Department of Pediatrics, State University of New York Downstate Medical Center, 450 Clarkson Avenue, Box 49, Brooklyn, NY 11203-2098, USA
Received 4 July 2001; returned 27 November 2001; revised 8 February 2002; accepted 18 February 2002.
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Abstract |
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Introduction |
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Materials and methods |
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Confluent HEp-2 cell monolayers were inoculated with a reference prototype isolate, strain C. pneumoniae TW-183 (ATCC VR-2282), and a relatively recent clinical isolate, CM-1 (ATCC VR-1360), which was isolated from an adult patient with pneumonia. Both cultures had been maintained for over 3.54 years without centrifugation or addition of cycloheximide or fresh cells, as previously described.9
Antibiotic activity assay
Continuously infected HEp-2 cells were seeded into 24 well plates the day before each experiment and incubated at 35°C. On day 0, the supernatants of all infected cells and uninfected control cells were replaced with medium with or without gemifloxacin or azithromycin at final concentrations of 0.25 mg/L (MIC of both antimicrobials) and 2.5 mg/L and the cells were incubated at 35°C for 30 days. The latter concentration is comparable to that achieved by both antimicrobials in the pulmonary epithelial lining fluid (ELF) of human volunteers.10,11 Every third day the medium was replaced with fresh medium containing the same antimicrobial concentrations. Infected cells were collected at 0, 6, 12, 18, 24 and 30 days by scraping the cell monolayers in sucrose/phosphate/glutamine (SPG) medium, and stored at 70°C. Supernatants were collected at the same time points and stored at 70°C. The inclusion-forming units per mL (ifu/mL) for every sample were determined as described previously.12 Briefly, infected cells were quickly defrosted at 35°C and ultrasonicated. Ten-fold dilutions from each sample were inoculated on to fresh HEp-2 cells in 96 well plates. The plates were centrifuged at 1700g and incubated for 72 h at 35°C. The cell monolayers were fixed in 90% ethanol, stained with FITC-conjugated monoclonal antibodies (Pathfinder Chlamydia Culture Conformation System; Bio-Rad, Redmond, WA, USA) and examined for the presence of inclusions according to the manufacturers instructions. The ifu/mL for every time point, isolate and drug was calculated. Cell cultures with no antimicrobials added were treated the same way and used as controls.
Cytokine production assay
Cytokines IL-4, IL-6, IL-8 and IL-12 (where IL stands for interleukin) were assayed by sandwich ELISA (Cytoscreen; Biosource International, Camarillo, CA, USA) according to the manufacturers instructions. Infected and uninfected HEp-2 cells with no antimicrobials added were used as controls.
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Results |
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Discussion |
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Gemifloxacin at concentrations of the MIC (0.25 mg/L) and ELF (2.5 mg/L) reduced the titre of C. pneumoniae in this model by 5 log10. The activity of azithromycin was similar. However, both antimicrobials failed to completely eliminate C. pneumoniae from continuously infected cells, even after 30 days of treatment. These results are similar to our previous findings with short-term 6 day treatment with 0.5 mg/L azithromycin and 4.0 mg/L ofloxacin (4 x MIC) and with prolonged 30 day treatment with 4 mg/L azithromycin, 64 mg/L clarithromycin and 16 mg/L levofloxacin (ELF concentrations), where titres of C. pneumoniae were also reduced, but not completely eradicated, from continuously infected cells.9,12 The MICs of these antimicrobials remained essentially the same.
Dreses-Werringloer et al.14 recently reported similar observations on the effect of ciprofloxacin and ofloxacin on Chlamydia trachomatis in HEp-2 cells using an established infection (23 days post-inoculation). They found that both drugs, at concentrations that exceeded the minimum bactericidal concentration (0.5 mg/L ciprofloxacin, and 1.0 and 2.0 mg/L ofloxacin), failed to eradicate C. trachomatis from infected cells and also induced persistent infection characterized by a low number of small aberrant inclusions present through 20 days of culture. After removal of ciprofloxacin from the medium on 10 or 14 days post-infection, the persistent chlamydiae reverted to overt growth.
