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" Detrction of rifampin resistsnce patterns in mycobacterium tuberculosis strains isolated in isfhan using PCR-SSCP and direct sequencing metods( 1381-83 ) "
; Akbar tavakoli, Hajeeh Ghasemian Safaee, Farah Taj Navabakbar, Mansoor Salehee
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Isfahan University of Medical Sciences
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| Document Type
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Latin Dissertation
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| Language of Document
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English
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| Record Number
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102564
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| Doc. No
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T9450
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| Call number
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QW,125.5,M9,N264d,2005
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| Main Entry
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Nasr isfahani, Bahram
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| Title & Author
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Detrction of rifampin resistsnce patterns in mycobacterium tuberculosis strains isolated in isfhan using PCR-SSCP and direct sequencing metods( 1381-83 )
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| College
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Schools, Medical
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| Date
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, 2005
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| Degree
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Bacteriology, Ph.D
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| Page No
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g,116 p.: dig., tab.
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| Note
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This thesis is also a research project with project ID: 83179
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Original works
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| Abstract
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ntroduction: Tuberculosis is the leading cause of death in the world from a single infectious disease. After a century of decline, tuberculosis is increasing, and multiple drug-resistant strains have emerged. Multidrug-resistant TB (MDRTB), associated with high death rates of 50 to 80 , spans a relatively short time (4 to 16 weeks) from diagnosis to death. Rifampin is one the most important antimycobacterial agents that interacts with the beta-subunit of RNA Polymerase (rpoB), thereby hindering transcription. Mutations in the rpoB locus confer conformational changes leading to defective binding of the drug to rpoB and consequently resistance in Mycobacterium tuberculosis species. In the past few years, genetic and molecular insights have unraveled the mechanisms involved in the acquisition of drug resistance by Mycobacterium tuberculosis (MTB). PCR-SSCP (Polymerase chain reaction-singlestrand conformation polymorphism) was established as a rapid screening test for the detection of mutation in rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations.Materials and Methods: A total of 37 Mycobacterium tuberculosis strains, 16 Rifampin susceptible (RIFs) and 15 Rifampin resistance (RIFr) strains from Isfahan and 6 RIFr strains from Pasteur Institute of Iran were used in this study. They were confirmed as M. tuberculosis by conventional methods (Staining by Ziehl Neelsen method, testing for their speed of growth and their colony morphology and nitrate reduction test) and PCR of DR gene specific amplification of DRs (Direct Repeats, a variable number of them are presents in the genomic DR region of M. tuberculosis complex strains and separated from others by a variety of spacer DNA sequence). Using 1 proportion method on Lowenstein-Jensen medium contained 41.tg/mL Rifampin, the sensitivity or resistance of M. tuberculosis isolates was determined. Using upstream primer (rpoB 105) and downstream primer (rpoB 273), a 193 - bp region of the rpoB gene was amplified and PCR-SSCP patterns of RIFr and RIFs strains were determined by electrophoresis on 10 acrylamide gel and then silver staining. Also 21 samples of 193-bp rpoB amplicons from RIFr and 10 from RIFs strains were sequenced and then analyzed using Clustalw, version 1.82, and software, EMBL-EBI. Each sequence was compared both with the control strain sequence and with the published rpoB sequence.Results: Staining by Ziehl Neelsen method, testing for speed of growth, colony morphology, nitrate reduction test and amplified specific region of DR gene confirmed species of M. tuberculosis.The PCR products of specific193 - bp region of rpoB gene of all RIFs and RIFr M. tuberculosis isolates showed identical bands similar to H37Rv strain on 2 agarose gel by electrophoresis. By the PCR-SSCP analysis, 7 PCR-SSCP distinguishable patterns were seen in the RIFr strains. Although 6 of these patterns were different but one pattern was identical to sensitive standard strain H37Rv. Six resistant strains from Pasteur institute of Iran demonstrated two different patterns. 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated different pattern. After determinations the rpoB sequences of the resistance strains, different mutations were seen in codons 516 (GAC/GTC), 526(CAC/TAC),531(TCG/TTG), 523 (GGG/GGT), 511(CTG/TTG) and 512(AGC/TGC. Conclusions: On the basis of the SSCP results, seven groups were determined: group one (4 bands, 4.75 ), group two (5 bands, 9.5 ), group three (5 bands, 4.75 ), group four (4 bands, 2.8 ), group five (4 bands, 23.8 ), group six 6(3 bands, 4.75 ) and group seven (3 bands, 9.5 ). This study demonstrated that frequencies of particular PCR-SSCP patterns in RMP-resistant M. tuberculosis isolates from Isfahan have some differences from those that have been reported for isolates of other geographic areas. The percentage of resistance was not correlated with the PCR-SSCP patterns (Pvalue>0.05), but was correlated with nationality (Pvalue<0.05).21 RIFr and 10 RIFs isolates were analyzed for DNA sequences. 85.8 showed a single mutation and 14.2 had no mutations in the 193-bp region of the rpoB gene. 47.6 had a mutation at codon 531 (TCG/TTG, Ser/Leu); 19 at codon 526 (CAC to GAC, His/Tyr); 4.75 at codon 516 (GAC to GTC, Asp/Val); 4.75 at codon 523 (GGG/GGT, LeufLeu); 4.75 at codon 511 (CTG/TTG, Leu/Leu) and 4.75 at codon 512 (AGC/TGC, Ser/Ser).Our data supported the common notion that Rifampin resistance genotypes with mutations at critical codon were the most frequently found in M. tuberculosis populations regardless of geographic origin. The results analysis showed that the mutations were correlated with the percentage of resistance (Pvalue<0.05), but not with the nationality (Pvalue>0.05)..
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| Descriptor
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1. Mycobacterium Tubercuiosis.- Descriptors: Tuberculosis
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Mycobacterium Tubercuiosis
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DNA
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Diagnotic Test, Routine
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Polymerase Chine Reaction
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Antitubercular Agents
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| Added Entry
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Tavakoli, Akbar, supervisor
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Hageeh ghasemian safaee, supervisor
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Navabakbar, Farahtaj, supervisor
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Salehee Mansoor, supervisor
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