Objectives:

Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol-resistant Mycobacterium tuberculosis isolates. The aim of this study was to improve or develop PCR–restriction fragment length polymorphism (RFLP) methods for rapid screening of ethambutol-resistant clinical M. tuberculosis isolates carrying substitutions at these codon positions.

Methods:

The M. tuberculosis H37Rv was used as the susceptible strain while well-characterised clinical isolates of M. tuberculosis with specific substitutions at embB codons 306 and 497 and iniA codon 501 were used as reference strains. The presence of mutations was detected by PCR amplification of the DNA region around the respective codon position followed by digestion with appropriate restriction enzymes to generate RFLPs.

Results:

The PCR–RFLP performed with Nla III with the susceptible strain carrying ATG and the mutant strains with GTG, ATT and CTG at embB codon 306 yielded DNA fragments of expected sizes. The restriction digestion performed with Hae III differentiated the strains with mutation at the first codon position (GTG and CTG) and those with mutations at the third codon position (ATT). The PCR–RFLP performed with AlwN I and Hpy99 I with the susceptible and mutant strains for embB codon 497 and iniA codon 501, respectively, also yielded expected DNA fragment patterns. In a preliminary application to clinical isolates, the established methods correctly identified mutations at embB codons 306 and 497 and iniA codon 501 in ethambutol-resistant M. tuberculosis strains and the results were confirmed by direct DNA sequencing.

Conclusions:

We have developed PCR–RFLP based methods for rapidly determining the substitutions at embB codons 306 and 497 and iniA codon 501. Since substitutions at these codon positions occur frequently in ethambutol-resistant clinical M. tuberculosis isolates, application of simple PCR–RFLP-based methods will result in rapid identification of resistant strains carrying these mutations.

Acknowledgement:

Supported by Kuwait University Research Administration grant MI 06/02.