Characterization of the embB gene in Mycobacterium tuberculosis isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array

Abstract

Ethambutol resistance has mostly been related to mutations in the embB gene. The objective of the present study was to characterize the embB gene in a collection of ethambutol-resistant and ethambutol-susceptible isolates of Mycobacterium tuberculosis complex (MTBC) from Barcelona, and to develop a DNA microarray for the rapid detection of embB mutations in our area. Fifty-three ethambutol-resistant and 702 ethambutol-susceptible isolates of MTBC were sequenced in internal 982-1495 bp fragments of the embB gene. In addition, a low-cost, low-density array was designed to include the embB codons identified as being most frequently mutated in our area (LD-EMB array). The global prevalence of embB mutations found among the ethambutol-resistant isolates was 77.4% (41/53). Substitutions in embB306 were the most common [53.7% (22/41)], followed by substitutions in embB406 [26.8% (11/41)]. The presence of mutations in embB406 was related to higher levels of ethambutol resistance and to multidrug resistance. Among unrelated isolates (from 24-locus MIRU-VNTR genotyping), the percentage of embB-mutated isolates was 72.9% (27/37)--59.3% (16/27) in embB306 and 25.9% (7/27) in embB406. None of the ethambutol-susceptible isolates studied showed a mutation in codon 306 or 406. The LD-EMB array showed 100% sensitivity and specificity in identifying the main embB substitutions in our area. Mutations at codons 306 and 406 of embB have a relevant role in resistance to ethambutol in our area. The LD-EMB array developed in this study would appear to be a good molecular test for rapid detection of ethambutol resistance.