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Antimicrobial Agents and Chemotherapy, December 2005, p. 5119-5122, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5119-5122.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Moxifloxacin and Azithromycin but not Amoxicillin Protect Human Respiratory Epithelial Cells against Streptococcus pneumoniae In Vitro when Administered up to 6 Hours after Challenge

Martina Ulrich,1 Cordula Albers,1 Jan-Georg Möller,2 Axel Dalhoff,3 Gisela Korfmann,4 Frank Künkele,4 and Gerd Döring1*

Institute of Medical Microbiology and Hygiene, Universitätsklinikum Tübingen, Tübingen,1 Bayer HealthCare, Wuppertal,2 Institute of Medical Microbiology and Virology, Universitätsklinikum Schleswig Holstein,3 Bayer Vital GmbH, Leverkusen, Germany4

Received 11 May 2005/ Returned for modification 7 July 2005/ Accepted 2 September 2005


   ABSTRACT
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We determined the protective effect of moxifloxacin, azithromycin, and amoxicillin against Streptococcus pneumoniae infection of respiratory cells. Moxifloxacin and azithromycin effectively killed intracellular S. pneumoniae strains and protected respiratory epithelial cells significantly even when given 6 h after S. pneumoniae challenge. Amoxicillin was less effective.


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Streptococcus pneumoniae is the leading gram-positive pathogen in patients with community-acquired pneumonia (CAP) (10) and is second after Haemophilus influenzae in patients with acute exacerbations of chronic bronchitis (AECB) (4, 11). In the treatment of CAP and AECB, penicillin- and macrolide-resistant pneumococci have become an increasing problem (5, 16). Based on in vitro data (2, 3, 9), animal models of pneumococcal infection (2), and several clinical studies (1, 4, 6, 10, 11, 22), moxifloxacin has been suggested for the treatment of these infections (7, 20). The rapid and positive clinical efficacy of moxifloxacin (10, 11, 18) can be explained by the high bactericidal capacity against leading pathogens in CAP and AECB (3) and the achievement of high drug concentrations at the site of infection as a consequence of adequate tissue drug penetration. Indeed, quinolone antibiotics penetrate well into various tissues, including the respiratory tract (8, 14, 16, 17, 23) and into phagocytic (12, 15) and epithelial cells (15, 19).

Whereas cell and tissue penetration of moxifloxacin has been widely determined, it is unclear how long one can wait before administering the drug once cells have been infected with S. pneumoniae. Therefore, we sought to assess the bactericidal effect of moxifloxacin on S. pneumoniae in a respiratory cell culture infection model in vitro and to determine the protective effect of moxifloxacin on respiratory epithelial cells (REC) when the drug was given at different time points after bacterial challenge of the respiratory cells. We compared moxifloxacin with azithromycin and amoxicillin.

S. pneumoniae strains ATCC 6303 and ATCC 27336 were cultured on blood agar overnight and subsequently grown in Trypton Soy Broth (TSB) with 5% CO2 at 37°C. To investigate the intracellular and intracellular-extracellular activity of moxifloxacin against S. pneumoniae, a primary vesicle cell culture model was used (21). The cells were preincubated with 5 µg/ml moxifloxacin for 30 min. For limiting moxifloxacin concentrations to the intracellular compartment, vesicles were washed five times with cell culture medium. The intracellular concentration of moxifloxacin was determined by high-performance liquid chromatography (13). In other experiments, vesicles were used without washing. Washed vesicles, unwashed vesicles, and vesicles without moxifloxacin were then challenged with S. pneumoniae. Viable cells of S. pneumoniae were grown to the mid-log phase, and 107 CFU/ml cell culture medium was incubated for 18 h with 105 respiratory cells/ml, grown in vesicles. Survival of vesicles was analyzed after 18 h by light and scanning electron microscopy.

In other experiments, monolayers of the alveolar epithelial cell line A549 were infected with 107 CFU/ml of the S. pneumoniae strains (2 to 3 h at 37°C and 5% CO2). One h, 3 h, 6 h, and 24 h after cells had been washed free of nonadherent bacteria, bacterial CFU were determined by routine methods after digestion of cells with 0.1% 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate (Sigma). Additionally, the alive or dead status of the epithelial cells was determined using the Syto13/Propidiumiodide (PI) Viability test (Molecular Probes, Leiden, The Netherlands). Ten fluorescence micrographs of each sample were taken at a magnification of x200 using the Zeiss Axio Vision program (Oberkochen, Germany). The number of dead cells (red fluorescence) was determined as the percentage of total cells (green fluorescence). In other experiments, 5 µg/ml moxifloxacin, 20 µg/ml azithromycin, or 10 µg/ml amoxicillin was added to A549 cells preincubated with bacteria and washed free of nonadherent bacteria at time zero and at later time points (1 h, 3 h, and 6 h) for 30 min each. Cells were washed again, and bacterial numbers were determined thereafter. Additionally, alive or dead status of the epithelial cells was determined as described above 24 h after time zero.

