四黄止痢复方醇提物(CSME)抗禽大肠杆菌的作用机制
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摘要
为了探讨四黄止痢复方醇提物(CSME)对禽大肠杆菌的抗菌机制。采用微孔-平板法检测其对禽大肠杆菌的最小抑菌浓度(MIC);用吸光光度法检测其对细菌生长曲线、细胞膜通透性和能量代谢活力的影响;荧光定量PCR法检测其对禽大肠杆菌4种主要毒力因子(tsh、iss、ibeA和fimC)表达的影响。结果显示,CSME对禽大肠杆菌(CVCC251)的MIC为125 g/L,1/2MIC就能显著抑制细菌生长,破坏细菌细胞膜结构,使核酸、蛋白质外泄,并抑制了琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)的活性;1/8MIC能显著抑制了禽大肠杆菌tsh、iss和fimC毒力因子基因的表达(P<0.01)。结果表明,CSME可以通过破坏细菌细胞膜结构、造成细菌能量代谢紊乱、抑制毒力基因表达发挥抗菌活性。
In order to study the antibacterial mechanism of CSME against avian Escherichia coli(E.coli),CSME was extracted with methanol.Minimum inhibitory concentration(MIC) and the effect of the extract against the E.coli growth curve was explored by micropore-plate and absorptiometry.Effect of the extract against cell membrane permeability and energy metabolism of avian E.coli was determined by spectrophotometry.The influence of the extract against virulence factors gene expression of tsh,iss,ibeA and fimC in avian E.coli,were evaluated by fluorescence quantitative PCR.The results showed that MIC of the extract against avian E.coli was 125 g/L and subinhibitory concentration could inhibit the growth of E.coli in the early stages.The extract could increase bacterial cell membrane permeability and lead to bacterial nucleic acid and protein macromolecular material overflow.The extract could inhibit the activity of SDH and MDH and the gene expression of tsh,iss and fimC in avian E.coli.CSME methanol extract could destroy cell membrane structure,causing energy metabolism disorder and inhibiting virulence gene expression.
In order to study the antibacterial mechanism of CSME against avian Escherichia coli(E.coli),CSME was extracted with methanol.Minimum inhibitory concentration(MIC) and the effect of the extract against the E.coli growth curve was explored by micropore-plate and absorptiometry.Effect of the extract against cell membrane permeability and energy metabolism of avian E.coli was determined by spectrophotometry.The influence of the extract against virulence factors gene expression of tsh,iss,ibeA and fimC in avian E.coli,were evaluated by fluorescence quantitative PCR.The results showed that MIC of the extract against avian E.coli was 125 g/L and subinhibitory concentration could inhibit the growth of E.coli in the early stages.The extract could increase bacterial cell membrane permeability and lead to bacterial nucleic acid and protein macromolecular material overflow.The extract could inhibit the activity of SDH and MDH and the gene expression of tsh,iss and fimC in avian E.coli.CSME methanol extract could destroy cell membrane structure,causing energy metabolism disorder and inhibiting virulence gene expression.
引文
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[3] 贾纪萍,熊玥,宋慧敏,等.四黄止痢颗粒中几种有效成分的薄层鉴别研究[J].中国畜牧兽医,2017(4):1233-1238.
[4] 彭齐,谢丽玲,谢俊.中药抑菌机制的研究方法[J].生物技术进展,2015,5(1):66-69.
[5] 马绪荣,苏德模.药品微生物学检验手册[M].北京:科学出版社,2000:210-219.
[6] 黄燕飞.黄连水提物对痢疾杆菌的抑菌机制研究[D].合肥:安徽农业大学,2016.
[7] 邵颖.APEC毒力岛基因irp2、fyua缺失对其毒力影响的研究[D].合肥:安徽农业大学,2014.
[8] SCHMITTGEN T D,ZAKRAJSEK B A,MILLS A G,et al.Quantitative reverse transcription-polymerasechain reaction to study mRAN decay:comparison of endpoint and real-time methods[J].Anal Biochem,2000,285(2):194-204.
[9] 郑全喜,苏显中,王河清,等.中草药体外抑菌作用的研究进展[J].中国医药生物技术,2009,4(4):295-298.
[10] 陈杖榴.兽医药理学[M].北京:中国农业出版社,2002:229-230.
[11] 李昌勤,赵琳,薛志平,等.隐丹参酮抑菌作用机制研究[J].中国药学杂志,2012,47(21):1706-1710.
[12] 贺常亮,张小艺,杨航,等.金银花提取物对金黄色葡萄球菌的抗菌作用研究[J].贵州畜牧兽医,2013,37(4):11-14.
[13] 李喜艳,王加启,卜登攀,等.多不饱和脂肪酸对细胞膜功能影响的研究进展[J].生物技术通报,2009(12):22-26.
[14] BOYD E F,NELSON K,WANG F S,et al.Molecular genetic basis of allelic polymorphism in malate dehydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enteric[J].Proc Natl Acad Sci USA,1994,91:1280-1284.
[15] 董向磊.禽致病性禽大肠杆菌的分离鉴定和分离株毒力基因与致病性相关性研究[D].江苏扬州:扬州大学,2014.
[16] 王利勤.鸡源致病性大肠埃希菌耐药基因及毒力基因检测研究[D].西安:西北农林科技大学,2012.
[17] 刘红玉,王君玮,王娟,等.禽大肠杆菌毒力因子的研究进展[J].中国动物检疫,2013,30(10):25-29.
[18] JOHNSON T J,WANNEMUEHLER Y M.,NOLAN L K.Evolution of the iss Gene in Escherichia coli[J].Appl Envir Microbiol,2008,74(8):2360-2369.
[19] EWERS C,JANSSEN T,KIESSLING S,et al.Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry[J].Vet Microbiol,2004,104(1/2):91-101.