Treatment of acute Chlamydia pneumoniae infection with telithromycin in C57BL/6J mice

L. Törmäkangas1,*, E. Saario2, D. Bem David3, A. Bryskier3, M. Leinonen1 and P. Saikku4

1 National Public Health Institute, P.O. Box 310, FIN-90101 Oulu; 2 Department of Internal Services, National Public Health Institute, Helsinki; 4 Department of Medical Microbiology, University of Oulu, Oulu, Finland; 3 Clinical Pharmacology Anti-infectives, Aventis Pharma, Paris, France

Received 10 November 2003; returned 18 December 2003; revised 17 February 2004; accepted 27 February 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The efficacy of telithromycin, a new ketolide antibiotic, was investigated in the treatment of acute Chlamydia pneumoniae infection in a mouse model.

Methods: C57BL/6J mice were inoculated intranasally, and the effects of three different doses of telithromycin (25, 50 and 100 mg/kg) were assessed after 5 and 10 days of treatment. Lungs for culture, PCR, histopathology, and blood for serum samples were collected immediately after each treatment period and at 3 weeks post-inoculation. C. pneumoniae-specific antibodies were analysed, and the effect of treatment was assessed by culture, detection of C. pneumoniae DNA and determination of histopathological inflammatory changes in mouse lungs.

Results: Culture negativity in the lungs was achieved with the higher doses, 50 and 100 mg/kg, after 10 days of treatment. C. pneumoniae DNA was not totally eradicated with the treatments, but the groups treated with 50 and 100 mg/kg doses for 10 days had the lowest DNA positivity rates (10%) 3 weeks after the inoculation. In lung histopathology, the efficacy of telithromycin on inflammatory changes was also dose-dependent: higher doses were more effective in reducing the inflammatory reaction. Overall, the 25 mg/kg dose had a weaker effect compared with the others.

Conclusions: Telithromycin had both time- and dose-dependent effects on the eradication of chlamydia and on reducing infection-induced inflammatory changes in mouse lungs.

Keywords: lung infections, ketolides, eradication


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Chlamydia pneumoniae is a common cause of acute respiratory tract infections, and ~10% of community-acquired pneumonias in adults are considered to be caused by this pathogen.1 C. pneumoniae, like the other chlamydial species, has a tendency to cause persistent infection, which has been associated with chronic diseases, such as asthma and coronary heart disease.2,3 Successful treatment of acute infection could have an impact on the course of such diseases.

In recent animal models, the treatment of acute C. pneumoniae infection with azithromycin,4,5 azithromycin plus rifampicin,6 doxycycline,4,5 amoxicillin,6 amoxicillin-clavulanate, erythromycin or fluoroquinolones5 has been studied. In all these experimental models, treatment markedly decreased the culture positivity of the organisms in lung tissue, but neither DNA nor antigens could be totally eradicated with any of these antimicrobial agents. The presence of pathogen DNA or antigens may be an indication of a latent, persistent infection, and re-activation of culture-negative infection in lung tissue has been demonstrated with immunosuppressive cortisone acetate in mice.7

Telithromycin, the first antimicrobial agent in the novel ketolide group, has been developed to treat respiratory tract infections caused by common and ‘atypical’ pathogens as well as macrolide- and penicillin-resistant bacteria.8 In vitro susceptibility studies have shown telithromycin to possess significant activity against different isolates of C. pneumoniae, particularly the most recent clinical isolates.9 The aim of the present work was to study the efficacy of different doses and treatment times of telithromycin in eradicating acute C. pneumoniae infection in C57BL/6J mice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In vitro susceptibility

The MIC and MBC of telithromycin against the C. pneumoniae isolate Kajaani 7 (K7) were determined in HL cell cultures. Cells were inoculated with equal amounts [~0.5 x 103 inclusion forming unit (IFU)/well] of chlamydia, and twofold dilutions of telithromycin were tested in triplicate. The cell culture medium was free of other antibiotics and contained cycloheximide 1 mg/L. Cells were incubated for 72 h, fixed and stained with fluorescein-conjugated genus-specific monoclonal antibody (Pathfinder, Sanofi Diagnostics Pasteur, France). MIC was defined as the concentration at which >95% of C. pneumoniae inclusion formation was inhibited after the first growth cycle. The concentration at which no inclusions were detected after a passage in antibiotic-free media was defined as MBC.

Animal model and treatment

Six-week-old female C57BL/6J mice were purchased from M&B A/S, Ry, Denmark. At the age of 8 weeks, the mice were inoculated intranasally with 6.0 x 105 IFU/mouse of isolate K7. Placebo or three different doses of telithromycin—25, 50 and 100 mg/kg—were administered subcutaneously once a day for 5 and 10 days, starting on the third day after the inoculation. Each treatment and placebo group consisted of 10 mice, and at every time point, four mice mock-inoculated with sucrose-phosphate-glutamic acid (SPG) buffer were also analysed. The animals were sacrificed and samples taken after 5 [day 8 post-inoculation (p.i.)] and 10 days (day 13 p.i.) of treatment, and at 21 days p.i. All procedures involving animals were approved by the Animal Care and Use Committee of the National Public Health Institute, Helsinki, Finland.

