[Evaluation of a newly developed broth microdilution test method to determine minimum inhibitory concentrations...

We developed a new broth microdilution antimycobacterial susceptibility test for determination of minimum inhibitory concentration (MICs) using an air-dried microplate containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The eight agents included were streptomycin (SM), isoniazid (INH), rifampicin (RFP), ethambutol (EB), kanamycin (KM), levofloxacin (LVFX), sparfloxacin (SPFX) and clarithromycin (CAM). Serial dilutions of the agents (128 micrograms/ml to 0.125 micrograms/ml) were prepared in microplates, and were reconstituted by inoculation of 0.2ml of cell suspensions (approximately 3 x 10(5) cells/ml). The test plates were incubated at 36 degrees C in 5% CO2, and the growth endpoints were read visually after 5-day, 7-day and 10-day incubations. Four ATCC reference strains, Mycobacterium tuberculosis, M. avium, M. kansasii and M. intracellulare, were repeatedly tested at three sites. Of 480 determination against eight agents, 455 (94.8%), 470 (97.9%) and 455 (94.8%) of the MICs read after 5-day, 7-day and 10-day incubations fell within 3log2 dilutions, respectively. The MICs gradually elevated during the incubation, however those of 7-day incubation were highly precise and easily determined. A total of 160 clinical isolates of M. tuberculosis and 114 of nontuberculous mycobacteria were tested against eight agents. As for the primary drugs (SM, INH, RFP and EB), most isolates of M. tuberculosis were highly susceptible with MIC90, < or = micrograms/ml. Both LVFX and SPFX were also active. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of RFP, LVFX, SPFX and CAM were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative and nonradiometric alternative for the determination of MICs in clinical mycobacteriology laboratories.