[Antimicrobial susceptibility testing for Mycobacterium tuberculosis by the bioluminescence assay of mycobacterial ATP using filamentous cell treatment].
The antimicrobial susceptibility testing for Mycobacterium tuberculosis by the bioluminescence assay of adenosine triphosphate(ATP) derived from living mycobacteria was improved introducing filamentous cell treatment(FCT) reported for beta-lactam susceptibility test of Pseudomonas aeruginosa by Hattori. Before ATP extraction, bacterial cells were treated with the FCT reagent for 30 minutes at room temperature. Adenosine phosphate deaminase in the FCT reagent simultaneously digested the extracted ATP and released ATP in a liquid culture of M. tuberculosis H37Rv and the RLU level was decreased markedly. Using this improved ATP method, we determined the ATP contents of M. tuberculosis inoculated into Middle-brook 7H9 broth medium with or without drugs. In ethambutol(EB) susceptibility, the ATP method reported previously, showed false-resistance when judged within 7 days. To eliminate false-resistance in EB susceptibility we applied the modified ATP method with FCT treatment to strains determined EB susceptible by reference methods. Using this modified ATP method, we could judge EB susceptibility of 5 ATCC reference strains within 3 days, and these of 15 clinical isolates of M. tuberculosis within 5 days. And all the results obtained were coincident between the ATP method and the reference methods. The reproducibility of this modified ATP method was evaluated with six ATCC reference strains at the concentrations of 0.1 microgram/ml of isoniazid(INH), 2.0 micrograms/ml of rifampicin(RFP), 2.5 micrograms/ml of EB, 2.0 micrograms/ml of streptomycin(SM), and 5.0 micrograms/ml of kanamycin(KM). The test was repeated six times. Reduction of ATP contents were observed in susceptible strains but not in resistant ones within 3 days of cultivation and susceptibilities to drugs could be determined within 3 days at every time when combined FCT to the ATP method. And highly reproducible results were obtained. It is strongly suggested that this modified method is simple, rapid, highly reproducible and nonradiometric, and could be used for the assessment of drug susceptibility for M. tuberculosis.
Yamazaki
T
T
Department of Bacteriology, National Institute of Infectious Diseases, Tokyo.
Sato
N
N
Yamashita
K
K
Okazawa
Y
Y
Tanno
K
K
D000255:Adenosine Triphosphate / Q000032:analysis*; D008163:Luminescent Measurements; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000737:chemistry*
D000255:Adenosine Triphosphate