山楂8种主要有效成分与组方消除ESBLs大肠杆菌耐药性的研究

A study on the elimination of drug resistance of ESBLs Escherichia coli by active components of Hawthorn

为提高抗生素对耐药菌的作用效果和减少抗生素使用剂量,本研究以齐墩果酸、槲皮素、柠檬酸、芦丁、熊果酸、牡荆素、山奈酚和山楂酸8种山楂主要有效成分为研究对象,以超广谱β-内酰胺酶(ESBLs)大肠杆菌为目的菌株,筛选大肠杆菌β-内酰胺类抗生素增敏剂。首先将亚抑菌浓度(1/2 MIC)的上述8种山楂有效成分分别与目的菌株共培养72 h,采用2倍稀释法测定头孢噻肟(CTX)对目的菌株最小抑菌浓度(MIC)的变化;其次利用琼脂糖凝胶电泳试验筛选能够消除ESBLs大肠杆菌耐药质粒的山楂有效成分;采用qPCR法筛选能够降低ESBLs大肠杆菌外排泵基因转录水平的山楂有效成分;采用ELISA方法筛选能够抑制ESBLs大肠杆菌及其生物被膜(BF)形成的山楂有效成分;然后采用牛津杯法和微量棋盘稀释法结合筛选出与CTX联用效果最好的药物组方;最后通过PCR法检测组方对8株野生多重耐药ESBLs大肠杆菌β-内酰胺类耐药基因(bla_(CTX-M)、bla_(TEM)、bla_(SHV)、bla_(OXA))的消除效果。结果显示:经柠檬酸和芦丁作用后,CTX对目的菌株的MIC由32.00 mg/mL降至8.00mg/mL;消除ESBLs大肠杆菌耐药质粒效果最显著的是柠檬酸,抑制ESBLs大肠杆菌外排泵AcrA mRNA、AcrB mRNA基因转录水平最显著的是柠檬酸、芦丁和山楂酸,抑制ESBLs大肠杆菌ESBLs活性最显著的是齐墩果酸和柠檬酸,抑制ESBLs大肠杆菌BF形成最显著的是芦丁和山奈酚;柠檬酸、芦丁(1:1)与CTX联合应用的中西药联合用药指数为0.53,有联合增效作用;柠檬酸、芦丁(1:1)对8株大肠杆菌耐药基因bla_(CTX-M)、bla_(TEM)、bla_(SHV)、bla_(OXA)的消除率为50%;这表明柠檬酸和芦丁(1:1)组方能够消除ESBLs大肠杆菌耐药性,增强ESBLs大肠杆菌对CTX敏感性。本研究首次验证了山楂有效成分中可作为大肠杆菌β-内酰胺类抗生素增敏剂的组方,为畜禽养殖生产中减少抗生素使用量和耐药菌感染的防控提供参考依据。

To improve the effects of antibiotics on drug-resistant bacteria and reduce the dosage of antibiotics used,we took 8main active ingredients of Hawthorn as research objects,including Oleanolic acid,Quercetin,Citric acid,Rutin,Ursolic acid Vitexin,Kaempferol and Crataegus acid.Extended-spectrumβ-lactam enzyme(ESBLs)E.coli was used as the target strain to screen for E.coliβ-lactam antibiotic sensitizers.First of all,the target strains were cultured for 72 hours with each of the 8 active ingredients of 1/2MIC(minimal inhibitory concentration),and the 96-well microplate assay was used to determine the MIC of cefotaxime(CTX)to the target strain.Then,the active components of Hawthorn,which can eliminate the multi-drug resistant plasmid of ESBLs,were screened by agarose gel electrophoresis.Secondly,we used qPCR to screen for the active ingredients of Hawthorn that can reduce the transcription level of ESBLs E.coli efflux pump gene,and ELISA to screen for the active ingredients of Hawthorn that can inhibit the formation of biofilm.Then,we selected the best combination of CTX and CTX by Oxford cup method and micro-chessboard dilution method.Finally,the elimination rate of ESBLs resistance genes(bla_(CTX-M),bla_(TEM),bla_(SHV),bla_(OXA))in 8 strains of wildtype E.coli was assessed by PCR.The MIC results showed that the MIC of CTX to the target strain decreased from 32.00mg/mL to 8.00mg/mL after treatment with Citric acid and Rutin.Citric acid was the most effective substance to eliminate the drug-resistant plasmid of ESBLs E.coli.Citric acid,Rutin,and Hawthorn acid were the most significant inhibitors of efflux pump gene AcrA m RNA and AcrB mRNA transcription levels of ESBLs E.coli.Oleanolic acid and Citric acid were the most significant inhibitors ofβ-lactamase activity of ESBLs E.coli,and Rutin and Kaempferol were the most significant inhibitors of biofilm formation of ESBLs E.coli.The FIC index(FICI)of Citric acid,Rutin(1:1)combined with CTX was 0.53,which had a synergistic effect.The resistant genes bla_(CTX-M),bla_(TEM),bla_(SHV)and bla_(OXA)of 8 E.coli strains were reduced to varying degrees after treatment with Citric acid and Rutin(1:1),and the elimination rates were 50%.These results indicate that Citric acid and Rutin(1:1)can eliminate the drug resistance of ESBLs E.coli and enhance the sensitivity of ESBLs E.coli to CTX.This study verified for the first time that the active ingredients of Hawthorn can be used as a sensitizers for E.coliβ-lactam antibiotics,and provided a theoretical basis for reducing the dosage of antibiotics used in livestock and poultry production as well as preventing and controlling drug-resistant bacterial infections.