Detection and Molecular Characterization of Mutations in rpoB, katG, and gyrA Genes of Mdr-Tb Isolates of the Philippine General Hospital

Abstract

The emergence of multidrug-resistant tuberculosis (MDR-TB) poses a significant challenge to prevailing TB therapeutic management. Drug resistance could arise from the mutation of several genes such as rpoB for rifampicin, katG for isoniazid and gyrA for ciprofloxacin, a fluoroquinolone drug. To detect and characterize the mutations of selected Mycobacterium tuberculosis isolates from the Infectious Disease Section of the Philippine General Hospital, 30 isolates were subjected to PCR amplification and sequencing and their sequences compared to the wild type strain H37Rv. The rpoB sequences revealed mutations in codons 512, 513, 516, 522, 526, 531 and 533.The katG region showed mutations in codons 280, 281, 315 and 333 while gyrA showed mutations in codons 90 and 94. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94 respectively. The clustering analysis of the mutations showed seven clusters for rpoB, four clusters for katG and three for gyrA. The isolates changed location from one cluster to another when they had different mutations in different genes. Eight clusters obtained from the combined sequences of the three genes possibly represents potential eight genotypes of local M. tuberculosis isolates wherein one cluster may possibly represents the genotype of the wild type strains and another cluster potentially represents a genotype of XDR strains. The remaining clusters may possibly correspond to six different genotypes of the MDR strains. Most of the mutated isolates showed confirmation with phenotypic resistance. A likely structural change associated with one or more mutations to the target enzymes would account for the conferred resistance. Similarly, much of the non-mutated samples prove to be susceptible to the drugs. Papers reviewed from other countries showed similar information except for katG and argued against the role of geographical isolation in developing resistance. Predictive efficiency (PE) for each gene was relatively high except for katG. RpoBpE was found to be at 90%, katGpE at 76.67% and gyrApz at 86.67%. The calculated predictive efficiency for MDR-TB and extensively drug resistant tuberculosis (XDR-TB) were estimated to be 68.4% and 58.82% respectively.