Journal of Antimicrobial Chemotherapy (2000) 46, 51-55
© 2000 The British Society for Antimicrobial Chemotherapy
Antibacterial effect of telithromycin (HMR 3647) and comparative antibiotics against intracellular Legionella pneumophila
Infectious Disease Section, Stratton VA Medical Center and Albany Medical College, Albany, NY 12208, USA
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Abstract |
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The activity of the ketolide telithromycin (HMR 3647) against intracellular Legionella pneumophila strain L-1033 was compared with the activities of erythromycin and levofloxacin. To assay intracellular antibacterial activity, human monocytes were allowed to adhere to wells in 24-well tissue culture plates and were then exposed to L. pneumophila cells for 1 h to allow phagocytosis to occur. Antibiotics were added to the wells after removal of unphagocytosed bacteria. Quantitative bacterial cell counts were made from lysed monocytes at 0, 24, 48, 72 and 96 h. The antibacterial effects of antibiotics against intracellular L. pneumophila L-1033 were concentration and time dependent; at 10 x MIC the activity of telithromycin was greater than that of erythromycin and was less than that of levofloxacin (P < 0.01); telithromycin–rifampicin combinations showed no synergy or interference; and removal of telithromycin from assays at 24 h did not affect its intracellular antibacterial activity. In conclusion, the ketolide telithromycin has excellent activity against intracellular L. pneumophila strain L-1033 and should be evaluated for therapy of legionnaires' disease.
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Introduction |
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Legionella pneumophila can cause serious infections in humans. The mortality rate among patients with these infections continues to be high, especially in bacteraemic, immunocompromised and intubated patients.1 Currently, legionnaires' disease is most often treated with erythromycin.2 Whereas the activity of erythromycin is mainly inhibitory, rifampicin and fluoroquinolones have been shown to have bactericidal activity.2–6
Ketolides are new semi-synthetic erythromycin A derivatives characterized by a 3-keto function instead of the L-cladinose moiety.7 A number of analogues, including telithromycin (HMR 3647), have been synthesized by substituting the C11-C12 carbamate by different alkyl-aryl chains. Ketolides have activity against many microbial pathogens, including penicillin-resistant Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Enterococcus faecium, Mycoplasma spp. and anaerobes.8–19 Furthermore, they can be bactericidal for many pathogens, and their activity is stable at low pH.5,7 In addition, ketolides lack the ability to induce MLSB resistance phenotypes.20
In this study, the effect of increasing concentrations of telithromycin on intracellular L. pneumophila was investigated. The activity of this drug was compared with the activities of erythromycin, rifampicin and levofloxacin. The effects of rifampicin on telithromycin action and on its intracellular activity following removal from the assay system were also investigated.
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Materials and methods |
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Bacterial strain
L. pneumophila strain L-1033, serogroup 1, isolated from the sputum of a patient with pneumonia, was obtained from the Wadsworth Center, New York State Department of Health, Albany, NY, USA. A stock culture was stored in skimmed milk at –70°C. For each experiment, L. pneumophila strain L-1033 was subcultured on buffered charcoal yeast extract (BCYE) agar supplemented with 5% 
-ketoglutarate (BBL Microbiology Systems, Cockeysville, MD, USA), and incubated at 35°C in air. Before each experiment, three to four colonies from a 48 h culture were subcultured from BCYE agar to buffered yeast extract (BYE) broth, and incubated at 35°C for 18 h in a shaking water bath. Cells were diluted to 1 x 107 cfu/mL in RPMI 1640 containing 20% fetal calf serum (FCS) and the suspensions kept at 4°C until use. Final bacterial counts (cfu/mL) were confirmed in duplicate using the standard bacterial plate count method and BCYE agar. Plates were incubated for 48 h at 35°C in air before counting.
Antimicrobial agents
Standard powders of the antimicrobial agents were obtained from the companies indicated: telithromycin, Hoechst Marion Roussel, Romainville, France; levofloxacin, R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA; erythromycin and rifampicin, Sigma Chemical Co., St Louis, MO, USA. Antibiotic solutions were prepared, filter sterilized (pore size, 0.45 µm; Lab Product Sales, Rochester, NY, USA) and used the same day. By the macrodilution technique in BYE broth, the MICs in µg/mL, for L. pneumophila strain L-1033 were as follows: telithromycin, 0.25; levofloxacin, 0.03; erythromycin, 0.50; rifampicin, 0.001.
