a Clinical Microbiology Laboratory and b Department of Infection Control, Oulu University Hospital; and c Department of Medical Microbiology, Universityof Oulu, Oulu; d The National Veterinary and Food Research Institute, Helsinki, Finland
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Abstract |
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Introduction |
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Materials and methods |
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Thirty-eight strains of F. tularensis were examined, 20 from human samples and 18 strains from dead wild animals (16 hares and two muskrats). F. tularensis tularensis ATCC 6223 and F. tularensis palaearctica ATCC 29684, the live vaccine strain kindly donated by Dr Anders Sjöstedt, Umeå, Sweden, were used as control strains.
Identification of F. tularensis strains
F. tularensis was incubated for 37 days at 35°C in a 5% CO2 atmosphere on chocolate agar plates. Preliminary identification of the strains was done by Gram's stain, catalase test with 15% H2O2, oxidase test, API 20E (bioMérieux, Lyon, France) and slide agglutination with a pooled human serum with a high titre (1:640) of anti-F. tularensis antibodies and indirect fluorescence staining with the same anti- F. tularensis serum and fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (Sigma, St Louis, MO, USA).
DNA was extracted from pure cultures of F. tularensis strains using a commercial DNA extraction kit (QIAamp Tissue Kit; Qiagen GmbH, Hilden, Germany). The primer sequences and amplification conditions for the membrane protein of F. tularensis were according to Long et al.7 and those for the 17 kDa lipoprotein gene fragment according to Sjöstedt et al.8 The PCR products (250 and 400 bp, respectively) were visualized by ethidium bromide staining after gel electrophoresis in a gel containing 1.5% agarose. A Gene Ruler 100 bp DNA Ladder (MBI Fermentas, Vilnius, Lithuania) was run in parallel with PCR products.
Antibiotic susceptibility tests
The susceptibility of the F. tularensis strains to the 17 antibiotics listed in the Table was determined using Etests (Biodisk, Solna, Sweden) on Bacto cysteine heart agar plates with 2% haemoglobin (Difco Laboratories, Detroit, MI, USA). The inoculum was adjusted to the density of a McFarland 0.5 turbidity standard and resulted in confluent growth. The MICs were read after incubation overnight or for 2 nights at 35°C in a 5% CO2 atmosphere.
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Results |
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All the strains studied grew well on chocolate agar, yielding smooth, opaque, grey or greyish green colonies about 2 mm in diameter after incubation for 37 days. F. tularensis are small, punctiform Gram-negative bacilli, weakly catalase-positive, oxidase-negative, give no reactions in the API20E panel and positive results in both slide agglutination and indirect fluorescent antibody (FA) staining tests. The PCR products for both species-specific primer pairs were obtained for all the F. tularensis strains tested. No false-positive or false-negative fragments were observed. The control strains, ATCC 6223 and ATCC 29684, gave PCR products of identical size (Figure).
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The Etest MICs are presented in the Table with the criteria of NCCLS for Enterobacteriaceae.9 All the clinical F. tularensis strains were highly resistant to ß-lactam antibiotics, including piperacillintazobactam (>256 mg/L), ceftriaxone (>32 mg/L), ceftazidime (>256 mg/L), cefpirome (>256 mg/L), meropenem (>32 mg/L) and imipenem (>32 mg/L), and to azithromycin (>256 mg/L). The strains were susceptible to aminoglycosides (gentamicin, tobramycin and streptomycin), quinolones (ciprofloxacin, levofloxacin, grepafloxacin and trovafloxacin), tetracycline, rifampicin and chloramphenicol. The MICs for the F. tularensis ATCC control strains were generally lower than those for the clinical strains, with the exception of azithromycin (Table
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Discussion |
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In our series, the MIC50s and MIC90s of streptomycin were identical to those reported previously from the USA for 13 strains of type A and two strains of type B.10 Similar results, with marginally higher MICs for the type B strains, have been reported from Norway.6 Thus, according to in vitro studies, type B infections with F. tularensis could also be treated with streptomycin, tetracycline and chloramphenicol.
