Vol. 35, No. 3
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1991, p. 462-470
0066-4804/91/030462-09$02.00/0 Copyright © 1991, American Society for Microbiology
Extracellular and Intracellular Activities of Clarithromycin Used Alone and in Association with Ethambutol and Rifampin against Mycobacterium avium Complex NALIN RASTOGI* AND VALERIE LABROUSSE Unite de la Tuberculose et des Mycobacteries, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France Received 21 September 1990/Accepted 7 January 1991
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.
cin) and the hydroxyl group at C-6 (clarithromycin [CLA] [10]). All three of these new macrolides have been recently tested against both Mycobacterium tuberculosis and MAC organisms (5, 7, 15). Among these three drugs, CLA attracted our attention as being unlike most of the drugs tested thus far against mycobacteria, which in general are more active against M. tuberculosis than against MAC organisms (1); this drug showed activity against MAC organisms 10 times higher than that against tubercle bacilli (5, 7). Moreover, among all three new macrolides tested, CLA was most effective in the treatment of M. avium-infected macrophages (16), M. avium-infected beige mice (5), and Mycobacterium leprae-infected HSD nulnu mice (6). For these reasons, we determined the MICs and MBCs of CLA against 10 MAC strains by using BACTEC radiometry. We also investigated whether the antibacterial action of CLA used at a sublethal concentration (1 ,g/ml) was enhanced by sublethal concentrations of EMB and/or rifampin (RIF; 1 ,ug/ml). Finally, all three drugs were used at concentrations obtainable in serum, and their activities against the type strain ATCC 15769 when grown both in 7H9 broth and BACTEC vials (extracellular action) and inside J-774 macrophages (intracellular action) were compared. CLA was highly bactericidal against M. avium, regardless of whether the bacteria were grown in 7H9 broth in a confined atmosphere using the BACTEC radiometric method or whether the bacteria were multiplying actively inside infected macrophages, and the concomitant use of CLA with EMB and RIF resulted in enhanced bactericidal activity. Electron
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 acid degradation (6, 10) and relatively low plasmatic levels (6, 11). Thus, the in vitro activities of the parent drug erythromycin are highly pH dependent, with activity decreasing with lower pHs. Recently, new erythromycin derivatives which are stable under acid conditions were synthesized by modifying those functional groups which participated in the degradation reaction, namely the ketone at C-9 (roxithromycin and a 15-member ring-expanded derivative, azithromy*
Corresponding author. 462
VOL. 35, 1991
microscopic studies using specific cytochemical staining of the bacterial OL were fruitful in establishing the sites of action of CLA and of CLA in combination with EMB and RIF.
MATERIALS AND METHODS Bacteria and growth. Ten strains of the MAC (type strains ATCC 13950 and ATCC 15769 and 8 recent clinical isolates [3 pulmonary, 3 extrapulmonary, and 2 disseminated isolates]) and 12 strains of M. tuberculosis (type strain H37Rv and 11 recent clinical isolates [9 pulmonary and 2 extrapulmonary isolates]) used in this study were from our own culture collection. The bacteria were grown in complete 7H9 broth (containing 0.05% [vol/vol] Tween 80; Difco Laboratories, Detroit, Mich.) to an optical density of 0.15 (measured at 650 nm with a Coleman Junior II spectrophotometer) at 37°C, which corresponded to about 108 viable counts per ml. Drug susceptibility testing. The growth of bacteria was monitored radiometrically by using a BACTEC 460-TB apparatus (Becton Dickinson, Towson, Md.) in a confined atmosphere as reported earlier (20, 21). Bacterial growth was measured as a function of their ability to catabolize 14Clabeled palmitate in the 7H12a broth during growth, which resulted in the release of 14C-labeled CO2. The growth was expressed as a numerical value called the growth index (GI), which ranged from 1 to 999. The method used by us for MIC determinations using the 1% proportion criterion for bacterial growth has been described in detail elsewhere (20, 21). MBCs were established by plating serial dilutions of the bacterial cultures from BACTEC vials after 7 days of drug addition onto 7H11 agar medium, and the bacterial counts were enumerated after 21 days of incubation at 37°C. MBC was the minimal concentration which effectively reduced the bacterial counts by 99%, compared with those of untreated controls. Combined drug action. The combined drug action (using two drugs against MAC bacteria) was evaluated by using the X/Y quotient criterion reported earlier (8, 9, 21). Briefly, the combined drug action was equal to X/Y, where X was the BACTEC GI obtained with the combination of two drugs and Y was the lowest GI obtained at the same time for any of the drugs used alone. An X/Y value of 1 indicated that there was no interaction between the two drugs, an X/Y of 2.0 indicated the presence of antagonism between the two. In the case of a three-drug combination, an X/Y quotient of
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1991, p. 462-470
0066-4804/91/030462-09$02.00/0 Copyright © 1991, American Society for Microbiology
Extracellular and Intracellular Activities of Clarithromycin Used Alone and in Association with Ethambutol and Rifampin against Mycobacterium avium Complex NALIN RASTOGI* AND VALERIE LABROUSSE Unite de la Tuberculose et des Mycobacteries, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France Received 21 September 1990/Accepted 7 January 1991
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.