Measurement of C. pneumoniae antibody levels

Antibodies were measured by the microimmunofluorescence test with purified, formalin-fixed whole elementary bodies of C. pneumoniae K7 as antigen. IgG serum antibodies were detected using FITC-conjugated anti-mouse IgG [F(ab')2 fragments, Serotec].

Culture and detection of C. pneumoniae DNA in lung tissues

Lobes from the right lung were homogenized in SPG buffer, and chlamydia culture was performed as described before.10 For a PCR test, 0.2 mL of lung tissue homogenate was lysed and purified using a commercially available QIAamp tissue kit. Primers for the C. pneumoniae omp1 gene (135 bp product) were used for DNA amplification, and the PCR product was analysed by time-resolved fluorescence (TRF)-based hybridization assay (L. Törmäkangas, H. Alakärppä, D. Bem David, H. Leinonen & P. Saikku, unpublished results). Briefly, biotinylated PCR product was attached to streptavidin-coated microtitration wells, denatured and incubated with europium-labelled hybridization probe specific for the amplified sequence. After washing the plates several times, TRF was measured with a fluorometer.

Histopathology of the lungs

The formalin-fixed left lung was embedded in paraffin, and 4 µm sections were cut and stained with haematoxylin and eosin. Sections were evaluated for the severity of broncho-interstitial pneumonia on a scale 0–4 as follows: grade 0, no inflammation; grade 1, slight perivascular and peribronchial lymphocyte and plasma cell infiltration with occasional neutrophilic and eosinophilic granulocytes; grade 2, moderate changes; grade 3, marked changes; grade 4, severe changes with patchy consolidation of lung tissue with alveolar macrophages and plasma cells.

Statistical analyses

Statistical analyses for IgG antibody titres were performed using a non-parametric Mann–Whitney U-test and analyses for histopathological results using Fisher’s exact test (SPSS for Windows, version 10.0.5).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Telithromycin MIC for the C. pneumoniae isolate K7 was 0.03 mg/L (inhibiting 99% of the formation of chlamydial inclusions), and MBC was 0.5 mg/L. The in vitro susceptibility of the isolate K7 to telithromycin in our study was in agreement with the values obtained in an earlier study with several C. pneumoniae isolates.9 There is no standardized method available for evaluating the susceptibility of C. pneumoniae to antimicrobial agents, and the slight differences may be due to different cell culture methods, in addition to the difference in the isolates tested.

At 8 days p.i., after 5 days of treatment, most of the mice in the antibiotic-treated groups had no C. pneumoniae IgG antibodies, whereas eight of the 10 mice in the untreated control group already had antibody titres between 8 and 32. On day 21 p.i., the titres in the telithromycin-treated groups were significantly lower than the untreated controls in all groups, with the exception of the mice treated with 25 mg/kg for 5 days. The geometric means of the titres per group are presented in Table 1. No anti-C. pneumoniae antibodies were detected in the mock-inoculated mice. Therefore, telithromycin treatment, especially the higher doses tested, significantly decreased the IgG antibody response in these mice. The effects of the treatment on serum antibody levels have not been reported in the previous mouse models with different antibiotics.


View this table:
[in this window]
[in a new window]
 
Table 1. Positive C. pneumoniae culture, PCR findings and geometric means of C. pneumoniae IgG antibodies per telithromycin treatment group
 
Viable chlamydiae were detected by culture in the telithromycin-treated groups, although with very low inclusion counts, in 60%–80% of the lung samples on day 8. On days 13 and 21 p.i., some weakly positive samples were found by culture only in the groups treated with 25 mg/kg of telithromycin. At the same time points, respectively, 100%, 90% and 60% of the placebo-treated mice were culture-positive with high inclusion counts. PCR positivity was 89%–100% on day 8 and 40% on day 13 p.i. in the treated groups, and no differences between the telithromycin doses were detected. All mice (100%) in the placebo groups were positive at these earlier time points. On day 21 p.i., 10%–40% of the mice in the telithromycin-treated groups and 50% in the placebo-treated group were still PCR-positive. All uninfected, mock-inoculated mice were culture- and PCR-negative. Both culture positivity and the geometric means of inclusion counts (in 2 mL of lung homogenate) per treatment group, as well as the PCR results, are presented in Table 1. Although the 10 days of treatment with the 50 and 100 mg/kg doses completely eliminated culture positivity from lung tissue homogenates, the lung tissue in 10% of these mice remained PCR positive. These results are in accordance with the previous mouse models of C. pneumoniae infection treated with various antibiotics.46 Complete eradication of chlamydial antigens or DNA was not achieved either, and in some cases antigens and DNA could still be detected 60 days after the primary C. pneumoniae inoculation and treatment with a combination of azithromycin and rifampicin.6

