摘要
伪结核棒状杆菌(Corynebacterium pseudotuberculosis,Cp)感染可影响家畜尤其是反刍动物羊的生产性能,本文旨在筛选对Cp有抑杀活性的植物精油并研究其作用机制,为Cp感染防控提供参考。通过纸片扩散法从4种植物精油中筛选出能高效抑杀Cp的香柠檬精油(bergamot essential oil, BEO),采用刃天青指示剂微量肉汤稀释法测定BEO对Cp不同菌株的最小抑菌浓度(minimal inhibitory concentration, MIC)。通过体外试验评价BEO对Cp感染J774A.1巨噬细胞的影响。通过测定脏器载菌量、脾抗菌肽基因表达量和存活率综合评价BEO对Cp感染昆明系小鼠的保护作用。采用扫描电镜观察BEO作用后Cp菌体形态变化,并检测BEO作用后Cp的DNA泄漏以及毒力基因表达情况,初步研究BEO抗Cp机制。结果显示:BEO具有较强的抗Cp作用,其MIC为0.78~1.56μL·mL^(-1)。BEO可降低Cp感染小鼠肾、肝和腹水的细菌负荷(P<0.01),上调Cp感染小鼠脾中抗菌肽Cramp、Bpifal 1、mBD2、mBD3和mBD4的mRNA表达水平(P<0.01)。BEO处理可降低Cp对J774A.1巨噬细胞的感染率,提高被Cp感染小鼠的存活率(P<0.05)。BEO处理使Cp细胞膜凹陷,菌体内DNA外渗增加(P<0.05),以及下调毒力基因磷脂酶D(phospholipase D,pld)的表达(P<0.01)。BEO能抑杀Cp,改善Cp对小鼠的感染程度,其机制可能与BEO损伤Cp细胞膜造成DNA泄漏并降低毒力基因pld的mRNA表达,以及促进Cp感染小鼠抗菌肽Cramp、Bpifal 1、mBD2、mBD3和mBD4的表达有关。
Corynebacterium pseudotuberculosis(Cp)infection highly impairs the production performance of livestocks,especially for the small ruminants(sheep/goats).This experiment was conducted to screen the essential oils against Cp,and to evaluate its antibacterial mechanism.Bergamot essential oil(BEO)was screened from four kinds of essential oils by K-B disk diffusion method,and the minimal inhibitory concentration(MIC)of BEO against different strains of Cp was determined by resazurin based broth dilution method.The effect of BEO on Cp infection of J774A.1 macrophages were detected through in vitro experiments.The bacterial load in visceral organs,antimicrobial peptide genes expression in spleens and survival rates were tested to evaluate the protective effects of BEO on Cp-infected Kunming mice in vivo.Then the antibacterial mechanism of BEO was explored via the changes of cell morphology,cellular nucleotide leakage and the expression of virulence genes of Cp.The MIC value of BEO against Cp is 0.78-1.56μL·mL^(-1).The treatment of BEO significantly reduced the bacterial loads in kidney,liver and ascites of Cp-infected mice(P<0.01),up-regulated the mRNA expression levels of antibacterial peptides genes including Cramp,Bpifal 1,mBD2,mBD 3 and mBD 4 in spleens(P<0.01),decreased infection rate of Cp in J774A.1 macrophages and reduced mortality of Cp-infected mice(P<0.05).Scanning electron microscope analysis showed that the cell membrane was destroyed,the nucleotide was significantly leaked in Cp treated with BEO,and the mRNA expression level of virulence gene phospholipase D(pld)was significantly decreased in BEO treated Cp(P<0.01).BEO possesses strong antibacterial activity against Cp,and alleviates the infectious degree of Cp in mice,the mechanism of which may be related to the disruption of membrane integrity and inhibition of pld mRNA expression of Cp treated with BEO.
作者
符雪珍
李鑫灿
钱虹宇
吕红
吴婵玉
王晓涵
王小华
王芝英
周作勇
FU Xuezhen;LI Xincan;QIAN Hongyu;L Hong;WU Chanyu;WANG Xiaohan;WANG Xiaohua;WANG Zhiying;ZHOU Zuoyong(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;Essence Pharmaceutical(Chengdu)Co.,Ltd,Chengdu 641423,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2024年第3期1217-1227,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家级大学生创新训练计划项目(202210635102)
重庆市自然科学基金(cstc2021jcyj-msxmX0884)
横向项目(F2023285)。
关键词
伪结核棒状杆菌
香柠檬精油
抗菌作用
机制
essential oil
Corynebacterium pseudotuberculosis
antibacterial activity
mechanism
