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 susceptibility 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, the extraction of bacterial ATP is not a trivial problem. Lysing the cells will 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 extractants we will know something about the intracellular ATP level. In the present study various extractants were compared for the extraction of ATP from Mycobacterium bovis (BCG) cultures. Dodecyl trimethyl ammonium bromide (DTAB) in Tris-buffer with EDTA resulted at 100 degrees C in an ATP yield that was approximately twice as high as the same buffer without DTAB. The optimum temperature was 80-100 degrees C. With the optimized extraction procedure the coefficient of variation for the entire assay of ATP in BCG cultures was 5%. The analytical interference from DTAB with the firefly reaction was obviated by neutralization with alpha-cyclodextrin, making it possible to increase the sensitivity by assaying 0.5 mL rather than 0.01 mL extract.