Gentamicin has been considered an acceptable parenteral alternative to streptomycin for the treatment of tularaemia.4 The MICs of gentamicin and tobramycin found in the present study were identical to those reported from the USA.
At least in Europe, F. tularensis of type B usually causes quite mild infections, while ulceroglandular tularaemia with primary ulceration and regional lymphadenopathy is the most common form of tularaemia. These infections are not usually severe enough to require hospitalization, so oral antibiotic treatment at home would be ideal. Our in vitro studies, as well as some earlier reports, show that quinolones are a promising alternative for such treatment of tularaemia.5,6 Ciprofloxacin, levofloxacin, grepafloxacin and trovafloxacin had similar low MIC90s, of <0.05 mg/L.
All the type B F. tularensis strains studied here were resistant to ß-lactams. A similar finding has been reported previously.6 In contrast, type A strains have been reported to be sensitive to some ß-lactams.10 Although type A strains have been reported to be sensitive to erythromycin, at least the Scandinavian type B strains are usually resistant to macrolides in that resistance has been described to erythromycin, roxithromycin and clarithromycin6 and, in the present study, also to azithromycin.
Both type A strains10 and type B strains (this study) of F. tularensis are sensitive to rifampicin, which might be useful, for example, in combination with aminoglycosides or quinolones, in severe cases of tularaemia.
In conclusion, our results suggest that, apart from traditional drugs (tetracycline and chloramphenicol), tularaemia might also be treated orally with quinolones, which would allow ambulatory treatment.
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Acknowledgments |
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Notes |
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References |
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2 . Cross, J. T. & Penn, R. L. (2000). Francisella tularensis (tularemia). In Principles and Practice of Infectious Diseases, 5th edn, (Mandell, G. L., Bennett, J. E. & Dolin, R., Eds), pp. 2393402. Churchill Livingstone, New York, NY.
3 . Evans, M. E., Gregory, D. W., Schaffner, W. & McGee, Z. A. (1985). Tularaemia: a 30-year experience with 88 cases. Medicine 64, 25169.[ISI][Medline]
4 . Enderlin, G., Morales, L., Jacobs, R. F. & Cross, J. T. (1994). Streptomycin and alternative agents for the treatment of tularaemia: review of the literature. Clinical Infectious Diseases 19, 427.[ISI][Medline]
5 . Syrjälä, H., Schildt, R. & Räisänen, S. (1991). In vitro susceptibility of Francisella tularensis to fluoroquinolones and treatment of tularemia with norfloxacin and ciprofloxacin. European Journal of Clinical Microbiology and Infectious Diseases 10, 6870.[ISI][Medline]
6 . Scheel, O., Hoel,T., Sandvik, T. & Berdal, B. P. (1993). Susceptibility pattern of Scandinavian Francisella tularensis isolates with regard to oral and parenteral antimicrobial agents. Acta Pathologica Microbiologica et Immunologica Scandinavica 101, 336.
7 . Long, G. W., Oprandy, J. J., Narayanan, R. B., Fortier, A. H., Porter, K. R. & Nacy, C. A. (1993). Detection of Francisella tularensis in blood by polymerase chain reaction. Journal of Clinical Microbiology 31, 1524.[Abstract]
8 . Sjöstedt, A., Eriksson, U., Berglund, L. & Tärnvik, A. (1997). Detection of Francisella tularensis in ulcers of patients with tularemia by PCR. Journal of Clinical Microbiology 35, 10458.[Abstract]
9 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility TestingEighth Informational Supplement M100-S8. NCCLS, Wayne, PA.
10 . Baker, C. N., Hollis, D. G. & Thornsberry, C. (1985). Antimicrobial susceptibility testing of Francisella tularensis with a modified MuellerHinton broth. Journal of Clinical Microbiology 22, 2125.[ISI][Medline]
Received 11 November 1999; returned 6 February 2000; revised 27 March 2000; accepted 7 April 2000