Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil

Abstract

We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide -48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G(269)A substitution. Although KatG(315) substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.

FIG. 1.

SSCP analysis of katG₃₁₅, inhA (promoter and structural ORFs), oxyR-ahpC intergenic region, and three regions of kasA in INH-resistant M. tuberculosis. All PCR products were denatured in the presence of 95% deionized formamide and electrophoresed by using specific conditions shown in Table 2 and by using a GenePhor electrophoresis system (Amersham Biosciences). (A) katG₃₁₅ (145-bp PCR products). Lanes: 3 and 10, wild-type control (H₃₇Rv); 1, W₃₄₁S; 2, S₃₁₅R; 4 and 6, S₃₁₅N; 7, S₃₁₅G; 5 and 8, wild type (susceptible isolates); and 9, S₃₁₅T. (B) inhA promoter (187-bp PCR products). Lanes: 1, wild-type control (H₃₇Rv); 2 and 4, C_-17T; 3, C_-15T. (C) inhA structural gene (codon 16) (222-bp PCR products). Lanes: 1 and 6, wild-type control (H₃₇Rv); 2, I₂₁V; 3, wild type (susceptible isolate); 4, L₄₄L; 5, I₂₁T. (D) oxyR-ahpC intergenic region (264-bp PCR products). Lanes: 1, 7, and 13, wild-type control (H₃₇Rv); 2, oxyR; 5, C₋₁₅T; 6, C₋₃₉T; 8, C₋₁₀T; 9, G₋₄₈A; 10, C₋₁₂T; 11, G_-9A; 12, C_-39T; 3, and 4, wild-type (susceptible isolates). (E) kasA region 3 (269-bp PCR products). Lanes: 1, wild-type control (H₃₇Rv); 2, 4, and 5, H₁₈₀H; 3, G₁₄₉G; and 6, wild type (susceptible isolates). (F) kasA region 4 (263-bp PCR products). Lanes: 1, wild-type control (H₃₇Rv); 2 and 3, G₂₆₉S. (G) kasA region 5 (223-bp PCR products). Lanes: 1, wild-type control (H₃₇Rv); 2, G₃₁₈G; and 3, wild type (susceptible isolates).