1 Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, USA; 2 Daiichi Pharmaceutical Company, Ltd, Tokyo, Japan
Received 7 June 2002; returned 2 August 2002; revised 9 August 2002; accepted 20 August 2002
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Abstract |
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Keywords: sitafloxacin, Mycobacterium ulcerans, ofloxacin, rifampicin
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Introduction |
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Despite the promising results in vitro4 and in laboratory animals,5 the treatment of these ulcers has been disappointing, especially in patients with extensive ulcers; large surgical excision of the necrotic tissue followed by skin grafting is, at present, the only treatment. Even though M. ulcerans is resistant to many antituberculosis drugs in vitro, antimicrobials could play a key role in the prevention of post-surgical recurrence of Buruli ulcers, which occurs frequently.
A new fluoroquinolone, sitafloxacin (DU-6859a), has been shown to be more potent than ofloxacin against mycobacteria, including M. tuberculosis.6 Thus, the aim of this study was to determine the activity of sitafloxacin, along with standard quinolones, used singly or in combination with rifampicin, against M. ulcerans.
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Materials and methods |
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Two type strains of M. ulcerans were used: ATCC 19423 and ATCC 35840. Six other M. ulcerans strains isolated from six countries were also included in this study. Isolates and type strains were maintained on LowensteinJensen medium. Whenever needed, colonies from LowensteinJensen medium were subcultured in Middlebrook 7H9 broth containing OADC enrichment (where OADC stands for oleic acid + albumin + dextrose + catalase).
Antimicrobial agents
Sitafloxacin, ()-7[(7S)-amino-5-azaspiro{2,4}heptan-5-yl]-8-chloro-6-fluoro[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid sesquihydrate, also known as DU-6859a, was obtained from Daiichi Pharmaceutical Company, Tokyo, Japan. Stock solutions of sitafloxacin, and levofloxacin and ofloxacin (both from R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA) were prepared fresh in 0.1 M NaOH. Stock solution of ciprofloxacin (Miles Laboratories, West Haven, CT, USA) was prepared in distilled water. Stock solution of rifampicin (Sigma Chemical Company, St Louis, MO, USA) was prepared by dissolving first in small volumes of methanol and diluting further in water. Stock solutions were further diluted appropriately in distilled water to prepare working solutions that were filter sterilized through a GA-6 membrane filter (pore size 0.22 µm; Gelman Sciences, Ann Arbor, MI, USA).
The procedures described by Heifets et al.7 (BACTEC method and plate counts) were followed to determine the MICs and MBCs, and also to assess the combined inhibitory effects. To assess the combined inhibitory effects, each drug was tested at concentrations below its MIC in serial two-fold dilutions (e.g. 2+2, 2+4 and 2+8, etc.), and the interaction indicator or fractional inhibitory concentration (FIC) was calculated. For each sample, control as well as with drug, triplicate assays were carried out in each case and statistical significance was determined by the Students t-test.
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Results |
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Discussion |
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The non-AIDS-associated infections caused by non-tuberculosis mycobacteria are increasing. Among these, infection caused by M. ulcerans poses the greatest public health threat and thus is rapidly becoming the third most prevalent mycobacterial disease after tuberculosis and leprosy.
Among the various antimycobacterial drugs that have already been tested in vitro4 against M. ulcerans, only rifampicin was evaluated in mice and found to be effective.5 However, pre-ulcerative and early ulcerative, but not advanced ulcers, can be effectively treated with rifampicin.8 Recently, Thangaraj et al.9 have shown superiority of sparfloxacin over ofloxacin and ciprofloxacin in inhibiting the in vitro growth of M. ulcerans. However, sparfloxacin was later found to be ineffective against M. ulcerans infection in mice.10 Most Buruli ulcer patients carry massive bacterial loads at some stage of the disease, thus creating an ideal situation for the selection of drug-resistant mutants. In such a situation, combined therapy, as in any other mycobacterial infection, will be highly advantageous. Since combination of sitafloxacin and rifampicin is effective in both in vitro as well as in vivo studies, such a combination could have potential for the chemotherapeutic treatment of advanced ulcers.
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Acknowledgements |
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Footnotes |
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References |
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2 . Dodge, O. G. & Lunn, H. F. (1962). Buruli ulcer: a mycobacterial skin ulcer in a Ugandan child. Journal of Tropical Medicine and Hygiene 65, 13942.[ISI][Medline]
3 . World Health Organization. (2000). Buruli Ulcer: Mycobacterium Ulcerans Infection (Asiedu, K. R., Scherpbier, R. & Raviglione, M., Eds). WHO/CDS/CPE/GBUI/1, WHO, Geneva, Switzerland.
4 . Schroeder, K. H. (1975). Investigation of the relationship of Mycobacterium ulcerans to Mycobacterium buruli. American Review of Respiratory Diseases 111, 55962.
5 . Srivastava, A., Singh, N. B. & Gupta, S. K. (1984). Prospective treatment for Mycobacterium ulcerans infection in rats and mice through combined therapy with rifampicin and septran. Current Science 53, 4878.[ISI]
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Tomioka, H., Sato, K., Akaki, T., Kajitani, H., Kawahara, S. & Sakatani, M. (1999). Comparative in vitro antimicrobial activities of the newly synthesized quinolone HSR-903, sitafloxacin (DU-6859a), gatifloxacin (AM-1155) and levofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Antimicrobial Agents and Chemotherapy 43, 30014.
7 . Heifets, L. B., Iseman, M. D. & Lindholm-Levy, P. J. (1988). Combinations of rifampicin or rifabutin plus ethambutol against Mycobacterium avium complex. Bactericidal synergistic, and bacteriostatic additive or synergistic effects. American Review of Respiratory Diseases 137, 7115.
8 . Meyers, W. M., Shelly, W. M. & Connor, D. H. (1974). Heat treatment of Mycobacterium ulcerans infections without surgical incision. American Journal of Tropical Medicine and Hygiene 23, 9249.[ISI][Medline]
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Thangaraj, H. S., Adjei, O., Allen, B. W., Portaels, F., Evans, M. R. W., Banerjee, D. K. et al. (2000). In vitro activity of ciprofloxacin, sparfloxacin, ofloxacin, amikacin and rifampicin against Ghanaian isolates of Mycobacterium ulcerans. Journal of Antimicrobial Chemotherapy 45, 2313.
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Dega, H., Robert, J., Bonnafous, P., Jarlier, V. & Grosset, J. (2000). Activities of several antimicrobials against Mycobacterium ulcerans infection in mice. Antimicrobial Agents and Chemotherapy 44, 236772.
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