KATG GENE MUTATIONS IN ISONIAZID RESISTANT MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES

• Main Author: Rosa Pane, Elfira
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/6041
• Institution: Institut Teknologi Bandung

ABSTRACT :<br /><br /><br /> Tuberculosis (TB) is an infected diseased caused by Mycobacterium tuberculosis, and treatment with anti-tuberculosis drugs (OAT) could cured the disease. Inappropriate medication could cause TB drugs resistance. Multi-drug Resistant TB (MDR-TB) has been define by World Health Organization as strains of tuberculosis that are resistant to at least the two main first-line drugs, namely isoniazid (INH) and rifampicin (RIF). Resistance to RIF has been associated with mutations in rpoB, the gene coding for the ? subunit of RNA polymerase, and the highest frequency in codon 526 and 531. INH resistance is most frequently associated with a single mutation in katG, a gene that encodes the catalase-peroxidase enzyme in M. tuberculosis. INH is a pro-drug and requires catalytic activation into its active form, which done by the M. tuberculosis catalase-peroxidase enzyme. One particular substitution reported to be the most frequent that confers resistance to INH is G944C, in codon 315 which repalced, AGC to ACC, hence Ser mutated to Thr. Our research group in KK Biokimia ITB has six clinical isolates (L4, L7, L10, L18, L19, and R2) MDR-TB, which genotypic-ally showed mutation in rpoB gene codon 526 and 531, but no mutation found in katG315 based on PCR experiment assays. The aim of our research is to find the genotype information of the above resistance INH in six clinical isolates.<br /><br /><br /> Our research methods consist of Polymerase Chain Reaction (PCR) multiplex specific alleles katG assays, gel agarose electrophoresis, nucleotides sequencings, and in silico analysis. The PCR multiplex assay employed three primers, two outer primers KF and KR; and one inner reverse primer K315. DNA fragments of PCR product were confirmed by sequencing method. In silico analysis of nucleotides using DNA star program was used to detect the position of mutations in six clinical isolates with wild-type H37Rv as a reference, and the Pymol program was exploited for protein modeling.<br /><br /><br /> Multiplex PCR of all isolates showed DNA fragment of 0.43kb and 0.29kb bands. Electroforegram of 0.43kb katG gene fragment were compared to the same fragment of M. tuberculosis wild type. Homology analysis showed three isolates (L10, L18, and L19) have mutation in nucleotide 946, G to T; and an isolate (R2) has mutation in