MUTATION OF CODON 306 embB GENE CLINICAL ISOLATES R9 AND L25 MULTIDRUG RESISTANT Mycobacterium tuberculosis

• Main Author: Nugraha Mazaafrianto, Donny
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35418
• Institution: Institut Teknologi Bandung

Tuberculosis (TB) is an infectious and contagious disease caused by a bacterial pathogen Mycobacterium tuberculosis (M. tuberculosis). The bacteria are spread through the air when patients cough, sneeze, talk or spit droplets contaminated with M. tuberculosis. A person can be exposed simply by inhaling small amounts of bacteria. According to WHO, a third of the world population is infected with TB and Indonesia ranks at the thirds in the world's TB cases. The emergence of multidrug- resistant strains of M. tuberculosis (MDR-M. tuberculosis) due to irregular and exhaustive in treatment, adding difficulties to overcome this disease. TB bacteria are classified as MDR-M. tuberculosis strains that were resistant to at least two drugs, rifampicin and isoniazid. Strains of MDR-M. tuberculosis can also resistant to other drugs, such as pyrazinamide, ethambutol, and streptomycin. Based on literature study and the results of previous researchers, the nature of ethambutol resitance allegedly incurred due to a gene mutation encoding a arabinosil transferase. This enzyme plays a role in the formation of cell walls of M. tuberculosis. Biochemistry Laboratory at ITB had a collection of 42 clinical isolates of MDR-M. tuberculosis that are resistant to the nature of phenotypes showed first-line TB drugs by genotype but not yet fully characterized, especially in isolates that are resistant to ethambutol. The purpose of this study to obtain information on the cause of ethambutol resistance in isolates R9 and L25, MDR-M. tuberculosis from our collection. This research begins with a target of gene amplification by the polymerase chain reaction method (PCR) of the embB gene, agarose gel electrophoresis, nucleotide sequencing, and in silico analysis. The embB gene amplification used primers, embF and embR. DNA fragments of its nucleotide sequence determined by sequencing Dideoksi Sanger method., Seoul, South Korea. Analysis in silico using the program DNASTAR are EditSeqTM, MegAlignTM and SeqManTM to detect the position of mutations in isolates compared with the nucleotide sequence as the standard H37Rv wild type bacteria. PCR fragments of the embB gene for each isolate showed a single band of 0.8 kb size on agarose gel electrophoresis. Data electrophoregram results of nucleotide sequencing isolates R9, L25, and H37Rv respectively 710-bp, 748-bp and 670-bp. Alignment analysis of nucleotide sequence of all isolates produced mutation (A916G) for isolate R9 and (G918A) isolates L25. Analysis of mutations at the amino acid level showed that the isolate R9 containing ATG to GTG alteration, corresponding to the methionine to valine, respectively. Whereas, the isolate L25 showed ATG to ATA, related to methionine to isoleucine. Both mutations were found at the same codon 306. These results are in accordance with previous researches, regarding to ethambutol resistant of M. tuberculosis, which is closely associated with the codon 306 mutation of embB. These mutations are likely causing the area surrounding the protein produced by the embB gene becomes more hydrophobic. Consequently, hydrophobic area of the enzyme is not compatible with polar substrate ethambutol. The analysis results were expected to provide information about the position and type of mutations in the gene of embB MDR-M. tuberculosis isolates R9 and L25. Further research is needed to analyze the protein properties of MDR-M. tuberculosis especially related to ethambutol resistant. In addition, the study of the embA and embC genes genotypically is also needed to provide more complete information about the causative agent of resistance to ethambutol.