Gene mutations analysis in resistant Mycobacterium tuberculosis isolates

Abstract

Objective To investigate the relationship between the phenotypes and the patterns of genetic mutations in the corresponding resistance genes (rpoB, katG, inhA, ahpC, rrs, rpsL, embB and gyrA) in resistant Mycobacterium tuberculosis (MTB) isolates. Methods Rifampicin-resistant gene (rpoB), isoniazid-resistant genes (katG, inhA, ahpC), streptomycin-resistant genes (rrs, rpsL), ethambutol-resistant gene (embB) and quinolinone-resistant gene (gyrA) were amplified by PCR with sequence-specific primers, then mutants screened by single-stranded conformation polymorphism (SSCP) were sequenced. Results rpoB mutation with predominant Ser450Trp pattern was 94. 9% (56/59) in 59 rifampicin-resistant isolates;katG mutation rate was 38. 9% (35/90) and the main pattern was Ser315Thr, but only 3 inhA mutants and no ahpC mutation were determined in 90 isoniazid-resistant isolates;gyrA mutation with main Asp94Gly then Ala90Val pattern was 82.4% (28/34) in 34 quinolinone-resistant isolates;the total mutation rate was 77.4% in 31 streptomycin-resistant isolates, of which 15 isolates mutated in rrs with main pattern A514C or A1041G, 10 isolates mutated in rpsL Lys88Arg;and embB mutation with main Met306Val accounted for 19.4% (6/31) in 31 ethambutol-resistant isolates. Conclusions The results showed that resistance of resistant MTB may be complicated, and DNA sequencing-based mutation analysis could efficiently detect the molecular makers such as rpoB, katG, gyrA, rrs, rpsL and embB in resistant MTB isolates. Meanwhile, it is notable that the rpoB mutation pattern in our isolates is different from previous report, further effort are needed to confirm the characteristics. The spectrum of potential resistance-related mutations in MTB clinical isolates may lay substantial foundation for the rapid molecular diagnosis and rational use of drug to MTB patients. Key words: Mycobacterium tuberculosis; Drug resistance, bacterial; Polymerase chain reaction; Mutation; Sequence analysis,DNA