亚抑菌浓度卡泊芬净对白念珠菌生物膜形成的影响及机制研究

摘要：目的 初步探讨亚抑菌浓度(sub-MIC)卡泊芬净对白念珠菌生物膜形成的影响，为清除生物膜策略的制定提供理论 基础。方法 用微量肉汤稀释法测定卡泊芬净对白念珠菌的活性，并研究sub-MIC卡泊芬净对白念珠菌生长的影响；运用菌落 计数、XTT染色和扫描电镜分析sub-MIC卡泊芬净作用下形成的白念珠菌生物膜特点；采用实时荧光定量RT-PCR测定Ece1， Can2，Hip1，GAP1，GCN4和Stp2等白念珠菌生物膜相关基因的mRNA表达水平。结果 12株白念珠菌对卡泊芬净的MIC值范 围为0.625~0.875 μg/mL，sub-MIC卡泊芬净对白念珠菌的生长有不同程度的抑制作用。针对白念珠菌生物膜的形成，1/4 MIC和 1/8 MIC卡泊芬净皆对生物膜中的菌体数量、菌体代谢活性及生物膜结构有抑制作用，而1/16 MIC卡泊芬净则对生物膜无明显影 响。1/4 MIC和1/8 MIC卡泊芬净对白念珠菌生物膜相关基因的表达水平也具有抑制作用，1/16 MIC卡泊芬净则对基因表达水平 无影响。结论 sub-MIC卡泊芬净对白念珠菌生物膜的形成有一定抑制作用。但白念珠菌在sub-MIC卡泊芬净作用下仍能形成生 物膜，临床上需要谨防出现卡泊芬净处于亚抑菌浓度的情况。

Influence and mechanism of caspofungin at sub minimal inhibitory concentration on the biofilm formation of Candida albicans

Abstract Objective To investigate the influence of sub minimal inhibitory concentration (sub-MIC) of caspofungin on the biofilm formation of Candida albicans, which may provide a theoretical basis for the strategies of removing biofilms in the future. Methods Susceptibility testing of C. albicans to caspofungin was determined using the broth microdilution method. Then we studied the impact of caspofungin at sub-MIC on the growth of C. albicans. In addition, colony counting, XTT staining, and scanning electron microscopy were performed to analyze the characteristics of C. albicans biofilms, which were under the action of caspofungin at sub-MIC. In addition, the expression levels of Ece1, Can2, Hip1, GAP1, GCN4, and Stp2 were analyzed by Real-time PCR. Results The MICs of twelve C. albicans strains to caspofungin were in the range of 0.625~0.875 μg/mL. We found that the growth of C. albicans was inhibited by different sub-MICs of caspofungin at varying degrees. As for the biofilm formation of C. albicans, both 1/4 MIC and 1/8 MIC of caspofungin had inhibitory effects on the number, metabolic activity of C. albicans cells within biofilm, and the structure of biofilm, while the biofilm seemed not to be significantly altered by1/16 MIC of caspofungin. The expression levels of C. albicans biofilm formation-related genes (Ece1, Can2, Hip1, GAP1, GCN4 and Stp2) were significantly decreased when treated with 1/4 MIC or 1/8 MIC of caspofungin, but stayed the same after the treatment of 1/16 MIC caspofungin. Conclusion Sub-MIC of caspofungin had an inhibitory effect on the biofilm formation of C. albicans. However, the biofilm of C. albicans can still be formed under the action of caspofungin at sub-MIC. It is crucial to beware of the subinhibitory concentration of caspofungin, which may occur in clinical practice.