绿原酸、c-di-GMP、人杀菌肽LL-37对铜绿假单胞菌生物被膜耐药性的影响

目的 探究铜绿假单胞菌（pseudomonas aeruginosa,PA）的生物被膜（Biofilm,BF）形成与耐药性中绿原酸（Chlorogenic acid）、c-di-GMP、人杀菌肽LL-37的作用。 方法 选取铜绿假单胞菌野生型株（pseudomonas aeruginosa strain，PAO1）展开研究，采用肉汤稀释法测定绿原酸、c-di-GMP、人杀菌肽LL-37对PA最低抑菌浓度（minimum inhibitory concentration，MIC），采用结晶紫定量法检测PA的BF形成中绿原酸、c-di-GMP、人杀菌肽LL-37的清除作用，采用PA运动试验检测绿原酸、c-di-GMP、人杀菌肽LL-37对PA运动能力的影响，荧光显微镜下观察绿原酸、c-di-GMP、人杀菌肽LL-37对PA的BF结构及存活情况的影响。 结果 人杀菌肽LL-37MIC值为64 mg/L、绿原酸MIC值为256 mg/L、c-di-GMPMIC值为32 mg/L，相比于c-di-GMP组，人杀菌肽LL-37组、绿原酸组、空白组的OD590nm值明显降低，且人杀菌肽LL-37组、绿原酸组明显降低（P＜0.05）。相比阴性对照组，人杀菌肽LL-37组、绿原酸组、c-di-GMP组的PA运动试验直径均明显降低，且相比于c-di-GMP组，人杀菌肽LL-37组、绿原酸组明显降低（P＜0.05）。空白组、c-di-GMP组的PA存活情况优于人杀菌肽LL-37组、绿原酸组，空白组、c-di-GMP组主要为绿色荧光，人杀菌肽LL-37组、绿原酸组主要为红色荧光、橙色荧光。相比c-di-GMP组，人杀菌肽LL-37组、阴性对照组、绿原酸组的耐药率明显降低，且相比于阴性对照组，人杀菌肽LL-37组、绿原酸组明显降低（P＜0.05）。 结论 绿原酸、人杀菌肽LL-37对PA的BF均可以发挥一定的清除作用，c-di-GMP可通过抑制细胞运动能力对PA的BF形成起到一定的调控作用，绿原酸、人杀菌肽LL-37以及c-di-GMP均可作为后续防治PA的BF的新方向。

Effect of chlorogenic acid,c-di-GMP and human bactericidal peptide LL-37 on drug resistance of Pseudomonas aeruginosa biofilm

Objective To investigate the role of chlorogenic acid, c-di-GMP and human bactericidal peptide LL-37 in biofilm formation(BF) of Pseudomonas aeruginosa(PA) and drug resistance.Methods Wild-type pseudomonas aeruginosa strain(PAO1) was selected to carry out the study, and the minimum inhibitory concentration(MIC) of chlorogenic acid, c-di-GMP and human germicide LL-37 on PA was determined by broth dilution method. Crystal violet quantitative method was used to detect the scavenging effects of chlorogenic acid, c-di-GMP and human germicidal peptide LL-37 in PA-BF formation, and PA motion test was used to detect the effects of chlorogenic acid, c-di-GMP and human germicidal peptide LL-37 on PA motion ability. The effects of chlorogenic acid, c-di-GMP and human bactericidal peptide LL-37 on the structure and survival of PA-BF were observed under fluorescence microscope.Results The value of MIC of human germicidal peptide LL-37, chlorogenic acid and c-di-GMP was 64 mg/L, 256 mg/L, and 32 mg/L, respectively. Compared with the c-divGMP group, the value of OD590nm human germicidal peptide LL-37 group, chlorogenic acid group and blank group significantly decreased, which was significantly lower in human bactericidal peptide LL-37 group and chlorogenic acid group(P＜0.05). Compared with the negative control group, the PA diameter in exercise testing of the human germicidal peptide LL-37 group, the chlorogenic acid group and the c-di-GMP group significantly decreased, which was significantly lower in human germicidal peptide LL-37 group and the chlorogenic acid group, as compared with the c-di-GMP group(P＜0.05).The survival of PA in blank group and c-divGMP group was better than that in human germicidal peptide LL-37 group and chlorogenic acid group. The fluorescence in blank group and c-di-GMP group was mainly green, while the fluorescence in human germicidal peptide LLv37 group and chlorogenic acid group was mainly red and orange.Compared with c-di-GMP group, the drug resistance rate of human germicidal peptide LL-37 group, negative control group and chlorogenic acid group significantly decreased, and compared with the negative control group, the drug resistance rate of human germicidal peptide LL-37 group and chlorogenic acid group significantly decreased(P＜0.05).Conclusion Both chlorogenic acid and human bactericidal peptide LL-37 can clear PA-BF to a certain extent, and c-di-GMP can regulate the BF formation of PA by inhibiting cell motor ability. Chlorogenic acid, human bactericidal peptide LL-37 and c-di-GMP can be used as a new direction for subsequent prevention and control of PA-BF.