Genetic analysis of the embB gene revealed mutations in 17 (68%) of 25 ethambutol (EMB) resistant isolates (M306I, M306V, M306L, Q497R) but also in 4 (20%) of 20 EMB-susceptible isolates of Mycobacterium tuberculosis, namely, an ATG3ATM substitution resulting in M306I, G406N, and the novel alterations M423I and A659T. Ethambutol (EMB) [(S,SЈ)-2,2Ј-(ethylenediimino)di-1-butanol] is a first-line drug used for antituberculosis therapy. It is often used in combination with isoniazid, rifampin, pyrazinamide, and streptomycin. Membrane-associated arabinosyl transferases have been implicated as the targets for EMB (2,3,14,15). The Mycobacterium tuberculosis emb operon is a gene cluster of three contiguous genes, namely, embC, embA, and embB, which encode mycobacterial arabinosyl transferases (26). These enzymes are involved in the polymerization of the cell wall arabinan (4,6,9,24,25,32). Inhibition of arabinan synthesis by EMB results in the accumulation of mycolic acids, leading to cell death.Alterations at codon 306 of embB have been identified as being the most common alteration in EMB-resistant M. tuberculosis clinical isolates (8, 12, 17-20, 23, 29). Initial work on 51 EMB-resistant isolates had shown that 89% of these isolates had alterations at residue 306 of embB, but these alterations were not detected in 30 EMB-susceptible isolates (23). A subsequent study confirmed this high frequency of embB306 alterations, with 67% of 75 EMB-resistant isolates having mutations not found in EMB-susceptible strains (19). This led to several groups developing targeted strategies for the detection of embB306 alterations (7,16,21,30). Amino acids within the EMB resistance-determining region of EmbB proteins are well conserved among mycobacterial species, including those from M. tuberculosis, M. leprae, and M. smegmatis (2), and mutations within this region have been detected in EMB-resistant isolates of M. tuberculosis.The aim of this present work was to screen all regions of the embB gene with previously reported mutations in order to assess the contribution of mutations within this gene to EMB resistance in M. tuberculosis clinical isolates from Singapore.Drug susceptibility testing was done using the BACTEC 460 radiometric method (Becton Dickinson, Towson, Md.) (2.5 g/ml). Twenty-five consecutive M. tuberculosis isolates resistant to EMB and 20 EMB-susceptible isolates from Singapore were collected as previously described (5, 10).DNA extracted from the isolates was analyzed by amplifying four fragments, using the PCR primers shown in Table 1. The PCR products were purified (QIAquick PCR purification kit or QIAquick gel extraction kit; QIAGEN) and directly sequenced using the BigDye Terminator sequencing kit and the ABI PRISM 377 automated sequencer (PE Biosystems, Branchburg, N.J.). Confirmation of mutations was done by reamplification and resequencing.IS6110 profiling was done according to standard procedures to determine if the isolates were epidemiologically independent (28). All isolates with the same nucleotide substit...