Department of Microbiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohta-ku, Tokyo 143-8540, Japan
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Abstract |
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Introduction |
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In this study, we examined the in vivo antimicrobial activities of glycylcycline against penicillin-resistant S. pneumoniae (PRSP) in a non-compromised mouse model of pneumonia.
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Materials and methods |
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Clinical isolates of S. pneumoniae from Toho University Hospital (Tokyo, Japan) collected from 1992 to 1994 were used for in vitro antibiotic susceptibility testing. Isolates for which the MIC of ampicillin was 0.06 mg/L were designated as penicillin susceptible, whereas those with MICs of ampicillin >1 mg/L were designated as penicillin resistant. Strain TUM19, a penicillin-resistant pneumococcus (MIC of penicillin G, 2 mg/L), was used for the preparation of the mouse model of pneumonia.
Antibiotics used
The following drugs were kindly provided by the following companies: glycylcycline, tetracycline and minocycline, Lederle Japan, Tokyo, Japan; penicillin G and ampicillin, Meiji Seika, Tokyo; erythromycin, Shionogi & Co., Osaka, Japan.
Antimicrobial susceptibility testing
MICs of antibiotics were determined by a broth dilution method using MuellerHinton broth (Difco, Detroit, MI, USA) supplemented with 5% lysed horse blood. An inoculum of 5.0 x 104 cfu/well was incubated with the antibiotic in microtitre plates at 35°C for 18 h. The MIC was defined as the lowest concentration of the antimicrobial agent that inhibited visible growth of S. pneumoniae.4
Mouse pneumonia model
Non-compromised CBA/J mice (5-week-old females, Charles River Japan) were used for experimentally induced pneumonia, as described previously.5,6 Briefly, the bacterial suspension (40 µL) was inoculated intranasally in anaesthetized mice. Antibiotics (10 mg/kg) were injected sc into mice daily for 3 days commencing 48 h after infection. Twenty-four hours after treatment, the mice were killed and the lungs were removed immediately. The lungs were homogenized in saline, and 0.1 mL of serial 10-fold dilutions of the homogenate was plated on blood agar for determination of viable bacterial counts. Results were expressed as the mean log cfu per lungs ± s.d. (n = 5 for each group).
Pharmacokinetic studies
Serum and lung samples were collected from three mice in each group, at 5, 15, 30, 60, 120, 240 and 360 min following sc injection of 50 mg/kg body weight of each antibiotic. Antibiotic concentrations in the serum and lung homogenates were determined by the paper disc method using Bacillus subtilis ATCC6633 for minocycline and penicillin G and Bacillus cereus ATCC11778 for glycylcycline as bioassay reference strains.
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Results |
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Glycylcycline showed the most potent antibacterial activity (Table I). Regardless of susceptibility to penicillin, MICs of glycylcycline against all strains examined were
0.06 mg/L. The MIC90 of glycylcycline was 100-fold lower than that of minocycline (8 mg/L) or tetracycline (32 mg/L).
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The MICs of glycylcycline, minocycline and penicillin G against S. pneumoniae TUM19 were, respectively, 0.031, 8 and 2 mg/L. Glycylcycline decreased bacterial counts in the lungs from 106 cfu to below 102 cfu after a single administration. In contrast, no apparent reduction in the bacterial count was observed with minocycline or penicillin G, even after three injections.
Effect of various doses of glycylcycline on pneumonia caused by S. pneumoniae TUM-19
We also examined the effect of three different doses of glycylcycline, 0.4, 2 and 10 mg/kg, each injected sc daily for 3 days from 2 days after infection. A clear dose-dependent effect on lung bacterial counts was noted after administration of glycylcycline. Thus, 10 mg/kg of glycylcycline induced a >4 log decrease in bacterial count in the lungs after a single administration, which was followed by complete clearance of bacteria 6 days after infection. On the other hand, a significant, but less marked reduction of bacterial numbers, approximately 2 log decrease, was also induced by treatment with glycylcycline at 2 mg/kg body weight. However, only a minimal effect was observed in mice treated with 0.4 mg/kg of glycylcycline.
Pharmacokinetic studies
The half-life of glycylcycline in the lungs was 5.56 h, which was about 1.6 and 31 times longer than that for minocycline and penicillin G, respectively (Table II). The AUC of glycylcycline in the lungs was about 7 and 13 times that for minocycline and penicillin G, respectively. Importantly, the ratio of AUC in the lung/serum was higher for glycylcycline (2.2), than for minocycline (1.0) and penicillin G (0.6). Another feature of glycylcycline was its preferential distribution in the lungs compared with the other antibiotics examined.
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Discussion |
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We have recently developed a lethal mouse model of PRSP pneumonia, in which non-compromised CBA/J mice are infected with the bacteria following intranasal instillation.5,6 This model provided us with the opportunity to investigate the in vivo efficacy of antibiotics against PRSP pneumonia in healthy individuals. Interestingly, a single injection of glycylcycline induced a marked reduction in bacterial count in the lungs from 106 cfu to <102 cfu. In contrast, no apparent reduction in bacterial count was observed when the same dose of minocycline or penicillin G was used, even after three injections.
The MIC of glycylcycline against S. pneumoniae TUM19 was 0.031 mg/L, which was 285- and 64-fold lower than that of minocycline and penicillin G, respectively. The potent in vitro antibacterial effects of glycylcycline were also confirmed against several clinical isolates of pneumococci. Moreover, clear differences in several pharmacokinetic factors were observed between the antibiotics examined in the present study. In particular, the half-life of glycylcycline in the lungs was superior to that of minocycline or penicillin G.
There are substantial differences in pharmacokinetic parameters between mice and humans. In addition, any immediate or late adverse reactions due to glycylcycline may limit the clinical usefulness of this antibiotic. Further animal and clinical studies using PRSP infection are warranted to test the effectiveness of this new compound and also its derivatives.
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Notes |
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References |
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2 . Weiss, W. J., Jacobus, N. V., Petersen, P. J. & Testa, R. T. (1995). Susceptibility of enterococci, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae to the glycylcyclines. Journal of Antimicrobial Chemotherapy 36, 22530.[Abstract]
3 . Wise, R. & Andrews, J. M. (1994). In vitro activities of two glycylcyclines. Antimicrobial Agents and Chemotherapy 38, 1096102.[Abstract]
4 . National Committee for Clinical Laboratory Standards. (1991). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Second Edition: Approved Standard M7-A2. NCCLS, Villanova, PA.
5 . Takashima, K., Tateda, K., Matsumoto, T., Ito, T., Iizawa, Y., Nakao, M. et al. (1996). Establishment of a model of penicillinresistant Streptococcus pneumoniae pneumonia in healthy CBA/J mice. Journal of Medical Microbiology 45, 31922.[Abstract]
6 . Tateda, K., Takashima, K., Miyazaki, H., Matsumoto, T., Hatori, T. & Yamaguchi, K. (1996). Noncompromised penicillin-resistant pneumococcal pneumonia CBA/J mouse model and comparative efficacies of antibiotics in this model. Antimicrobial Agents and Chemotherapy 40, 15205.[Abstract]
Received 14 September 1999; returned 22 November 1999; revised 11 April 2000; accepted 30 May 2000