柠檬醛体外诱导白念珠菌耐药的实验研究

Studies on Resistant Induction of Citral to Candida albicans in vitro

目的探讨柠檬醛体外诱导白念珠菌耐药性的反应情况，进一步评价柠檬醛的临床应用价值。方法①参照NC—CLS M27-A方案中的微量液基稀释法测定柠檬醛和氟康唑对原始白念珠菌的最低抑菌浓度（MIC）和最低杀菌浓度（MFC）；②采用多步诱导法，观察在含梯度浓度（1～50MIC）柠檬醛培养基上连续转种的白念珠菌MIC的变化情况，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MIC值：④测定柠檬醛对氟康唑敏感性下降诱导株的MIC值。结果①柠檬醛和氟康唑对原始白念珠菌的MIC值分别为：0.162μg／ml和2．0μg／ml；柠檬醛对原始白念珠菌的MFC值为0．324μg／ml，氟康唑在实验的最高浓度（64μg/ml）未发现有杀菌作用。②在多步诱导培养过程中，柠檬醛组的MIC值始终保持不变，未诱导出对柠檬醛耐药的白念珠菌菌株，菌株转种至含10MIC柠檬醛的培养基时，即停止生长，而在含50MIC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，MIC值逐渐升高，转种至50MIC时，其MIC（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MIC为20．16μg／m，与FR相比，差异无显著性（P〉0．05）；④柠檬醛对FR的MIC值为0．189μg／ml，与其相应的原始菌株无差异。结论柠檬醛不易产生耐药性，且对氟康唑敏感性下降的诱导株仍敏感。柠檬醛是一种有良好开发前景的抗系统性念珠菌感染的天然药物。

Objective To investigate whether it is likely to induce Citral - resistant Candida albicans mutations in vitro, in order to give further evaluations of its antifungal actions for clinical use, Methods ①The minimum inhibitory concentration（MIC） and the minimum fungicidal concentration（MFC） of citral against C. albicans were measured by broth microdilution method according to document M27 -A published by the National Committee for Clinical Laboratory Standards（NCCLS） ; ② A C. albicans strain was induced by serial subcultures on agar medium containing different concentrations of Citral （equivalent to 1MIC - 50MIC ） , and 3 groups were set as control groups which were fluconazole group （positive control） , 1.5% alcohol group（ solvent control）, drug- free group, respectively. The susceptibilities （MIC） of this parental strain and its respective derivatives to Citral and fluconazole were measured. ③ The induced less susceptible derivatives were subcultured on drag - free medium for 10 times consecutively, then their MICs were tested. ④ The MIC of induced fluconazole less susceptible derivative to Citral was also measured. Results ① MIC of citral and fluconazole against C. albicans were 0. 162μg/ml and 2.0μg/ml, respectively; MFC of citral was 0. 324μg/ml and fluconazole with maximum concentration in this experiment was not detected to have fungicidal action. ②After subcuhured to the medium containing 10MIC Citral, there was no visiable colony. No Citral-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 fluconazole （up to 12-fold the parental MIC, designated FR） with the increase of inducing passages. ③After FR was subcuhured repeatedly on drag - free medium, we got FR＇which showed no significant MIC difference with FR. ④The MIC of FR to Citral was still the similar to that of the parental one. Conclusion Citral is a potential antifungal Chinese herb, and might be used for the treatment in Candidiasis.