When vesicles were incubated with moxifloxacin, high intracellular concentrations were detected (moxifloxacin, 54.7 µg/ml ± 17.7 µg/ml), corresponding to a ~10-fold drug accumulation and supporting previous reports (15, 19). When cells were infected with S. pneumoniae strains, 95% of the inoculated bacteria were killed (Fig. 1A, column 1). The intracellular concentration of moxifloxacin rescued 41.9% ± 9.8% of epithelial cells (Fig. 1B, column 1) compared to 1.3% ± 5.5% of controls (Fig. 1B, column 3). When REC were not washed free of extracellular moxifloxacin, 100% of both S. pneumoniae strains were killed (Fig. 1A, column 2) and 79% ± 1.7% of epithelial cells were rescued (Fig. 1B, column 2).



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  		FIG. 1. Moxifloxacin accumulates in primary respiratory epithelial cells and protects these cells against S. pneumoniae. Primary respiratory epithelial cells were incubated for 30 min with 5 µg/ml moxifloxacin and infected with S. pneumoniae. Numbers of surviving bacteria (A) or epithelial cells (B) were determined in experiments in which epithelial cells were washed before bacterial challenge (column 1) or left unwashed (column 2). Infected cells without moxifloxacin served as control (column 3). (C) Scanning electron micrographs of S. pneumoniae-infected respiratory epithelial cells with or without moxifloxacin preincubation. The arrow depicts a single damaged cell protruding out of the cell vesicle.

 
After preincubation of S. pneumoniae strains with A549 cells, nonadhering bacteria were washed away and the incubation was continued for an additional 24 h. Bacterial cell numbers increased slightly to ~1 x 108 CFU/ml (Fig. 2A and B) and caused the death of 100% of A549 cells at that time point (Fig. 2C and D). Addition of moxifloxacin or azithromycin at time point zero (after preincubation of bacteria with epithelial cells) and 1 h, 3 h, and 6 h later resulted in complete killing of S. pneumoniae strains (Fig. 2E and F). Similar incubations with amoxicillin resulted in incomplete bacterial killing (Fig. 2E and F).



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  		FIG. 2. Moxifloxacin and azithromycin but not amoxicillin protect A549 epithelial cells, even when given 6 h after infection with S. pneumoniae strains. A549 epithelial cells were infected with S. pneumoniae strain ATCC 6303 or ATCC 27336 for 2 to 3 h, nonadhering bacteria were washed away, and the incubation was continued for an additional 24 h. Bacterial numbers (A and B) and surviving epithelial cells (C and D) were determined. In other experiments, moxifloxacin (mox), azithromycin (az), or amoxicillin (amox) was added after epithelial cells had been washed free of nonadhering bacteria at time zero and 1 h, 3 h, and 6 h later. Bacterial numbers (E and F) and surviving epithelial cells (G and H) were determined 24 h after time zero. (I and J) Alive or dead status of the epithelial cells in incubations with antibiotics as described for panels G and H.

 
Importantly, moxifloxacin and azithromycin stopped epithelial cell damage (Fig. 2G and H). In contrast to incubations without moxifloxacin where cell damage was 100%, only 18.1% of cell damage was observed at 24 h when moxifloxacin was given 6 h after the challenge of epithelial cells with S. pneumoniae strains. A similar percentage of rescued cells was obtained in incubations with azithromycin, whereas amoxicillin was far less effective.

Taken together, our data demonstrate the excellent rescue of respiratory epithelial cells by moxifloxacin and support the rapid antibacterial effect of moxifloxacin in clinical studies (18, 22), leading to the concept of a reduced therapy interval in CAP or AECB. Thus, moxifloxacin but not amoxicillin is a valid alternative to azithromycin in these clinical conditions.


   ACKNOWLEDGMENTS
 
The study was supported by Bayer Vital GmbH with a grant to M.U.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology and Hygiene, Universitätsklinikum Tübingen, Wilhelmstrasse 31, D-72074 Tübingen, Germany. Phone: (49) 7071-298-2069. Fax: (49) 7071-29-3011. E-mail: gerd.doering{at}med.uni-tuebingen.de. Back


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Antimicrobial Agents and Chemotherapy, December 2005, p. 5119-5122, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5119-5122.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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