抗菌肽MPX对肠出血性大肠杆菌的体外抑菌作用及其作用机制的初步研究  

A preliminary study on the antibacterial activity and the mechanism of antibacterial peptide MPX against enterohemorrhagic Escherichia coli in vitro

作　　者：黄俊杰 赵雪芹[1,2] 赵娅娅 刘双双 朱春玲 夏小静 张守平[1] 罗维玉[1] 徐彦召 杭柏林[1] 孙亚伟[1] Hanna Fotina[2] 胡建和 王磊[1] HUANG Jun-jie;ZHAO Xue-qin;ZHAO Ya-ya;LIU Shuang-shuang;ZHU Chun-ling;XIA Xiao-jing;ZHANG Shou-ping;LUO Wei-yu;XU Yan-zhao;HANG Bo-lin;SUN Ya-wei;Hanna Fotina;HU Jian-he;WANG Lei(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Xinxiang 453003,China;Faculty of Veterinary Medicine,Sumy National Agrarian University,Sumy 40021,Ukraine)

机构地区：[1]河南科技学院动物科技学院,河南新乡453003 [2]Faculty of Veterinary Medicine,Sumy National Agrarian University,Sumy 40021

出　　处：《中国预防兽医学报》2022年第7期763-770,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省青年人才托举工程项目(2020HYTP041);河南省高等学校重点科研项目(21A230004);河南省高等学校青年骨干教师培养计划(2020GGJS162)。

摘　　要：为研究抗菌肽MPX(Mastoparan-X)对肠出血性大肠杆菌(EHEC)的体外抑菌作用及作用机理。本研究通过琼脂扩散法、最小抑菌浓度(MIC)的测定、杀菌曲线的绘制以及扫描电镜观察MPX对EHEC的抑菌效果,结果显示MPX对EHEC的抑菌圈直径为9.7 mm,大于抗菌肽PR39但小于恩诺沙星(Enro);其对EHEC的MIC为31.25μg/mL;杀菌曲线结果显示,4 MIC MPX作用6 h后,EHEC菌液OD值降至0.1,极显著低于阴性对照组(只加入ddHO的EHEC,下同)(P<0.01),活菌数量降至阴性对照的千分之一(P<0.05),且4 MIC MPX的抑菌效果优于抗菌肽PR39与Enro;电镜观察结果显示,MPX作用后的菌体明显变小,细菌内容物渗漏。MPX稳定性试验结果显示,酸、碱、温度、一价阳离子等对MPX的抑菌效果影响较小,二价阳离子极显著抑制MPX的抑菌活性(P<0.01)。上述结果表明,MPX在体外有较强的抑菌效果,且稳定性较强。采用BCA法测定不同浓度MPX(0.5 MIC、1 MIC、2 MIC)作用后的EHEC上清中的蛋白含量;采用SYTO 9/PI染色后经激光共聚焦显微镜观察上述不同浓度MPX作用后的EHEC菌体荧光颜色;采用苯基萘胺(NPN)探针、碘化丙啶(PI)染料和3,3-二丙基硫代二碳氰碘化物[DiSC3(5)]探针分别检测不同浓度MPX(1 MIC、2 MIC、4 MIC)作用后的EHEC菌液的荧光强度,以分析MPX对EHEC细胞膜通透性的影响。BCA结果显示,1 MIC及2 MIC MPX作用后的菌液上清蛋白含量均显著高于阴性对照组(P<0.05);激光共聚焦试验结果显示,与阴性对照组相比,只有2 MIC MPX作用后的EHEC中的红色荧光明显增多;NPN探针和DiSC3(5)探针检测结果显示,不同浓度MPX作用后(0~2 min)EHEC的荧光强度均明显增强,其后不再变化;PI染料检测结果显示,不同浓度MPX作用后(0~4 min)EHEC的荧光强度均明显增强,其后逐渐降低。上述结果表明,MPX可以破坏EHEC的外膜和细胞膜,使膜的通透性增加,进而导致细菌死亡,且该作用与MPX的浓度基本呈正相关。分别利To study the antibacterial effects and the mechanism of antimicrobial peptide MPX (Mastoparan-X) on enterohemorrhagic Escherichia coli (EHEC) in vitro.In this study,the bacteriostatic effect of MPX on EHEC was tested by agar diffusion method,minimum inhibitory concentration (MIC) determination,bactericidal curve drawing and scanning electron microscope.The results showed that the inhibition zone diameter of MPX on EHEC was 9.7mm,which was larger than that of the antimicrobial peptide PR39 treatment group and smaller than that of the enrofloxacin (Enro) treatment group.The MIC of MPX on EHEC was 31.25μg/mL.The results of the bactericidal curve showed that the effect of 1MIC MPX was weaker than that of the positive control group in the first 4 hours;after 6 hours of 4MIC MPX treatment,the ODvalue of EHEC decreased to 0.1which was significantly lower than that of the negative control group (EHEC with only ddHO,the same below)(P<0.01),and the number of viable bacteria decreased to one thousandth of the negative control (P<0.05).The effect of 4MIC MPX was better than that of PR39 and Enro.Observation under electron microscope showed that EHEC treated with 1MIC MPX were obviously smaller and the bacterial contents leaked.The results of MPX stability test showed that acid,alkali,temperature and monovalent cations had little effect on the bacteriostatic effect of MPX,and the bivalent cations significantly inhibited the antibacterial activity of MPX(P<0.01).The above results showed that 4MIC MPX could significantly reduce the ODvalue and viable bacterial concentration of the EHEC in vitro,and its effect was better than that of the Enro and PR39 positive control groups,and its stability was stronger The protein content in the supernatant of EHEC treated with different concentrations of MPX (0.5MIC,1MIC,and 2MIC) was determined by BCA kit.The color of EHEC treated with the above different concentrations of MPX was observed under laser confocal microscope after staining with SYTO 9/PI.N-phenyl-naphthylamine (NPN),propidium

关 键 词：抗菌肽MPX 肠出血性大肠杆菌 体外试验 

分 类 号：S852.61[农业科学—基础兽医学]