Aycobacterium avium strains susceptible to clarithromycin and azithromycin contain mutants resistant to these macrolides with a frequency of 1.1 x 10-10 to 1.2 x 10-6. Cross-resistance between clarithromycin and azithromycin was demonstrated with mutants selected in the laboratory as well as with resistant strains isolated from patients. The susceptibility-resistance patterns of the macrolide-resistant strains with drugs other than macrolides were the same as those of the onrginal susceptible strains. The emergence of clarithromycin resistance in patients was a result of multiplication of the preexisting resistant mutants that survived the elimination of bacteria during the initial period of treatment and was an exclusive cause of the relapse of bacteremia.Clarithromycin, a new macrolide, has been shown to have activity against Mycobacterium avium in various models including experiments in culture media (1,2,4,7,9,12,17,22), macrophages (16,18,19,21,24), and mice (7,13,14). The effectiveness of this drug in the elimination of M. avium bacteremia in patients with AIDS has been shown in controlled clinical trials (3, 6, 10). Another macrolide, azithromycin, has also been shown to be effective in the therapy of M. avium infections in patients with AIDS (23,26). Clinical trials with both macrolides have had limited periods of observation, usually less than 12 weeks. Recent reports on prolonged monotherapy with clarithromycin or azithromycin showed that after a period of elimination of the bacteria, a relapse of bacteremia took place in many patients (3,5,23,26). Those reports also implied that prolonged monotherapy may lead to the emergence of drug resistance.The goals of the present study were to address the following various aspects of drug resistance of M. avium to new macrolides, especially clarithromycin: (i) the frequency of clarithromycin-and azithromycin-resistant mutants in susceptible M. avium strains and the degree of resistance of these mutants, (ii) cross-resistance between clarithromycin and azithromycin, (iii) comparison of the drug susceptibilityresistance patterns between the original strains and their resistant mutants in experiments with other drugs, and (iv) drug susceptibilities of strains isolated from patients with AIDS and bacteremia at the beginning of therapy and at the time of relapse. In the present study, we analyzed the bacteriological response to clarithromycin monotherapy and presented a general description of the phenomenon of the emerging resistance of M. avium to this drug. The genetic basis of macrolide resistance in M. avium was not among the goals of the present study. Neither did we make an attempt to address such issues as the rate of mutation or mechanisms of resistance.MATERIALS AND METHODS Antimicrobial agents. Clarithromycin was obtained from Abbott Laboratories (Abbott Park, Ill.). The drug was dissolved in dimethyl sulfoxide (DMSO; 100 mg/10 ml). This stock solution was diluted further with DMSO to make * Corresponding author. appropriate working solutions, which were ke...

Levofloxacin exhibited twofold greater inhibitory and bactericidal activities than ofloxacin against either extracellular or intracellular tubercle bacilli. The activities of both drugs against extracellular and intracellular bacteria were about the same, despite the accumulation of the drugs in macrophages at a level four-to fivefold greater than that in the extracellular medium. The activities of both drugs against intracellular bacteria were largely associated with the short, 2-h pulsed exposures of the infected macrophages to the concentrations which correspond to those attainable in blood during the period of the maximum concentration of drug in serum.Among the quinolones, ofloxacin was the first agent used to treat patients with tuberculosis (16). Comparison of the in vitro activities of various quinolones indicated that ofloxacin and ciprofloxacin had better potentials than norfloxacin, amifloxacin, perfloxacin, and other compounds of this class for the treatment of tuberculosis (1, 2, 5, 7, 9, 13-15, 17, 18). The MICs of both ofloxacin and ciprofloxacin in various types of liquid media and in 7H10 or 7H11 agar ranged from 0.12 to 2.0 ,ug/ml (10). The MBCs of these two drugs determined in 7H12 broth were 2.0 ,ug/ml, giving MBC/MIC ratios of 2 to 4 (10, 15). Levofloxacin, the optically active L-isomer of ofloxacin, has shown high levels of antibacterial activity both in vitro and in mice against a number of bacterial species, being either as active as or up to fourfold more active than ofloxacin (8).The aim of the study described here was the preclinical evaluation of the activity of levofloxacin in comparison with that of ofloxacin against Mycobacterium tuberculosis in human monocyte-derived macrophages as well as in cell-free medium.Antimicrobial agents. Ofloxacin and levofloxacin were obtained from the R. W. Johnson Pharmaceutical Research Institute (Springhouse, Pa.). Stock solutions were made in phosphate buffer (pH 6.5). These stock solutions were diluted further to make appropriate working solutions.Test strains. Studies in macrophages were conducted with two M. tuberculosis strains: H37Rv, which is susceptible to all antituberculosis drugs, and a Vertullo strain (2937-0), one of the quality control strains in our laboratory, which is resistant to isoniazid, rifampin, streptomycin, and ethambutol. The same two strains and an additional nine clinical isolates were used for determination of the MICs in vitro. Subcultures in 7H9 broth were preserved in aliquots frozen at -70°C. For each experiment, a subculture of frozen stock was made in 7H9 broth. After S to 6 days of cultivation at 37°C, the culture was forced through a 27-gauge needle and was then centrifuged at 250 x g for 5 min to remove large clumps of bacteria.Model of the M. tuberculosis-infected macrophages. The technique for preparation of the human monocyte-derived macrophage monolayers was as described previously by Crowle and colleagues (3, 4), with some modifications given in our * Corresponding author. Mailing address: National Jewish C...

