Intracellular activity of ABT-773 and other antimicrobial agents against Legionella pneumophila

Rose Jung1, Larry H. Danziger2 and Susan L. Pendland2,*

1Department of Pharmacy Practice, University of Colorado Health Science Center, Denver, CO; 2Microbiology Research Laboratory, Department of Pharmacy Practice, The University of Illinois at Chicago, Chicago, IL, USA

Received 2 July 2001; returned 31 October 2001; revised 2 January 2002; accepted 1 February 2002.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The intracellular activity of ABT-773 against Legionella pneumophila was compared with azithromycin and ciprofloxacin using HL-60 cells. Against L. pneumophila ATCC 33152 and three clinical isolates the MICs (mg/L) were ABT-773 0.015, ciprofloxacin 0.03 and azithromycin 0.03. At 48 h, the mean percentage inhibition was as follows: 28.5 ± 5.9% and 32.6 ± 4.6% at 8 x and 16 x MIC of ABT-773; 38.1 ± 8.6% and 48.2 ± 7.0% at 8 x and 16 x MIC of ciprofloxacin; and 26.3 ± 9.9% and 28.5 ± 9.9% at 8 x and 16 x MIC of azithromycin. In this study, all three agents were highly active, with ABT-773 demonstrating similar activity to azithromycin against L. pneumophila.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the majority of cases, Legionnaires’ disease is caused by Legionella pneumophila. The ability of the organism to multiply intracellularly in alveolar macrophages is regarded as a major virulence factor in this pneumonic disease. By surviving inside the macrophages, L. pneumophila can escape the killing activity of human polymorphonuclear leucocytes, which play an important role in host defence against this pathogen. Thus, antibiotics capable of accumulating and exerting antimicrobial activity in phagocytes are crucial in the treatment of Legionnaires’ disease.14

Previously, a retrospective analysis had supported the choice of erythromycin for the treatment of confirmed cases of Legionnaires’ disease. However, use of this agent can be limited by major side effects, including a requirement for a large volume of fluid for parenteral administration, phlebitis, gastrointestinal intolerance and ototoxicity. The search for better-tolerated agents led to studies with the fluoroquinolones and newer macrolide/azalide antimicrobials. These agents achieve high intracellular concentrations and demonstrate more potent in vitro and in vivo activities than erythromycin against L. pneumophila.14 In the class of macrolide/azalide antimicrobials, azithromycin has shown superiority over erythromycin in the treatment of L. pneumophila pneumonia.2,4 Although many new fluoroquinolones have demonstrated excellent in vitro activity, ciprofloxacin is the only agent from this class with documented clinical efficacy in the treatment of Legionnaires’ disease.1,4 Therefore, the current recommendations have shifted their emphasis to agents that are more potent and better tolerated, such as azithromycin or ciprofloxacin.4

ABT-773 is a novel ketolide derived from erythromycin A. The potent antibacterial activity of ABT-773 extends beyond that of erythromycin, including excellent in vitro activity against macrolide-resistant Streptococcus pneumoniae.5 This agent has been shown to be as active or more active than azithromycin against extracellular Legionella spp. (0.5–1 versus 0.12–2 mg/L).5,6 Since Legionnaires’ disease is caused by an intracellular pathogen, studies of the intracellular activity of ABT-773 are necessary. In this study, the intracellular activity of ABT-773 against L. pneumophila was compared with azithromycin and ciprofloxacin. These agents were selected for comparison because of their excellent in vitro activity and documented clinical efficacy in the treatment of Legionnaires’ disease.14


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial agents

ABT-773 (Abbott Laboratories, North Chicago, IL, USA) was prepared according to the manufacturer’s recommendations, with methanol as the solvent and potassium phosphate buffer (pH 6.5) as the diluent. Azithromycin (Pfizer Inc., New York, NY, USA) was prepared according to the manufacturer’s recommendations, with ethanol as the solvent and sterile water as the diluent. Ciprofloxacin (United States Pharmacopeia, Rockville, MD, USA) was prepared as per NCCLS guidelines, with sterile water as the solvent and diluent.

Bacterial strains

Three clinical isolates of L. pneumophila (UIC 1, UIC 9 and UIC 11) were obtained from the Microbiology Laboratory at the University of Illinois at Chicago Medical Center. The control strain, ATCC 33152, was purchased from the American Type Culture Collection (Rockville, MD, USA). The organisms were stored at –70°C in skimmed milk, and underwent three subcultures on buffered charcoal yeast extract (BCYE) agar (Remel, Leneza, KS, USA).

MIC procedure

Microbroth dilution MICs were carried out in duplicate using buffered yeast extract (BYE) broth.7 Final concentrations tested were 0.00175–2 mg/L for all antibiotics. MIC microtitre plates were incubated aerobically at 35°C and read at 48 h. The MIC was defined as the lowest concentration at which there was no visible growth.

