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

Ryu, Sung Weon; Kim, Hyun Ho; Bang, Mun Nam; Park, Young Kil; Park, Sue Nie; Shim, Young Soo; Kang, Seongman; Bai, Gill Han

Abstract

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.