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Antimicrobial Agents and Chemotherapy, September 2004, p. 3556-3558, Vol. 48, No. 9
0066-4804/04/$08.00+0 DOI: 10.1128/AAC.48.9.3556-3558.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Kuzell Institute, California Pacific Medical Center Research Institute, San Francisco, California,1 Department of Biomedical Science, College of Veterinary Medicine,2 Department of Microbiology, College of Science, Oregon State University, Corvallis, Oregon,3 Southern Research Institute, Birmingham, Alabama4
Received 11 December 2003/ Returned for modification 7 March 2004/ Accepted 16 May 2004
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Mefloquine (MFQ; a derivative of 4-quinoline-methanol) is an antimicrobial agent widely used for prophylaxis of chloroquine-resistant Plasmodium falciparum malaria (7). It has been demonstrated to have anti-M. avium activity in an experimental mouse model (MIC of 16 µg/ml) (2, 3). Moxifloxacin (MXF) is a fluoroquinolone with activity against a wide range of gram-positive and gram-negative bacteria, including Mycobacterium tuberculosis (11). Recently, we have shown that MXF is active against M. avium in the beige mouse model (MIC of 4 µg/ml) (1). Thiacetazone is a thiosemicarbazole antimicrobial agent that has been widely used for therapy of tuberculosis. SRI-286, a thiosemicarbazole analog, also has good activity against M. avium both in vitro and in vivo (MIC of 2 µg/ml) (4). In vivo, SRI-286 has been shown to be bacteriostatic against M. avium (4). In contrast, thiacetazone, the parent compound, has no effect on M. avium in mice (4). Although thiacetazone has a synergistic effect with isoniazid against M. tuberculosis, neither of the single compounds is active against M. avium (2, 4, 9). We now extend our previous observation to evaluate the effect of SRI-286 in combination with MFQ and MXF, two compounds with activity in vivo against M. avium (1, 3).
Mycobacteria. MAC 101 is a well-characterized human isolate that is susceptible to macrolides and has been used in many previous in vivo studies (1, 4). M. avium organisms were cultured on Middlebrook 7H11 agar (BBL) supplemented with oleic acid-albumin-dextrose-catalase (Hardy Diagnostics) for 10 days at 37°C. Transparent colony morphotypes were harvested and suspended in Hanks' buffered salt solution to a concentration of 3 x 108 CFU/ml for murine challenge. The inoculum was confirmed by colony plating onto 7H11 agar.
Antibiotics. Antimicrobial preparations for therapy were made by suspending the agent with 2.5% gum arabic (Sigma) in 0.2% Tween 80 (Sigma). MFQ was purchased from the hospital pharmacy with a prescription. SRI-286 was provided by the Southern Research Institute (Birmingham, Ala.). MXF was provided by Bayer AG.
Experimental design. C57BL/6J-bgj/bgj female mice, aged 8 to 10 weeks, were obtained from Jackson Laboratories for challenge studies. Mice were infected through the caudal vein with 3 x 107 CFU of MAC 101. After 7 days, all mice were bled for quantitative blood culture to ensure an adequate baseline level of infection. Treatment was then initiated with MFQ at 40 mg/kg, SRI-286 at 40 or 80 mg/kg, or MXF at 100 mg/kg daily as oral monotherapy; all permutations of two-drug therapy; or all three drugs in combination. Each treatment group contained at least 12 evaluable animals. In addition, an experimental group of 10 mice was harvested after 7 days of infection in order to establish a baseline level of infection in tissues. Animals were then treated daily for an additional 4 weeks and then euthanized after allowing 2 days without therapy to reduce carryover. In the original work (3), it was determined that there was no carryover effect. The livers and spleens of all of the mice were aseptically dissected, weighed, and homogenized in 5 ml of 7H9 broth containing 20% glycerol. Tissue suspensions were serially diluted and plated onto 7H11 agar plates to quantitate viable organisms. Plates were incubated at 37°C for 10 days.
We defined bactericidal activity as a reduction in the number of bacteria in the liver and spleen after 4 weeks of treatment (5 weeks) to a number smaller than the number of bacteria in those organs prior to treatment (control at 1 week). One also needs to consider that once a drug decreases the number of viable bacteria, the host immune system would participate in the control of the infection.
Statistical analysis. The statistical significance of the difference between tissue bacterial loads was assessed by analyses of variance. Differences between experimental groups were considered significant at the P < 0.05 level.
Treatment of mice with MFQ alone resulted in bactericidal activity in 5 weeks. These results agree with previously published information demonstrating that MFQ is bactericidal for M. avium in both the liver and the spleen (3, 7). MXF at 100 mg/kg had bacteriostatic activity against M. avium, which has been observed previously (1). SRI-286 at 40 mg/kg, a dose comparable to the dose of thiacetazone in mice, was bacteriostatic, with activity comparable to that of MXF. When administered at 80 mg/kg/day, it had bactericidal activity in the liver and bacteriostatic activity in the spleen (Table 1). No toxicity was revealed by visual observation of the mice.
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Infections caused by the MAC are difficult to treat, and only regimens containing macrolides or azalides have been shown to be effective (6, 12, 15). More recently, we have shown that in mice the combination of MFQ, MXF, and ethambutol was as effective as the macrolide- and azalide-containing regimens (2). In fact, it was the first time that an alternative to a macrolide-containing regimen was proposed.
Now, we have shown that a regimen consisting of MFQ, MXF, and SRI-286 is also as active as macrolide-containing regimens against M. avium (8, 10). Although no side-by-side comparison was performed, we have experience of more than 70 experiments using macrolide therapy. SRI-286 is bacteriostatic against M. avium, while thiacetazone has no activity (4). Thiacetazone, however, is bacteriostatic against M. tuberculosis but is synergistic with isoniazid (5). It appears that SRI-286, when combined with MFQ, had an increasing effect on M. avium.
The triple regimen may be superior to the combination of two compounds because of the smaller chance that antibiotic resistance will develop. It can also be an alternative to treat infections resistant to a macrolide-containing regimen or for patients who cannot tolerate macrolide therapy.
One of the unknown aspects of the MFQ regimen is whether the drug would be well tolerated. When MFQ is used for prophylaxis of P. falciparum malaria, it is administered once a week. Almost certainly, for the treatment of M. avium infection, more frequent doses will be required. Recently, a report of a case of M. avium infection successfully treated with MFQ has been published (14). No side effects were observed (14). The definitive answer to this question awaits future studies with humans.
MFQ may also have another advantage in the treatment of M. avium infection. Thus far, despite trying several strategies, no resistant clone has been identified by attempting induction in vitro, by treating mice for 12 weeks, and by chemical mutagenesis (unpublished data), which suggests that the drug target in M. avium might be a lethal target or may indicate that killing does not occur by a simple mode of action. Future studies will attempt to address this possibility.
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ACKNOWLEDGMENTS |
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This research was supported by National Institute of Allergy and Infectious Diseases contract NO1-AI-25104.
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| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
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