[Basic evaluation for new antimicrobial susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay...

It has been reported that the number of living bacteria is correlated to their ATP contents. Based on this, ATP measurement was applied to the susceptibility test for Mycobacterium tuberculosis to antimicrobial agents. ATP was extracted from the bacterial suspension prepared from M. tuberculosis H37Rv grown on 1% Ogawa medium and measured by bioluminescent assay. The highest relative light units (RLU) was obtained when ATP was extracted with the reagent supplied by Kikkoman Inc. (Chiba, Japan) at 100 degrees C for 3 minutes. The amounts of ATP recovered was constant at 100 degrees C for 8 minutes. The ATP contents correlated well with the number of bacteria expressed as CFU. The ATP contents of M. tuberculosis H37Rv inoculated into the Middlebrook 7H9 broth medium containing antituberculous agents were measured at days of 0, 3, 5 and 7. The control culture showed the time-dependent increase in the RLU values, while cultures supplemented with antimicrobial agents reduced their ATP contents concomitant with the concentrations of drugs. The growth of tubercle bacilli was expressed as RLU ratio, the ratio of RLU in the drug-containing cultures to those in drug-free ones. RLU ratio of 0.5 or less was determined as sensitive and the ratio of more than 0.5 as resistant to drugs. The inoculum size of bacteria did not affect the days giving RLU ratio below 0.5 or 0.3. Within 7 days, susceptibilities to drugs could be determined. In conclusion, this test is simple, rapid, sensitive and highly reproducible and useful for the assessment of susceptibility.