Studies On Mutations Of RpoB And KatG Genes In Strains Of Drug-resistant Mycobacterium Tuberculosis

Objectives To study the mutations of rpoB and katG genes in drug-resistantmycobacterium tuberculosis(MTB) isolates from Tianjin, and to investigate themolecular mechanisms of rifampin-and isoniazid-resistant MTB.Methods 60 MTB clinical isolates from reference laboratary of Tianjin TuberculosisControl Center were analysed, included 50 RFP and(or) INH-resistant isolatesbetween July 1, 2005 and April 30, 2006 and 10 isolates sensitive to both drugsbetween January 1, 2006 and February 30, 2006. Drug susceptibility testing wasperfomed by BACTEC MGIT960. The isolates were identified their mycobacterialspecies by PNB and TCH growth media. The PCR-SSCP, reverse dot-blothybridization (RDBH) and PCR-DNA sequencing (PCR-DS) were porformed todetecte the mutations in the rpoB and katG genes from those clinical isolates. H37Rvstandard strain was used as a control.Results Among 60 MTB clinical i.solates, 27 were found to be RFP-resistant, 33were RFP-sensitive, 43 were INH-resistant, 17 were INH-sensitive.1 The results of rpoB gene mutation:305bp fragments of rpoB genes from 60 MTB isolates were amplified by PCR andanalyzed by SSCP. Compared with the H37Rv, 7(26％) of 27 RFP-resistant isolatesand 1 of 33 RFP-sensitive isolates had different SSCP patterns.240bp fragments of rpoB genes from 7 RFP-resistant MTB isolates whichdisplayed different SSCP patterns were amplified by PCR and analyzed by RDBH, 4isolates had mutations at condon 531TCG→TTG(Ser→Leu), 1 at 526CAC→TAC(His→Tyr), 1 at 516GAC→GGC(Asp→Gly) and 533CTG→CCG(Leu→Pro), one of the isolates was not found mutation by RDBH andthen detected by PCR-DS carrying mutation at 526 CAC→CGC(His→Arg), 4condons and 5 types of mutations were found in 81-bp "core region" of rpoB in all.2 The results of katG gene mutation: 273bp fragments of katG genes from 60 MTB isolates were amplified by PCRexcept 1 INH-resistant isolate, and then analyzed by SSCP. Compared with theH37Rv, 17 of 42 INH-resistant isolates had different SSCP patterns. 18 (42％) of 43INH-resistant isolates were found mutations in katG.273bp fragments of katG genes from 17 INH-resistant MTB isolates wereamplified by PCR and analyzed by RDBH., 13 isolates had mutations at condons of315 AGC→ACC (Ser→Thr), 1 at 31.5 AGC→AAC(Ser→Asn), 2 at 315 and thenrevealed by PCR-DS carrying mutations ofACG(Thr), ACA(Thr) respectively, I wasnot found mutation by RDBH and then detected by PCR-DS carrying mutation at306ACC→CCC (Thr→Pro). 2 condons and 5 types of mutations were found in all.Conclusions Mutations at condon 531 in rpoB gene and 315 in katG gene are themost frequent in MTB isolates from Tianjin, mutations of rpoB and katG genes areassociated with RFP and INH resistance, respectively.