大蒜素对铜绿假单胞菌生物膜早期黏附及胞外多糖的影响

目的研究大蒜素对铜绿假单胞菌PA01菌株生物膜（biofilm，BF）早期黏附及胞外多糖复合物（Extracellular Polymeric Substances，EPS）的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGF-Puv转化PA01菌株的荧光强度，计算黏附率以表示干预对不同时间点细菌黏附的影响，利用荧光显微镜下定性观察细菌的黏附量；应用异硫氰酸标记的刀豆蛋白A（FITC conjugated concanavalin A，FITC-conA）特异性结合细菌EPS，荧光显微镜下定性观察各组EPS的变化；利用硫酸-苯酚法定量各组细菌EPS的产量。结果6h组，大蒜素高浓度干预后细菌的黏附率由0．70±0．03下降至0．50±0．01，t=15．014，P〈0．05，大蒜素低浓度干预后黏附率也有下降，但不及高浓度组明显；除了9h组其他时间组趋势与6h组大致相似，可能与大蒜素含量下降有关。荧光显微镜观察可见生理盐水对照组细菌菌落分布，干预组黏附的细菌稀疏散在分布，以高浓度为甚。FITC-conA可使胞外多糖显色，在荧光显微镜下观察可见大蒜素干预后EPS减少，稀薄；EPS定量实验，EPS总量大蒜素高浓度干预组（181．19±1．59）μg较生理盐水对照组（602．66±21．94）μg有明显降低，t=60．589，P〈0．05。结论大蒜素可显著减少PA01菌株黏附及产EPS的能力。

Effects of allitride on adhesion and exopolysaccharide of Pseudomonas aeruginosa biofilms

Objective To investigate the effects of allitride on adhesion of Pseudomonas aeruginosa biofilm at early stage and Extracellular Polymeric Substances （EPS）. Methods Multifunction fluorometer was used to measure fluorescence of green fluorescent protein（GFP） in pGFPuv transformated PA01 in 96-well plate. The adhesion ratio of PA01 was calculated to determine the effects of allitride on PA01 biofilm adhesion,and qualitative study adherence of GFP-PA01 was performed by fluorescence microscope. The fluorescein isothiocyanate （FITC） conjugated concanavalin A （FITC-conA） was used to stain the EPS, in order for the qualitation of EPS by fluorescence microscope; the phenol-sulfuric acid method was used to quantitate the EPS in each group. Results Six-hour group：after intervention with high dose of allitride,the adhesion ratio was decreased from 0.70±0.03 to 0.50±0.01 ,t = 15. 014,P 〈0.05. Low dose of allitride could also affect the adhesion ratio, but not so obvious as high dose. Other time groups except the 9-hour group had an approximate tendency as the 6-hour group, which may be related with unstability of allitride. Compared with normal saline control group, the colony distribution of GFP-PA01 became rarefactive after intervention,especially with high doses. The EPS in biofilm was significantly decreased after intervention with allitride, which was visualized by fluorescence microscope, based on the FITC-conA staining. Quantification of EPS showed that total EPS decreased from （602.66±21.94） P4g to （ 181.19±1.59 ） μg after high dose of allitride intervention （ t = 60.589, P 〈 0.05 ）. Conclusions Allitride could decrease the adhesion and EPS producing ability of PA01 significantly.