Introduction

HIV and tuberculosis, as well as HIV and M.avium/M. intracellulare coinfections are leading global causes of mortality and morbidity. Rifabutin is a semisynthetic ansamycin antibiotic derived from rifamycin S. It has similar therapeutic efficacy to rifampicin, but induces cytochrome P-450 3A metabolism in a significantly lower level, and that’s why it may be a useful component of drug regiments for treatment of HIV/mycobacterial coinfections. It may preserve the quality of life of patients with AIDS.

Changes in Mycobacterium tuberculosis sigB, sigE, and sigF genes mRNA level after exposure to rifabutin were studied.

Materials and methods

Gene expression was analyzed in two M. tuberculosis strains cultures: H37Rv strain sensitive to rifabutin and 35/5 strain resistant to the drug (both the M. tuberculosis strains were obtained from the National Tuberculosis and Lung Diseases Research Institute in Warsaw). The cells were standardized, and incubated in the Middlebrook 7H9 medium in presence of rifabutin concentrations of 0,5, 20, 30 and 40 µg/ml for 0, 72, and 96 hours. Parallel control cultures were provided. Concentration and quality of extracted total RNA, OD of broth cultures as well as CFU number on L-J solid medium were assessed.

Total RNA was extracted by a modified guanidine-phenol-chloroform method.

QRT-PCR reaction was done with ABI-PRISM 7700 sequence detection system (TaqMan). Sequence specific PCR primers and oligonucleotide detector probes labeled with FAM and TAMRA were used.

Results

Potentially useful in the molecular diagnostics of Mycobacterium tuberculosis infection changes in sigB and sigE genes mRNA level were detected after exposure to selected concentrations of rifabutin:

/1/ sigB, 0,5 µg/ml; H37Rv: 10x decrease of mRNA level after 72 h exposure to the drug, and 1,5x increase of mRNA copy number after 96 h; 35/5: 20x increase of mRNA level after 72 h, and then 2x decrease after 96 h,

/2/ sigE, 20 µg/ml; H37Rv: 3,5x increase after 72 h exposure to rifabutin, and 11x decrease of of mRNA level after 96 h; 35/5: 1,5x decrease after 72 h exposure, and 5x increase after 96 h of exposure to rifabutin.

The results obtained in studies of sigF gene mRNA were not clear.

Conclusions

1. From the diagnostic point of view extremely interesting results were obtained in studies of sigB and sigE genes mRNA level changes after exposure to different concentrations of rifabutin.

2. Quantitative analysis of sigB and sigE mRNA potentially may help in the discrimination between rifabutin susceptible and resistant M. tuberculosis strains.

3. Further studies on a large spectrum of rifabutin susceptible and resistant M. tuberculosis clinical strains are needed.

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