香芹酚抑制高毒力肺炎克雷伯菌生物被膜形成机制的研究

Mechanism of carvacrol on inhibiting biofilm formation of hypervirulent Klebsiella pneumoniae

目的探究香芹酚抑制高毒力肺炎克雷伯菌生物被膜形成的潜在作用机制。方法通过检测香芹酚对高毒力肺炎克雷伯菌生物被膜形成和形态学的影响、胞外多糖及荚膜多糖含量的改变,以及生物被膜相关基因rmp A2、mag A、mrk A、mrk B、tre C表达量的变化,分析其作用的可能机制。结果香芹酚对高毒力肺炎克雷伯菌的最低抑菌浓度为512μg/mL,且对该菌生物被膜形成有明显的抑制作用,其效应呈浓度依赖性;扫描电镜观察显示,香芹酚干预状态下,生物被膜结构松散,细胞之间连接不致密;刚果红吸附试验与间羟基联苯比色法测定表明,香芹酚可降低高毒力肺炎克雷伯菌荚膜多糖含量而不影响胞外多糖总含量;荧光定量PCR显示,在亚抑菌浓度香芹酚作用下,有关荚膜多糖合成、糖转运系统及菌毛黏附相关基因表达量降低均超过50%。结论香芹酚对高毒力肺炎克雷伯菌生物被膜形成具有明显的抑制作用,其机制可能与降低细菌荚膜多糖合成、糖转运系统及菌毛黏附等生物被膜相关基因的表达有关。

Objective To explore the potential mechanism of carvacrol on inhibiting the formation of biofilm of hypervirulent Klebsiella pneumoniae(hvKP).Methods The possible mechanisms of carvacrol were analyzed based on the detection of its effects on the formation and morphology of biofilms,changes in extracellular polysaccharide and capsule polysaccharide content,as well as changes in the expression levels of biofilm-related genes rmp A2,mag A,mrk A,mrk B,and tre C of hvKP.Results The minimum inhibitory concentration of carvacrol on hvKP was 512μg/mL,with an obvious inhibitory effect on the biofilm formation of hvKP,presenting a concentration-depen-dent effect.Under the scanning electron microscope,it was observed that the biofilm structure was loose and the intercellular connections were not dense under the intervention of carvacrol.The Congo Red adsorption test and m-hydroxybiphenyl colorimetric method showed that carvacrol could reduce the content of capsule polysaccharides of hvKP,but didn’t affect the total extracellular polysaccharide content.Fluorescence quantitative polymerase chain reaction(PCR)showed that under the effect of carvacrol at sub-inhibitory concentration,the synthesis of capsule polysaccharide,expression levels of sugar transport system and pili adhesion-related genes all decreased by more than 50%.Conclusion Carvacrol has a significant inhibitory effect on the formation of biofilm in hvKP,and its mechanism may be related to the decrease of synthesis of capsule polysaccharide as well as expression of biofilm-related genes,such as sugar transport system and pili adhesion.