MUTATIONS IN KATG GENE-SEQUENCES IN ISONIAZID-RESISTANT CLINICAL ISOLATES OF MYCOBACTERIUM-TUBERCULOSIS ARE RARE

In this study, a battery of oligonucleotides was directed toward the k atG gene and PCR-single-stranded conformation polymorphism (SSCP) anal ysis was used to search for katG gene deviations in clinical isolates of Mycobacterium tuberculosis from different geographical regions. Sin ce a complete deletion of the katG gene was not found, it is suggested that deletion is not a major mechanism of isoniazid (isonicotinic aci d hydrazide; INH) resistance in these isolates. However, 7 of 39 isola tes (4 of 25 from South Africa and 3 of 14 from other geographical reg ions) showed mobility shifts by SSCP analysis, suggesting aberrations in the katG gene. Direct sequence analysis confirmed that the mobility shifts were due to Thr-275-->Ala (Thr275Ala), Arg309Ala, Arg463Leu, a nd Asp695Ala mutations and a 12-bp deletion in the 5' region of the ka tG gene. Mutations at codons 275, 463, and 695 created altered restric tion sites for HhaI, MspI, and HaeIII, respectively, and sequence resu lts, supported by restriction fragment length polymorphism analysis, s uggested that the PCR-SSCP procedure is a good indicator of mutations in PCR-amplified fragments. Identical mutations at codons 463 and 275 were found in isolates from different geographical regions. This may s uggest a common evolutionary event, but one of the control isolates (s usceptible to INH [3%; n = 30]) also had a mutation at codon 463. The results suggest that variations in the katG coding gene sequences of I NH-resistant isolates of M. tuberculosis are infrequent and that defec ts in other regions of the M. tuberculosis genome are of equal or grea ter importance in contributing to the acquisition of resistance to INH .