Although gemifloxacin is 24 times more active in vitro than levofloxacin (MIC90 0.25 versus 0.51.0 mg/L),15 it was not more effective than levofloxacin in eliminating C. pneumoniae from persistently infected cells. Lower MICs do not always correlate to higher microbiological efficacy. Clarithromycin, which is 10100-fold more potent than erythromycin in vitro, has not been found to be more effective in eradicating C. pneumoniae from the respiratory tract in patients with community-acquired pneumonia.2
Recent ultrastructural studies of the continuously infected cells have demonstrated the presence of a subpopulation of aberrant chlamydial inclusions very similar to those seen after treatment with -interferon.16 These forms generally do not appear to replicate and therefore may not be susceptible to antimicrobials. The existence of a persistent form raises an important issue for the treatment of C. pneumoniae infections and associated diseases. The results of two pneumonia treatment studies in adults, which evaluated levofloxacin and moxifloxacin, found eradication rates of only 7080% in patients with culture-documented C. pneumoniae.7,17 It is possible that these microbiological failures and the ability of C. pneumoniae to survive antibiotic treatment in our experiments may be directly related to this persistent state. These findings may also have important implications in the ongoing use of antimicrobials for secondary prevention of cardiac morbidity. The dosages of antimicrobials being used would be unlikely to eliminate a persistent C. pneumoniae infection from an intravascular focus.
C. pneumoniae can stimulate production of cytokines, chemokines and adhesion molecules in various endothelial and epithelial cell lines.1822 These immunologically active molecules are able to induce and sustain inflammatory processes that may play an essential role in the pathogenesis of asthma and atherosclerosis,23 diseases that have recently been linked with chronic C. pneumoniae infection. In this study C. pneumoniae stimulated significant production of IL-6 and IL-8 in the continuously infected HEp-2 cells. These cytokines have been detected in fibrous plaques, which is suggestive of their involvement in the development of atherosclerosis.24
Quinolones have been shown to possess immunomodulatory properties independent of their antimicrobial activity.2530 Levofloxacin was found to increase IL-2 production and suppress IL-1ß production by peripheral blood mononuclear cells stimulated with phytohaemagglutinin and lipopolysaccharide (LPS), respectively. At high concentrations, levofloxacin suppressed the granulocyte-macrophage colony stimulating factor (GM/CSF), soluble IL-2 receptor and tumour necrosis factor- (TNF-
) production in these cells. IL-8 production was barely affected.25 Grepafloxacin has been demonstrated to inhibit production of IL-1
and IL-1ß, and to down-regulate mRNA expression for IL-1
, IL-1ß, TNF-
, IL-6 and IL-8 in LPS-stimulated human peripheral blood cells.26 Ciprofloxacin decreased IL-6 mRNA and increased IL-8 mRNA expression in human endothelial cells.27,28 In another study, ciprofloxacin increased levels of mRNA expression for IL-1
, IL-2, IFN-
, IL-3, IL-4, GM/CSF and TNF-
in peripheral blood lymphocytes.29
In this study, treatment with both antimicrobials increased levels of IL-12 in CM-1 cultures at higher concentrations. This cytokine has been shown to be critically involved in protection against an array of intracellular pathogens, including Chlamydia spp.31,32 IL-12 is produced early during infection and plays the major role in cellular immunity against chlamydia lung infection, mostly through its induction of IFN- and Th1 responses. Treatment with IL-12 has been demonstrated to reduce the severity of chlamydial pneumonia, abolish mortality and significantly reduce numbers of chlamydial organisms in lungs.33 Both antimicrobials decreased levels of IL-6 and IL-8 in C. pneumoniae cultures, but this effect appeared to be related to their anti-chlamydial activity rather than a direct anti-inflammatory effect. The anti-inflammatory properties of gemifloxacin may have a beneficial impact on the treatment of chlamydial infections and should be further investigated.
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Footnotes |
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References |
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2 . Block, S., Hedrick, J., Hammerschlag, M. R., Cassell, G. H. & Craft, C. (1995). Mycoplasma pneumoniae and Chlamydia pneumoniae in pediatric community-acquired pneumonia: comparative efficacy and safety of clarithromycin vs. erythromycin ethylsuccinate. Pediatric Infectious Disease Journal 14, 4717.[ISI][Medline]
3
.
Roblin, P. M. & Hammerschlag, M. R. (1998). Microbiologic efficacy of azithromycin and susceptibilities to azithromycin of isolates of Chlamydia pneumoniae from adults and children with community-acquired pneumonia. Antimicrobial Agents and Chemotherapy 42, 1946.
