香柠檬精油抗伪结核棒状杆菌作用及机制研究

doi:10.11843/j.issn.0366-6964.2024.03.033

摘要/Abstract

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0.78~1.56 μL·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。

关键词: 伪结核棒状杆菌, 香柠檬精油, 抗菌作用, 机制

Abstract: 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 0.78-1.56 μL·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, mBD3 and mBD4 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.

Key words: essential oil, Corynebacterium pseudotuberculosis, antibacterial activity, mechanism

中图分类号:

S859
S852.61

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. 香柠檬精油抗伪结核棒状杆菌作用及机制研究[J]. 畜牧兽医学报, 2024, 55(3): 1217-1227.

FU Xuezhen, LI Xincan, QIAN Hongyu, LÜ Hong, WU Chanyu, WANG Xiaohan, WANG Xiaohua, WANG Zhiying, ZHOU Zuoyong. Antibacterial Effect and Mechanism of Bergamot Essential Oil on Corynebacterium pseudotuberculosis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1217-1227.

参考文献

[1] DORELLA F A, PACHECO L G, SEYFFERT N, et al. Antigens of Corynebacterium pseudotuberculosis and prospects for vaccine development[J]. Expert Rev Vaccines, 2009, 8(2):205-213.
[2] STING R, GEIGER C, RIETSCHEL W, et al. Corynebacterium pseudotuberculosis Infections in alpacas (Vicugna pacos)[J]. Animals (Basel), 2022, 12(13):1612.
[3] BAIRD G J, FONTAINE M C. Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis[J]. J Comp Pathol, 2007, 137(4):179-210.
[4] 曹启航, 姜悦才, 张天亮, 等. 山羊伪结核棒状杆菌病流行现状和防控技术研究进展[J]. 动物医学进展, 2022, 43(7):74-78.
CAO Q H, JIANG Y C, ZHANG T L, et al. Progress on epidemic status, prevention and control techniques of caseous lymphadenitis in goats[J]. Progress in Veterinary Medicine, 2022, 43(7):74-78. (in Chinese)
[5] DOSOKY N S, SETZER W N. Biological activities and safety of Citrus spp. essential oils[J]. Int J Mol Sci, 2018, 19(7):1966.
[6] 陈红丽, 刘可, 杨小爱, 等. 葡萄柚精油及其分子蒸馏组分的抗菌活性研究[J]. 赣南师范大学学报, 2017, 38(3):81-84.
CHEN H L, LIU K, YANG X A, et al. Antibacterial activities of grapefruit oil and its molecular distillation fraction[J]. Journal of Gannan Normal University, 2017, 38(3):81-84. (in Chinese)
[7] KANG J M, JIN W Y, WANG J F, et al. Antibacterial and anti-biofilm activities of peppermint essential oil against Staphylococcus aureus[J]. LWT, 2019, 101:639-645.
[8] 吴均, 杨德莹, 李抒桐, 等. 甜橙精油的化学成分、抑菌和抗氧化活性研究[J]. 食品工业科技, 2016, 37(14):148-153.
WU J, YANG D Y, LI S T, et al. Study on chemical components, antimicrobial and antioxidant activity of essential oil from Citrus sinensis[J]. Science and Technology of Food Industry, 2016, 37(14):148-153. (in Chinese)
[9] 李娜. 紫苏精油提取及其防腐复合材料的制备和性能研究[D]. 太原:中北大学, 2018.
LI N. Study on extraction of perilla essential oil, preparation and properties of corresponding anticorrosive compounds[D]. Taiyuan:North University of China, 2018. (in Chinese)
[10] 朱梅芳, 唐宇, 郑琴, 等. 不同提取方式对连翘、荆芥、薄荷挥发油成分及抗菌活性的影响[J]. 中草药, 2018, 49(12):2845-2854.
ZHU M F, TANG Y, ZHENG Q, et al. Effects of different extraction methods on composition and antibacterial activity of volatile oil from Forsythiae Fructus, Schinzonepetae Herba, and Menthae Haplocalycis Herba[J]. Chinese Traditional and Herbal Drugs, 2018, 49(12):2845-2854. (in Chinese)
[11] TAN J M, YI W Y, WANG Z Y, et al. TRIM21 negatively regulates Corynebacterium pseudotuberculosis-induced inflammation and is critical for the survival of C. pseudotuberculosis infected C57BL6 mice[J]. Vet Microbiol, 2021, 261:109209.