The combined effect of clarithromycin and rifabutin against Mycobacterium avium multiplying either within human monocyte-derived macrophages or extracellularly in a liquid medium was additive: both MICs and MBCs were twofold lower for the combination than they were for each drug alone. Prolonged exposure for 4 weeks of M. avium-infected macrophages to combined concentrations that were only twofold greater than the MICs resulted in a 100-fold decrease in the number of viable bacteria, while in the drug-free controls a 100-fold or greater increase in comparison with the initial viable counts took place. Comparison of this effect with the results of the prolonged exposure to each drug alone suggested that under these experimental conditions rifabutin enhanced the antimicrobial activity of clarithromycin against intracellular bacteria. At the same time, inhibition of intracellular growth by a 2-h pulsed exposure of the infected macrophages to the combination of the two drugs was not different from the effect induced by clarithromycin alone. In conclusion, clarithromycin played the major role in the antimicrobial activity of the tested combination, while rifabutin may have enhanced this effect during a prolonged exposure of the intracellular bacteria to these two agents.Combinations of clarithromycin with a variety of other drugs have been investigated in vitro in cell-free culture media and macrophages (3,7,9,10,16,17). The overall analyses of these data indicated that the interaction in these combinations tested against Mycobacterium avium was additive rather than synergistic (2). The effect of this combination for the treatment and prevention of disseminated M. avium infection in patients with AIDS is under investigation. The aim of the present study was to evaluate the activity of clarithromycin in combination with rifabutin against M. avium in a cell-free culture medium and human monocyte-derived macrophages. The study included an evaluation of the antimicrobial activity during prolonged exposure and the effect of a drug combination given to infected macrophages by pulsed exposure.MATERUILS AND METHODS Antimicrobial agents. Rifabutin (Adria Laboratories, Pharmacia, Columbus, Ohio) was dissolved first in methanol (640 jig/ml) and was then diluted further in distilled water to make appropriate working solutions. Clarithromycin (Abbott Laboratories, Abbott Park, Ill.) was first dissolved in dimethyl sulfoxide (1,280 jig/ml) and was then diluted further in phosphate buffer (pH 6.5).Test strains. Three strains identified as M. avium by the Gen-Probe (San Diego, Calif.) technique were used for the studies. These strains, isolated from blood samples obtained from patients with AIDS, were used in our previous studies (8,12,13). They were preserved in frozen 7H9 broth culture aliquots at -70°C. For each experiment, a subculture in 7H9 broth was made from a frozen aliquot.Antimicrobial activity against extracellular bacteria. The MIC was defined as the lowest drug concentration that inhibited more than 99% of the bacteria...

Clarithromycin is known to accumulate in polymorphonuclear leukocytes, but no accumulation studies with macrophages have been reported. We exposed J774 macrophages, grown for 4-6 days, to clarithromycin 3.0 micrograms/ml for 2 hours. The cells were separated from the extracellular fluid, and the concentration of clarithromycin was determined in an agar diffusion bioassay. The accumulation of clarithromycin was 15.8-fold greater in the cells than it was in the extracellular fluid when the test was performed with noninfected cells, and 17.3-fold greater for cells infected with Mycobacterium avium. However, the ratio was substantially lower, only 3.7 for dead macrophages, suggesting that intracellular accumulation is probably an active process. These data may clarify the nature of the activity of clarithromycin against M. avium in macrophages.