Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, USA
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Abstract |
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Introduction |
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Despite promising results in vitro and in laboratory animals, the treatment of these ulcers has been disappointing. Surgery is widely regarded as the definitive treatment, removing necrotic tissue. Early pre-ulcerative lesions can be treated effectively with rifampicin alone or by heating at 40°C.2 However, rifampicin is not usually effective against advanced ulcerative lesions.
Pattyn & Ermengem3 had earlier demonstrated the sensitivity of M. ulcerans to dapsone. Our previous work revealed that inhibitors of folate biosynthesis had bactericidal activity against Mycobacterium leprae and also exhibited synergy when combined with dapsone.4 Thus, the objective of the current study was to evaluate the effects of these dihydrofolate reductase inhibitors, when used singly as well as in combination with dapsone, on in vitro growth of 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. In addition, six other M. ulcerans strains isolated from six countries were also included in this study. Isolates and strains were maintained on Lowenstein Jensen medium. Whenever needed, colonies from LowensteinJensen medium were subcultured in Middlebrook 7H9 broth containing OADC enrichment.
Antimicrobial agents
Epiroprim {2,4-diamino-5[3,5-diethoxy-4-(1-pyrrolyl)-benzyl]-pyrimidine} and brodimoprim [2,4-diamino-5-(4 bromo-3,5-dimethoxybenzyl) pyrimidine] were obtained from F. Hoffmann-La-Roche Ltd (Basel, Switzerland); K-130 [2,4-diamino-diphenyl sulphone-substituted 2,4-diamino-4-benzyl-pyrimidine] was obtained from Professor Seydel, Forschungszentrum Borstel (Borstel, Germany); trimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl) pyrimidine] and dapsone [4,4'-diaminodiphenyl sulphone] were obtained commercially from Sigma Chemical Co. (St Louis, MO, USA). Stock solutions were first prepared by dissolving the individual compounds in small quantities of dimethylsulphoxide and then diluting further with distilled water. Each working solution was then filter sterilized through a GA-6 membrane filter (pore size 0.22 µm; Gelman Sciences, Ann Arbor, MI, USA).
The procedures described by Heifets and co-workers5 were followed to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), and also to assess the combined inhibitory effects.
For each sample, control as well as with drug, triplicate assays were performed in each case, and statistical significance was determined by Student's t-test.
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Results |
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Discussion |
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We have previously shown excellent synergy between brodimoprim and dapsone against M. leprae, both in vitro and in mice.7 Similar findings were also reported recently with the combinations of K-130 and dapsone8 and of epiroprim and dapsone4 against M. leprae. Thus, it seems appropriate to evaluate further the effects of combining epiroprim and dapsone against M. ulcerans infection in mice, and we are in the process of undertaking these studies.
The non-AIDS-associated infections caused by non-tuberculous mycobacteria are increasing.9 Among these, infection caused by M. ulcerans poses the greatest public health threat. This infection is rapidly becoming the third most prevalent mycobacterial disease after tuberculosis and leprosy.10
At some stage of the disease, most of the patients with M. ulcerans infection carry massive bacterial loads and this creates an ideal situation for the selection of drug-resistant mutants. In such a situation, as in any other mycobacterial infection, combined therapy may be advantageous. Thus, the combination of epiroprim and dapsone, along with rifampicin, could have great potential in the clinical treatment of advanced ulcers.
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Acknowledgments |
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Notes |
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References |
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2 . Meyers, W. M., Shelly, W. M. & Connor, D. H. (1974). Heat treatment of Mycobacterium ulcerans infections without surgical excision. American Journal of Tropical Medicine and Hygiene 23, 9249.[ISI][Medline]
3 . Pattyn, S. R. & van Ermengem, J. (1968). DDS sensitivity of mycobacteria. Antagonistic effect of PABA for M. ulcerans and M. kansasii. International Journal of Leprosy and Other Mycobacterial Diseases 36, 42731.[Medline]
4 . Dhople, A. M. (1999). In vitro activity of epiroprim, a dihydrofolate reductase inhibitor, singly and in combination with brodimoprim and dapsone, against Mycobacterium leprae. International Journal of Antimicrobial Agents 12, 31923.[ISI][Medline]
5 . Heifets, L. B., Iseman, M. D. & Lindholm-Levy, P. J. (1988). Combinations of rifampin or rifabutin plus ethambutol against Mycobacterium avium complex. Bactericidal synergistic, and bacteriostatic additive or synergistic effects. American Review of Respiratory Disease 137, 7115.[ISI][Medline]
6 . Then, R. L., Bohni, E., Angehrn, P., Plozza-Nottebrock, H. & Stoeckel, K. (1982). New analogs of trimethoprim. Reviews of Infectious Diseases 4, 3727.[ISI][Medline]
7 . Dhople, A. M., Ortega, I., Seydel, J. K. & Gardner, G. D. (1990). Effect of brodimoprim on Mycobacterium leprae in vitro and in mouse foot-pads. Indian Journal of Leprosy 62, 7686.[Medline]
8 . Dhople, A. M. (1999). In vitro and in vivo activity of K-130, a dihydrofolate reductase inhibitor, against Mycobacterium leprae. Arzneimittel-Forschung/Drug Research 49, 26771.
9 . Horsburgh, C. R. (1996). Epidemiology of disease caused by nontuberculous mycobacteria. Seminars in Respiratory Infections 11, 24451.[Medline]
10 . World Health Organization. (1998). The World Health Organization targets untreatable ulcer: report from the first international conference on Buruli ulcer control and research. Yamousoukro (Cote d'Ivoire). Inter Press Service July 31, 1998.
Received 13 July 2000; returned 16 August 2000; revised 7 September 2000; accepted 27 September 2000
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