基于转录组学技术分析绿原酸对鸡源大肠杆菌抑菌及耐药消除的作用机制

doi:10.16431/j.cnki.1671-7236.2020.12.040

摘要/Abstract

摘要： 为探究绿原酸对鸡源抗生素耐药大肠杆菌抑菌及耐药消除的作用机制，以影印法分离绿原酸浓度为1.25 mg/mL（亚抑菌浓度，1/2 MIC）的LB肉汤培养的第3代禽源大肠杆菌耐药逆转菌株，以微板法测定耐药逆转菌株的左氧氟沙星最小抑菌浓度（MIC），并进一步通过转录组测序方法分析绿原酸消除大肠杆菌耐药性的分子机制。结果显示：耐药逆转菌株对左氧氟沙星的MIC由16 μg/mL降低至4~8 μg/mL，说明1.25 mg/mL的绿原酸可在一定程度上消除耐药大肠杆菌的左氧氟沙星耐药性。为进一步解析绿原酸对鸡源大肠杆菌耐药消除作用的分子机制，通过转录组测序方法对耐药性消除前后鸡源大肠杆菌基因表达水平进行对比分析发现：绿原酸作用后共有12个基因的表达量发生显著下调，通过荧光定量PCR验证结果基本一致。经过GO功能富集分析和KEGG代谢通路富集分析发现，差异表达基因在功能上集中于膜组成成分和DNA重组，在代谢通路富集中差异基因均与代谢相关，差异基因中bhsA、cysP为Coli-HB染色质转录RNA（GenBank登录号：CP020933），oqxA、oqxR、emaA、pinR、xerD、folA、repA、repC、adhP、IS26为Coli-HB质粒1转录RNA（GenBank登录号：CP020934）。从差异表达基因的分布可以看出，绿原酸可以抑制细菌DNA重组、使细菌抗逆性减弱、抑制耐药基因活性等，在一定程度上消除大肠杆菌的左氧氟沙星耐药性，起到抑菌及耐药消除作用。

关键词: 大肠杆菌; 绿原酸; 耐药消除; 转录组

Abstract: In order to explore the mechanism of chlorogenic acid on the inhibition and elimination of drug resistance of antibiotic resistant Escherichia coli from chicken,the third generation of avian Escherichia coli drug-resistant reverse strains,cultivated in the LB broth whose chlorogenic acid concentration was 1.25 mg/mL (1/2 MIC),was separated through photolithography,the minimal inhibitory concentration (MIC) of levofloxacin was determined through microplate method,and the molecular mechanism of chlorogenic acid eliminating drug resistance of Escherichia coli was further analyzed through transcriptome sequencing method.The results showed that the MIC of drug-resistant reverse strains to levofloxacin decreased from 16 μg/mL to 4-8 μg/mL,indicating that to a certain extent,chlorogenic acid of 1.25 mg/mL could eliminate the resistance of Escherichia coli to levofloxacin.To further analyze the molecular mechanism of chlorogenic acid on the elimination of drug resistance of Escherichia coli from chicken,transcriptome sequencing was used to compare the gene expression of this kind of Escherichia coli before and after the elimination of drug resistance.And it was found that the expression levels of 12 genes were significantly down-regulated after chlorogenic acid treatment,which the results of fluorescence quantitative PCR were basically consistent with.After GO functional enrichment analysis and KEGG metabolic pathway enrichment analysis,it was found that the differentially expressed genes were functionally concentrated in membrane composition and DNA recombination,and in metabolic pathway,the differentially expressed genes were related to metabolism.bhsA and cysP in the differentially expressed genes were Coli-HB chromatin transcribed RNA (GenBank No.:CP020933).oqxA,oqxR,emaA,pinR,xerD,folA,repA,repC,adhP,IS26 were Coli-HB plasmid 1 transcribed RNA (GenBank No.:CP020934).It could be seen from the distribution of differentially expressed genes that chlorogenic acid could weaken the resistance of bacteria,inhibit the activity of drug-resistant genes,and inhibit the recombination of bacterial DNA,etc.To some extent,it could eliminate the drug-resistance of Escherichia coli to levofloxacin and play a role in inhibiting the bacteria and eliminating the drug-resistance.

