INSERM U.411, Laboratoire de Microbiologie, Faculté de Médecine Necker-Enfants Malades 156, rue de Vaugirard, 75730 Paris Cedex 15, France
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Abstract |
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Introduction |
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Materials and methods |
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We used L. monocytogenes strain EGD (serovar 1/2a) grown in brainheart infusion broth (Difco, Detroit, MI, USA), harvested during log-phase (10 cfu/mL),8 dispensed in vials in 1 mL aliquots, and stored at -80°C until used. Bacteria were routinely plated in tryptocaseinsoy agar (bioMérieux, Marcy-l'Etoile, France).
Infection of gerbils and antibiotic therapy
Female Mongolian gerbils (8-week-old, 3545 g) from Janvier (Le Genest Saint-Isle, France), were anaesthetized by ketamine and xyloxine as described previously8 and inoculated percutaneously into the superior chamber of both middle ears with 25µL of a bacterial suspension. We tested seven antibiotics: amoxycillin, co-trimoxazole, rifampicin, imipenem, vancomycin, gentamicin and ciprofloxacin. The MICs for strain EGD determined by the dilution method on MuellerHinton agar (bioMérieux) were amoxycillin 0.03 mg/L, imipenem 0.03 mg/L, gentamicin 0.06 mg/L, rifampicin 0.12 mg/L, ciprofloxacin 1 mg/L and vancomycin 1.5 mg/L. Antibiotics were administered subcutaneously bd from day three to day four (three injections). To avoid any carry-over of antibiotics when bacteria were plated, organs were removed 24 h after the last administration of antibiotics in each treated group. Groups of five gerbils were killed by cervical dislocation on day three, just before antibiotic treatment and on day five. Organs (spleen, liver, brain, rhombencephalum) were aseptically removed and ground in 0.15 M NaCl. After sectioning the spinal cord, brains and rhombencephalum were extracted, separated by dissection and homogenized. One hundred microlitres of serial 10-fold dilutions of homogenates were plated on tryptocaseinsoy agar. Colonies were counted after 24 incubation at 37°C. The detection limits were 102 bacteria per liver and spleen (ground in a final volume of 10 mL), and 20 bacteria per cerebrum and rhombencephalum (ground in a final volume of 1 mL). Statistical comparisons were made by a one-way analysis of variance, and probability values <0.05 in Fisher's test were significant. This study was performed in accordance with the regulations regarding the care of laboratory animals in the European Communities.
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Results and discussion |
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Bacterial counts observed in untreated gerbils on day five were compared with those obtained from organs of antibiotic-treated gerbils. Amoxycillin, co-trimoxazole and rifampicin were the most bactericidal antibiotics in vivo (Table I). A doseresponse was observed with these antibiotics with a rapid decrease of bacteria (approximately 2.03.0 log10) in the spleen, the liver and the CNS (3.040 log10). Imipenem was less active in the CNS than amoxycillin (Table I). Gentamicin, vancomycin and ciprofloxacin alone had little or no activity (Table I). Vancomycin has been reported to be active against listeriosis with bacteraemia,6 but our results strongly suggest that vancomycin is unlikely to be effective in patients with CNS infection, as previously suspected.7 Ciprofloxacin was only weakly active in the spleen, liver and CNS, probably because of its high MIC (1 mg/L) and its weak intracellular activity against L. monocytogenes.4,9 However, a strong bactericidal activity has been reported recently for new fluoroquinolones (sparfloxacin, levofloxacin, clinafloxacin) in a model of HeLa cells in vitro.10 We tested the efficacy of three combinations, amoxycillinco-trimoxazole, amoxycillingentamicin and rifampicinco-trimoxazole (Table II). Gentamicin did not significantly improve the antibiotic activity of amoxycillin in the CNS, as previously reported.3 Similarly, co-trimoxazole and rifampicin only weakly improved the bactericidal activity of amoxycillin or co-trimoxazole, respectively (Table II). The results obtained with this new model of rhombencephalitis in gerbils reproducing the major features of human listeriosis strongly suggest that amoxycillin, co-trimoxazole and rifampicin can be used safely in humans in monotherapy or in combination. This model will be of great value for testing new antibiotics and studying new drugs which increase the intracerebral diffusion of antibiotics.
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Acknowledgments |
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Notes |
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References |
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2 . Cherubin, C. E., Appleman, M. D., Heseltine, P. N. R., Khayr, W. & Stratton, C. W. (1991). Epidemiological spectrum and current treatment of listeriosis. Review of Infectious Diseases 13, 11084.[ISI][Medline]
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4 . Michelet, C., Avril, J. P., Cartier, F. & Berche, P. (1994). Inhibition of intracellular growth of Listeria monocytogenes by antibiotics. Antimicrobial Agents and Chemotherapy 38, 43846.[Abstract]
5 . Spitzer, P. G., Hammer, S. M. & Karchmer, A. W. (1986). Treatment of Listeria monocytogenes infection with trimethoprimsulfamethoxazole: case report and review of the literature. Review of Infectious Diseases 3, 42730.
6 . Blatt, S. P. & Zajac, R. A. (1991). Treatment of Listeria bacteremia with vancomycin. Review of Infectious Diseases 136, 812.
7 . Baldassarre, J. S., Ingerman, M. J., Nansteel, J. & Santoro, J. (1991). Development of Listeria meningitis during vancomycin therapy: a case report. Journal of Infectious Diseases 164, 2212.[ISI][Medline]
8 . Blanot, S., Muffat-Joly, M., Vilde, F., Jaubert, F., Clement, O., Frijat, G. et al. (1997). A gerbil model for rhombencephalitis due to Listeria monocytogenes. Microbial Pathogenesis 23, 3948.[ISI][Medline]
9 . Van, Ogtrop, M. L., Mattie, H., Sekh, B. R., Van, Strijen E. & Furth, Van R. (1992). Comparison of the antibacterial efficacies of ampicillin and ciprofloxacin against experimental infections with Listeria monocytogenes in hyrocorticone-treated mice. Antimicrobial Agents and Chemotherapy 36, 237580.[Abstract]
10 . Michelet, C., Avril, L., Arvieux, C., Jacquelinet, C., Vu, N. & Cartier, F. (1997). Comparative activities of new fluoroquinolones, alone or in combination with amoxicillin, trimethoprimsulfamethoxazole, or rifampin, against intracellular Listeria monocytogenes. Antimicrobial Agents and Chemotherapy 41, Suppl. 1, 605.[Abstract]
Received 16 November 1998; returned 8 March 1999; revised 22 March 1999; accepted 31 May 1999