食源性大肠杆菌中可移动遗传元件（MGEs）介导的消毒剂与抗生素共同耐药及传播机制研究

Our previous study showed that disinfectant resistance in foodborne Escherichia coli has become serious in recent years. Most importantly, we found that the disinfectant resistance genes and antibiotic resistance genes frequently co-existed in mobile genetic elments (MGEs) in E. coli leading to the co-resistance to disinfectants and antibiotics, which brought new challenge for preventing pathogens and controlling resistance. However, the mechanism of co-resistance to disinfectant and antibiotic and its transmission was not understood. Therefore, further work is needed. In our study, above all, the prevalence of disinfectant and antibiotic resistance in foodborne E. coli will be investigated thoroughly in order to find out the regularity of epidemic. After gotten some sequences of MGEs mediated co-resistance to disinfectant and antibiotic previously, more sequences of MGEs will be obtained and species of MGEs which mediated disinfectant and antibiotic resistance should be detected. Meanwhile, the structures of MGEs will be analyzed and figured out by bioinfomatic softwares. Besides, gene knock out, RT-PCR and other technologies will be used to find out conseved segment's effect on co-resistance. By analyzing the species of MGEs, gene strutures of regulatory elements and measuring the gene expression level, the mechanism of different MGEs mediated high co-resistance level and regularity of disinfectant and antibiotic resistance selecting each other would be discussed. Finally, conjugation, gene knock out, serial passage and fitness cost will be carried out to find out the ability of co-resistance's transmission, condition of transmission and stability of inheritance. Thus, through our study we can explore the mechanism of co-resistance to disinfectant and antibiotic and its transmission in foodborne E. coli, providing new idea and scientific basis for the prevention of foodborne pathogens and antimicrobial resistance.

前期研究表明食源性大肠杆菌对消毒剂的耐药性问题日益突出，更重要的是，消毒剂耐药基因与抗生素耐药基因常共存于可移动遗传元件(MGEs)，使菌株呈现出对消毒剂和抗生素共同耐药的特点，然而，目前共同耐药及传播机制尚不清楚。鉴于此，本项目拟继续开展食源性大肠杆菌对消毒剂和抗生素耐药性调查，获取介导共同耐药的MGEs序列，探明耐药性流行规律、MGEs多样性及结构特征；应用基因敲除、RT-PCR等技术研究MGEs保守端序列对耐药性的影响；分析MGEs种类、调控元件等基因结构，测定基因表达水平，研究共同耐药菌株对消毒剂和抗生素高水平耐药机理及耐药性相互选择规律；通过接合试验、基因敲除、连续传代及适应性代价测定等，阐明共同耐药的传播能力、传播条件以及遗传稳定性等科学问题。通过以上研究，探讨食源性大肠杆菌中MGEs介导的消毒剂与抗生素共同耐药及传播机制，可为食源性病原菌及耐药性的防控提供新的思路和科学依据。

大肠杆菌（Escherichia coli）是食品污染中常见的细菌之一，在食品生产、加工、运输、零售过程中可能会发生大肠杆菌污染，对食品安全产生潜在的威胁。与此同时，抗生素、消毒剂的大量使用，造成食源性大肠杆菌对抗生素、消毒剂的耐药性日益严重。研究表明，消毒剂耐药基因与抗生素耐药基因常共存于可移动遗传元件，使菌株呈现出对消毒剂和抗生素共同耐药的特点，然而，大肠杆菌对抗生素、消毒剂的共同耐药及传播机制尚不清楚。. 本研究以食源性大肠杆菌为研究对象，对大肠杆菌的消毒剂和抗生素耐药性调查，发现大肠杆菌对苯扎氯铵（BC）的最小抑菌浓度在8-128μg/mL，对氯化十六烷基吡啶（CPC）的MIC在4-256 μg/mL；大肠杆菌对抗生素的耐药率为6.83%-90.92%。大肠杆菌的中qacEΔ1消毒剂耐药基因检出率为45.45%，sugE(p)和qacF的检出率为28.13%、11.36%。对分离得到的大肠杆菌进行PFGE电泳分型。通过BioNumerics软件对图像进行分析，发现大肠杆菌在养殖、食品中存在交叉污染的现象。构建了消毒剂耐药基因（qacEΔ1、sugE(p)）荧光表达载体及工程菌，从可视化角度观察qacEΔ1、sugE(p)基因介导对消毒剂的耐药。构建了重组qacEΔ1、sugE(p)的pET30b(+)表达载体及重组工程菌株，证实 qacEΔ1和sugE(p)基因可介导大肠对BC的耐药。通过二代测序完成25株大肠杆菌的测序，利用三代测序，完成共同耐药大肠杆菌的全基因组测序，发现大肠杆菌的质粒上存在着重金属（chrA）、消毒剂（qacEΔ1）、抗生素（blaCTX-M-55、oqxA、oqxB）耐药基因，可能使大肠杆菌同时产生对重金属、消毒剂、抗生素耐药性。耐药基因两侧存在大量的插入序列（IS26），表明其耐药基因可能存在转移的潜力。阐明大肠杆菌1整合子检出率和携带的基因盒种类，主要以携带3’端qacEΔ1-sul1，基因盒以dfrA17-aadA5，dfrA1-aadA1为主。接合试验说明供qacEΔ1耐药基因能在细菌间进行水平传播。对共同耐药菌株诱导，发现大肠杆菌对抗生素和消毒剂耐药性之间存在相互选择。本研究对防控消毒剂和抗生素耐药性与传播有着重要作用，可为消毒剂及抗生素的规范、合理使用提供科学依据。