青蒿琥酯联合左氧氟沙星对耐碳青霉烯类大肠埃希菌抗菌活性的影响

Effect of artesunate combined with levofloxacin on antibacterial activity of carbapenem-resistant Escherichia coli

目的 探讨青蒿琥酯与抗生素联用对耐碳青霉烯类大肠埃希菌(CRECO)抗菌活性的影响。方法 收集分离的CRECO 20株,采用肉汤稀释法测定抗菌物质青蒿琥酯(ASN)、左氧氟沙星(LEV)、头孢他啶(CAZ)、亚胺培南(IMP)最小抑菌浓度(MIC)。用棋盘法测定ASN与LEV、CAZ、IMP协同抗菌活性;用细菌生长曲线法观察不同处理组CRECO临床菌株(E5)生长的情况;用RT-PCR检测外排泵基因以及调控基因的表达。结果 抗菌物质ASN、LEV、CAZ、IMP的MIC50分别为>8 192、64、2 048、32μg/mL。协同抗菌试验显示:ASN (1 024μg/mL)与LEV联用时,LEV的MIC50由64μg/mL降至16μg/mL (即1/4 MIC50),联合作用效果以协同为主;与CAZ联用时,CAZ的MIC50由2 048μg/mL降至1 024μg/mL (即1/2 MIC50),联合作用效果以相加为主。生长曲线显示,ASN联合LEV能有效抑制CRECO生长,曲线走向平缓。20株菌株全部携带外排泵基因AcrA和AcrB基因。RT-PCR结果显示ASN联合LEV可降低外排泵基因AcrB表达。结论 ASN对CRECO无抗菌活性,但ASN联合LEV能有效增强抗生素的抑菌作用,其协同抑菌机制与降低外排泵基因AcrB表达相关。

Objective To investigate the effect of artesunate combined with levofloxacin on the antibacterial activity of carbapenem-resistant Escherichia coli(CRECO).Methods 20 strains of CRECO isolated from the laboratory were collected and the minimum inhibitory concentrations(MIC)of artesunate,levofloxacin,ceftazidime and imipenem were determined by broth microdilution methods.The synergistic antibacterial activity of artesunate with levofloxacin,ceftazidime and imipenem was determined by broth microdilution checkerboard methods.The effects of different treatment groups on the growth of clinical strain(E5)of CRECO were observed by bacterial growth curve method.The expression of efflux pump gene and regulatory gene was detected by RT-PCR.Results The MIC50 of artesunate,levofloxacin,ceftazidimeandimipenem were>8192,64,2048 and 32μg/mL,respectively.Synergistic antibacterial test showed that when artesunate was combined with levofloxacin at 1024μg/mL,the MIC50 decreased from 64μg/mL to 16μg/mL(i.e.,1/4 MIC50),and the combined effect was mainly synergistic.When combined with ceftazidime,its MIC50 decreased from 2048μg/mL to 1024μg/mL(i.e.,1/2 MIC50),and the combined effect was mainly additive.The results of the growth curve showed that artesunate combined with levofloxacin could effectively inhibit the growth of CRECO,and the curve tended to be flat.All 20 strains carried the efflux pump genes,AcrA and AcrB genes.RT-PCR results showed that artesunate combined with levofloxacin could reduce the expression of efflux pump gene AcrB.The decrease of its expression level was associated with the decreased expression of the regulatory gene AcrB.Conclusion Artesunate has no antibacterial activity against CRECO,but artesunate combined with levofloxacin could effectively enhance the antibacterial action of antibiotics,and its synergistic inhibitory mechanism may be related to the reduction of the expression of efflux pump gene AcrB.