1 Hôpital diInstruction des armées Bégin, Biologie médicale, Saint-mandé; 2 Centre détudes du Bouchet, Laboratoire de microbiologie, Vert-le-petit; 3 Centre de recherches du service de santé des armées, Laboratoire de microbiologie, La tronche, France
Received 10 April 2003; returned 21 May 2003; revised 13 August 2003; accepted 25 September 2003
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Abstract |
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Keywords: Y. pestis, antibiotic susceptibility, plague
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Introduction |
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Streptomycin is the drug of choice for the treatment of infection in humans but is no longer available in some countries. Tetracycline and chloramphenicol have been demonstrated to be effective alter-natives in humans.1 In animal models, doxycycline, ciprofloxacin and ofloxacin have demonstrated their effectiveness, but have not been employed as first-line options for plague treatment in humans. Penicillins and cephalosporins are not effective in vivo.2 Resistance to antibiotics is exceptional, but a high level of resistance to multiple antibiotics as a result of a self-transferable plasmid has been observed in a strain isolated from a patient with bubonic plague.3 Another isolate, highly resistant to streptomycin has also been described.4 Furthermore, Wong et al. published a percentage of 20.6% resistance to imipenem among 92 strains isolated over a 21-year period in the United States.5
As described by Inglesby et al.,6 the use of an aerosolized plague weapon could cause fever, cough, chest pain and haemoptysis with signs consistent with severe pneumonia 16 days after exposure. Rapid evolution of disease would occur in the 24 days after symptom onset and would lead to septic shock with high mortality without early treatment.6 Tetracycline, streptomycin, gentamicin and fluoroquinolone antibiotics have been recommended for post-exposure prophylaxis.6 There is, however, a need to continue to carry out in vitro susceptibility testing to identify potential alternative agents for the treatment of plague.
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Materials and methods |
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All the experiments were conducted in the BSL3 laboratory of the Centre dEtudes du Bouchet (Vert-le-petit, France), which is part of the French Ministry of Defence.
Bacterial strains
Ninety-four isolates of the collection of the Centre dEtudes du Bouchet (Vert-le-petit, France) were included in this protocol. Bacteria are stored in the BSL3 laboratory of the institute. Isolates were collected between 1964 and 1988 and were from Saigon (12, isolated in 1964), Dalat (45 isolated in 1966, 16 in 1967 and five in 1968), Madagascar (two isolated in 1981, and three in 1988), Kurdistan (one, isolated in 1979), Birmania (two, isolated in 1979), Java (two, isolated in 1979) and India (two, isolated in 1979). The other strains tested were CIP 6/69M, the vaccinal strain EV76 and two strains provided by the Pasteur Institute of Lille, France. Among these strains six were medievalis, six were orientalis type 1, and the other strains were orientalis type 2. Typing was done as described by Motin et al.7 The host of origin is unknown.
Susceptibility testing
Antibiotics tested were: amoxicillin (Inava, France), co-amoxiclav (clavulanic acid 2 mg/L) (Smith-Kline-Beecham, France), piperacillin (Dakota, France), piperacillin/tazobactam (tazobactam 4 mg/L) (Wyeth-Lederle, France), cefalothin (Panpharma, France), cefoxitin (Merck-Sharp-Dohme-Chibret, France), cefotaxime (Roussel-Diamand, France), ceftazidime (Glaxo-Wellcome, France), aztreonam (Sanofi-Winthrop, France), imipenem (Merck-Sharp-Dohme-Chibret, France), nalidixic acid (Sanofi-Winthrop, France), ofloxacin (Roussel-Diamand, France), pefloxacin (Bellon, France), ciprofloxacin (Bayer, France), norfloxacin (Glaxo-Wellcome, France), gatifloxacin (Grunenthal, France), trimethoprim/sulfamethoxazole (Roche, France), streptomycin (Pharmacie centrale des armies, France), gentamicin (Schering-Plough), amikacin (Bristol-Myers-Squibb, France), tobramycin (Lilly, France), doxycycline (Asta-medica, France), chloramphenicol (Chauvin, France) and colistin (Bellon, France). The concentrations of ß-lactamase inhibitors described were used with all dilutions of amoxicillin and piperacillin.
Antimicrobial agents were reconstituted according to manufacturers recommendations. Susceptibility testing was done by the agar-dilution method in MuellerHinton medium. After identification by routine laboratory technique (Biolog system, Hayward, CA, USA), three or four colonies of each isolate were plated on blood agar and incubated for 48 h at 28°C. Colonies from plates were then suspended in PBS in order to obtain a final inoculum of 108 cfu/mL. One hundred microlitres was added to 3.9 mL of MuellerHinton broth and incubated for 24 h in a water bath at 28°C. Final inoculum was prepared before inoculation and was 104 cfu/spot. After inoculation, agar-plates were incubated for 48 h at 28°C. Two operators read minimal inhibitory concentrations (MICs). Quality control was Escherichia coli ATCC 25922. Interpretative criteria were done according to the Comité de lAntibiogramme de la Société Française de Microbiologie.8 These recommendations are freely available at http://www.sfm.asso.fr.
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Results and discussion |
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In conclusion, Y. pestis remains susceptible to most antibiotics tested (except colistin) with a higher efficacy for fluoroquinolones, third-generation cephalosporins and aminoglycosides. All the strains tested were susceptible to the antibiotics recommended for post-exposure prophylaxis. However, further in vivo studies are needed for determining alternative antibiotic treatments in case of bioterrorist attack with strains resistant to recommended antibiotics.
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Acknowledgements |
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Footnotes |
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References |
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2
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Byrne, W. R., Welkos, S. L., Pitt, M. L. et al. (1998). Antibiotic treatment of experimental pneumonic plague in mice. Antimicrobial Agents and Chemotherapy 42, 67581.
3
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Galimand, M., Guiyoule, A., Gerbaud, G. et al. (1997). Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. New England Journal of Medicine 337, 67780.
4 . Guiyoule, A., Gerbaud, G., Buchrieser, C. et al. (2001). Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis. Emerging Infectious Diseases 7, 438.[ISI][Medline]
5
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Wong, J. D., Barash, J. R., Sandfort, R. F. et al. (2000). Susceptibilities of Yersinia pestis strains to 12 antimicrobial agents. Antimicrobial Agents and Chemotherapy 44, 19956.
6
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Inglesby, T. V., Dennis, D. T., Henderson, D. A. et al. (2000). Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. Journal of the American Medical Association 283, 228190.
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Motin, V. L., Georgescu, A. M., Elliott, J. M. et al. (2002). Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 genotyping and analysis of structural genes encoding glycerol-3-phosphate dehydrogenase (glpD). Journal of Bacteriology 184, 101927.
8 . Comité de lAntibiogramme de la Société Française de Microbiologie. (2003). Report 2003. International Journal of Antimicrobial Agents 21, 36491.[CrossRef][ISI][Medline]
9 . Frean, J. A., Arntzen, L., Capper, T. et al. (1996). In vitro activities of 14 antibiotics against 100 human isolates of Yersinia pestis from a southern African plague focus. Antimicrobial Agents and Chemotherapy 40, 26467.[Abstract]
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Rahalison, L., Guiyoule, A., Bonacorsi, S. P. et al. (2000). Failure of oily chloramphenicol depot injection to treat plague in a murine model. Journal of Antimicrobial Chemotherapy 45, 5415.