4 . Harris, J.-A., Kolokathis, A., Campbell, M., Cassell, G. H. & Hammerschlag, M. R. (1998). Safety and efficacy of azithromycin in the treatment of community acquired pneumonia in children. Pediatric Infectious Disease Journal 17, 86571.[ISI][Medline]
5 . Dean, D., Roblin, P. M., Mandel, L., Schachter, J. & Hammerschlag, M. R. (1998). Molecular evaluation of serial isolates from patients with persistent Chlamydia pneumoniae infections. In Chlamydial Infections: Proceedings of the Ninth International Symposium on Human Chlamydial Infection, (Stephens, R. S., Byrne, G. I., Christiansen, G. et al., Eds), pp. 21923. UCSF, San Francisco, CA.
6 . Hammerschlag, M. R., Chirgwin, K., Roblin, P. M., Gelling, M., Dumornay, W., Mandel, L. et al. (1992). Persistent infection with Chlamydia pneumoniae following acute respiratory illness. Clinical Infectious Diseases 14, 17882.[ISI][Medline]
7
.
Hammerschlag, M. R. & Roblin, P. M. (2000). Microbiological efficacy of levofloxacin for treatment of community-acquired pneumonia due to Chlamydia pneumoniae. Antimicrobial Agents and Chemotherapy 44, 1409.
8
.
Roblin, P. M., Reznik, T., Kutlin, A. & Hammerschlag, M. R. (1999). In vitro activity of gemifloxacin (SB-265805, LB20304) against recent clinical isolates of Chlamydia pneumoniae. Antimicrobial Agents and Chemotherapy 43, 28067.
9
.
Kutlin, A., Roblin, P. M. & Hammerschlag, M. R. (1999). In vitro activity of azithromycin and ofloxacin against Chlamydia pneumoniae in a continuous infection model. Antimicrobial Agents and Chemotherapy 43, 226872.
10 . Rodvold, K. A., Gotfried, M. H., Danziger, L. H. & Servi, R. J. (1997). Intrapulmonary steady-state concentrations of clarithromycin and azithromycin in healthy adult volunteers. Antimicrobial Agents and Chemotherapy 41, 1399402.[Abstract]
11
.
Gee, T., Andrews, J. M., Ashby J. P., Marshall G. & Wise R. (2001). Pharmacokinetics and tissue penetration of gemofloxacin following a single oral dose. Journal of Antimicrobial Chemotherapy 47, 4314.
12
.
Kutlin, A., Roblin, P. M. & Hammerschlag, M. R. (2002). Effect of prolonged treatment with azithromycin, clarithromycin and levofloxacin on Chlamydia pneumoniae in a continuous infection model. Antimicrobial Agents and Chemotherapy 46, 40912.
13 . Block, S., Hedrick, J., Hammerschlag, M. R., Cassell, G. H. & Craft, C. (1995). Mycoplasma pneumoniae and Chlamydia pneumoniae in pediatric community-acquired pneumonia: comparative efficacy and safety of clarithromycin vs. erythromycin ethylsuccinate. Pediatric Infectious Disease Journal 14, 4717.[ISI][Medline]
14
.
Dreses-Werringloer, U., Padubrin, I., Jurgens-Saathoff, B., Hudson, A. P., Zeidler, H. & Kohler, L. (2000). Persistence of Chlamydia trachomatis is induced by ciprofloxacin and ofloxacin in vitro. Antimicrobial Agents and Chemotherapy 44, 328897.
15
.
Hammerschlag, M. R. (2000). Activity of gemifloxacin and other new quinolones against Chlamydia pneumoniae: a review. Journal of Antimicrobial Chemotherapy 45, Suppl. S1, 359.
16
.
Kutlin, A., Flegg, C., Stenzel D., Reznik, T., Roblin, P. M., Mathews, S. et al. (2001). Ultrastructural study of Chlamydia pneumoniae in a continuous infection model. Journal of Clinical Microbiology 39, 37213.
