镁离子对黏液型铜绿假单胞菌生物膜形成过程的影响

目的探讨镁离子对黏液型铜绿假单胞菌早期黏附和生物膜形成过程的影响。方法荧光多功能酶标仪检测各组不同时间点96孔板底部黏附细菌的荧光强度，荧光探针FTTC-ConA染细菌胞外多糖（Extracellular Polymeric Substances，EPS）、荧光显微镜下观察各组多糖差别；SYTO9／PI染生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（Image Structor Analyzer，ISA）对各组生物膜结构参数进行定量分析。结果2d时，空白组和1mmol／L镁组的黏附细菌的荧光强度分别为1845．67±45．3和2254．78±42．45，t=-9．96，P〈0．05；0．1mmol／L的镁浓度下荧光强度也有增加，其余各时间组趋势与2d组相似；在荧光显微镜下观察可见随着镁浓度增加，EPS增多；激光共聚焦显微镜下可见随着镁浓度增加，生物膜活菌增加、菌落变密集；ISA软件分析结果示：空白组和1mmol／L镁组的6d生物膜厚度分别为（25．80±1．16）μm和（34．87±1．59）μm，t=-13．85，P〈0．05；区域孔率分别为0．96±0．05和0．90±0．04，t=2．48，P〈0．05；平均扩散距离分别为1．54±0．15和1．92±0．16，t=5．23，P〈0．05；结构熵分别为3．64±0．57和4．70±1．09，t=-2．6，P〈0．05，3d组生物膜也有相同的趋势。结论镁离子可以增强黏液型铜绿假单胞菌的早期黏附，影响随后生物膜的形成及结构。

Effects of Magnesium ions on the mucoid Pseudomonas aeruginosa biofilm

Objective To investigate the influence of different Mg^2＋ concentration on biofilm formation by mucoid Pseudomonas aeruginosa. Method Muhifunction fluorometer was used to measure fluorescence of PA at the bottom of 96- well plate. The EPS of PA was stained with FITC-ConA and detected by fluorescence microscope. The biofilm was stained with SYTO9/PI, monitored by Confocal Laser Scanning Microscope（CLSM）. Quantification of the effect was calculated by Image Structor Analyzer（ISA） sofeware. Result Two days group： after intervention with 1 mmol/L Mg^2＋ , the fluorescence intensity was increased from 1845.67 ± 45.39 to 2254.78± 42.45, t = - 9.96, P 〈 0.05 ; 0.1 mmol/L Mg^2＋ could also increase the fluorescence intensity; Other time groups showed approximate tendency as two days group; the EPS in biofilm was significantly increased after intervention with Mg^2＋ , which was visualized by fluorescence microscope, based on the FITC-ConA stain. The live bacteria in the biofilm increase and colony close-up after administration of Mg^2＋ ,when observed by CLSM, based on the SYTOg/PI stain. The quantitative data from ISA software showed that after administration of 1 mmol/L Mg^2＋, the depth of 6 day biofilm increase from （ 25.80 ± 1.16） μm to （34.87 ± 1.59） μm （ t = - 13.85, P 〈 0.05 ）. Areal porosity decrease from 0.96 ± 0.05 to 0.90± 0.04 （ t = 2.48,P 〈 0.05 ）, average diffusion distance increase from 1.54 ± 0. 15 to 1.92 ±0.16 （ t = 5.23 ,P 〈 0.05）, and textual entropy increase from 3.64 ±0.57 to 4.70 ± 1.09 （t = - 2.6 , P 〈 0.05 ）. The 3 day biofilm showed the same tendency. Conclusion Magnesium can increase the initial attach- ment of PA and alter the subsequent biofilm formation and structure.