盐酸多西环素诱导大肠埃希菌及其对替加环素体外交叉耐药性及相关耐药基因分析

Induction of cross resistance of Escherichia coli to tigecycline by doxycycline hydrochloride and identification of resistance genes

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、Y₆₄和Y_128-2，且耐药程度依次为Y_3.2-2<Y₆₄<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。

Abstract:

Objective To investigate the mechanism of the induced cross resistance of drug-resistant 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 drug-resistant E. coli mutants Y_3.2-2, Y₆₄, 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<Y₆₄<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 down-regulated. 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.