- Volume 62 , Number 1
- Page: 140–3
Minimal bactericidal concentration of clarithromycin against M. leprae
To the Editor:
The new macrolide antibiotic, clarithromycin, was discovered by Franzblau and Hastings (3) to inhibit the metabolism of Mycobacterium leprae in vitro and in M. leprae-infected mice to demonstrate bactericidal-type activity when administered at 0.01% in the diet. The bactericidal activity (96%) of dietary 0.1% clarithromycin for M. leprae was quantitated by the authors (8), as well as by Ji, et al. (10), (87%-95%), wherein mice were administered 12.5-50 mg/kg clarithromycin by gavage. Because levels in vivo of clarithromycin that were inactive against M. leprae had not been studied to date, we (6) established for a single M. leprae isolate that clarithromycin fed continuously in dietary concentrations of 0.001 %, 0.005%, 0.01%, 0.05%, and 0.1% consistently inhibited the growth of M. leprae , while 0.0001 % did not, establishing a minimal inhibitory mouse serum concentration of no more than 25 ng/ml and likely as little as 2.5 ng/ml.
Because actual bactericidal activity against M. leprae is critical to its use in patients, this current study was designed to establish clarithromycin's minimal bactericidal dietary concentration against the same M. leprae isolate used by us previously to establish its minimal inhibitory concentration. For these purposes, groups of generally 10 BALB/c female mice were infected in both hindfoot pads with 50, 500, and 5000 M. leprae and treated for the first 2 months (1) with various dietary concentrations of clarithromycin [0% (control), 0.01%, 0.05%, and 0.1%]. For these studies, clarithromycin was initially dissolved in 95% ethanol and evenly distributed in mouse chow by using a Patterson-Kelly twin-drum liquid-solid blender (Patterson-Kelly, East Stroudsburg, Pennsylvania, U.S.A.). Diets were made fresh every 2 weeks and stored in a refrigerator. One year after therapy had been discontinued, a time sufficient to detect multiplication of M. leprae from any bacilli surviving therapy, the number of foot pads wherein M. leprae survived (number of M. leprae /foot pad > 105) was assessed. (1) From these results the percentage of bacteria killed was quantitated by the method of Spearman and Kärber, described previously by Shepard (12), and the probability that actual killing occurred determined.
Both 0.05% and 0.1% clarithromycin were found to be bactericidal (88% ± 8%) for M. leprae (The Table). Furthermore, we observed that the probability that killing occurred at these two dietary concentrations was highly significant (p < 0.002). Unfortunately, in this study the control inoculum itself had low viability, perhaps accounting for why these high dietary concentrations were not found to be as bactericidal as previously demonstrated for other isolates. In these studies 0.01 % clarithromycin resulted in no significant killing of M. leprae .
It is noteworthy in this study that the growth of the M. leprae isolate used herein had been found to be consistently inhibited by the continuous dietary administration of clarithromycin of 0.001% and higher (6). Thus in this study we found a 50-fold greater concentration of clarithromycin was required to kill M. leprae than had been found previously to inhibit the same isolate's growth. It is further noteworthy that Walker and Shinnick (14) also found that for several M. leprae isolates, including "pan-susceptible," dapsone-resistant, and rifampin-resistant, a discrepancy between clarithromycin's minimal inhibitory dietary concentration and minimal bactericidal dietary concentration was generally demonstrable. Such discrepancies between the amount of drug necessary to inhibit and kill M. leprae have been demonstrated previously for active antimicrobials, dapsone (4), clofazimine (5), and ethionamide (5). On the other hand, the amount of minocycline required to inhibit and kill M. leprae was found previously to be remarkably similar (9). The relative activity of antimicrobials against M. leprae after standard doses (2) frequently has been compared with regard to the time in patients that inhibitory levels of the drug are maintained. Because of our general recognition now that bactericidal activity is critical to efficacious treatment of lepromatous patients and inhibitory bactericidal levels for several antimicrobials are discordant, we believe that the actual period of time that levels above those required for bactericide would be best substituted to such considerations.
