目的 考察紫苏叶Perillae Folium油对鸡大肠杆菌（avian pathogenic Escherichia coli，APEC）的抗菌活性、生物被膜形成以及群体感应相关系统表达的影响，为开发紫苏叶油为新型抗菌药物以替代饲用抗生素提供实验数据和理论基础。方法 采用微量稀释法测定紫苏叶油对4株APEC临床分离株的体外抗菌活性，筛选敏感株；活菌计数法测定紫苏叶油作用于APEC的时间-杀菌曲线。通过微板法构建APEC生物被膜体外模型，用CCK-8法及结晶紫半定量黏附法测定APEC的生物被膜生长动力及总量生长曲线，并测定紫苏叶油对APEC生物被膜的影响，用扫描电子显微镜确认微观形态变化。通过qRT-PCR检测紫苏叶油对APEC群体感应系统的影响。结果 紫苏叶油对APEC的最低抑菌浓度（minimum inhibitory concentration，MIC）为2.000～2.667 mg/mL，其中对紫苏叶油敏感性最强的是APEC-3。时间-杀菌曲线结果显示，紫苏叶油可延迟APEC-3进入对数生长期。生长曲线确定APEC-3的黏附、聚集及成熟期给药时间为6、24、48 h。2MIC、MIC紫苏叶油显著降低APEC-3生物被膜生长动力（P＜0.05、0.01），2MIC、MIC、1/2MIC紫苏叶油均可减少生物被膜总量（P＜0.05、0.01、0.001），紫苏叶油可下调成熟期APEC-3群体感应系统luxS和sdiA基因表达（P＜0.001）。结论 紫苏叶油对APEC有良好的抗菌活性，且对APEC-3不同生长阶段的生物被膜有一定的抑制作用。群体感应抑制可能是通过单独或同时下调细菌群体感应系统的luxS和sdiA来介导的。

Objective To investigate the antibacterial activity, biofilm formation, and modulation of quorum sensing-related systems of oil from Zisuye (Perillae Folium, PO) against avian pathogenic Escherichia coli (APEC), thereby providing experimental data and a theoretical foundation for developing PO as a novel antibacterial agent to replace feed antibiotics. Methods The microdilution method was used to determine the in vitro antibacterial activity of PO against four APEC clinical isolates and screen sensitive strains; The viable count method was employed to measure the time-kill curve of PO against APEC. An in vitro APEC biofilm model was constructed using the microplate assay. The biofilm growth dynamics and total growth curve of APEC were evaluated via the CCK-8 assay and crystal violet semi-quantitative adhesion assay, and the effect of PO on APEC biofilms was assessed. Scanning electron microscopy was applied to confirm microscopic morphological changes. The effect of PO on the quorum sensing system of APEC was analyzed by qRT-PCR.Results The minimum inhibitory concentration (MIC) of PO against APEC ranged from 2.000 to 2.667 mg/mL, with APEC-3 exhibiting the highest sensitivity to PO. Time-kill curve results demonstrated that PO delayed the entry of APEC-3 into the logarithmic growth phase. Growth curve analysis identified the adhesion, aggregation, and maturation phases of APEC-3 at 6 h, 24 h, and 48 h, respectively. PO at 2MIC and MIC significantly reduced the biofilm growth dynamics of APEC-3 (P < 0.05, 0.01). PO at 2MIC, MIC and 1/2MIC decreased the total biofilm biomass (P < 0.05, 0.01, 0.001). PO down-regulated the expression of the quorum sensing-related genes luxS and sdiA in APEC-3 during the maturation phase (P < 0.001).Conclusion PO exhibits potent antibacterial activity against APEC and effectively inhibits biofilm formation in APEC-3 across its different developmental stages. The quorum sensing inhibition is mediated by down-regulating the luxS and/or sdiA genes of the bacterial quorum sensing system, either individually or concurrently.

R285.5

国家重点研发计划“中医药现代化”重点专项项目（2018YFC1707100）