Key words: Escherichia coli; chlorogenic acid; drug resistance elimination; transcriptome

中图分类号:

S853.74

姚姗姗, 张石磊, 梁存军, 张铁, 王春光. 基于转录组学技术分析绿原酸对鸡源大肠杆菌抑菌及耐药消除的作用机制[J]. 中国畜牧兽医, 2020, 47(12): 4156-4165.

YAO Shanshan, ZHANG Shilei, LIANG Cunjun, ZHANG Tie, WANG Chunguang. Analysis of Bacteriostatic and Drug Resistance Elimination Mechanisms of Chicken-derived Escherichia coli by Chlorogenic Acid Based on Transcriptome Technology[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 4156-4165.

参考文献

[1] 陆继爽,李波,单春乔,等.抗生素耐药性研究进展[J].中国兽医学报,2019,39(10):2088-2095. LU J S,LI B,SHAN C Q,et al.Progress of solutions against antibiotic resistance[J].Chinese Journal of Veterinary Science,2019,39(10):2088-2095.(in Chinese)
[2] 王云鹏,马越.养殖业抗生素的使用及其潜在危害[J].中国抗生素杂志,2008,33(9):519-523. WANG Y P,MA Y.Potential public hazard of using antibiotics in livestock industry[J].Chinese Journal of Antibiotics,2008,33(9):519-523.(in Chinese)
[3] DUARTE G,PEREIRA A A,FARAH A.Chlorogenic acids and other relevant compounds in Brazilian coffees processed by semi-dry and wet post-harvesting methods[J].Food Chemistry, 2010,118(3):851-855.
[4] LEE B,LEE D G.Depletion of reactive oxygen species induced by chlorogenic acid triggers apoptosis-like death in Escherichia coli[J]. Free Radical Research, 2018,52(5):605-615.
[5] LI G,WANG X,XU Y,et al.Antimicrobial effect and mode of action of chlorogenic acid on Staphylococcus aureus[J].European Food Research and Technology,2014,238(4):589-596.
[6] LI Y,WANG Q,YAO X,et al.Induction of CYP3A4 and MDR1 gene expression by baicalin,baicalein,chlorogenic acid,and ginsenoside Rf through constitutive androstane receptor- and pregnane X receptor-mediated pathways[J].European Journal of Pharmacology, 2010,640(1):46-54.
[7] BAGDAS D,GUL Z,MEADE J A,et al.Pharmacologic overview of chlorogenic acid and its metabolites in chronic pain and inflammation[J].Current Neuropharmacology, 2020,18(3):216-228.
[8] LOU Z,WANG H,ZHU S,et al.Antibacterial activity and mechanism of action of chlorogenic acid[J].Journal of Food Science, 2011,76(6):M398-M403.
[9] 张石磊,翟向和,王春光,等.小檗碱对禽源大肠杆菌抑菌作用及耐药消除作用的转录组学分析[J].中国畜牧兽医,2017,44(5):1518-1525. ZHANG S L,ZHAI X H,WANG C G,et al.Study on antimicrobial effect of berberine sulfate on avian Escherichia coli and elimination of leyofloxacin resistance based on transcriptome sequencing[J].China Animal Husbandry & Veterinary Medicine, 2017,44(5):1518-1525.(in Chinese)
[10] MATTILA P,HELLSTROM J.Phenolic acids in potatoes,egetables,and some of their products[J].Journal of Food Composition & Analysis,2007,20(3):152-160.
[11] 屈景年,莫运春,刘梦琴,等.金银花中绿原酸一步提取法及绿原酸抗菌活性[J].化学世界,2005,46(3):167-169. QU J N,MO Y C,LIU M Q,et al.Extraction of chlorogenic acid from Flos lonicerae by one-step-method and antimicrobial abilities of chlorogenic acid[J].Chemical World,2005,46(3):167-169.(in Chinese)
