Characterization Of The KatG, InhA, AhpC, KasA And OxyR Gene Mutations In Isoniazid-resistant And Susceptible Stains Of Mycobacterium Tuberculosis By Automated DNA Sequencing

Objective: To elucidate the characterization of katG, inhA, ahpC, kasA, and oxyR gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis, and discuss the value of judging the susceptibility of Mycobacterium tuberculosis strains to isoniazid by DNA sequencing. Method: A total of 101 isoniazid-resistant and 43 susceptible strains of Mycobacterium tuberculosis were analyzed by PCR and sequence analysis of their katG, inhA, ahpC, kasA, oxyR genes. Results: (1) Sequencing of katG from 101 INH-resistant strains showed point mutations, small deletions or insertions in 81 isolates(80.20%), but no complete deletions were identified. The mutations at 16 positions were found for the first time. Point mutations at position 315 including AGC→ACC(Ser-to-Thr) and AAC(Ser-to-Asn) substitutions were found in the genomes of 38.61%(39/101) of isoniazid-resistant strains. Among the 39 strains the substitutions of Ser-to-Thr were identified in 31 isolates and the substitutions of Ser-to-Asn were detected in 8 isolates. Low level isoniazid resistant strains (lug/ml) had higher mutation frequency at 315-Ser than high level isoniazid resistant strains (10ug/ml) (P<0.05). Mutations at position 463 causing an Arg-to-leu substitution were detected in 58 (57.43%) isoniazid-resistant strains, combinative mutation rate is 75.86%(44/58). mutations at 463 were independent of isoniazid-resistant. Partial gene deletions were found in 12 of 101 isoniazid-resistant strains, Frameshift insertion was detected only in 1 of 101 isoniazid-resistant isolates combining with synonymous mutation and Arg4631eu. 7 of 12 strains having small deletions showed low level isoniazid resistant including 6 strains at 1716 bp position deletion, and 1 isolates having insertion showed high level isoniazid resistant. In addition, Other types missense mutations were identified in 16 of 101 (15.84%) isolates. Arg4631eu was also present in 23 of 43 susceptible strains, In addition, 1 susceptible strains had synonymous mutation. (2) Among the 101 isoniazid-resistant clinical isolates, mutations in inhA genes were identified in 5 isolates ( Ser94Ala[n=2], Ile21Thr[n=2], small deletion[n=1]) (4.95%). Among the 5 isolates, 4 strains showing missense mutations all combined with Arg4631eu , and 1 strain having deletion combined with Ser315Thr. An Ser-to-Ala substitution at position 94 was detected in 2 isolates showing low level isoniazid resistant, An Ile-to-Thr substitution at posotion 21 was found in 2 isolates showing high level isoniazid resistant, and small deletion was identified in 1 isolates showing high level isoniazid resistant. None of the susceptible strains contained any mutation in inhA genes. (3) Only 3 isolates in the 101 (2.97%) isoniazid-resistant clinical isolates had mutations in ahpC genes (Ala195Asp[n=2], Glnl69Arg[n=l]); All the 3 isolates showed high level isoniazid resistant. 1 strain showing Ala 195Asp combined with Arg4631eu substitution. Glnl69Arg was first identified. No mutations were identified inthe ahpC genes in 43 isoniazid-susceptible isolates. (4) Mutations in kasA genes were present in 17 of 101 (16.83%) isoniazid-resistant isolates (Glyl21Lys[n=2], Gly312Ser[n=7], Gly387Asp[n=3], Phe413Leu[n=1], Asp66Asn[n=4] ). These strains all showed low level isoniazid resistant. 5 strains concluding Gly312Ser combined with Arg4631eu mutation. No mutations in katG, inhA, ahpC, oxyR genes were identified in 10 strains showing other types mutations. However, G312S was also present in 3 of 43 susceptible strains. (5) None of the isoniazid-resistant strains and susceptible isolates contained oxyR gene mutation. (6) Summarize: 85 of 101(84%) isoniazid-resistant strains had mutations in katG, inhA, ahpC, kasA genes associated with drug resistance. Conclusion: Genotypic analysis of resistant to isoniazid in Mycobacterium tuberculosis is complex due to the various genes potencially invovled. These studies further support the association between katG, inhA, ahpC, kasA gene mutations and INH resistant in Mycobacterium tu...