亚抑菌浓度氟苯尼考作用下携带cfr基因质粒对大肠杆菌竞争力的影响及相关机制研究

Conferring resistance to five chemically unrelated antimicrobial classes, the cfr gene has received international and domestic attentions on its spread and diffusion in recent years. Previous studies have focused on the transfer of cfr gene mediated by transferable elements such as plasmid and insertion sequence, and there is still no report on the effect of cfr-carrying plasmid on the bacterial competitiveness. In our previous study, we have obtained the complete sequence of three cfr-carrying plasmids from Escherichia coli of animal origin. Based on the preliminary achievement, we will research the effect of cfr-carrying plasmid on the competitiveness of E. coli by detecting the minimal selective concentration (MSC) of florfenicol with the bacterial competitiveness evaluation model constructed with flow cytometry and E. coli K12 labeled with fluorescent protein, analyze the relationship of bacterial competitiveness with transcription and protein expression of cfr gene and plasmid copy number according to the MSC value, and finally explore the underlying mechanism by transcriptome sequencing analysis. The results of the studies will further elucidate the transmission mechanism of cfr-carrying plasmid based on bacterial competitiveness, and thus provide the foundation for risk assessment and control of the residue of veterinary drug and bacterial drug resistance.

多重耐药基因cfr做为目前发现的第一种可同时介导对五类抗菌药物耐药的可转移基因，它的传播与扩散已成为国内外研究的热点。目前对cfr基因的研究多集中于质粒、插入序列等可移动元件介导cfr基因及其耐药性的传播，还未见针对cfr基因及其质粒影响细菌竞争力导致耐药性扩散的报道。在前期工作中，我们已从动物源大肠杆菌中获得了3个携带cfr基因质粒及其全序列。本项目拟在已有研究基础上，通过Red同源重组技术构建标记荧光蛋白的大肠杆菌K12突变株，并结合流氏细胞术构建细菌竞争力评价模型；通过细菌竞争力评价模型测定氟苯尼考对携带质粒菌株的最小筛选浓度(MSC)，分析亚抑制浓度氟苯尼考作用下携带cfr基因质粒对细菌竞争力的影响；比较不同质粒对MSC的影响，分析质粒拷贝数、cfr基因转录表达量与大肠杆菌竞争力的关系；并采用转录组测序分析阐明携带cfr基因质粒影响大肠杆菌竞争力的相关机制。研究结果将从细菌竞争力的角度深入阐明cfr基因及其质粒扩散与传播机制，为兽药残留和细菌耐药性的风险评估和控制提供依据。

多重耐药基因cfr是发现的第一种可同时介导对五类抗菌药物耐药的可转移基因，目前对cfr基因传播的研究多集中于质粒、插入序列等可移动元件介导cfr基因及其耐药性的传播，而鲜有针对cfr基因及其质粒影响细菌竞争力导致耐药性扩散的报道。从细菌竞争力的角度深入阐明cfr基因及其质粒扩散与传播机制，将为兽药残留和细菌耐药性的风险评估和控制提供依据。.本项目计划的研究内容分3个部分，包括细菌竞争力评价模型的建立、亚抑菌浓度氟苯尼考作用下携带cfr基因质粒对大肠杆菌竞争力的影响、携带cfr基因质粒影响大肠杆菌竞争力的相关机制研究，现已全部完成。.本研究以绿色荧光蛋白标记菌株E. coli MG1655-sfGFP与同源菌株E. coli MG1655为竞争对，通过流式细胞术成功构建细菌竞争评价模型。以该模型为基础，测得氟苯尼考对携带cfr阳性质粒pSD5耐药菌的最小筛选浓度仅为相应无质粒敏感菌MIC的1/43。通过比较分析生长速率推测氟苯尼考对携带质粒pSD11耐药菌的最小筛选浓度仅为相应无质粒敏感菌MIC的1/100。这表明极低浓度的氟苯尼考对携带cfr阳性质粒pSD5或pSD11的宿主大肠杆菌可产生选择优势。转录组分析结果表明携带质粒pSD5的耐药菌株主要通过硫代谢途径和磷酸糖转移酶系统等途径来增强其竞争力。研究结果阐明了亚抑菌浓度氟苯尼考作用下，携带cfr基因质粒对宿主大肠杆菌竞争力的影响及其相关机制，为携带cfr基因质粒在环境中的传播风险评估及氟苯尼考的临床应用提供科学依据。