Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin

Abstract

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 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 embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 microg/ml to 7, 7, 8.5, and 14 microg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 microg/ml or 28 microg/ml, respectively, to 3 microg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 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 embB306 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 embB306 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.

FIG. 1.

Differential growth rates of the isogenic mutant NJT210CTG and M. tuberculosis strain 210. Strains 210 (closed symbols) and NJT210CTG (open symbols) were cultured in different sub-MICs of INH (A) and RIF (B) (concentrations are shown below the graphs and are in micrograms per milliliter). The daily growth index for each culture is shown. The 1:100 data represent results for no-antibiotic control cultures inoculated after a 1:100 dilution in the medium. The means and standard deviations of three independent experiments are shown.

FIG. 2.

Differential growth rates of M. tuberculosis strains 30167 and NJT30167ATG. Strains 30167 (open symbols) and NJT30167ATG (closed symbols) were cultured in different sub-MICs of INH (concentrations are shown below the graph and are in micrograms per milliliter). The daily growth index for each culture is shown. The 1:100 data represent results for no-antibiotic control cultures inoculated after a 1:100 dilution in the medium. In both Fig. 1 and 2, the strains with the embB306 ATG allele show repressed growth at sub-MICs. The means and standard deviations of three independent experiments are shown.

FIG. 3.

Competition assays at sub-MICs of INH. Equal numbers of strains 210 and NJT210 CTG were cultured with no antibiotics or in different sub-MICs of INH, as shown. Significant differences in growth are indicated as follows: *, P < 0.01; **, P = 0.01. The means and standard deviations of three independent experiments are shown.