Comparative antimycobacterial activities of difloxacin, temafloxacin, enoxacin, pefloxacin, reference fluoroquinolones, and a new macrolide, clarithromycin

120

Mycobacterium avium complex bacteria are opportunistic human pathogens, and their chemotherapy remains a challenge since these organisms are resistant to a majority of routine antituberculous drugs. Recently, a wide range of new macrolide antibiotics has been developed, among which the drug clarithromycin appears to have a selective action against M. avium bacteria. In the present study, we have investigated the action of clarithromycin alone (MIC and MBC determinations) and in association with the routine antimycobacterial drugs ethambutol and rifampin at sublethal concentrations (1 ,Ig/ml; below concentrations obtainable in human serum) against M. avium. Our viable count data showed that clarithromycin was bactericidal against all 10 strains of M. avium studied and that its activity was enhanced by ethambutol (in 8 of 9 strains) and rifampin (in 3 of 9 strains). The use of all three drugs in association resulted in higher bactericidal effects than found with any of the drugs used alone or in two-drug combinations in seven of nine strains. The bactericidal effects of various drugs used alone and in combination at concentrations obtainable in human serum were investigated against the type strain ATCC 15769 by using 7H9 broth and BACTEC radiometry (extracellular action) and a J-774 macrophage cell line (intracellular action). A good agreement between the extracellular and intracellular activities was found. Electron microscopy using a ruthenium red cytochemical staining of the bacteria showed that clarithromycin disorganized the outer wall layer and the cytoplasmic membrane in the mycobacterial cell envelope and resulted in formation of large vacuoles inside the cytoplasm, with solubilization of ribosomal structures and consequent plasmolysis. Its association with ethambutol and rifampin resulted in more drastic alterations in the bacterial morphology than were seen with any of the drugs used alone, leading to the removal of the bacterial outer layer, homogenization of cytoplasm, complete cell lysis, and formation of ghosts.Adequate therapy of disease caused by Mycobacterium avium complex (MAC) bacteria has long been a dilemma because of their multiple-drug resistance (4), which is supposedly caused by their cell envelope architecture acting as a barrier excluding drugs (1, 17). We have attempted to circumvent this problem by using a wide range of strategies, e.g., testing new drugs against MAC bacteria (2, 19), using some of the drugs in association with cell wall and/or outer layer (OL) inhibitors such as ethambutol (EMB) and m-fluorophenylalanine (21), synthesizing new amphipathic derivatives of existing hydrophilic drugs such as isoniazid (23), and lastly, correlating the extracellular drug activities to their intracellular efficacy by developing an in vitro macrophage cell line model (24).Among the macrolides, the parent molecule erythromycin has not been widely used as an antimycobacterial agent, and only a few reports have screened its antimycobacterial activity (2,12,14). Two of the reasons are its rapid ...

Section: Resultssupporting

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Extracellular and intracellular activities of clarithromycin used alone and in association with ethambutol and rifampin against Mycobacterium avium complex

Rastogi

Labrousse

1991

120

“…These facts led us to Clarithromycin and its metabolite, either alone or in combination, had very little activity against multidrug-resistant M. tuberculosis. This is in agreement with previous studies which have shown a MIC 90 of Ͼ10 g/ml for M. tuberculosis (13,26) and only modest activity in a murine model of tuberculosis (21).…”

Section: Discussionsupporting

confidence: 93%

Synergistic activities of clarithromycin and antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis

