Recent reports of embB306 mutations in ethambutol-resistant and ethambutol-susceptible drug-resistant strains of Mycobacterium tuberculosis have questioned the significance of these mutations in conferring resistance to ethambutol (EMB).

To determine the occurrence of embB306 mutations in all EMB-resistant and -susceptible drug-resistant M. tuberculosis strains isolated during a specified period at a single geographical location.

Twenty-five pansusceptible, all EMB-resistant and -susceptible M. tuberculosis strains resistant to other first-line drugs isolated in Kuwait during 2000–2003 were analyzed. The embB306 mutations were detected by PCR-restriction fragment length polymorphism and DNA sequencing. The PCR-based methods were also used for determining strain relatedness.

None of the pansusceptible M. tuberculosis strains contained a mutation at embB306. Fifteen of 50 (30%) EMB-resistant strains but only three of 122 (2%) EMB-susceptible isolates resistant to other first-line drugs contained a mutated embB306. The EMB-susceptible isolates with embB306 mutation were resistant to isoniazid plus other drug(s). The isolates carrying similar mutations were genotypically distinct strains.

The frequency of embB306 mutations in EMB-resistant strains compared to EMB-susceptible M. tuberculosis isolates resistant to other drugs was 15 times higher. Association of embB306 mutations in EMB-susceptible strains with isoniazid resistance and inherent problems associated with phenotypic EMB susceptibility testing suggest that these strains may actually be EMB-resistant.