Infectious Disease Section, Stratton VA Medical Center and Albany Medical College, Albany, NY 12208, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.819 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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
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).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Edelstein, P. H. (1995). Antimicrobial therapy for Legionnaires' disease: a review. Clinical Infectious Diseases 21, Suppl. 3, S526576.
3 . Baltch, A. L., Smith, R. P. & Ritz, W. (1995). Inhibitory and bactericidal activities of levofloxacin, ofloxacin, erythromycin, and rifampin used singly and in combination against Legionella pneumophila. Antimicrobial Agents and Chemotherapy 39, 16616.[Abstract]
4
.
Baltch, A. L., Smith, R. P., Franke, M. A. & Michelsen, P. B. (1998). Antibacterial effect of levofloxacin, erythromycin and rifampin in a human monocyte system against Legionella pneumophila. Antimicrobial Agents and Chemotherapy 42, 31536.
5 . Smith, R. P., Baltch, A. L., Ritz, W., Franke, M. & Glezerman, I. (1997). Legionella pneumophila (L.pn.) susceptibilities and postantiobiotic effect (PAE) of ketolide RU64004 and five comparative antibiotics. In Program and Abstracts of the Ninety-Seventh General Meeting, American Society for Microbiology, Miami Beach, FL. Abstract A145, p. 26. American Society for Microbiology, Washington, DC.
6 . Barker, J. E. & Farrell, I. D. (1990). The effects of single and combined antibiotics on the growth of Legionella pneumophila using timekill studies. Journal of Antimicrobial Chemotherapy 26, 4553.[Abstract]
7 . Bryskier, A., Agouridas, C. & Chantot, J. F. (1997). Ketolides: New semi-synthetic 14-membered-ring macrolides. In Expanding Indications for the New Macrolides, Azalides and Streptogramins, (Zinner, S. H., Young, L. S., Acar, J. F. & Neu, H. C., Eds), pp. 3950. Marcel Dekker Inc, New York.
8 . Agouridas, C., Bonnefoy, A. & Chantot, J. F. (1997). Antibacterial activity of RU 64004 (HMR 3004), a novel ketolide derivative active against respiratory pathogens. Antimicrobial Agents and Chemotherapy 41, 214958.[Abstract]
9 . Barry, A. L., Fuchs, P. C. & Brown, S. D. (1997). In vitro activity of the new ketolide HMR 3004 compared to an azalide and macrolides against Streptococcus pneumoniae and Haemophilus influenzae. European Journal of Clinical Microbiology and Infectious Diseases 16, 7679.[ISI][Medline]
10 . Biedenbach, D. J., Barrett, M. S. & Jones, R. N. (1998). Comparative antimicrobial activity and kill-curve investigations of novel ketolide antimicrobial agents (HMR 3004 and HMR 3647) tested against Haemophilus influenzae and Moraxella catarrhalis strains. Diagnostic Microbiology and Infectious Diseases 31, 34853.
11 . Doern, G. V., Brueggemann, A., Holley, H. P. & Rauch, A. M. (1996). Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients in the United States during the winter months of 1994 to 1995: results of a 30-center national surveillance study. Antimicrobial Agents and Chemotherapy 40, 120813.[Abstract]
12 . Ednie, L. M., Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1997). Susceptibilities of 228 penicillin- and erythromycin-susceptible and -resistant pneumococci to RU 64004, a new ketolide, compared with susceptibilities to 16 other agents. Antimicrobial Agents and Chemotherapy 41, 10336.[Abstract]
13 . Hamilton-Miller, J. M. T. & Shah, S. (1998). Comparative in-vitro activity of ketolide HMR 3647 and four macrolides against grampositive cocci of known erythromycin susceptibility status. Journal of Antimicrobial Chemotherapy 41, 64953.[Abstract]
14 . Jones, R. N. & Biedenbach, D. J. (1997). Antimicrobial activity of RU 66647, a new ketolide. Diagnostic Microbiology and Infectious Diseases 27, 712.[ISI][Medline]
15
.
Pankuch, G. A., Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1998). Susceptibilities of penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, compared with susceptibilities to 17 other agents. Antimicrobial Agents and Chemotherapy 42, 62430.
16 . Schülin, T., Wennersten, C. B., Moellering, R. C. & Eliopoulos, G. (1997). In vitro activity of RU 64004, a new ketolide antibiotic, against Gram-positive bacteria. Antimicrobial Agents and Chemotherapy 41, 1196202.[Abstract]
17 . Smith, R. P., Baltch, A. L. & Ritz, W. (1998). Antibacterial effects of HMR 3647 and four comparative antibiotics, used singly and in combination, against vancomycin-resistant (VRE) and vancomycin-susceptible (VSE) E. faecium. In Program and Abstracts of the Thirty-Eighth Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. Abstract A109, p. 34. American Society for Microbiology, Washington, DC.
18
.
Bébéar, C. M., Renaudin, H., Aydin, M. D., Chantot, J. F. & Bébéar, C. (1997). In-vitro activity of ketolides against mycoplasmas. Journal of Antimicrobial Chemotherapy 39, 66970.
19 . Ednie, L. M., Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1997). Antianaerobic activity of the ketolide RU 64004 compared to activities of four macrolides, five ß-lactams, clindamycin, and metronidazole. Antimicrobial Agents and Chemotherapy 41, 103741.[Abstract]
20 . Bonnefoy, A., Girard, A. M., Agouridas, C. & Shantot, J. F. (1997). Ketolides lack inducibility properties of MLSB resistance phenotype. Journal of Antimicrobial Chemotherapy 40, 8590.[Abstract]
21 . Stuart, A. & Ord, J. K. (1991). Kendall's Advanced Theory of Statistics, volume 2, pp. 110153. Oxford University Press, New York.
22 . Horwitz, M. A. (1982). Phagocytosis of microorganisms. Reviews of Infectious Diseases 4, 10423.[ISI][Medline]
23 . Horwitz, M. A. (1987). Characterization of a virulent mutant Legionella pneumophila that survive but do not multiply within human monocytes. Journal of Experimental Medicine 166, 131028.[Abstract]
24 . Smith, R. P., Baltch, A. L., Franke, M., Hioe, W., Ritz, W. & Michelsen, P. (1997). Effect of levofloxacin, erythromycin, or rifampicin pretreatment on growth of Legionella pneumophila in human monocytes. Journal of Antimicrobial Chemotherapy 40, 6738.[Abstract]
25
.
Vazifeh, D., Preira, A., Bryskier, A. & Labro, M. T. (1998). Interaction between HMR 3647, a new ketolide, and human polymorphonuclear neutrophils. Antimicrobial Agents and Chemotherapy 42, 194451.
26 . Baltch, A. L., Smith, R. P., Ritz, W., Franke, M. & Glezerman, I. (1998). Legionella pneumophila susceptibilities and postantibiotic effect (PAE) of ketolide RU 66647 and five comparative antibiotics. In Program and Abstracts of the Ninety-Eighth General Meeting, American Society for Microbiology, Atlanta, GA. Abstract A24, p. 42. American Society for Microbiology, Washington, DC.
Received 21 July 1999; returned 10 November 1999; revised 2 December 1999; accepted 11 February 2000