Ghannoum M A, Ibrahim A S, Fu Y, Shafiq M C, Edwards J E, Criddle R S
Department of Medicine, Harbor-UCLA Medical Center, Torrance 90509.
J Clin Microbiol. 1992 Nov;30(11):2881-6. doi: 10.1128/jcm.30.11.2881-2886.1992.
We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cells per ml yielded optimal growth in BYNB 7 and YNB 5.4, whereas 10(5) cells per ml was optimal in RPMI and SAAMF. Furthermore, variation of inocula from 10(3) to 10(5) cells per ml showed small but significant inoculum effects in determining MICs of fluconazole, amphotericin B, and flucytosine for C. neoformans. Therefore, 10(4) cells per ml was chosen as the optimal inoculum for susceptibility testing in this study. Mean MICs of fluconazole, amphotericin B, and flucytosine for 21 crytococcal isolates in RPMI and BYNB 7 were low (for example, fluconazole had mean MICs of 1.2 and 1.3 micrograms/ml in RPMI and BYNB 7, respectively) and differed significantly from medium to medium. In contrast, the MICs obtained in SAAMF were significantly higher (e.g., fluconazole had a mean MIC of 2.2 micrograms/ml). Variance in MICs was large with fluconazole and flucytosine but small with amphotericin B, irrespective of the medium used. A microtiter system employing BYNB 7 as the medium, 48 h as the incubation period, and 10(4) cells per ml as the final inoculum is a simple, accurate, and reproducible method for the testing of C. neoformans susceptibility to fluconazole, amphotericin B, and flucytosine.
我们在微量滴定系统中研究了一系列测试条件，以确定测定新型隐球菌对抗真菌药物敏感性的最佳方法。将21株新型隐球菌在四种化学成分明确的培养基中培养24或48小时：酵母氮碱（BYNB 7）；RPMI 1640；合成氨基酸培养基-真菌（SAAMF），在pH 7.0缓冲以选择最能支持这种苛求酵母生长的培养基；以及pH 5.4的酵母氮碱（YNB 5.4）。新型隐球菌在35℃下的最大生长量在YNB 5.4中获得，其次是BYNB 7、SAAMF和RPMI中的生长水平。所有培养基中24小时的生长均较差且缺乏可重复性。相比之下，培养48小时生长良好且标准差较低，因此选择48小时作为本研究的最佳培养时间。对8株隐球菌分离株的生长动力学与初始接种量之间的关系进行比较，结果显示每毫升10⁴个细胞在BYNB 7和YNB 5.4中产生最佳生长，而每毫升10⁵个细胞在RPMI和SAAMF中最佳。此外，接种量从每毫升10³到10⁵个细胞变化时，在确定新型隐球菌对氟康唑、两性霉素B和氟胞嘧啶的最低抑菌浓度（MIC）时显示出虽小但显著的接种量效应。因此，本研究选择每毫升10⁴个细胞作为药敏试验的最佳接种量。在RPMI和BYNB 7中，21株隐球菌分离株对氟康唑、两性霉素B和氟胞嘧啶的平均MIC较低（例如，氟康唑在RPMI和BYNB 7中的平均MIC分别为1.2和1.3微克/毫升），且不同培养基之间差异显著。相比之下，在SAAMF中获得的MIC显著更高（例如，氟康唑的平均MIC为2.2微克/毫升）。无论使用何种培养基，氟康唑和氟胞嘧啶的MIC差异很大，而两性霉素B的差异很小。采用BYNB 7作为培养基、48小时作为培养时间以及每毫升10⁴个细胞作为最终接种量的微量滴定系统是一种简单、准确且可重复的方法，用于测试新型隐球菌对氟康唑、两性霉素B和氟胞嘧啶的敏感性。