Intracellular susceptibility testing

Intracellular susceptibility testing was conducted using HL-60 cells. The cells were maintained in Iscove’s modified medium supplemented with 20% heat-inactivated fetal calf serum. The cells were collected and suspended in Iscove’s medium with 10% heat-inactivated human serum and 10 mg/L phorbol 12-myristate 10-acetate (PMA) at a concentration of 5 x 105 cells/mL. One millilitre was added to each well of 24 well tissue culture plates and incubated for 24 h at 35°C in 5% CO2. L. pneumophila was added to the monolayer using a bacteria:cell ratio of 10:1. Cells were incubated for 24 h and the extracellular bacteria were removed by three washes. Media containing 8 x and 16 x MIC of ABT-773, azithromycin and ciprofloxacin were added and the cells were incubated for 72 h. L. pneumophila incubated without antimicrobials was used as a control. Antimicrobial agents were also added to uninfected wells to assess the cytotoxic activity of ABT-773 against the HL-60 cells. Infected cells were lysed with sterile distilled water and plate counts were carried out on BCYE agar at 0, 24, 48 and 72 h after addition of the antimicrobial agents. All experiments were carried out in triplicate.

Results are expressed as percentage inhibition at 24, 48 and 72 h. Percentage inhibition was defined as the fraction of inhibition with agents divided by the total number of organisms without agent x 100. Thus, the higher value represents greater inhibition of growth.

Statistical analysis

Analysis of variance (ANOVA) was used to determine significant differences in mean percentage inhibition between agents. A P value of <0.05 was considered significant. Dunnett’s test was used for pairwise comparison with ABT-773.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility testing

The MICs of ABT-773, azithromycin and ciprofloxacin were similar for the L. pneumophila strains. Against all four organisms, the MICs of ABT-773, azithromycin and ciprofloxacin were 0.015, 0.03 and 0.03 mg/L, respectively.

Inhibition of intracellular multiplication

The intracellular growth of the L. pneumophila strains is depicted in Figure 1. The percentage inhibition of ABT-773, azithromycin and ciprofloxacin on intracellular multiplication of L. pneumophila is shown in Figure 2. All three antibiotics demonstrated inhibitory activity. The percentage inhibition at 8 x MIC was similar to results at 16 x MIC for ABT-773 and azithromycin. However, ciprofloxacin at 16 x MIC resulted in greater inhibition than at 8 x MIC against all four strains of L. pneumophila.



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Figure 1. Growth of L. pneumophila in HL-60 cells versus days of incubation after infection. (a) UIC 1, serogroup 1; (b) UIC 9, serogroup 3; (c) UIC 11, serogroup 6; (d) ATCC 33152. All points represent the means for triplicate wells and error bars represent 95% confidence intervals. Open circles, drug-free control; filled triangles, ABT-773 8 x MIC (0.12 mg/L); open triangles, ABT-773 16 x MIC (0.24 mg/L); filled squares, ciprofloxacin 8 x MIC (0.24 mg/L); open squares, ciprofloxacin 16 x MIC (0.48 mg/L); filled diamonds, azithromycin 8 x MIC (0.24 mg/L); open diamonds, azithromycin 16 x MIC (0.48 mg/L).

 


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Figure 2. Percentage inhibition of the intracellular multiplication of L. pneumophila caused by ABT-773 (ABT), ciprofloxacin (CIP) and azithromycin (AZI). (a) UIC 1, serogroup 1; (b) UIC 9, serogroup 3; (c) UIC 11, serogroup 6; (d) ATCC 33152. All bars represent the means of triplicate values and error bars represent 95% confidence intervals. Black bars, at 1 day; white bars, at 2 days; grey bars, at 3 days.

 
At both 8 x and 16 x MIC, ABT-773 demonstrated greater inhibitory activity than azithromycin against L. pneumophila isolates UIC 1 and UIC 9 (P < 0.05). The activity of this new ketolide was similar to azithromycin against the other strains (P >= 0.05). Ciprofloxacin demonstrated more potent activity than azithromycin and ABT-773 at both concentrations (P < 0.05). Cytotoxicity, assessed by microscopic examination of cellular morphology, was not detected in uninfected wells exposed to ABT-773.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Owing to the high morbidity and mortality associated with S. pneumoniae and L. pneumophila infections, empirical treatment for community-acquired pneumonia often includes therapy against these pathogens. Macrolides, azalide and fluoroquinolones have demonstrated excellent activity against L. pneumophila.14 However, the increasing frequency of resistant S. pneumoniae mandates judicious use of currently available agents, as well as the development of new compounds with enhanced activity against resistant pathogens.

ABT-773 is a novel ketolide possessing an extended spectrum of activity in comparison with macrolides.5 Despite structural similarity, the activity of ABT-773 against S. pneumoniae does not depend on susceptibility to erythromycin. Extracellular studies have shown ABT-773 to be as active or more active than azithromycin or erythromycin against Legionella species.5,6 In a recently published study, ABT-773 was reported to be as active as erythromycin in an animal model of L. pneumophila pneumonia.8 However, results were confounded by the high number of animals whose death was attributed to peritonitis in the ABT-773 treatment group. Ten out of 15 guinea pigs treated with the ketolide survived for 13 days post-infection compared with 14 out of 15 treated with erythromycin. Maximum plasma concentrations in the ABT-773 treatment group were 490 and 300 ng/mL, respectively, at 0.5 and 1 h post-administration.

