Improved extraction and assay of mycobacterial ATP for rapid drug susceptibility testing

The firefly luciferase assay of ATP is a rapid and convenient technique for monitoring growth of mycobacteria. The time needed to obtain a drug suscep tibility pattern can be reduced to less than 1 week as compared to 4 weeks with conventional methods. The ATP assay is simple and reliable. However, t he extraction of bacterial ATP is not a trivial problem. Lysing the cells w ill immediately activate ATP-degrading enzyme systems. The extractant must therefore lyse the cells and simultaneously inactivate ATP-degrading enzyme systems. Only by comparing the ATP yields obtained with different extracta nts we will know something about the intracellular ATP level. In the presen t study various extractants were compared for the extraction of ATP from My cobacterium bovis (BCG) cultures. Dodecyl trimethyl ammonium bromide (DTAB) in Tris-buffer with EDTA resulted at 100 degrees C in an ATP yield that wa s approximately twice as high as the same buffer without DTAB. The optimum temperature was 80-100 degrees C. With the optimized extraction procedure t he coefficient of variation for the entire assay of ATP in BCG cultures was 5%. The analytical interference from DTAB with the firefly reaction was ob viated by neutralization with alpha-cyclodextrin, making it possible to inc rease the sensitivity by assaying 0.5 mt rather than 0.01 mt extract. Copyr ight, 1999 John Wiley & Sons, Ltd.