THE SUBSTITUTION OF C1349T rpoB GENE IN MULTIDRUG-RESISTANT Mycobacterium tuberculosis CLINICAL ISOLATE L18 AND R9

• Main Author: KUSNANDAR (NIM: 20507001), BERTHA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/11915
• Institution: Institut Teknologi Bandung

Mycobacterium tuberculosis (M. tb) causes a contiguous disease, called tuberculosis (TB), which is widespread through patient's sputum. Until now, Indonesia is one of the developing countries, which has huge numbers of TB cases and at the third in the world for its mortality rates. The TB-treatment using one or more antibiotics (rifampin, isoniazid, canamycin, streptomycin, ethambutol, and pyrazinamide) takes at least six months. The inadequate and unfinished treatment emerge Multidrug-resistant strains of M. tb (MDR-M. tb). World Health Organization (WHO) defines that MDR-M. tb is a strain that is resistant to at least two drugs, namely rifampin and isoniazid. Mutations on the resistance-determining region (RRDR) on the rpoB gene were hypothesized to be one of surrogate markers for MDR-M. tb. The Biochemistry Laboratory of ITB has a collection of 42 clinical isolates MDR-M. tb. Phenotypic properties of these clinical isolates against antituberculosis drugs had been characterized. In contrast, genotypic properties of most isolates are unknown. The aim of this study is to sequence DNA fragments of the rpoB gene of L18 and R9 isolates MDR-M. tb, which have been analysed genotypically by multiplex allele-specific (MAS) rpoB Polymerase Chain Reaction (PCR).<p>Several methods were used in this study to obtain the research objective. Initially, amplifications of the DNA fragment of the rpoB gene of the L18 and R9 isolates MDR-M. tb were carried out by Polymerase Chain Reaction (PCR) using RF and RR primers. The PCR product was then visualized using agarose gel electrophoresis. Nucleotide sequence of the PCR product was then sequenced by Macrogen Inc., Seoul, Korea by employing the dideoksi Sanger method. Finally, in silico analysis was done by employing SeqmanTM and Meg align, associated to the DNASTAR program. A 0,25 kb DNA fragment of the samples was produced by PCR. This product was confirmed by electrophoresis with DNA pUC19/HinfI as a marker. The sequencing results showed that L18, R9 isolates and H37Rv have nucleotides 215 pb, 190 pb and 207 pb in sizes, respectively. The presence of mutated nucleotides is known by aligning sequences of both isolates and M. tb H37Rv as the wild type strain, using the SeqManTMII and MegAlignTM DNASTAR programs.<p>The alignment between the isolates, L18 and R9, with the H37Rv showed the same mutation at 1349 position as genotipic information, reported previously. The mutations of both isolates are substitution nucleotide base of cytosine to thymine (C1349T). As reported, the C1349T mutation in the rpoB gene in the RRDR is generally and highly frequency for the M. tb isolates which are resistant rifampin. Rifampin is an antibiotic that binds to the B-subunit of RNA polymerase (rpoB) of M. tb bacteria resulting in inhibition of transcription initiation. As conclusion of this study and the previous researches, the C1349T subtitution in rpoB gene is assumed as one of the causative agents for phenotypic alteration in L18 and R9 MDR-M. tb isolates. The intact sequences of the rpoB gene from L18 and R9 can give more information about a position and type mutation for genotipic properties of the rpoB gene of M. tb. Further researches, which analysis genotypic characterization of other isolates from the biochemistry laboratory of ITB can lead to give more information on MDR-M. tb.