Owing to the potent in vitro activity of ABT-773, the concentrations (120 and 240 ng/mL) tested in our study were very low. Published data on the pharmacokinetics of ABT-773 are limited. Maximum serum concentrations reported following single doses of 100, 200 and 400 mg were 141 ± 60, 180 ± 75 and 614 ± 309 ng/mL, respectively.9 Based on data currently available, the ABT-773 concentrations tested in our study should be clinically achievable.

In this study, the HL-60 cell line was used to compare the intracellular activity of ABT-773 with azithromycin and ciprofloxacin against L. pneumophila. The HL-60 human leukaemic cell line has been well characterized and has been shown to have many of the same properties as normal human monocyte-macrophages. Once differentiated to monocyte/macrophage lineage, these cells have been shown to be suitable hosts for the study of Legionella intracellular parasitism and pathogenesis.10 Furthermore, this cell line has been used successfully to determine the effects of many antimicrobials on the ability of L. pneumophila to survive and replicate within phagocytic cells.2,3

Using the HL-60 cell line, ciprofloxacin demonstrated greater inhibition of L. pneumophila multiplication compared with ABT-773 and azithromycin. Other studies using intracellular models have also shown that fluoroquinolones have greater inhibitory activity against L. pneumophila than erythromycin.3,4

In summary, the intracellular activity of ABT-773 against L. pneumophila appears to be similar to azithromycin. Based on this study, ABT-773 may provide good coverage against L. pneumophila when used empirically in patients suspected of community-acquired pneumonia.


    Acknowledgements
 
The authors thank Donghua Li for excellent technical assistance. Financial support was provided by Abbott Laboratories, Abbott Park, IL, USA. This work was presented in part at the poster session of the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 1999.


    Footnotes
 
* Correspondence address. The University of Illinois at Chicago, College of Pharmacy, Department of Pharmacy Practice (M/C 886), 833 South Wood Street, Chicago, IL 60612, USA. Tel: +1-312-996-8639; Fax: +1-312-413-1797; E-mail: Pendland{at}uic.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Unertl, K. E., Lenhart, F. P., Forst, H., Vogler, G., Wilm, V., Ehret, W. et al. (1989). Ciprofloxacin in the treatment of legionellosis in critically ill patients including those cases unresponsive to erythromycin. American Journal of Medicine 87, S128–31.[Medline]

2 . Stout, J. E., Arnold, B. & Yu, V. L. (1998). Activity of azithromycin, clarithromycin, roxithromycin, dirithromycin, quinupristin/dalfopristin and erythromycin against Legionella species by intracellular susceptibility testing in HL-60 cells. Journal of Antimicrobial Chemotherapy 41, 289–91.[Abstract]

3 . Stout, J. E., Arnold, B. & Yu, V. L. (1998). Comparative activity of ciprofloxacin, ofloxacin, levofloxacin, and erythromycin against Legionella species by broth microdilution and intracellular susceptibility testing in HL-60 cells. Diagnostic Microbiology and Infectious Disease 30, 37–43.[ISI][Medline]

4 . Edelstein, P. H. (1995). Antimicrobial chemotherapy for Legionnaires’ disease: a review. Clinical Infectious Diseases 21, S265–76.[ISI][Medline]

5 . Nilius, A. M., Bui, M. H., Almer, L., Hensey-Rudloff, D., Beyer, J., Ma, Z. et al. (2001). Comparative in vitro activity of ABT-773, a novel antibacterial ketolide. Antimicrobial Agents and Chemotherapy 45, 2163–8.[Abstract/Free Full Text]

6 . Dubois, J. & St-Pierre, C. (2001). In vitro susceptibility study of ABT-773 against Legionella spp. In Program and Abstracts of the Twenty-second Interscience Congress of Chemotherapy, Amsterdam, The Netherlands, 2001. Abstract P19.012. The British Society for Antimicrobial Chemotherapy, Birmingham, UK.

7 . Ristroph, J. D., Hedlund, K. W. & Allen, R. G. (1980). Liquid medium for growth of Legionella pneumophila. Journal of Clinical Microbiology 11, 19–21.[ISI][Medline]

8 . Edelstein, P. H., Higa, F. & Eldelstein, M. A. C. (2001). In vitro activity of ABT-773 against Legionella pneumophila, its pharmacokinetics in guinea pigs, and its use to treat guinea pigs with L. pneumophila pneumonia. Antimicrobial Agents and Chemotherapy 45, 2685–90.[Abstract/Free Full Text]

9 . Gustavson, L., Pradhan, R., Zhang, Y., Zhang, J. & Paris, M. (2001). Single oral dose pharmacokinetics and safety of ABT-773 in healthy subjects. In Program and Abstracts of the Twenty-second Interscience Congress of Chemotherapy, Amsterdam, The Netherlands, 2001. Abstract P9.002. The British Society for Antimicrobial Chemotherapy, Birmingham, UK.

10 . Marra, A., Horwitz, M. A. & Shuman, H. A. (1990). The HL-60 model for the interaction of human macrophages with the Legionnaires’ disease bacterium. Journal of Immunology 7, 2738–44.