大蒜素对铜绿假单胞菌生物膜群体密度感应系统调控毒力因子表达的影响

目的通过体外测定铜绿假单胞菌（Pseudomonas aeruginosa）群体密度感应（Quorum Sensing，QS）系统调控的毒力因子表达，比较大蒜素干预前后毒性因子表达的差异以及对铜绿假单胞菌PAO1成熟生物膜（biofilm，BF）的影响。方法应用ELISA法比较处理前后外毒素A的含量差异；利用弹性蛋白一刚果红染色的方法，测定处理前后弹性蛋白酶活性；用蒽酮一硫酸法测定鼠李糖脂；利用荧光酶标仪检测青脓素含量变化；利用激光共聚焦显微镜观察干预前后大蒜素对铜绿假单胞菌成熟BF的影响。结果大蒜素干预后与生理盐水对照组比较，外毒素A、弹性蛋白酶、鼠李糖脂和青脓素表达分别由（19．630±0．573）pg/μl、（0．467±0．003）、（2．009±0．063）g／L、（9325．833±367．675）下降到（6．529±0．289）pg／μl、（0．032±0．001）、（0．269±0．009）g／L、（7819．167±111．800）。激光共聚焦显微镜观察可见生理盐水对照组细菌菌落云集呈蘑菇状分布，干预组黏附的细菌稀疏散在平坦分布。结论大蒜素可抑制铜绿假单胞菌群体密度感应系统调控的毒力因子表达，减弱其毒力，干扰BF分化成熟。

Effects of allitride on quorum sensing-controled virulence factors production in Pseudomonas aeruginosa biofilm

Objective Quorum-sensing （QS） system was a key regulator of virulence and biofilm development in P. aerug/nosa. Allitride was examined in this study for its effects on P. aeruginosa virulence factors and biofilm formation in vitro. Method Sensitive sandwich enzyme-llnked immunosorbent assay （ELISA） systems for the quantification of exotoxin A. Expression of elastase and pyoverdin were assessed semi-quantitatively using elastin Congo red and fluorescence respectively. Production of rhamnolipid was determined by H2SO4-anthrone analysis at the absorbance of 553 nm. Confocal laser scanning microscope （CKSM） was used to study the effects of allitride on mature biofilm in vitro. Result AUitride down regulated virulence factors exotoxin A, elastase, rhamnolipid and pyoverdin deceased from （ 19.630±0.573） pg/μl, （0.467 ± 0. 003）, （2. 009 ± 0. 063） g/L, （9325. 833 ±367. 675） to （6. 529 ± 0. 289） pg/μl, （0. 032 ± 0. 001 ）, （0.269 ±0.009）g/L and （7819. 167 ± 111. 800） respectively. Under the CLSM,P. aerug/nosa in control group formed mature mushroom structures, while it formed flat undifferentiated biofilm, susceptible to treatment with allitride in the intervention group. Conclusion Allitride attenuates the vinalence of P. aeruginosa significantly and supresses it biofilm formation by intervening the QS system of P. aeruginosa.