肉桂醛体外诱导白色念珠菌耐药的实验

Resistant induction of candida albicans to cinnamaldehyde in vitro

目的:观察肉桂醛体外诱导白色念珠菌耐药性的反应情况。方法:实验于2005-02/12在赣南医学院病原生物学教研室完成。①参照美国国家临床实验室标准化委员会推荐的M27-A方案中的微量液基释法测定肉桂醛和氟康唑对原始白色念珠菌的最低抑菌浓度和最低杀菌浓度。②采用多步诱导法,观察在含梯度浓度(1～50倍最低抑菌浓度)肉桂醛培养基上连续转种的白色念珠菌的最低抑菌浓度值变化情况,以氟康唑作为阳性对照,同时设空白对照和溶媒对照。③将敏感性下降的诱导株在无药培养基上连续转种10次后,检测其最低抑菌浓度值。④测定肉桂醛对氟康唑敏感性下降诱导株的最低抑菌浓度值。结果:①肉桂醛和氟康唑对原始白色念珠菌的最低抑菌浓度值分别为:0.014mg/L和2.0mg/L;肉桂醛对原始白色念珠菌的最低杀菌浓度值为0.056mg/L,氟康唑在本实验的最高浓度为64mg/L,未发现有杀菌作用。②在多步诱导培养过程中,肉桂醛组的最低抑菌浓度值始终保持不变,未诱导出对肉桂醛耐药的白色念珠菌菌株,菌株转种至含10倍最低抑菌浓度肉桂醛的培养基时,即停止生长,而在含50倍最低抑菌浓度氟康唑的培养基上菌株仍能生长。氟康唑诱导组菌株在培养过程中,其最低抑菌浓度值逐渐升高,转种至含50倍最低抑菌浓度氟康唑的培养基时,其最低抑菌浓度值(命名为FR株)是其亲株的12倍。③FR在无药培养基上连续转种10次后得到FR’,氟康唑在FR’的最低抑菌浓度值为20.16mg/L,与FR相比,差异无显著性(P>0.05)。④肉桂醛对FR的最低抑菌浓度值为0.014mg/L,与其相应的原始菌株相同。结论:肉桂醛不易产生耐药性,且对氟康唑敏感性下降的诱导株仍敏感。

AIM： To investigate the reaction of candida albiccans tolerance induced by cinnamaldehyde in vitro. METHODS： The experiment was perforated at the Department of Pathogenic Biology, Gannan Medical College from February to December 2005. ①The minimum inhibitory concentration （MIC） and the minimum fungicidal concentration （MFC） of cinnamaldehyde and fluconazole against eandida albicans were measured by broth microdilution method aceording to document M27-A published by the National Committee for Clinical Laboratory Standards （NCCLS）. ②A C. albieans strain was induced by serial subcultures on agar medium containing different concentrations of cinnamaldehyde （equivalent to 1 minimal inhibitory concentration （MIC）-50 MIC）, and 3 groups were set as control groups which were flueonazole group （positive control）, 1.5% alcohol group （solvent control）, blank control group, respectively. ③The susceptibilities （MIC） of this parental strain and its respective derivatives to cinnamaldehyde and fluconazole were measured. The induced less susceptible derivatives were subcultured on drug-free medium for 10 times consecutively, and then their MICs were tested. ④The MIC of induced fluconazole less susceptible derivative to cinnamaldehyde was also measured. RESULTS：①MIC of cinnamaldehyde and fluconazole against candida albicans were 0.014 mg/L and 2.0 mg/L, respectively; Minimal bacteriocidal concentration （MBC） of cinnamaldehyde was 0.056 mg/L and fluconazole with maximum concentration （64 mg/L,） in this experiment was not detected to have fungicidal action. ②After subcultured to the medium containing 10 MIC einnamaldehyde, there was no viable colony would be seen. No ein namaldehyde-resistant mutants could be induced （the MICs of serial subcultrues were similar to that of the parental one）. While the strain could acquire some resistance to flueonazole （up to 12-fold the parental MIC, designated FR） with the increase of inducing passages. ③After FR＇ was subcultured repeatedly on drug-free medium, we got FR＇ which showed no significant MIC （20.16 mg/L） difference from FR （P 〉 0.05）. ④The MIC （0.014 mg/L） of FR to cinnamaldehyde was still the same as the parental one. CONCLUSION： The resistance to cinnamaldehyde is not easy to induce; moreover, fluconazole less susceptible derivative is still sensitive to cinnamaldehyde.