摘要
【目的】研究体外诱导的大肠埃希菌耐药突变株对替加环素的交叉耐药机制。【方法】采用防耐药突变浓度(mutant prevention concentrations,MPC)诱导方法,使用盐酸多西环素对大肠埃希菌ATCC 25922进行分步耐药突变诱导,并对诱导所得耐药突变株进行耐药谱测定;通过全基因组二代测序分析ATCC 25922及最高倍耐药突变株的关键差异耐药基因突变情况,并根据全基因测序结果使用RT-PCR技术对最高倍耐药突变株的关键差异耐药基因进行转录量测定;采用siRNA技术分别干扰关键差异耐药基因在最高倍耐药突变株中的表达。【结果】经分步耐药突变诱导获得3株不同程度对替加环素耐药的大肠埃希菌耐药突变株,分别为Y_(3.2-2)、Y_(64)和Y_(128-2),且耐药程度依次为Y_(3.2-2)<Y_(64)<Y_(128-2),其耐药谱均表现出多重耐药性;共检出14种耐药基因,均出现不同程度的碱基突变和氨基酸突变;在高倍耐药突变株Y_(128-2)中,acrA、acrE、acrF、acrS、plsC、rpsJ、acrB和macA基因上调,而tolC、marA、sdiA和macB基因下调;在1×MIC和1/2×MIC替加环素浓度下成功干扰Y_(128-2)的rpsJ和plsC耐药基因表达,菌株恢复对替加环素的敏感性。【结论】体外诱导所得的高倍耐药突变株Y_(128-2)对替加环素产生耐药性的主要作用机制是,核糖体结合位点rpsJ和细菌细胞膜通透性相关耐药基因plsC过量表达。
[Objective]To investigate the mechanism of the induced cross resistance of drugresistant mutants of Escherichia coli to tigecycline in vitro.[Methods]We used doxycycline hydrochloride and the mutation preventive concentration(MPC)method to induce the drug resistance mutation of Escherichia coli ATCC 25922,and the drug resistance spectra of the mutants were determined.Genome-wide next-generation sequencing was utilized to analyze the mutations of key differentially expressed resistance genes of ATCC 25922 and the mutant with the highest resistance index.RT-PCR was used to determine the transcription levels of the key differentially expressed resistance genes of the mutant with the highest resistance index according to the whole genome sequencing results.The expression of key differentially expressed resistance genes in the mutant with the highest resistance index was knocked down by siRNA.[Results]Three drugresistant E.coli mutants Y_(3.2-2),Y_(64),and Y_(128-2) with different degrees of resistance to tigecycline were obtained after stepwise induction of drug resistance mutation,with the resistance following the order of Y_(3.2-2)<Y_(64)<Y_(128-2).All the mutants showed multi-drug resistance.Fourteen resistance genes were detected with varying degrees of base mutations and amino acid mutations.In the mutant Y_(128-2) with the highest resistance index,the expression of acrA,acrE,acrF,acrS,plsC,rpsJ,acrB,and macA was up-regulated,while that of tolC,marA,sdiA,and macB was downregulated.The resistance genes rpsJ and plsC in Y_(128-2) were successfully interfered with at tigecycline concentrations of 1×MIC and 1/2×MIC,and the strain regained sensitivity to tigecycline.[Conclusion]Y_(128-2) develops resistance to tigecycline by the overexpression of the ribosome binding site gene rpsJ and the bacterial cell membrane permeability-related resistance gene plsC.
作者
黄歆如
郭刘玲
吴俊伟
唐鑫
邓开锋
HUANG Xinru;GUO Liuling;WU Junwei;TANG Xin;DENG Kaifeng(College of Veterinary Medicine,Southwest University,Chongqing,China;Chongqing Bull Animal Pharmaceutical Co.,Ltd.,Chongqing,China)
出处
《微生物学报》
北大核心
2025年第8期3524-3539,共16页
Acta Microbiologica Sinica
关键词
替加环素
大肠埃希菌
耐药机制
干扰
tigecycline
Escherichia coli
drug resistance mechanism
interference
