Yamane Nobuhisa, Onaga Sayuri, Saitoh Hiroshi, Toyoshima Shunkoh, Shimojima Masahiro, Kawahara Shin, Takashima Tetsuya, Yamashita Tomonari
Department of Laboratory Medicine, Faculty of Medicine, University of the Ryukyus, Nakagami-gun, Okinawa-pref. 903-0215.
Rinsho Byori. 2002 Apr;50(4):381-91.
We developed a new broth microdilution susceptibility test for nontuberculous mycobacteria to determine minimum inhibitory concentrations(MICs). The test method utilized air-dried microplates containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The nine agents tested were rifampicin, isoniazid, ethambutol, streptomycin, kanamycin, levofloxacin, clarithromycin, ethionamide and amikacin. The test plates were reconstituted by inoculation of 0.1 ml of cell suspensions prepared in distilled water by a 1:100 dilution of the 0.5 McFarland suspension. After inoculation, the isolates resulted in incubation at 36 degrees C with clarithromycin at pH 7.4, and with the remaining agents at pH 6.6. The growth endpoints were visually read after 7-day and 10-day incubations for slowly growing nontuberculous mycobacteria, and after 3-day and 5-day incubations for rapidly growing mycobacteria, respectively. The reproducibility was evaluated with the five ATCC reference strains of nontuberculous mycobacteria. Of the 1,287 repeated tests of the four ATCC slowly growing strains(M. avium, M. intracellulare, M. kansasii and M. gordonae), 1,200(93.2%) of the MICs read after 7-day incubation fell within 3 log 2 dilutions. Also, a strain of rapidly growing mycobacteria, M. fortuitum, was repeatedly tested, and the reproducibility was estimated to be 93.3% after 3-day incubation. A total of 728 clinical isolates of nontuberculous mycobacteria comprising 14 species were tested against nine agents. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of rifampicin, levofloxacin, clarithromycin were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative means to determine MICs for nontuberculous mycobacteria in clinical laboratories.
我们开发了一种用于非结核分枝杆菌的新型肉汤微量稀释药敏试验，以确定最低抑菌浓度（MICs）。该试验方法使用含有系列稀释抗菌剂的空气干燥微孔板和改良的Middlebrook 7H9肉汤。所测试的九种药物为利福平、异烟肼、乙胺丁醇、链霉素、卡那霉素、左氧氟沙星、克拉霉素、乙硫异烟胺和阿米卡星。通过接种0.1 ml由0.5麦氏浊度标准液按1:100稀释于蒸馏水中制备的细胞悬液来重构试验平板。接种后，对于缓慢生长的非结核分枝杆菌，在36℃、pH 7.4条件下用克拉霉素培养，对于其余药物在pH 6.6条件下培养；对于快速生长的分枝杆菌，分别在3天和5天培养后观察生长终点。用五株非结核分枝杆菌的ATCC参考菌株评估重复性。在对四株ATCC缓慢生长菌株（鸟分枝杆菌、胞内分枝杆菌、堪萨斯分枝杆菌和戈登分枝杆菌）进行的1287次重复试验中，7天培养后读取的MICs中有1200次（93.2%）落在3个对数2稀释范围内。此外，对一株快速生长的分枝杆菌偶然分枝杆菌进行了重复试验，3天培养后的重复性估计为93.3%。对总共728株包含14个种的非结核分枝杆菌临床分离株进行了九种药物的测试。非结核分枝杆菌的MICs分布范围很广，利福平、左氧氟沙星、克拉霉素的活性更强。这些结果表明，这种新开发的试验方法是临床实验室中确定非结核分枝杆菌MICs的一种实用、快速、定量的手段。
