绿原酸、异绿原酸对烟曲霉生物膜抑制作用的体外研究

摘要

目的：通过在体外复制烟曲霉的生物膜模型，研究绿原酸（chlorogenic acid，CRA）、异绿原酸（isochlorogenic acid， IRC）对生物膜的影响。方法选取临床分离的烟曲霉构建体外生物膜模型，微量液体稀释法测定最低抑菌浓度（MIC），将菌株分为空白对照组、CRA实验组（1024 mg/L、512 mg/L、256 mg/L），IRC实验组（1000 mg/L、500 mg/L、250 mg/L），用扫描电子显微镜（scanning electron microscopy，SEM）、激光共聚焦扫描显微镜（laser confocal scanning microscope，CLSM）定性观察生物膜；结晶紫染色法半定量生物膜量。结果37℃培养24 h烟曲霉形成早期生物膜，48 h形成成熟期生物膜。SEM 及CLSM 观察到实验组烟曲霉生物膜均较其相应空白对照组空洞、稀疏，但菌丝未见明显减少，死活菌数也无明显差别。CRA、IRC实验组早期结晶紫半定量结果为1．05±0．19、1．14±0．26、0．99±0．14、1．39±0．06、1．41±0．06、1．60±0．04，均少于空白对照组1．91±0．17，差异具有统计学意义（P＜0．05）；CRA、IRC实验组晚期结晶紫半定量结果为2．25±0．05、2．27±0．05、2．31±0．03、2．26±0．02、2．27±0．02、2．29±0．04，少于空白对照组2．36±0．01，差异具有统计学意义（P＜0．05）。结论CRA、IRC 对体外烟曲霉生物膜形成有抑制作用。%Objective To investigate the in vitro destructive effect of chlorogenic acid (CRA)/isochlorogenic acid (IRC)on Aspergillus fumigatus biofilm in a model of biofilm formation.Methods The in vitro biofilm model was established using clinical isolates of A.fumigatus.The minimum inhibitory concentrations (MIC)of antimicrobial agents against A.fumigatus were determined by microdilution method.Scanning electron microscope (SEM)and laser confocal scanning microscope (CLSM)were used to characterize the biofilm.Crystal violet staining method was used for biofilm quantitation.Results After reaction with CRA/IRC for 24 h or 48 h,observation of biofilm showed that A.fumigatus extracellular matrix was thinner than the control group.Biofilm quantitation showed that the optical densities were 1.05±0.19,1.14±0.26,0.99±0.14 for CRA group (1 024 mg/L,512 mg/L,256 mg/L);1.39±0.06,1.41±0.06,1.60±0.04 for IRC group (1 000 mg/L,500 mg/L,250 mg/L)at 24 h,and 1.91±0.17 for control group (P<0.05).The quantitation of biofilm showed that the optical densities were 2.25±0.05,2.27±0.05,2.31±0.03 for CRA group,2.26 ± 0.02,2.27±0.02,2.29±0.04 for IRC group at 48 h,and 2.36±0.01 for control group (P<0.05).Conclusions CRA/IRC has some inhibitory effect on the formation of A.fumigatus biofilm.