首页|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

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

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为了探究中药对大肠杆菌抗生素耐药性的消除机制,以小檗碱为处理药物,使用小檗碱浓度为250μg/mL(1/2最小抑菌浓度(MIC))的LB肉汤培养临床分离的禽源耐药大肠杆菌,每隔24 h传一代,共传3代.对第3代菌液以影印法分离突变菌,以微板法测定突变菌的左氧氟沙星MIC.经测定发现突变菌对左氧氟沙星的MIC由16μg/mL降至8μg/mL,说明对左氧氟沙星的耐药性具有消除作用.为了解小檗碱作用大肠杆菌的分子机制,通过转录组测序方法对耐药性消除前后禽源大肠杆菌基因表达水平进行对比分析.结果显示,小檗碱作用后共有45个基因的表达量发生显著变化,其中有30个基因表达量上调,15个基因表达量下调.经过GO功能富集分析和KEGG代谢通路富集分析,发现上调主要为:与色氨酸合成有关的基因,磷酸吡哆醛结合相关3个基因,表达转酮酶基因;下调主要为:双组份系统中多个基因,十一异戊二烯焦磷酸磷酸酶(UppP)编码基因ybj G,酰基辅酶A脱氢酶合成有关的基因.推测大肠杆菌体内酶活性降低是小檗碱抑菌的主要机制;大肠杆菌多重耐药外排泵表达降低、细胞膜和细胞壁成分的改变是小檗碱耐药性消除的主要机制.
Study on Antimicrobial Effect of Berberine Sulfate on Avian Escherichia coli and Elimination of Levofloxacin Resistance Based on Transcriptome Sequencing
In order to explore the mechanism of traditional Chinese medicine eliminated the antibi-otic resistance of Escherichia coli ,with berberine as drug treatment,the antibiotic-resistant Escherichia coli isolated from clinical isolates was cultured in LB broth with a concentration of 250 μg/mL (1/2 MIC)of berberine,and passaged for 3 generations every 24 h.The MIC of the mutant was determined by microplate method.The MIC of the mutant was determined by photo-lithography.The MIC of levofloxacin was reduced from 16 to 8 μg/mL,and the drug resistance to levofloxacin was eliminated.In order to understand the molecular mechanism of berberine's effect on Escherichia coli ,the gene expression level of avian Escherichia coli was analyzed by transcriptome sequencing method before and after drug resistance elimination. The results showed that the expression level of 45 genes was significantly changed after treated with berber-ine,among which 30 genes were up-regulated and 15 genes were down-regulated.The enrichment analysis of GO function and enrichment analysis of KEGG metabolic pathways showed that up-regulated genes were mainly included the synthesis of tryptophan,the binding of pyridoxal phos-phate,transketolase.Down-regulation of multiple genes were in the two-component system,theybjG of the undecylpodophosphate phosphatase (UppP)gene and acyl-CoA dehydrogenase syn-thesis related genes.Escherichia coli in vivo enzyme activity might be the main mechanism of in-hibition of berberine.Decreased activity of the multi-drug resistant efflux pump and the changes of Escherichia coli cell membrane and cell wall might be the main mechanisms for the elimination of berberine resistance.

Escherichia coliberberinedrug resistance eliminationtranscriptomeReal-time quantitative PCR

张石磊、翟向和、王春光、陈谏、贾泽、张铁

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河北农业大学动物医学院,保定 071000

大肠杆菌 小檗碱 耐药消除 转录组 实时荧光定量PCR

国家自然科学基金河北省重点研发计划项目

3157256016222701D

2017

中国畜牧兽医
中国农业科学院北京畜牧兽医研究所

中国畜牧兽医

CSTPCD北大核心
影响因子:0.72
ISSN:1671-7236
年,卷(期):2017.44(5)
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