Cavalieri

Biehle

Sanders

1995

The rise of multidrug-resistant Mycobacterium tuberculosis has complicated therapy for tuberculosis and led us to search for a potentially active combination of drugs against these strains. The susceptibilities of 12 strains of multidrug-resistant M. tuberculosis to standard antituberculous drugs (isoniazid, rifampin, ethambutol, and pyrazinamide), clarithromycin, and its metabolite, 14-hydroxyclarithromycin, were determined by use of the BACTEC radiometric method. All strains were resistant to at least two of the antituberculous drugs. Clarithromycin and 14-hydroxyclarithromycin MICs were in the range indicating resistance at >8.0 g/ml for all strains. Combination testing by the BACTEC method was performed with various concentrations of isoniazid, rifampin, and ethambutol, and with clarithromycin/14-hydroxyclarithromycin at fixed concentrations of 2.0/0.5 g/ml, respectively. Addition of clarithromycin/14-hydroxyclarithromycin to these antituberculous drug mixtures resulted in a 4-to 32-fold reduction in MICs of isoniazid, rifampin, and ethambutol and made resistant strains susceptible. Fractional inhibitory concentrations ranged from 0.23 to 0.50 for all strains, suggesting a synergistic interaction between standard antituberculous drugs and clarithromycin/14-hydroxyclarithromycin. The ability of clarithromycin/14-hydroxyclarithromycin to enhance the activities of isoniazid, ethambutol, and rifampin in vitro suggests that this combination may be efficacious in the treatment of multidrug-resistant M. tuberculosis infections.In recent years there has been renewed interest in infections caused by Mycobacterium tuberculosis. This has been due in part to the resurgence of tuberculosis cases nationwide and an alarming increase in multidrug resistance in M. tuberculosis isolates from these infections (2,5,6,12,14,27,29). Multidrug resistance in M. tuberculosis is especially prevalent in certain geographic localities in the United States, including New York City and Florida (2,9,12,14,29). In addition, certain populations, including those infected with human immunodeficiency virus, recent immigrants to the United States, and patients with a history of treatment for tuberculosis (2,10,14,19,27), have an increased incidence. Most of these strains are resistant to the primary antituberculous drugs isoniazid and/or rifampin (2,10,12,29). Multidrug resistance complicates therapeutic options for the clinician. Indeed, patients infected with multidrug-resistant M. tuberculosis must be treated for a much longer period of time than are patients infected with susceptible strains and response rates are decreased (7,9,12,19).Clarithromycin is a new, acid-stable macrolide antibiotic which has good bioavailability after oral dosing (11,15,25). Peak serum and lung clarithromycin concentrations are 2 to 4 g/ml and 17.5 g/g, respectively (11, 15). Clarithromycin is metabolized to 14-hydroxyclarithromycin, which also has antibacterial activity and may act synergistically with the parent compound (18, 25). Clarithromycin has broad-spectr...

“…The MIC was defined as the drug concentration at which the bacterial growth was reduced to 1% or less of that of the drug-free control culture (18). For five quinolones (sitafloxacin, norfloxacin, temafloxacin, fleroxacin, and enoxacin), the MICs were taken from the literature (11,12,36,39,42).…”

Section: Methodsmentioning

confidence: 99%

Mycobacterium tuberculosis DNA Gyrase: Interaction with Quinolones and Correlation with Antimycobacterial Drug Activity

Aubry

Pan

Fisher

et al. 2004

223

172

Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in Mycobacterium tuberculosis. Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of M. tuberculosis have precluded direct purification of its gyrase and detailed analysis of quinolone action. To address these issues, we separately overexpressed the M. tuberculosis DNA gyrase GyrA and GyrB subunits as His-tagged proteins in Escherichia coli from pET plasmids carrying gyrA and gyrB genes. The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity. The drug concentration that inhibited DNA supercoiling by 50% (IC 50 ) was measured for 22 different quinolones, and values ranged from 2 to 3 g/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 g/ml (pipemidic acid and nalidixic acid). By comparison, MICs measured against M. tuberculosis ranged from 0.12 g/ml (for gatifloxacin) to 128 g/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC 50 s (R 2 ‫؍‬ 0.9). Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion. Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (R 2 ‫؍‬ 0.7). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against M. tuberculosis. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting M. tuberculosis gyrase.Fluoroquinolones are active against Mycobacterium tuberculosis and are the first new antimycobacterial drugs to be available since the discovery of rifampin (7,13,40). Fluoroquinolones are part of the drug regimens now recommended for treating rifampin-resistant tuberculosis (6, 10). Ofloxacin and ciprofloxacin have a bacteriostatic antimycobacterial activity (13,20,40), but several new fluoroquinolones, such as sparfloxacin and moxifloxacin, show high bactericidal activity against M. tuberculosis that compares with that of major antituberculous drugs in animal models (20). By contrast, other new fluoroquinolones, such as gemifloxacin and trovafloxacin, are less active than ofloxacin against M. tuberculosis.Studies of other bacterial species suggest that the differences in intrinsic activity observed between quinolones are mainly related to differences in quinolone inhibition of the targets. This has been demonstrated for the differences in activities of several compounds against a given bacterial species, e.g., nalidixic acid and ciprofloxacin against Escherichia coli (17), and of a given compound against several species, e.g., ciprofloxacin against...