[12] TANG C L, CHEN J L, ZHANG L X, et al. Exploring the antibacterial mechanism of essential oils by membrane permeability, apoptosis and biofilm formation combination with proteomics analysis against methicillin-resistant staphylococcus aureus[J]. Int J Med Microbiol, 2020, 310(5):151435.
[13] WU Y P, BAI J R, ZHONG K, et al. A dual antibacterial mechanism involved in membrane disruption and DNA binding of 2R, 3R-dihydromyricetin from pine needles of Cedrus deodara against Staphylococcus aureus[J]. Food Chem, 2017, 218:463-470.
[14] ZHOU Z Y, YANG H Y, LI H X, et al. Sodium butyrate ameliorates Corynebacterium pseudotuberculosis infection in RAW264. 7 macrophages and C57BL/6 mice[J]. Microb Pathog, 2019, 131:144-149.
[15] LI H X, YANG H Y, ZHOU Z Y, et al. Isolation, antibiotic resistance, virulence traits and phylogenetic analysis of Corynebacterium pseudotuberculosis from goats in southwestern China[J]. Small Rumin Res, 2018, 168:69-75.
[16] 崔乃菠, 马境谦, 余河水, 等. 基于刃天青微平板法检测金黄色葡萄球菌对天然产物的敏感性[J]. 天津中医药大学学报, 2022, 41(6):774-779.
CUI N B, MA J Q, YU H S, et al. Susceptibility tests of Staphylococcus aureus to natural products using the resazurin method[J]. Journal of Tianjin University of Traditional Chinese Medicine, 2022, 41(6):774-779. (in Chinese)
[17] 李宙阳, 周英. 刃天青显色法检测铜绿假单胞菌对几种抗菌药物的敏感性[J]. 山地农业生物学报, 2010, 29(3):237-241.
LI Z Y, ZHOU Y. A Resazurin-based assay for evaluation of anti-pseudomonas aeruginosa drugs sensitivity[J]. Journal of Mountain Agriculture and Biology, 2010, 29(3):237-241. (in Chinese)
[18] CIRMI S, BISIGNANO C, MANDALARI G, et al. Anti-infective potential of Citrus bergamia risso et poiteau (bergamot) derivatives:a systematic review[J]. Phytother Res, 2016, 30(9):1404-1411.
[19] VALUSSI M, DONELLI D, FIRENZUOLI F, et al. Bergamot oil:botany, production, pharmacology[J]. Encyclopedia, 2021, 1(1):152-176.
[20] 张卓, 聂德超, 赵琛, 等. 柑橘类植物精油的抗菌活性及其作用机制[J]. 动物营养学报, 2022, 34(2):758-764.
ZHANG Z, NIE D C, ZHAO C, et al. Antibacterial activity of citrus essential oils and its mechanism[J]. Chinese Journal of Animal Nutrition, 2022, 34(2):758-764. (in Chinese)
[21] SONG X Y, LIU T, WANG L, et al. Antibacterial effects and mechanism of mandarin (Citrus reticulata L.) essential oil against Staphylococcus aureus[J]. Molecules, 2020, 25(21):4956.
[22] 崔海英, 周慧, 宋方平, 等. 丁香精油对单核细胞增生性李斯特菌的抗菌性能及杀菌机制研究[J]. 中国食品添加剂, 2015(12):65-69.
CUI H Y, ZHOU H, SONG F P, et al. Antibacterial activity and mechanism of clove oil against Listeria monocytogenes[J]. China Food Additives, 2015(12):65-69. (in Chinese)
[23] 周作勇, 李和贤, 杨浩钺, 等. 伪结核棒状杆菌毒力因子的研究进展[J]. 中国人兽共患病学报, 2017, 33(12):1115-1119.
ZHOU Z Y, LI H X, YANG H Y, et al. Research advances on the virulence factors of Corynebacterium pseudotuberculosis[J]. Chinese Journal of Zoonoses, 2017, 33(12):1115-1119. (in Chinese)
[24] LAKSHMAIAH NARAYANA J, CHEN J Y. Antimicrobial peptides:possible anti-infective agents[J]. Peptides, 2015, 72:88-94.
[25] 郭志强, 杨奉珠, 雷岷, 等. 抗菌肽对肉兔小肠黏膜形态、盲肠菌群和免疫功能的影响[J]. 动物营养学报, 2012, 24(9):1778-1784.
GUO Z Q, YANG F Z, LEI M, et al. Effects of antibacterial peptides on intestinal mucosal morphology, caecal microflora and immune function of meat rabbits[J]. Chinese Journal of Animal Nutrition, 2012, 24(9):1778-1784. (in Chinese)
[26] CHONG K T, THANGAVEL R R, TANG X H. Enhanced expression of murine β-defensins (MBD-1, -2, -3, and-4) in upper and lower airway mucosa of influenza virus infected mice[J]. Virology, 2008, 380(1):136-143.
[27] 韩晓芳. 羊皮下脓肿病原分离鉴定及天蚕素AD防治试验[D]. 杨凌:西北农林科技大学, 2021.
HAN X F. Isolation and identification of pathogen of subskin abscess and control of cecropin AD[D]. Yangling:Northwest A&F University, 2021. (in Chinese)