cin) and the hydroxyl group at C-6 (clarithromycin [CLA] [10]). All three of these new macrolides have been recently tested against both Mycobacterium tuberculosis and MAC organisms (5, 7, 15). Among these three drugs, CLA attracted our attention as being unlike most of the drugs tested thus far against mycobacteria, which in general are more active against M. tuberculosis than against MAC organisms (1); this drug showed activity against MAC organisms 10 times higher than that against tubercle bacilli (5, 7). Moreover, among all three new macrolides tested, CLA was most effective in the treatment of M. avium-infected macrophages (16), M. avium-infected beige mice (5), and Mycobacterium leprae-infected HSD nulnu mice (6). For these reasons, we determined the MICs and MBCs of CLA against 10 MAC strains by using BACTEC radiometry. We also investigated whether the antibacterial action of CLA used at a sublethal concentration (1 ,g/ml) was enhanced by sublethal concentrations of EMB and/or rifampin (RIF; 1 ,ug/ml). Finally, all three drugs were used at concentrations obtainable in serum, and their activities against the type strain ATCC 15769 when grown both in 7H9 broth and BACTEC vials (extracellular action) and inside J-774 macrophages (intracellular action) were compared. CLA was highly bactericidal against M. avium, regardless of whether the bacteria were grown in 7H9 broth in a confined atmosphere using the BACTEC radiometric method or whether the bacteria were multiplying actively inside infected macrophages, and the concomitant use of CLA with EMB and RIF resulted in enhanced bactericidal activity. Electron
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 acid degradation (6, 10) and relatively low plasmatic levels (6, 11). Thus, the in vitro activities of the parent drug erythromycin are highly pH dependent, with activity decreasing with lower pHs. Recently, new erythromycin derivatives which are stable under acid conditions were synthesized by modifying those functional groups which participated in the degradation reaction, namely the ketone at C-9 (roxithromycin and a 15-member ring-expanded derivative, azithromy*
Corresponding author. 462
VOL. 35, 1991
microscopic studies using specific cytochemical staining of the bacterial OL were fruitful in establishing the sites of action of CLA and of CLA in combination with EMB and RIF.
MATERIALS AND METHODS Bacteria and growth. Ten strains of the MAC (type strains ATCC 13950 and ATCC 15769 and 8 recent clinical isolates [3 pulmonary, 3 extrapulmonary, and 2 disseminated isolates]) and 12 strains of M. tuberculosis (type strain H37Rv and 11 recent clinical isolates [9 pulmonary and 2 extrapulmonary isolates]) used in this study were from our own culture collection. The bacteria were grown in complete 7H9 broth (containing 0.05% [vol/vol] Tween 80; Difco Laboratories, Detroit, Mich.) to an optical density of 0.15 (measured at 650 nm with a Coleman Junior II spectrophotometer) at 37°C, which corresponded to about 108 viable counts per ml. Drug susceptibility testing. The growth of bacteria was monitored radiometrically by using a BACTEC 460-TB apparatus (Becton Dickinson, Towson, Md.) in a confined atmosphere as reported earlier (20, 21). Bacterial growth was measured as a function of their ability to catabolize 14Clabeled palmitate in the 7H12a broth during growth, which resulted in the release of 14C-labeled CO2. The growth was expressed as a numerical value called the growth index (GI), which ranged from 1 to 999. The method used by us for MIC determinations using the 1% proportion criterion for bacterial growth has been described in detail elsewhere (20, 21). MBCs were established by plating serial dilutions of the bacterial cultures from BACTEC vials after 7 days of drug addition onto 7H11 agar medium, and the bacterial counts were enumerated after 21 days of incubation at 37°C. MBC was the minimal concentration which effectively reduced the bacterial counts by 99%, compared with those of untreated controls. Combined drug action. The combined drug action (using two drugs against MAC bacteria) was evaluated by using the X/Y quotient criterion reported earlier (8, 9, 21). Briefly, the combined drug action was equal to X/Y, where X was the BACTEC GI obtained with the combination of two drugs and Y was the lowest GI obtained at the same time for any of the drugs used alone. An X/Y value of 1 indicated that there was no interaction between the two drugs, an X/Y of 2.0 indicated the presence of antagonism between the two. In the case of a three-drug combination, an X/Y quotient of
