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[摘要] 目的 探讨金银花活性成分异绿原酸对早期、成熟期烟曲霉生物被膜的体外作用。 方法 采用微量液基稀释法测定异绿原酸对受试烟曲霉菌株最低抑菌浓度（MIC）和最低杀菌浓度（MFC）；构建体外早期、成熟期静止生物被膜模型并用不同浓度（64、128、256、512、1024 μg/mL）的异绿原酸组分别作用48 h，激光共聚焦显微镜（CLSM）检测生物被膜内存活菌；扫描电镜（SEM）观察生物被膜形态学改变；结晶紫染色法定量生物被膜。比较各组MIC和MFC。 结果 异绿原酸对受试烟曲霉菌株的MIC和MFC均大于1024 μg/mL；不论早期或成熟期，空白对照组和异绿原酸各浓度组生物被膜内菌体在CLSM下均以绿色的活菌为主；电镜观察证实异绿原酸可破坏早期、成熟期的生物被膜结构，使胞外基质减少，菌体轮廓清晰；当异绿原酸浓度达到256 μg/mL时，可以使载体上不同时期生物被膜的量明显降低，256、512、1024 μg/mL异绿原酸组生物被膜定量与空白对照组及组间两两比较，差异均有统计学意义（P < 0.05）。 结论 异绿原酸在体外不能杀死烟曲霉生物被膜内菌体，但可对烟曲霉早期和成熟期生物被膜产生破坏作用，其作用呈浓度依赖性。

[关键词] 烟曲霉；生物被膜；异绿原酸

[中图分类号] R282.71 [文献标识码] A [文章编号] 1673-7210（2014）11（b）-0004-04

[Abstract] Objective To investigate the effect of isochlorogenic acid on the biofilm of Aspergillus Fumigatus in early and mature stages in vitro. Methods The minimum inhibitory concentration （MIC） and the minimum fungicidal concentration （MFC） for isochlorogenic acid were measured by broth microdilution method according to CLSI M38-A2 guideline. The biofilm models in early and mature stages established under static condition were affected by different concentration gradients （64， 128， 256， 512， 1024 μg/mL） of isochlorogenic acid for 48 h in vitro， then biofilm quantitation was performed by crystal violet staining assay. Scanning electron microscope （SEM） was used to observe the morphological change of biofilm structure. The viability of Aspergillus Fumigatus hyphae in biofilm was detected by confocal laser scanning microscope （CLSM）. The MIC and MFC of different groups were compared. Results Both the MIC and the MFC of isochlorogenic acid on Aspergillus Fumigatus isolate were above 1024 μg/mL. The results of CLSM demonstrated that isochlorogenic acid showed hardly any fungicidal effect to the mycelia in early or mature stage of biofilm. Under SEM， the structure of Aspergillus Famigatustus biofilm treated with isochlorogenic acid was destructed and the outline of mycelia appeared to be more clear than that without isochlorogenic acid due to the reduction of extracellular matrix. The in vitro action was performed while the concentration of isochlorogenic acid achieved the level of 256 μg/mL， within the concentrations of 256， 512， 1024 μg/mL， results of biofilm quantities indicated significant differences between serially increasing isochlorogenic acid concentration groups and untreated control group， with statistical significance （P < 0.05）. Conclusion Isochlorogenic acid exhibits destructive effect on the early and mature stages of Aspergillus Famigatustus biofilm in vitro， which shows a concentration-dependent manner.endprint

[Key words] Aspergillus Fumigatus； Biofilm； Isochlorogenic acid

烟曲霉是导致临床免疫力低下患者肺部真菌感染的常见病原体之一。近年来虽然新型抗曲霉菌药物的研发取得了一定的进展，但唑类耐药和多重耐药菌株的出现，使烟曲霉感染的患者病死率依旧很高。体外研究发现，生物被膜形成是烟曲霉产生耐药的重要机制之一，且生物被膜一旦成熟，其耐药性将提高几十、几百甚至上千倍[1]，控制难治性烟曲霉感染成为当前临床治疗上面临的一大难题。……