17 . Hammerschlag, M. R. & Roblin, P. M. (2000). Microbiologic efficacy of moxifloxacin for the treatment of community-acquired pneumonia due to Chlamydia pneumoniae. International Journal of Antimicrobial Agents 15, 14952.[ISI][Medline]
18 . Gaydos, C. A. (2000). Growth in vascular cells and cytokine production by Chlamydia pneumoniae. Journal of Infectious Diseases 181, S4738.[ISI][Medline]
19 . Gaydos, C. A., Summersgill, J. T., Sahney, N. N., Ramirez, J. A. & Quinn, T. C. (1996). Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. Infection and Immunity 64, 161420.[Abstract]
20 . Kaukoranta-Tolvanen, S. S., Teppo, A. M., Laitinen, K., Saikku, P., Linnavuori, K. & Leinonnen, M. (1996). Growth of Chlamydia pneumoniae in cultured human peripheral blood mononuclear cells and induction of a cytokine response. Microbial Pathogenesis 21, 21521.[ISI][Medline]
21 . Netea, M. G., Selzman, C. H., Kullberg, B. J., Galama, J. M. D., Weinberg, A., Stalenhoef, A. F. H. et al. (2000). Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells. European Journal of Immunology 30, 5419.[ISI][Medline]
22 . Roblin, P. M., Kutlin, A. & Hammerschlag, M. R. (1996). Production of IL-4 and IL-6 in HEp-2 cells infected with Chlamydia pneumoniae. Infectious Diseases in Obstetrics and Gynecology 4, 1956.
23 . Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362, 8018.[ISI][Medline]
24 . Rus, H. G., Vlaicu, R. & Niculescu, F. (1996). Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall. Atherosclerosis 127, 26371.[ISI][Medline]
25 . Yoshimura, T., Kurita, C., Usami, E., Nakao, T., Watanabe, S., Kobayashi, J. et al. (1996). Immunomodulatory action of levofloxacin on cytokine production by human peripheral blood mononuclear cells. Chemotherapy 42, 45964.[ISI][Medline]
26
.
Ono, Y., Ohmoto, Y., Ono, K., Sakata, Y. & Murata, K. (2000). Effect of grepafloxacin on cytokine production in vitro. Journal of Antimicrobial Chemotherapy 46, 914.
27
.
Galley, H. F., Dhillon, J. K., Paterson, R. L. & Webster N. R. (2000). Effect of ciprofloxacin on the activation of the transcription factors nuclear factor B, activator protein-1 and nuclear factor-interleukin-6, and interleukin-6 and interleukin-8 mRNA expression in a human endothelial cell line. Clinical Science 99, 40510.[ISI][Medline]
28 . Galley, H. F., Nelson, S. J., Dubbels, A. M. & Webster, N. R. (1997). Effect of ciprofloxacin on the accumulation of interleukin-6, interleukin-8, and nitrite from a human endothelial cell model of sepsis. Critical Care Medicine 25, 13925.[ISI][Medline]
29
.
Riesbeck, K., Sigvardsson, M., Leanderson, T. & Forgsten, A. (1994). Superinduction of cytokine gene transcription by ciprofloxacin. Journal of Immunology 153, 34352.
30 . Zakeri, S. M., Meyer, H., Meinhardt, G., Reinisch W., Schrattebauer K., Knoefler, M. et al. (2000). Effects of trovafloxacin on the IL-1-dependent activation of E-selectin in human endothelial cells in vitro. Immunopharmacology 48, 2734.[ISI][Medline]
31 . Lu, H., Yang, X., Takeda, K., Zhang, D., Fan, Y., Luo, M. et al. (2000). Chlamydia trachomatis mouse pneumonitis lung infection in IL-18 and IL-12 knockout mice: IL-12 is dominant over IL-18 for protective immunity. Molecular Medicine 6, 60412.[ISI][Medline]
32
.
Geng, Y., Berencsi, K., Gyulai, Z., Valyi-Nagy T., Gonczol, E. & Trinchieri, G. (2000). Roles of interleukin-12 and interferon in murine Chlamydia pneumoniae infection. Infection and Immunity 68, 224553.
33
.
Huang, J., Wang, M. D., Lenz, S., Gao, D. & Kaltenboeck, B. (1999). IL-12 administered during Chlamydia psittaci lung infection in mice confers immediate and long-term protection and reduces macrophage inflammatory protein-2 level and neutrophil infiltration in lung tissue. Journal of Immunology 162, 221726.