Detection of embB Gene Mutation of Mycobacterium tuberculosis by Reverse Hybridization Assay / 결핵및호흡기질환

BACKGROUND: Ethambutol (EMB) is one of important first-line drug in the treatment of tuberculosis. Molecular techniques to detect embB gene mutations have been considered as an method to define the EMB resistance. We investigated the mutation rate within embB gene among EMB resistant strains using reverse hybridization techniques. METHODS: We made 11 probes that had wild or mutated sequences containing codons 306, 406, or 497 within embB gene respectively. These probes were reverse-hybridized with PCR products amplified from embB gene which were isolated from 149 ethambutol resistant strains and 50 pan-susceptible strains. RESULTS: Out of 149 ethambutol resistant strains, one hundred (67.1%) had mutation at least one base at codon 306, 406, or 497 in embB gene. Mutation at codon 306, 406, 497 were demonstrated in 75 (50.3%), 16 (10.7%), and 13 strains (8.7%) respectively. There were four strains that showed multi-mutation at codon 306 and codon 406 simultaneously. A high proportion (8.1%) had single mutation at codon 406. There was no mutation observed in embB gene among 50 pan-susceptible strains. CONCLUSION: Reverse hybridization will be useful technique for detection of gene mutation correlated to ethambutol resistance.

Trial for Drug Susceptibility Testing of Mycobacterium tuberculosis with Live and Dead Cell Differentiation / 결핵

BACKGROUND: The resurgence of tuberculosis and outbreaks of multidrug resistant (MDR) tuberculosis have increased the emphasis for the development of new susceptibility testing of the Mycobacterium tuberculosis for the effective treatment and control of the disease. Conventional drug susceptibility testings, such as those using egg-based or agar-based media have some limits, such as the time required and difficulties in determining critical inhibitory concentrations, but these are still being used in many diagnostic laboratories because of no better alternatives, considering cost and accuracy. To overcome these limits, a rapid and simple method for new susceptibility testing, using live and dead assays, was applied for a bacterial cell viability assay to distinguish dead from live bacterial cells based on two-color fluorescence. MATERIALS AND METHODS STRAINS: Forty strains were used in this study, 20 susceptible to all antituberculosis drugs and the other 20 resistant to the four first line antituberculosis drugs isoniazid, rifampicin, streptomycin and ethambutol. ANTIBIOTICS: The four antibiotics were dissolved in 7H9 broth to make the following solutions: 0.1micro gram isoniazid(INH)/ml, 0.4micro gram rifampicin(RMP)/ml, 4.0micro gram streptomycin(SM)/ml and 4.0micro gram ethambutol(EMB)/ml. RESULTS: Live and dead Mycobacterium tuberculosis cells fluoresced green and red with the acridin (Syto 9) and propidium treatments, respectively. These results are very well accorded with conventional drug susceptibility testing by proportional method on Lowensen-Jensen media (L-J) containing 4 drugs (INH, RMP, EMB and SM), showing a 93.7 % accordance rate in susceptible strains and 95% in resistant strains. CONCLUSION: The results of the drug susceptibility testing using the live and dead bacterial cell assay showed high accordance rates compared with the conventional proportion method on L-J. This finding suggests that the live and dead bacterial cell assay can be used as an alternative to conventional drug susceptibility testing for M. tuberculosis strains.

Analysis of the Cell Lysate Proteome of a Korean Mycobacterium tuberculosis Isolate K01 with H37Rv and H37Ra Strains

Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.

Rapid Drug Susceptibility Testing for Isoniazid and Rifampicin by Reverse Hybridization Assay / 결핵

BACKGROUND: Development of rapid drug susceptibility testing provides the opportunity for rapid identification of individuals with drug resistant tubercle bacilli, allowing selection of appropriate therapeutic regimens. METHODS: A total of 502 drug resistant isolates were subjected to reverse blot hybridization assay to detect mutations within genes (rpoB, katG, inhA, and ahpC) associated with rifampicin (RMP) and isoniazid (INH) resistance. RESULTS: Among the 264 RMP resistant strains (RMPR) tested, the most prevalent mutation was the Ser531Leu seen in 121 strains (46%). The second common mutation occurred in 84 strains (32%) at codon 526. And 27 strains (10%) showed the mutation at codon 516. Among all 469 INH resistant strains (INHR), the katG mutation was responsible for INH. The inhA mutation was present in 88 strains (19%). In 11 isolates (2%), coexisting of the katG and inhA mutations were identified. Reverse hybridization assay successfully detected over 80% of INHR and over 92% of RMPR among Korean isolates. CONCLUSION: Reverse hybridization was useful for rapid detection of INHR and RMPR.