Transfer of embB Codon 306 Mutations into Clinical Mycobacterium tuberculosis Strains Alters Susceptibility to Ethambutol, Isoniazid, and Rifampin

Abstract

ABSTRACT Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis , mutations in codon 306 of the embB gene ( embB 306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB 306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB 306 mutations in any type of drug resistance. Replacing wild-type embB 306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB 306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 μg/ml to 7, 7, 8.5, and 14 μg/ml, respectively. Replacing embB 306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 μg/ml or 28 μg/ml, respectively, to 3 μg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB 306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB 306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB 306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.