桂皮醛对禽致病性大肠杆菌生物被膜形成影响的研究

Effects of cinnamaldehyde on biofilm formation by avian pathogenic Escherichia coli

为探究桂皮醛对禽致病性大肠杆菌(APEC)生物被膜(BF)的形成及其相关基因转录水平的影响,本研究利用牛津杯法分别检测桂皮醛对3株BF强阳性(E-39、E-40、E-41)和3株中等阳性(E-27、E-30、E-42)APEC的抑菌圈,分析桂皮醛对APEC的抑菌活性;采用二倍稀释法测定桂皮醛对上述6株APEC的最小抑菌浓度(MIC);根据该结果,进一步采用96孔板结晶紫染色法测定MIC、1/2 MIC和1/4 MIC桂皮醛对6株APEC BF形成能力的抑制作用;采用qPCR法分别检测1/2 MIC和1/4 MIC桂皮醛对6株APEC BF形成相关基因转录水平的影响。结果显示:桂皮醛对6株APEC的抑菌圈直径为16.12±0.13 mm~23.80±0.23 mm;桂皮醛对BF强阳性和中等阳性APEC菌株的MIC均为256μg/mL;BF形成能力的检测结果显示,与未用桂皮醛处理的APEC相比,MIC、1/2 MIC和1/4 MIC的桂皮醛均极显著抑制上述6株APEC BF的形成(P<0.01),且该抑制作用与桂皮醛的浓度呈正相关;经1/2 MIC和1/4 MIC桂皮醛处理3 d后,与未经桂皮醛处理的APEC阳性对照组相比,3株BF强阳性菌株组氨酸激基因qseC、鞭毛主调节器基因flhC和外膜外排蛋白基因tolC的转录水平均显著降低(P<0.05),代谢基因bdcR的转录水平均显著升高(P<0.05),3株BF中等阳性菌株反应调控蛋白基因qseB、qseC及flhD、flhC和tolC基因的转录水平均显著降低(P<0.05)。本研究首次证实桂皮醛在体外对APEC有较强的抑菌活性,并且可通过影响APEC qseB、qseC、flhD、flhC、tolC和bdcR基因的转录水平进而抑制其BF的形成。本研究为临床防治大肠杆菌病及清除其BF提供了参考依据。

In order to explore the effects of cinnamaldehyde on the biofilm formation by avian pathogenic Escherichia coli(APEC)and the transcription level of related genes,the Oxford cup method was used to detect the inhibitory circles of three strongly positive(E-39,E-40,E-41)and three moderately positive(E-27,E-30,E-42)APECs,and the antibacterial activity of cinnamaldehyde on APEC was analyzed.The minimum inhibitory concentration(MIC)of cinnamaldehyde on the above six APEC strains was determined by 2-time serial dilution.According to the results,the inhibitory effects of MIC,1/2MIC and 1/4MIC cinnamaldehyde on the formation of biofilm in six APEC strains were further determined by 96-well plate crystal violet staining method.The effects of 1/2MIC and 1/4MIC cinnamaldehyde on the transcription levels of genes related to the formation of APEC biofilms were determined by qPCR,respectively.The results showed that the inhibition zone of cinnamaldehyde on 6 APEC strains was 16.12±0.13-23.80±0.23,and the MIC of cinnamaldehyde for biofilm-positive and moderately positive APEC strains was 256μg/mL.The results of the biofilm formation ability showed that compared with APEC not treated with cinnamaldehyde,the formation of the above six APEC biofilms(P<0.01)was significantly inhibited by MIC,1/2MIC and 1/4MIC cinnamaldehyde,and the inhibitory effect was positively correlated with the concentration of cinnamaldehyde.After 3 days of treatment with 1/2MIC and 1/4MIC cinnamon barrexaldehyde,compared with the APEC positive control group without cinnamaldehyde,the transcription levels of qseC,flhC and tolC genes of the three biofilm-positive strains were significantly reduced(P<0.05);the transcription levels of the metabolic gene bdcR were significantly increased(P<0.05);and the transcription levels of qseB,qseC,flhD,flhC and tolC genes of the three moderately positive strains with biofilm were significantly reduced(P<0.05).This study is the first to confirm that cinnamaldehyde has strong bacteriostatic activity against APEC in vitro,and can inhibit the formation of biofilm by affecting the transcription levels of APEC qseB,qseC,flhD,flhC,tolC and bdcR genes.This study provides a reference for the clinical prevention and treatment of E.coli disease and the removal of its biofilm.