[12] COSTA V,APRILE M,ESPOSITO R,et al.RNA-Seq and human complex diseases:Recent accomplish-ments and future perspectives[J].European Journal of Human Genetics,2013,21(2):134-142.
[13] 李小白,向林,罗洁,等.转录组测序(RNA-seq)策略及其数据在分子标记开发上的应用[J].中国细胞生物学报,2013,35(5):168-188. LI X B,XIANG L,LUO J,et al.The strategy of RNA-seq,application and development of molecular marker derived from RNA-seq[J].Chinese Journal of Cell Biology,2013,35(5):168-188.(in Chinese)
[14] 蔡元锋,贾仲君.基于新一代高通量测序的环境微生物转录组学研究进展[J].生物多样性,2013,21(4):401-410. CAI Y F,JIA Z J.Progress in environmental transcriptomics based on next-generation high-throughput sequencing[J].Biodiversity Science,2013,21(4):401-410.(in Chinese)
[15] 周志娥,罗秋水,熊建华,等.绿原酸、异绿原酸A对大肠杆菌的抑菌机制[J].食品科技,2014,39(3):228-232. ZHOU Z E,LUO Q S,X J H,et al.Antimicrobial mechanisms of 3-O-caffeoyl quinic acid and 3.5-di-o-caffeoyl quinic acid against Escherichia coli[J].Food Science and Technology.2014,39(3):228-232.(in Chinese)
[16] KHIL P P,R DANIEL C O.Over 1000 genes are involved in the DNA damage response of Escherichia coli[J].Molecular Microbiology, 2010,44(1):89-105.
[17] 许钦坤,赵翠燕.金银花提取物对4种病原菌的体外抑菌效果研究[J].韶关学院学报,2019,40(12):58-60. XU Q K,ZHAO C Y.Study on antibacterial effect of honeysuckle extract on four pathogenic bacteria in vitro[J].Journal of Shaoguan University,2019,40(12):58-60.(in Chinese)
[18] SUZANNE B D,JEAN-PHILIPPE L,KATHLEEN G,et al.Differential contribution of AcrAB and OqxAB efflux pumps to multidrug resistance and virulence in Klebsiella pneumoniae[J].Journal of Antimicrobial Chemotherapy,2015,70(1):81-88.
[19] DONLAN R M,J WILLIAM C.Biofilms:Survival mechanisms of clinically relevant microorganisms[J].Clinical Microbiology Reviews,2002,15(2):167-193.
[20] SUBRAMANYA H S,ARCISZEWSKA L K,BAKER R A,et al.Crystal structure of the site-specific recombinase,XerD[J]. The EMBO Journal,2014,16(17):5178-5187.
[21] STEWARD K F,HARRISON T,ROBINSON C,et al.PinR mediates the generation of reversible population diversity in Streptococcus zooepide-micus[J]. Microbiology,2015,1(1):1-11.
[22] GHOSAL D,SAEDLER H.Isolation of the mini insertions IS 6 and IS 7 of E.coli[J].Molecular & General Genetics,1977,158(2):123-128.
[23] ZHANG X S,GARCÍA-CONTRERAS R,WOOD T K.YcfR (BhsA) influences Escherichia coli biofilm forma-tion through stress response and surface hydropho-bicity[J].Journal of Bacteriology,2007,189(8):30-51.
[24] HENRY G S.Sequence and structural analogies between glyceraldehyde-3-phosphate dehydrogenase of homo sapiens and the CysP periplasmic binding protein from Escherichia coli[J].Microbiology, 2000,146(4):8-15.
[25] THOMAS L M,HARPER A R,MINER W A,et al.Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD[J].Acta Crystallographica,2013,69(7):730-732.
[26] SHAFQAT J,HOOG J L,OPPERMANN U,et al.An ethanol-inducible MDR ethanol dehydrogenase/acetaldehyde reductase in Escherichia coli:Structural and enzymatic relationships to the eukaryotic protein forms[J].The FEBS Journal,1999,263(2):305-311.