Mutations in the embB Locus among Korean Clinical Isolates of Mycobacterium tuberculosis Resistant to Ethambutol

Resistance of Mycobacterium tuberculosis to ethambutol (EMB) has been assigned to an operon, embCAB, which has been proposed to be a structural gene for mycobacterial arabinosyl transferases. Recently, genetic events resulting in structural mutations at embB have been proposed as major contributors to the EMB-resistance of isolates whose minimum inhibitory concentration (MIC) level is higher than 20 microgram/ml. On the contrary, isolates with a MIC level lower than 20 microgram/ml do not seem to contain any sequence alterations. In this study, in an effort to understand the role of embB mutations at a low-level of EMB resistance, we investigated the sequence polymorphisms of clinical isolates whose MIC levels are lower than 10 microgram/ml. Accordingly, the sequence alterations of a 312-bp region of the embB gene containing the 306th codon, which has been assigned as a hot-spot for EMB-resistance related mutations, were determined for 21 EMB-resistant and 5 EMB-susceptible clinical isolates. In brief, among 21 EMB- resistant isolates examined, 12 (57.1%) contained mutations in embB (10 at the 306th codon and 2 at other sites), and the remaining isolates 9 contained no mutations in any region of embB. The observed mutations included M306V, M306I, and M306L substitutions that have been reported previously. However, 3 were novel types, which included M306T, A313G and Y322C, D331Y double substitutions. On the other hand, all of the EMB-susceptible isolates were found to be free of mutations. In conclusion, our findings suggest that sequence polymorphism of embB may play a pivotal role in the EMB- resistance of M. tuberculosis.

Evaluation of PCR-SSCP vs. PCR - Sequence Analysis for Detecting Rifampicin Resistance of Mycobacterium tuberculosis Clinical Isolates

In the present study, we made an attempt to compare polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with PCR-direct sequence analysis for their accuracy and sensitivity in detecting resistance to rifampicin (RMP). A total of 32 clinical isolates of Mycobacterium tuberculosis including 22 resistant and 10 sensitive isolates, whose drug susceptibility have been tested by conventional proportion method, were analyzed by using PCR-SSCP and PCR-sequence analysis. Among 22 RMP resistant isolates, 16 isolates showed SSCP profiles different from that of a RMP sensitive control strain, M. tuberculosis H37Rv indicating the possible existence of a sequence alteration in this region of the rpoB gene, while 6 resistant isolates displayed SSCP profiles indistinguishable from the sensitive control strain. On the other hand, all of 10 RMP sensitive isolates showed SSCP profiles similar to that of the sensitive control strain. Therefore, overall agreement rste between conventional proportion method and PCR-SSCP reached 81%. Subsequently, all of 32 clinical isolates were subjected to sequence analysis. The results from the sequence analysis revealed that all of 22 resistant isolates indeed contain mutations in the stretch of 81 bp region of rpoB gene, while none of 10 sensitive isolates contain any sequence alterations. Therefore, this study suggests that PCR-sequence analysis works more efficiently and accurately than PCR-SSCP analysis for rapid screening of RMP-resistant M. tuberculosis clinical isolates.