Opsonization
Pooled heat-inactivated human serum (PHS) obtained from the blood of four healthy donors was diluted in RPMI 1640 to 15% and used immediately to opsonize L. pneumophila cells for 30 min at 35°C.
Preparation of human monocytes
Monocytes were prepared from heparinized blood of healthy human donors who had signed an informed consent form approved by the Institutional Review Board of the Albany Medical College/Stratton VA Medical Center, Albany, NY, USA. Monocytes were separated from whole blood using Histopaque 1077 (Sigma, Cincinnati, OH, USA). Separated monocytes were resuspended in RPMI 1640 plus 20% FCS to a concentration of 2 x 106 cells/mL. Cell viability was 
98% by the trypan blue test.
Effect of antibiotics on intracellular L. pneumophila
Aliquots (1 mL) of human monocytes (2 x 106 cells/mL) were added to the wells of 24-well tissue culture plates (Corning/Costar Corp., Cambridge, MA, USA) and allowed to adhere for 2 h. Non-adherent cells and media were removed by aspiration. The adherent cell layer was then washed gently once with RPMI 1640 containing 20% FCS. Opsonized L. pneumophila L-1033 cells (1 mL at 1 x 107 cells/mL) were added to the wells containing adherent monocytes. After 1 h of phagocytosis, the medium containing opsonized L. pneumophila cells was removed by aspiration and the monolayer was washed once with RPMI 1640 containing 20% FCS. RPMI 1640 containing FCS and antibiotics at increasing concentrations (0.1, 0.25, 1.0, 2.0, 5.0 and 10 x MIC) were then added to duplicate wells. Combinations of antibiotics were studied only at 10 x MIC. At each time point (0, 24, 48, 72 and 96 h) the supernatants were removed from duplicate wells, monocytes were lysed with distilled water and viable L. pneumophila in the lysates were enumerated in duplicate using the standard plate count method. Experiments were performed from three to seven times for each assay condition.
In a separate series of experiments designed to test the effect of antibiotic removal on the regrowth of intracellular L. pneumophila strain L-1033, the antibiotic previously used at 10 x MIC was removed from the incubation medium in half of the wells at 24 h. The experiment then proceeded as described above.
Results are expressed as percentage of viable count: the numbers of cfu/mL at 0, 24, 48, 72 and 96 h were divided by the corresponding count at zero time and multiplied by 100.
Statistical analyses
Statistical analyses were performed using the difference in log10 units of the number of cfu/mL (day specified minus day zero) and analysis of variance.21 The level of significance was 0.05.
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Results |
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Figure 1
shows the antimicrobial effect of increasing concentrations (0.25, 2, 5 and 10 x MIC) of telithromycin on the survival of intracellular L. pneumophila strain L-1033. Even at the lowest concentration studied (0.25 x MIC), a significant effect was observed compared with the control (P < 0.01). Greater antimicrobial effects were observed with higher telithromycin concentrations (P < 0.01).
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), 2 x MIC (
), 5 x MIC (
), 10 x MIC (
).Figure 2
shows the antimicrobial effects of telithromycin, erythromycin, rifampicin and levofloxacin at 10 x MIC on the survival of intracellular L. pneumophila strain L-1033. At day 1, the cfu/mL in untreated monocytes and in monocytes treated with telithromycin, erythromycin or rifampicin increased significantly (P < 0.01), while the number of viable bacteria in levofloxacin-treated monocytes decreased (P < 0.01). From day 1 to day 4, the rates of decrease in per cent cfu/mL were similar for erythromycin-treated, levofloxacin-treated and untreated monocytes. However, the decline of the percentage cfu/mL was significantly more rapid for monocytes treated with telithromycin or rifampicin (P < 0.01). At day 4, the percentage cfu/mL remained lower in monocytes treated with each antibiotic than in the untreated control monocytes (P < 0.01).
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) at 10 x MIC on intracellular Legionella pneumophila strain L-1033. Control (•).Figure 3
demonstrates the effects of removal of telithromycin and levofloxacin after 1 day on the growth of intracellular L. pneumophila strain L-1033. No significant differences were observed from days 2–4 when telithromycin was removed from the assay. In contrast, removal of levofloxacin was associated with a significant increase in the percentage cfu/mL over days 3 and 4 (P < 0.01).
