大蒜素对铜绿假单胞菌生物膜形成的影响及结构定量化分析

目的利用激光共聚焦显微镜（Confocal Laser Scanning Microscopy, CLSM）观察大蒜素对铜绿假单胞菌PA01菌株生物膜（biofilms，BF）形成过程的影响，并用ISA（Image Structure Analyzer）软件对BF结构进行定量化分析。方法体外建立1d．3d和7d组PA01菌株BF模型，每组分3小组，分别用生理盐水、大蒜素128μg／mL、大蒜素10μg／mL作用6h。通过荧光探针SYT09／PI标记，利用CLSM进行动态观察，并用ISA软件对其BF结构进行定量分析。结果（1）生理盐水对照组铜绿假单胞菌生物膜厚度随时间的增加而增加，但在后3d增加的速度减慢；同时区域孔率（Areal Porosity，AP）呈下降趋势，平均扩散距离（Average Diffusion Distance，ADD）有逐渐增加趋势，结构熵（Textural Entropy，TE）有增加的趋势。（2）7d组模型中，大蒜素128μg／mL作用后，BF厚度由（28．83±0．67）μm减低到（16．50±0．69）μm；AP由0．76±0．10增加到0．92±0．02；TE由6．78±0．93减低到5．61±0．55。10μg／mL大蒜素干预后，有同样趋势，但效应不如高浓度明显。（3）1d、3d模型组，大蒜素作用后同对照组相比，厚度、AP、TE的差异均有统计学意义，趋势同7d组。结论通过ISA软件对干预前后的BF进行结构定量分析，大蒜素对各个时间段BF的形成均有影响，高浓度效果更显著。

Effects of allitride on formation and quantification of structure of Pseudomonas aeruginosa biofilm

Objective To investigate the effect of allitride on biofilms forming process of Pseudomonas aeruginosa PAO1 by Confocal Laser Scanning Microscopy(CLSM),and quantify the BF structure with Image Structure Analyer(ISA) software.Method In vitro models of 1 d,3 d and 7 d of PAO1 biofilms were established.The effects of normal saline(NS,control groμp) and allitride at concentrations of both 128 μg/mL and 10 μg/mL on BF were monitored by CLSM respectively,based on fluorophores from PAO1 with nucleic acid SYTO9/PI stained.Quantification of the BF was calculated by Image Structure Analyzer(ISA) software.Results(1) In NS control group,the thickness of biofilm showed a decreasing tendency;the Average Diffusion Distance(ADD) tended to increase slightly;the Textual Entropy(TE) had an increasing tendency.(2) In BF models at 7 d after administration of 10 μg/mL allitride,the BF thickness was decreased from(28.83±0.67)μm to(16.50±0.69)μm;AP was increased from 0.76±0.128 to 0.92±0.02;TE was decreased from 6.78±0.93 to 5.61±0.55;All the differences are significant comparing to control group.The effect of allitride at concentration of 10 μg/mL showed the same tendency as the 128 μg/mL group,but not so obviously.(3) After administration of allitride,the BF models at 1 d and 3 d showed the same tendency as that of the 7 d model.Conclusion ISA software can quantify the structure of PAO1 biofilms at different stages.Allitride can affect PAO1 biofilm at different stages;the effect of 128 μg/mL allitride was more significant than 10 μg/mL allitride group.