Allelic Exchange and Mutant Selection Demonstrate That Common Clinical EmbCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol In Mycobacterium tuberculosis
Antimicrob Agents Chemother. 2009 Oct 12;
Mutations within the 306 codon of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) minimum inhibitory concentrations (MICs). To identify other causes of EMB-resistance and to identify causes of high-level resistance we generated EMB-resistant M. tuberculosis in-vitro and performed allelic exchange studies of embB406 and embB497 mutations. In-vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13 with EMB MICs of 8 or 14 mug/ml, 5 mug/ml, 12 mug/ml, 3 mug/ml, and 4 mug/ml respectively; and mutations at embB320, embB324 and embB445 that have not been identified in clinical M. tuberculosis with EMB MICs of 8 mug/ml, 8 mug/ml, and 2-8 mug/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB-susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497CAG codon in EMB-susceptible M. tuberculosis strain 210 for embB497CGG; and the wild-type embB406GGC codon for either embB406GCC, embB406TGC, embB406TCC, or embB406GAC. These new mutants showed a 6 fold and a 3 to 3.5-fold increase in EMB MIC, respectively. In contrast to embB306 mutants, isogenic embB497 and embB406 mutants did not have preferential growth in sub-MIC concentrations of isoniazid or rifampin. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MIC. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB-resistance is multi-genic and high level EMB-resistance requires mutations in currently unknown loci.