorly by antimicrobials.1 L. pneumophila grow in specialized endosomes of non-activated macrophages and are able to prevent the fusion of these endosomes with lysosomes, which would kill the intracellular organisms.2

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Compounds

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 Mueller–Hinton 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{alpha} broth and glycerol (1:1) at –70°C. For each experiment a fresh aliquot was thawed and grown on BCYE{alpha} 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{alpha} 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{alpha} 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{alpha} agar plates. Colonies were counted after 1 week of incubation, because these released bacteria grew more slowly than after a normal passage on agar.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Broth dilution susceptibility

L. pneumophila strains 6 and 26 were tested for their susceptibility to azithromycin, ciprofloxacin, ceftizoxime, imipenem–cilastatin and amoxycillin–clavulanic acid (TableGo). For both L. pneumophila strains, ciprofloxacin and imipenem were more active than azithromycin or amoxycillin–clavulanic acid.


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Table. Broth microdilution susceptibility of L. pneumophila
 
To estimate the potencies of azithromycin and ciprofloxacin in the different media employed in this study, their MICs for S. aureus ATCC 29213 were determined in BYE{alpha} broth (pH 6.9), in tissue culture medium used for infection experiments (pH 7.4), in MHB (pH 6.9) and in MHB adjusted to pH 7.4 as standard. The MIC of ciprofloxacin in all four media corresponded to standard values,13 but the MIC of azithromycin varied markedly with pH, being 8 mg/L in BYE{alpha} broth (pH 6.9) and MHB (pH 6.9), but 0.5 mg/L in tissue culture medium.

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 1Go. After only 1 day of incubation with either azithromycin or ciprofloxacin, the number of viable legionellae decreased. Ceftizoxime, imipenem and amoxycillin–clavulanic acid did not affect intracellular replication when compared with untreated cells.



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Figure 1. Effect of antibiotics with different intracellular activity. Mono Mac 6 cells were infected with L. pneumophila strain 6 and incubated for 1 day. Subsequently, on day 0, different antibiotics were added. The number of viable cfu is expressed on a log10 scale; error bars represent standard deviations, which, unless shown, were smaller than the height of the symbol representing the mean. Symbols: {blacksquare}, azithromycin 1 mg/L; {blacktriangleup}, ciprofloxacin 0.015 mg/L; {square}, amoxycillin–clavulanic acid 1 mg/L; {triangleup}, ceftizoxime 4 mg/L; x, imipenem 0.015 mg/L; •, none.

 
Dose-dependent effect of azithromycin on intracellular growth of L. pneumophila

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 2Go. In both cases, there was a similar dose–response 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 0.125–0.25 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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Figure 2. Dose-dependent effect of azithromycin on intracellular replication of L. pneumophila strains 6 (a) and 26 (b). Mean changes are calculated from cfu from day 0 to day 2 of intracellular legionellae and expressed on a log10 scale; error bars represent s.e. The figures (n) above the bars indicate numbers of independent experiments at each concentration.

 
Dose-dependent effect of ciprofloxacin on intracellular growth of L. pneumophila

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 3Go. Compared with untreated cells, ciprofloxacin significantly inhibited intracellular growth at a concentration of 0.004–0.008 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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Figure 3. Dose-dependent effect of ciprofloxacin on intracellular replication of L. pneumophila. Mean and s.e. of three independent experiments with Legionella strain 6 values are calculated as described in Figure 2Go.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The primary goal in the antimicrobial therapy of legionnaires' disease is to stop detrimental intracellular growth of legionellae in non-activated alveolar macrophages. Numerous in vitro infection models, based on macrophages derived from guinea-pigs or cell lines, have been used to investigate the effect of antimicrobial agents on phagocytosed legionellae.5,15,1720 In experiments presented here, we used Mono Mac 6 cells as they were more differentiated compared with myeloid cell lines used in other models.9 This infection model has already been used successfully to investigate de novo protein synthesis of phagocytosed legionellae, different growth rates of various Legionella spp. and inflammatory reaction after infection.10,12,21 The cell line was recently used to examine the effect of rifampicin in Mycobacterium tuberculosis-infected macrophages.22

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 (0.125–0.5 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{alpha} agar, is 1 mg/L,25 but this may be erroneously high, since the charcoal in BCYE{alpha} 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{alpha} 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{alpha} 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{alpha} 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.


    Acknowledgments
 
The authors thank Deborah Lawrie-Blum for assistance with the manuscript. This work was supported by a grant from Pfizer Pharmaceuticals, New York, NY.


    Notes
 
* Corresponding author. Tel: +49-761-270-5445; Fax: +49-761-270-5485; E-mail: djonas{at}iuk3.ukl.uni-freiburg.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 5 January 2000; returned 20 March 2000; revised 2 May 2000; accepted 10 May 2000