摘要
为探究黄芩水煎剂能否增强头孢噻呋对产超广谱β-内酰胺酶基因bla_(CTX-M)阳性大肠杆菌的抗菌活性,本试验首先分别制备携带bla_(CTX-M)-55和bla_(CTX-M)-14的重组菌(C600-1和C600-9),绘制时间-杀菌曲线评价1最小抑菌浓度(MIC)头孢噻呋和1 MIC黄芩水煎剂联用对bla_(CTX-M)阳性重组菌的体外抑杀活性;使用不加黄芩水煎剂(阴性对照)、加1/4 MIC、1/2 MIC剂量的黄芩水煎剂继代培养重组菌7 d,通过测定头孢噻呋对重组菌的MIC和重组菌的bla_(CTX-M)表达,分析黄芩水煎剂对重组菌的头孢噻呋耐药发展的干预作用;使用不加黄芩水煎剂(阴性对照)、加1/4 MIC、1/2 MIC、1 MIC剂量的黄芩水煎剂培养C600、C600-1和C600-9菌株2 h,通过荧光染色法和逆转录实时荧光定量聚合酶链式反应(RT-qPCR)测定细胞内、外膜通透性和外排泵活性,探索黄芩水煎剂逆转耐药性的机制。结果显示,与头孢噻呋单药相比,黄芩水煎剂与其联用后对大肠杆菌C600和重组菌C600-1、C600-9具有明显的协同抑杀活性;不同剂量黄芩水煎剂与bla_(CTX-M)阳性重组菌共培养7 d后,头孢噻呋对重组菌的MIC下降8~16倍,重组菌中bla_(CTX-M)的mRNA相对表达量均极其显著下降(P<0.001);不同剂量黄芩水煎剂与bla_(CTX-M)阳性重组菌共培养2 h后,大肠杆菌C600和重组菌C600-1、C600-9的内、外膜通透性均极显著增加(P<0.01或P<0.001),外排泵活性均极其显著下降(P<0.001),其中在1/2 MIC和1 MIC黄芩水煎剂的共培养下,主要膜孔蛋白相关基因ompF、ompC和ompR的mRNA相对表达量均极其显著升高(P<0.001),主要外排泵相关基因arcA、acrB和tolC的mRNA相对表达量均极其显著降低(P<0.001)。结果表明,黄芩水煎剂与bla_(CTX-M)阳性重组菌共培养后,不仅能抑制重组菌的bla_(CTX-M)表达,还能明显增加细胞膜通透性并降低外排泵活性。本试验结果为我国临床从中药中挖掘超广谱β-内酰胺酶抑制剂提供了新思路。
To investigate whether Scutellaria baicalensis decoction can enhance the antibacterial activity of ceftiofur against Escherichia coli harboring the extended-spectrumβ-lactamase(ESBL)gene blactx-,recombinant strains C600-1 and C600-9carrying bla_(CTX-M-55) and bla_(CTX-M-14),respectively,were constructed.Time-kill curves were plotted to evaluate the in vitro bactericidal activity of ceftiofur at 1 MIC alone or in combination with S.baicalensis decoction at 1 MIC.The recombinant strains were serially passaged for 7 days in media containing 1/4 MIC or 1/2 MIC of the decoction,while the group without the decoction served as the negative control,and ceftiofur MIC and blacrx-m expression levels were determined to assess the effect of S.baicalensis decoction on resistance development.Additionally,the wild-type(C600)and recombinant strains(C600-1,and C600-9)were treated for 2 h with 1/4 MIC,1/2 MIC,or 1 MIC of the decoction,while the group without the decoction served as the negative control.Changes in cell membrane permeability and efflux pump activity were examined by reverse transcription real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and fluorescent staining.The results showed that compared to ceftiofur alone,the combination with S.baicalensis decoction significantly enhanced the bactericidal activity against all three E.coli strains.After 7 days of co-culture with the decoction,ceftiofur MICs decreased by 8 to 16-fold,and blacrx-m mRNA levels were significantly reduced(P<O.O01).After 2 hours of co-culture with the decoction,both inner and outer membrane permeability of all three E.coli strains significantly increased(P<0.01 or P<0.001),while efflux pump activity was markedly reduced(P<0.001).Notably,co-treatment with 1/2 MIC or 1 MIC decoction significantly upregulated the mRNA expression of major porin-related genes(ompC,ompF,ompR)and significantly downregulated efflux-related genes(acrA,tolC,acrB)(all P<O.O01).These findings suggest that S.baicalensis decoction not only suppresses blacrx-m gene expression but also enhances membrane permeability and decreases efflux pump activity in recombinant strains.This study provides a novel strategy for identifying broad-spectrumβ-lactamase inhibitors from traditional Chinese medicine for clinical use in China.
作者
陈秋如
许慧
郝振鑫
张浩则
张超君
邢少川
马小元
胡功政
苑丽
CHEN Qiuru;XU Hui;HAO Zhenxin;ZHANG Haoze;ZHANG Chaojun;XING Shaochuan;MA Xiaoyuan;HU Gongzheng;YUAN Li(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)
出处
《中国兽医杂志》
北大核心
2025年第11期102-111,共10页
Chinese Journal of Veterinary Medicine
基金
河南省高校科技创新团队支持计划项目(23IRSTHN021)
河南省自然科学基金重点项目(232300421111)。
作者简介
共同第一作者:陈秋如(2000-),女,硕士生,研究方向为基础兽医学,E-mail:2416731992@qq.com;共同第一作者:许慧(1997-),女,硕士生,研究方向为基础兽医学,E-mail:610013943@qq.com;通信作者:苑丽,E-mail:yuanli-hn@163.com。
