Molecular characterization of drug-resistant and extensive-drug resistant isolates of Mycobacterium tuberculosis in Shenzhen of China

Abstract

To study the molecular characterization of drug-resistant and extensive-drug resistant isolates of Mycobacterium tuberculosis (MTB) in Shenzhen of China during 2007 - 2008. According to the standards of WHO and International Union Against Tuberculosis and Lung Disease (IUATLD), 136 strains of MTB were collected by performing drug sensitivity test (DST) to isoniazid, rifampicin, streptomycin, ofloxacin and kanamycin on Lowenstein-Jensen in 1% proportion method. Genetic mutations in the corresponding resistance genes (rpoB, katG, rpsL, rrs((1)), gyrA/B, rrs((2))) in these MTB isolates were identified by PCR, followed by DNA sequencing of the purified PCR products. A total of 123 isolates were collected. Seventy-three isolates were drug resistant, and 50 isolates were drug susceptible. Among the isolates that were resistant to isoniazid, rifampicin, streptomycin, ofloxacin and kanamycin, the proportion of isolates that harboured mutations in the respective genes was 44/52, 44/47, 28/41, 11/11, and 11/18, respectively. For katG gene, the mutation detected was S315T or S315N. For rpoB, the most frequently found changes were S531L (30/44) and H526D (9/44) or H526R (1/44). For the reported mutations related with streptomycin-resistant strains, K43R and K88Q were found in the rpsL locus, and 491C-->T and 513A-->C were found in the rrs gene. For gyrA, all gyrA mutations were clustered in codons 90, 91, and 94 apart from the S95T that was natural polymorphism, accounting for 9/11 of the ofloxacin-resistant isolates, and condon 91 was the most frequently mutated. No mutations were found in gyrB. The most frequent substitutions were 1400A-->G (9/11) and 1483G-->T (2/11) in a specific region of the rrs gene related with kanamycin-resistant strains. No mutations except S95T of gyrA was detected in the drug- susceptible isolates. The mutation characterization of drug-resistant and drug- susceptible isolates of MTB was shown to vary according to geographic regions, and these findings can help understanding the molecular characterization and establishing a novel method for the detection of drug-resistant MTB.