The inflammatory reaction in lung tissue was also decreased in a dose-dependent manner. All treatment groups at all time points had milder inflammatory changes than the infected untreated groups, and the most severe changes (grade 4) were detected only in the untreated control animals (Table 2). On days 8 and 13 p.i., the inflammation in the mice treated with 100 mg/kg was significantly (P < 0.05) milder than that in the mice treated with 25 mg/kg. No differences were seen between the treated groups 21 days p.i. (data not shown). These effects, as well as the decrease in the IgG antibody titres discussed above, were most probably due to the bacteriostatic/bacteriocidal activity of telithromycin and the diminished chlamydial load in the lung tissue, as shown by the culture results. This is also likely to be the reason for the delay in the development of C. pneumoniae IgG antibodies in the telithromycin-treated groups on day 8 p.i. Based on the results of this study, we cannot, however, exclude a possible anti-inflammatory effect of telithromycin.


View this table:
[in this window]
[in a new window]
 
Table 2. Inflammatory changes in lung histopathology in telithromycin-treated and control groups of mice
 
In this study, we tested the in vitro susceptibility of C. pneumoniae K7 to telithromycin in cell cultures, and the in vivo efficacy of different doses and treatment times of telithromycin compared with placebo in acute C. pneumoniae infection in mice. We found that telithromycin had a significant dose- and time-dependent effect on the presence of C. pneumoniae, as indicated by both culture and PCR in mouse lungs after an acute infection. Complete eradication was not achieved with the doses and treatment times assessed here. Our results are in agreement with those obtained in previous animal studies with different antimicrobial agents. Further studies using several different antibiotics and their combinations in a similar research setting are needed to find the optimal treatment regimen for the eradication of C. pneumoniae infection.


    Acknowledgements
 
This study was funded by Aventis Pharma, Paris, France.


    Footnotes
 
* Corresponding author. Tel: +358-8-537-6231 ; Fax: +358-8-537-6251 ; E-mail: liisa.tormakangas{at}ktl.fi Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Hahn, D. L., Azenabor, A. A., Beatty, W. L. et al. (2002). Chlamydia pneumoniae as a respiratory pathogen. Frontiers in Bioscience 7, e66–76.[ISI][Medline]

2 . Hahn, D. L., Dodge, R. W. & Golubjatnikov, R. (1991). Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset asthma. Journal of the American Medical Association 266, 225–30.[Abstract]

3 . Danesh, J., Collins, R. & Peto, R. (1997). Chronic infections and coronary heart disease: is there a link? Lancet 350, 430–6.[CrossRef][ISI][Medline]

4 . Malinverni, R., Kuo, C. C., Campbell, L. A. et al. (1995). Effects of two antibiotic regimens on course and persistence of experimental Chlamydia pneumoniae TWAR pneumonitis. Antimicrobial Agents and Chemotherapy 39, 45–9.[Abstract]

5 . Masson, N. D., Toseland, C. D. & Beale, A. S. (1995). Relevance of Chlamydia pneumoniae murine pneumonitis model to evaluation of antimicrobial agents. Antimicrobial Agents and Chemotherapy 39, 1959–64.[Abstract]

6 . Bin, X. X., Wolf, K., Schaffner, T. et al. (2000). Effect of azithromycin plus rifampin versus amoxicillin alone on eradication and inflammation in the chronic course of Chlamydia pneumoniae pneumonitis in mice. Antimicrobial Agents and Chemotherapy 44, 1761–4.[Abstract/Free Full Text]

7 . Malinverni, R., Kuo, C. C., Campbell, L. A. et al. (1995). Reactivation of Chlamydia pneumoniae lung infection in mice by cortisone. Journal of Infectious Diseases 172, 593–4.[ISI][Medline]

8 . Bryskier, A. (2000). Ketolides-telithromycin, an example of a new class of antibacterial agents. Clinical Microbiology and Infection 6, 661–9.[CrossRef][ISI][Medline]

9 . Roblin, P. M. & Hammerschlag, M. R. (1998). In vitro activity of a new ketolide antibiotic, HMR 3647, against Chlamydia pneumoniae. Antimicrobial Agents and Chemotherapy 42, 1515–6.[Abstract/Free Full Text]

10 . Erkkila, L., Laitinen, K., Laurila, A. et al. (2002). Experimental Chlamydia pneumoniae infection in NIH/S mice: effect of reinoculation with chlamydial or cell preparation on culture, PCR and histological findings of lung tissue. Vaccine 20, 2318–24.[CrossRef][ISI][Medline]