Investigation of the Mechanisms of Resist Ance to Streptomycin in Mycobacterium tuberculosis

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Addis Ababa University


MIC value of streptomycin resistant M. tuberculosis strains was detemlined using tlle proportional method. Sequence analysis of IpsL and rrs genes was performed using botll tlle radioactive and non-radioactive cycle sequencing protocols. For functional analysis ofrRNA of M. tuberculosis, M. smegmatis strain mc2 155 was used as a model system. There were 34 streptomycin resistant Mycobacterium tuberculosis strains subjected for MIC value dete~ation. This included 18 strains from Sweden and 16. strains from Etlliopia. Their streptomycin resistance character had been previously confirmed by culruring in BACTEC media contailling 4~lg/ml streptomycin. Eleven strains growing on 7HI0 plates containing ~ 160 )lg/ml streptomycin were classed as high level resistant, 13 strains growing on 5-10 )lg/ml streptomycin classed low level resistant and 10 strains were found to be susceptible to streptomycin. DNA was extracted from tlle 11 high level and from 10 of tlle 13 low level streptomycin resistant strains. The extracted DNA was PCR amplified by targeting the rpsL and rrs genes. PCR products of all the high level and 6 of the low level resistant strains were sequence analyzed. For the rpsL gene, 9 of the high level resistant strains had the previously documented mutation at codon 43, and one strain has mutation at codon 88. One of the high level streptomycin resistant strains was negative for PCR amplification of the rpsL gene. All sequence analyzed low level streptoniycin resistant strains were wild type for the rpsL and rrs genes. Since the low level resistant strairis did not show any of the docwnented mutations, it is likely that they nlight have mutations in other ribosomal genes. Tills possibility was explored by manipulating the mycobacterial relative M. smegmatis for functional analysis of the rRNA. M. s111egmatis with oniy one functional rRL'IA operon was generated by replacing the second rRNA operon with plasntid derived inactivated rRNA operon. For replacing one of the rRNA opeions, a replacement plasntid was constructed. The replacement plasntid contained one rRi'lA operon inactivated by kanamycin resistant marker. As a counter selectable marker, the plasntid is equipped with the SacB gene which is lethal to mycobacteria in the presence of 15 % sucrose. After transformation, colonies were selected on LB plates contailling kanamycin 25)lg/ml and different sucrose concentrations. M. smegmatis transformants resulted from double crossover and homologous recombinations were identified by their kanamycin and sucrose resistant phenotypes. To check for replacements of one of tlle rRNA operon, transfonnants were analyzed by Southern blotting. Competent cells of M. slllegmatis with one functional rRi'lA operon were prepared and transfOlmed with plasntid construct that contained the wild type rpsL gene. Transfonnants were selected on LB plates contailling 25)lg/ml streptomycin. Determination of MIC value of the streptomycin resistant M. smegmatis transformants and sequencing of rrs, rpsL and the entire genes of the rRNA operon will need to be detennilled. This will be necessary to establish tlle mutation induced. If established the hitherto undocumented mutation will be useful to trace the mechanisms of resistance in low level streptomycin resistant M. tuberculosis strains.