Effect of drug concentration on emergence of macrolide resistance in Mycobacterium avium

The emergence of antibiotic resistance in mycobacteria involves the selection of mutant variants within a susceptible bacterial population. However, it is unclear whether antimycobacterial drugs act just as selective agents or can influence the rate of appearance of resistant mutants. The present study was initiated to address this issue by monitoring the effects of antimicrobial agents on the appearance and growth of clarithromycin (CLR)-resistant (CLR(r)) bacilli in broth cultures of Mycobacterium avium. Preexposure of M. avium to CLR had a significant dose effect on the emergence of resistance, with concentrations of 4 to 8 microg/ml resulting in a maximal (approximately 10(4)-fold) increase in the number of CLR(r) bacilli after a 4-day incubation. In addition, a dose effect was found with azithromycin. The use of combinations of CLR with either ethambutol (EMB) or rifabutin (RFB) resulted in fewer resistant bacilli compared to the use of CLR alone. The lowest active concentration of EMB (4 microg/ml) was equivalent to the EMB MIC (4 to 8 microg/ml) for the parental CLR(s) strain and the emergent CLR(r) variants, and thus, the antiresistance effect was probably the result of the bacteriostatic effect of EMB on CLR(r) bacilli. However, RFB was an order of magnitude more active (0.05 microg/ml) at reducing resistance than suggested by the MIC of this agent (0.5 to 1 microg/ml). These results indicate that the emergence of resistance was not simply the selection of a preexisting subpopulation of resistant bacilli. Further analysis suggested that early events in the emergence of resistance involved organisms (progenitors) that acquired a resistance phenotype. In addition, the progenitors appeared to be in a transient state, able to develop into a stable resistant lineage in the presence of CLR, or able to revert to the wild type in nonselective conditions.