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The antimicrobial effects of telithromycin and erythromycin (at 10 x MIC) when human monocytes were exposed to inocula of 1 x 104 or 1 x 105 cfu/mL of L. pneumophila strain L-1033 were also investigated. For each of the antibiotics the average percentage cfu/mL was less when the inoculum was 1 x 104 cfu/mL than when it was 1 x 105 cfu/mL, although the differences were not statistically significant.
In assays that included telithromycin or rifampicin singly, or in combination at 10 x MIC, the viable counts of L. pneumophila L-1033 following exposure to telithromycin alone did not differ significantly from viable counts following exposure to telithromycin plus rifampicin. However, compared with rifampicin alone the combination of telithromycin and rifampicin lowered viable counts significantly (P < 0.01).
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Discussion |
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L. pneumophila is known to be resistant to the killing effects of serum, monocytes, polymorphonuclear leucocytes and alveolar macrophages.22,23 It has been demonstrated that L. pneumophila cells pretreated with antibiotics are capable of multiplication within human monocytes.24 Currently, patients with legionnaires' disease have a high mortality rate even when they are treated with antimicrobials.1,2 Treatment of L. pneumophila infections appears to have the best results with antimicrobials capable of penetration into phagocytic cells.2,4 Telithromycin is highly concentrated by human neutrophils, with resulting intracellular-to-extracellular concentration ratios of 31 ± 4.2 at 5 min, which reach 348 ± 27.1 at 180 min.25 Ketolides have excellent in vitro activity against most respiratory pathogens, including penicillin-sensitive and -resistant S. pneumoniae, H. influenzae, M. catarrhalis and Mycoplasma spp.8–17 They remain effective at pH values lower than 7.0 and in anaerobic conditions, and do not induce MLSB resistance.5,6,20,26
This study demonstrates that telithromycin is active against intracellular L. pneumophila strain L-1033. This antibacterial activity is evident with concentrations as low as 0.25 mg/L, and it increases with an increase in the intracellular drug concentration. At 10 x MIC, telithromycin, erythromycin and rifampicin allowed growth of L. pneumophila strain L-1033 on day 1 of the assay, but this growth was significantly diminished when compared with the control (P < 0.01). In contrast, levofloxacin markedly decreased the growth rate of L. pneumophila strain L-1033 on day 1 of the assay (P < 0.01). After day 1, however, the rates of decline in the numbers of viable intracellular bacteria were similar with all drugs tested except for telithromycin and rifampicin. The rates of decline in the number of viable intracellular bacteria exposed to telithromycin or rifampicin were similar and significantly greater than the rates of decline resulting from exposure to the other drugs (P < 0.01). By day 4 of the assay, the activities of telithromycin, erythromycin, rifampicin and levofloxacin were similar. One log10 unit increase in the inoculum used in the assay had no statistically significant effect on the intracellular antimicrobial activity of the ketolide. There was no evidence that rifampicin increased or interfered with the intracellular activity of telithromycin. In contrast to levofloxacin, where removal of the antibiotic from the assay on day 1 was associated with rapid regrowth of intracellular L. pneumophila strain L-1033, removal of telithromycin did not affect the continued antimicrobial activity of the monocytes. The rapid efflux of fluoroquinolones once they are removed from the surroundings may account for the rapid regrowth of the intracellular organisms, whereas such rapid efflux does not occur with macrolide drugs, including ketolides. Thus, the prolonged intracellular activity of the ketolides, as well as their prolonged post-antibiotic effects, may make these compounds more effective than erythromycin, the macrolide currently used most frequently for treatment of legionellosis.5,26 As we have demonstrated previously for fluoroquinolones, this study with ketolides failed to show any benefit from the addition of rifampicin to the intracellular L. pneumophila assay system.5,26
In summary, our results indicate that telithromycin, a new ketolide, has excellent activity against intracellular L. pneumophila strain L-1033 in a human monocyte assay. These studies, as well as our previously reported in vitro studies,5,26 demonstrate excellent ketolide activity against L. pneumophila and indicate the need for further evaluation of this subclass of macrolides against infections caused by L. pneumophila.
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Acknowledgments |
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This study was supported by Hoechst Marion Roussel and in part by the Medical Research Service, Office of Research and Development, Department of Veterans Affairs, USA.
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Notes |
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* Corresponding author. Tel: +1-518-462-3311, ext. 3080; Fax: +1-518-462-3350; E-mail: baltch.aldona_{at}albany.va.gov
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References |
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Received 21 July 1999; returned 10 November 1999; revised 2 December 1999; accepted 11 February 2000
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