甘草甜素对铜绿假单胞菌生物被膜形成的抑制作用  

Inhibitory Effect of Glycyrrhizin on Biofilm Formation of Pseudomonas aeruginosa

作　　者：黄盼 王志鹏 肖琛闻[2] 季权安[2] 鲍国连 刘燕[2] HUANG Pan;WANG Zhi-Peng;XIAO Chen-Wen;JI Quan-An;BAO Guo-Lian;LIU Yan(College of Chemistry and Life Science,Zhejiang Normal University,Jinhua 321004,China;Institute of Animal Husbandry and Veterinary Medicine,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China)

机构地区：[1]浙江师范大学化学与生命科学学院,金华321004 [2]浙江省农业科学院畜牧兽医研究所,杭州310021

出　　处：《农业生物技术学报》2022年第3期570-579,共10页Journal of Agricultural Biotechnology

基　　金：国家兔产业技术体系项目(CARS-43-C-2);浙江省畜禽新品种选育项目(2016C02054-10)。

摘　　要：铜绿假单胞菌(Pseudomonas aeruginosa)具有多重耐药特性,生物被膜的形成是其重要的耐药机制之一。甘草甜素(glycyrrhizin,GLY)具有抗菌功效。为了探讨GLY对多重耐药铜绿假单胞菌临床分离株的抗菌活性及生物膜形成的作用效果,本研究从角膜炎病兔(浙江嵊州'白中王'长毛兔)(Leporidae angora)眼分泌物中分离获得铜绿假单胞菌G1株(GenBank No.MZ683158)和G2株(GenBank No.MZ683159),进一步采用微量稀释法测定GLY对G1株和G2株的最小抑菌浓度(minimum inhibitory concentration,MIC),利用结晶紫法定量生物被膜,利用扫描电子显微镜初步观察GLY对铜绿假单胞菌生物被膜形态的影响,采用qPCR方法检测GLY对铜绿假单胞菌生物被膜合成相关调控基因pelA、algA、rhlI、rhlR和pslA表达的影响。结果显示,GLY对G1株和G2株的MIC分别为40和20 mg/mL;G1株和G2株都能形成生物被膜,G1株的形成能力较强;结晶紫染色显示,20 mg/mL GLY处理使G1株和G2株生物被膜的形成量显著减少(P<0.01)。扫描电子显微镜观察发现,5 mg/mL GLY处理使生物被膜的形成显著减少,10 mg/mL GLY处理使菌体分散且未见明显生物被膜结构。qPCR检测结果显示,5 mg/mL GLY处理后,pelA基因表达量升高,但差异不显著;algA、rhlI、pslA和rhlR的基因表达量均显著降低(P<0.01);10 mg/mL GLY处理后,pelA、algA、rhlI、rhlR和pslA的基因表达量均显著降低(P<0.01)。上述结果提示,GLY对铜绿假单胞菌有抗菌活性,对生物被膜的合成具有负调控作用,呈剂量依赖关系,可能通过下调相关基因表达来抑制生物被膜的形成。本研究为深入探讨GLY对铜绿假单胞菌生物被膜的影响和兔铜绿假单胞菌角膜炎治疗提供参考。Pseudomonas aeruginosa has multiple drug resistance,and the formation of biofilm is one of its important drug resistance mechanisms.Glycyrrhizin(GLY)has antibacterial effect.In order to investigate the effects of antibacterial activity and biofilm formation of GLY on clinical isolates of multidrug resistant P.aeruginosa,G1 strain(GenBank No.MZ683158)and G2 strain(GenBank No.MZ683159)were isolated from the eye secretions of rabbits suffering from keratitis(Zhejiang Shengzhou wool rabbit'White King',Leporidae angora).Further,the minimum inhibitory concentration(MIC)of GLY on G1 and G2 strains was determined by microdilution method,crystal violet staining was used to quantify biofilm,the effect of GLY on biofilm morphology was observed by scanning electron microscope(SEM),and the expression of pelA,algA,rhlI,rhlR and pslA was analyzed by qPCR.The results showed that MICs of GLY against G1 and G2 strain were 40 and 20 mg/mL,respectively;both G1 and G2 strains could form biofilm,and G1 strain had stronger ability.The biofilm formation of G1 and G2 strains decreased significantly with the treatment of 20 mg/mL GLY(P<0.01).SEM observation showed that 5 mg/mL GLY significantly reduced the biofilm formation;under the treatment of 10 mg/mL GLY,the bacteria were dispersed and no obvious biofilm structure was found.The expression of pelA increased after 5 mg/mL GLY treatment without signifcant difference,while those of algA,rhlI,pslA and rhlR were significantly decreased(P<0.01).After 10 mg/mL GLY treatment,the relative expression of pelA,algA,rhlI,rhlR and pslA were significantly decreased(P<0.01)compared with the control.In conclusion,GLY had a inhibitory regulatory effect on the synthesis of P.aeruginosa biofilm in a dose-dependent manner,which might be due to the down regulating of related genes.This study provides a reference for in-depth study of the effect of GLY on P.aeruginosa biofilm and the treatment of P.aeruginosa keratitis in rabbits.

关 键 词：铜绿假单胞菌 甘草甜素(GLY) 生物被膜 长毛兔 分离鉴定 

分 类 号：S-3[农业科学]