In vitro effects of antimicrobial agents on Mycobacterium leprae in mouse peritoneal macrophages

The in vitro activities of a variety of aminoglycosides, lincosamides, and rifamycins against Mycobacterium leprae were evaluated with the BACTEC 460 system. At 20 ,g/ml, gentamicin, kanamycin, tobramycin, streptomycin, and amikacin were inactive. Lincomycin was active at 20 ,ug/ml, and clindamycin was active at 0.31 ,ug/mi. Rifamycin SV, rifabutin, and rifampin were active at 3.1, 3.1 to 12.5, and 200 ng/ml, respectively. The M. leprae inoculum was prepared from infected nude mouse footpads and partially purified by differential centrifugation as previously described (6).All drugs, except for rifabutin (Farmitalia Carlo ERB, Milan, Italy), were obtained from Sigma Chemical Co. (St. Louis, Mo.). Aminoglycosides and lincosamides were dissolved in water, and rifamycins were dissolved in ethanol. All were adjusted for potency, filter sterilized, and diluted in Middlebrook 7H9 broth to 40 times the final desired concentrations.Susceptibility testing was performed with the BACTEC 460 as previously described (3). In brief, i07 M Ieprae organisms were inoculated into BACIEC 12B medium, and drugs were added in 0.1-ml aliquots to quadruplicate vials. All drugs were tested at fourfold dilutions; aminoglycosides and lincosamides were tested at 0.31 to 20 ,ug/ml, and rifamycins were tested at 3.1 to 200 ng/ml. Ampicillin and amphotericin B were added to control occasional contaminants. Vials were flushed with 2.5% 02-10% CO2 (balance, N2) and incubated at 33°C. The growth index (14C02 evolution) was measured at 1-week intervals with the BACTEC 460. Drugs at various concentrations were considered to be active if they effected a significant (Student's t test) reduction in the growth index during the reading interval of days 7 to 14, relative to that of drug-free controls. When these experiments were repeated a second time, essentially identical results were obtained (data not shown).None of the aminoglycosides effected a significant reduction in the growth index at concentrations of 20 ,ug/ml or less after 2 weeks of incubation (Fig. 1). Subsequent readings taken at 3, 4, and 5 weeks postincubation gave similar results (data not shown). In contrast, lincomycin was active at 20 ,ug/ml, and clindamycin was active at 0.31 ,g/ml. At 20 ,ug/ml, clindamycin effected a reduction of 61% in 14CO2evolution.The rifamycins were tested at ¼lo of the concentrations of the aminoglycosides and lincosamides. As previously noted, rifampin was active at 200 ng/ml (Fig. 2). Both rifabutin and rifamycin SV showed significant activity at 3.1 to 200 ng/ml, although the latter effected a greater reduction in the growth index at the lower concentrations.Gelber et al. (10) demonstrated the bactericidal activities of streptomycin, kanamycin, and amikacin at high (100 to 150 mg/kg) daily doses against M. leprae in the footpads of mice. Gentamicin and tobramycin at 20 mg/kg were much less active. Reducing the dose or frequency of administration resulted in a reduction of the activity of streptomycin and a loss of the bactericidal activity of kanamycin (9...

mentioning

confidence: 67%

In vitro activities of aminoglycosides, lincosamides, and rifamycins against Mycobacterium leprae

Franzblau

1991

“…This study and those of others (11,19) have demonstrated that clarithromycin has superior activity against M. leprae compared with erythromycin; these activities were also found against M. avium (6). Clarithromycin is more lipophilic than erythromycin (6), and perhaps its consistently superior activity against mycobacteria in vitro and in vivo may also be a result of its ability to penetrate the outer mycobacterial lipid-rich cell wall, a situation we found previously (13) to be the likely cause of the unique activity of minocycline among the tetracyclines against M. leprae.…”

mentioning

confidence: 68%

“…The higher levels in serum found in the previous study (11) and this one and the higher levels of clarithromycin found in footpad tissues in this study compared with those of roxithromycin may, at least partially, account for the superiority in vivo of clarithromycin vis-a-vis roxithromycin. Indeed, Ramasesh et al (19), using an in vitro assay quantitating the metabolic activity of M. leprae within mouse peritoneal macrophages based on the incorporation of radiolabeled palmitic acid into its unique phenolic glycolipid I, found clarithromycin and roxithromycin to be equally active.…”

mentioning

confidence: 99%

Activities of various macrolide antibiotics against Mycobacterium leprae infection in mice

