百里酚对奇异变形杆菌生物膜的抑制作用及其机制研究

Inhibitory effect and mechanism of thymol on biofilm formation of Proteus mirabilis

摘要:

目的 研究百里酚对奇异变形杆菌(Proteus mirabilis, PM)生物膜的抑制作用及其潜在机制。方法 采用微量肉汤稀释法测定百里酚的最低抑菌浓度(minimal inhibitory concentration, MIC)和最低生物膜抑制浓度(minimal biofilm inhibitory concentration, MBIC)；采用菌落计数法测定百里酚的时间-杀菌曲线；结晶紫染色法测定百里酚对生物膜形成的抑制作用；测定百里酚对奇异变形杆菌群集运动和泳动能力的影响；测定百里酚对生物膜胞外聚合物(胞外多糖、胞外蛋白、eDNA)的影响；采用实时荧光定量PCR法测定百里酚对奇异变形杆菌生物膜相关基因表达量的影响，涉及的基因包括rsbA (双组分系统中组氨酸激酶感应蛋白基因)、flhD (鞭毛一级调控子基因)和mrpA (耐甘露糖样变形杆菌菌毛基因)。结果 百里酚对奇异变形杆菌的MIC为0.156 mg/mL，MBIC为0.313 mg/mL，可显著抑制生物膜的形成。百里酚抑制奇异变形杆菌的运动能力，抑制生物膜胞外多糖、蛋白质和eDNA的形成，同时抑制rsbA、flhD和mrpA基因的表达。结论 百里酚可以抑制奇异变形杆菌生物膜的形成，其机制可能与抑制细菌运动能力、胞外多聚物的形成及菌毛黏附等生物膜相关基因表达有关。

Abstract:

Objective To investigate the inhibitory effect and mechanism of thymol on the biofilm formation of Proteus mirabilis (PM).Methods The minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC) of thymol were determined by the broth micro-dilution method. The time-kill curve of thymol was established by the colony counting method. Crystal violet staining was performed to determine the inhibitory effect of thymol on the biofilm formation of PM. The effects of thymol on the swarming and swimming of PM and the extracellular polymeric substances (extracellular polysaccharides, extracellular proteins, and eDNA) of the biofilm were determined. RT-qPCR was employed to investigate the effects of thymol on the expression levels of PM biofilm-related genes: rsbA (encoding histidine-containing phosphotransmitter of the bacterial two-component system), flhD (encoding the flagellar regulon), and mrpA (encoding mannose-resistant Proteus-like fimbriae).Results The MIC of thymol against PM was 0.156 mg/mL. Thymol significantly inhibited the biofilm formation of PM, with the MBIC of 0.313 mg/mL. Thymol inhibited the motility of PM, reduced the formation of extracellular polysaccharides, proteins, and eDNA in PM biofilm, and decreased the expression levels of rsbA, flhD, and mrpA.Conclusion Thymol inhibits the biofilm formation of PM by suppressing the motility and formation of extracellular polymers and down-regulating the expression of biofilm-related genes.