It has been found previously for dapsone (13) and minocycline (7) that sensitive M. leprae isolates are inhibited consistently by a very narrow range of antimicrobial concentrations. On the other hand, we previously reviewed for rifampin that there is a broad range of published results of both its minimal inhibitory concentration (MIC) and its minimal bactericidal concentration (MBC), suggesting a broader range of susceptibility among isolates (4). Franzblau and Hastings (3) found for an M. leprae isolate that 0.01% dietary clarithromycin inhibited its growth and was fully bactericidal. While we (6) found that 0.001% inhibited an isolate, it did not result in significant killing of that same isolate herein, this requiring 0.05% or greater. On the other hand, for another isolate Walker and Shinnick (14) found that 0.01% clarithromycin neither inhibited nor killed, killing itself for several M. leprae isolates being accomplished consistently only by increasing the dietary concentration to 0.1%. Thus, there appears a significant range of susceptibility among M. leprae isolates to the activity of clarithromycin. We (8) found previously that 0.1% clarithromycin was bactericidal and that this level of dietary clarithromycin results in mouse serum levels one third of that of the peak obtained in man by a very small (400 mg) dose. It is encouraging that the found range of susceptibility of several M. leprae isolates does not limit clarithromycin's general applicability to the treatment of leprosy patients, although the exact time/concentration level for optimal clarithromycin therapy remains to be determined. Such considerations may be especially critical to the application of clarithromycin to certain intermittent schedules, particularly once monthly (11), that are now being seriously entertained.
- Robert H. Gelber, M.D.
Lydia P. Murray
Patricia Siu, B.S.
Mabel Tsang, B.S.
Kuzell Institute for Arthritis and Infectious
Diseases
Medical Research Institute of San Francisco
1600 Webster Street
San Francisco, California 94115, U.S.A.
GWL Hansen's Disease Center
5445 Point Clair Road
Carville, Louisiana 70721, U.S.A.
REFERENCES
1. COLSTON, M. J., HILSON, G. R. and BANERJEE, D. The "proportional bactericidal test": a method for assessing bactericidal activity in drugs against Mycobacterium leprae in mice. Lepr. Rev. 49(1978)7-15.
2. ELLARD, G. A. and GAMMON, P. T. Drugs for combined therapy: experimental studies on the antileprosy activity of ethionamide and prothionamide, and a general review. Lepr. Rev. 49(1978)115-126.
3. FRANZBLAU, S. G. and HASTINOS, R. C. In vitro and in vivo activities of macrolidcs against Mycobacterium leprae . Antimicrob. Agents Chemother. 32(1988)1758-1762.
4. GELBER, R. H. The killing of Mycobacterium leprae in mice by various dietary concentrations of dapsone and rifampin. Lepr. Rev. 57(1986)347-353.
5. GELBER, R. H. The killing of Mycobacterium leprae in mice by various dietary concentrations of clofazimine and ethionamide. Lepr. Rev. 58(1987)407-411.
6. GELBER, R. H., MURRAY, L. P., Siu, P. and TSANG, M. Clarithromycin at very low levels and on intermittent administration inhibits the growth of M. leprae in mice. Int. J. Lepr. 60(1992)485-487.
7. GELBER, R. H., SIU, P., TSANG, M., ALLEY, P. and MURRAY, L. P. Effect of low-level and intermittent minocycline therapy on the growth of Mycobacterium leprae in mice. Antimicrob. Agents Chemother. 35(1991)992-994.
8. GELBER, R. H., SIU, P., TSANG, M. and MURRAY, L. P. Activities of various macrolide antibiotics against Mycobacterium leprae infection in mice. Antimicrob. Agents Chemother. 35(1991)760-763.
9. GELBER, R. H., SIU, P., TSANG, M. and MURRAY, L. P. Minimal bactericidal dietary concentration of minocycline for Mycobacterium leprae -infected mice is very low and similar to its minimal inhibitory dietary concentration. Int. J. Lepr. 60(1992)276-277.
10. Ji, B., PERANI, E. G. and GROSSET, J.-H. Effectiveness of clarithromycin and minocycline alone and in combination against experimental Mycobacterium leprae infection in mice. Antimicrob. Agents Chemother. 35(1991)579-581.
11. Ji, B., PERANI, E. G., PETINON, C. and GROSSET, J. H. Bactericidal activities of single or multiple doses of various combinations of new antileprosy drugs and/or rifampin against M. leprae in mice. Int. J. Lepr. 60(1992)556-561.
12. SHEPARD, C. C. Statistical analysis of results obtained by two methods for testing drug activity against Mycobacterium leprae . Int. J. Lepr. 50(1982)96-101.
13. SHEPARD, C. C, REES, R. J. W., LEVY, L., PATTYN, S. F., JI, B. and DELA CRUZ, E. C. Susceptibility of strains of Mycobacterium leprae isolated prior to 1977 from patients with previously untreated lepromatous leprosy. Int. J. Lepr. 54(1986)11-15.
14. WALKER, L. L., VAN LANDINGHAM, R. M. and SHINNICK, T. M. Clarithromycin is bactericidal against strains of Mycobacterium leprae resistant and susceptible to dapsone and rifampin. Int. J. Lepr. 61(1993)59-65.
Reprint requests to Dr. Gelber at 2211 Post St., Suite 301, San Francisco, California 94115, U.S.A.