Gelber¹,

Siu²,

Tsang³

et al. 1991

We evaluated the activities of several macrolide antibiotics against M. leprae infections in mouse footpads. Erythromycin and azithromycin were inactive, while both roxithromycin and clarithromycin were found to be consistently active and, in fact, bactericidal. By both methods, clarithromycin was found to be superior to roxithromycin, a finding which, at least in part, may be a consequence of the higher levels of clarithromycin at the site of infection.The World Health Organization (23) estimates that there are 10 million patients with leprosy, and yet there remains a compelling need for new antimicrobial agents to treat leprosy (2,14). At present, only dapsone, rifampin, and clofazimine are available to treat patients with leprosy. Because (i) Mycobacterium leprae resistance to dapsone and rifampin is encountered and is of considerable concern (18), (ii) toxicities and unacceptable side effects from each of these agents are not uncommon, and (iii) there is general consensus that lepromatous patients require multidrug therapy (25), other antimicrobial agents are certainly required to treat leprosy (2,14). A number of studies (9-11, 17, 19) performed with cell-free and macrophage in vitro culture systems demonstrated that many macrolide antibiotics inhibit the metabolic activity of M. leprae. However, to date there has been only one study that has confirmed that any macrolide has activity against the actual growth of M. leprae in vivo (11). In that study, clarithromycin, but not erythromycin or roxithromycin, was found to be effective against M. leprae in infected mice. Here we report results of studies on the activities of several macrolides against M. leprae-infected mice and findings that, while erythromycin and azithromycin were inactive, roxithromycin and clarithromycin were consistently active and, indeed, bactericidal.Unfortunately, M. leprae cannot be grown on artificial medium. In 1960, Shepard (20) demonstrated that M. leprae multiplies locally when it is injected into the footpads of mice: an inoculum of 5 x 10i M. leprae per footpad generally results in a plateau of 1 x 106 M. leprae per footpad in 6 months. For the past three decades, this animal model has proved extremely useful for evaluating the efficacy of antimicrobial agents against M. leprae; and in general, antimicrobial agents active in the mouse footpad model at clinically achievable levels, including dapsone, rifampin, clofazimine, ethionamide, and streptomycin, have proved to be effective clinically (2,14).For the initial studies of antimicrobial agents in this model, we used continuous therapy from the time of infection and did not distinguish between bacteriostatic and bactericidal activity. To avoid this problem in these studies, we used the kinetic method of Shepard et al. (22) footpads were infected with 5,000 M. leprae. At day 150 and intervals thereafter, the number of M. leprae in four feet (two mice) was assessed microscopically. The activity of a drug was determined by measuring the delay in the appearance of the bacterial p...

“…We and our colleagues have also used assays quantitating phenolic glycolipid-1 synthesis and adenosine triphosphate concentration (8) as indices of drug activity against both extracellular (16) and intracellular (20) M. leprae. However, the sensitivity, reproducibility, and automated nature of the radiorespirometric systems have resulted in their becoming the primary indices of M. Ieprae viability in our laboratory.…”

Section: Discussionmentioning

confidence: 99%

Fusidic acid is highly active against extracellular and intracellular Mycobacterium leprae

Franzblau

Biswas

Harris

1992

The activity of fusidic acid against Mycobacterium leprae was studied in axenic medium and in bacilli residing within mouse peritoneal macrophages. Activity was assessed by subsequent quantitation of bacillary radiorespirometric activity. Significant inhibition in both systems was observed at 0.156 micrograms/ml, and an approximately 50% reduction in activity occurred after exposure to 1.25 to 2.5 micrograms/ml. The excellent human pharmacokinetics and in vitro activity of fusidic acid against the leprosy bacillus warrant a clinical trial of this drug for leprosy.