香芹酚和月桂酰精氨酸乙酯盐酸盐联用对荧光假单胞菌抑菌作用的研究

Activity of Carvacrol and Ethyl-N^(α)-lauroyl-L-arginate Hydrochloride Against Pseudomonas fluorescens

为探究香芹酚和月桂酰精氨酸乙酯盐酸盐(ethyl-N^(α)-lauroyl-L-arginate hydrochloride,LAE)联用对荧光假单胞菌的抑菌效果与机制,通过最小抑菌浓度法(minimum inhibitory concentration,MIC)、棋盘法和生长曲线的测定研究抑菌效应;利用蛋白质和核酸泄漏、透射电子显微镜观察和胞内蛋白质电泳的方法分析抑菌机制;以生物被膜形成、群集和泳动为指标,测定群体感应活性。结果显示,香芹酚和LAE联用对荧光假单胞菌有相加抑菌作用。1/2 MIC香芹酚+1/16 MIC LAE抑菌效果最显著,24 h抑制率达97.9%。以上组合联用可使荧光假单胞菌的细胞膜通透性显著增强,蛋白质和核酸快速泄漏,1 h时OD280值和OD260值分别升高31.2倍和17.5倍;处理3 h后,菌体胞内电子密度下降,蛋白质浓度下降84.1%,且条带丰富度减弱。联合作用下,对生物膜形成、群集和泳动的抑制率分别达到97.0%、92.5%和65.0%。综上表明,香芹酚和LAE联用破坏荧光假单胞菌的细胞完整性,引起胞内物质泄漏,发挥强烈的抑菌作用。同时,二者联用表现出抑制群体感应活性的能力。

This study aimed to explore theactivity and mechanism of carvacrol combined with ethyl-N^(α)-lauroyl-L-arginate hydrochloride(LAE)against Pseudomonas fluorescens.Specifically,the activity was evaluated based on the minimum inhibitory concentration(MIC),checkerboard microdilution assay,and growth curve analysis.The antibacterial mechanism was explored through protein and nucleotide leakage,transmission electron microscopy,and electrophoresis of intracellular proteins.The quorum-sensing activity was evaluated with the indexes of biofilm formation,and swarming and swimming motility.The results showed that carvacrol and LAE displayed synergic activity against P.fluorescens,particularly the combination of carvacrol at 1/2 MIC and LAE at 1/16 MIC which achieved the inhibition rate of 97.9% at 24 h.The above combination could significantly enhance the permeability of cell membrane,thus leading to the rapid leakage of proteins and nucleic acids.As a result,the OD_(280)and OD_(260)values were 31.2 and 17.5 folds higher at 1 h,respectively.At3 h,the intracellular electron density decreased and protein concentration declined by 84.1%,with bands of proteins becoming obviously shallow or even disappearing.The inhibition rates of biofilm formation,swarming motility,and swimming motility were 97.0%,92.5% and 65.0%,respectively.In conclusion,the combination of carvacrol and LAE can destroy P.fluorescens cells,leading to leakage of intracellula substances and thus exerting strong inhibitory effect on the bacteria.Moreover,the combination suppresses the quorum-sensing activity.