IncA/C质粒介导食源性副溶血弧菌ESBLs基因获得及进化的分子机制研究

Vibrio parahaemolyticus is one of the major organisms affecting the quality and safety of aquatic raw materials and it is also a major foodborne pathogen. Due to the emergence of the plasmid mediated extended spectrum β-lactamases (ESBLs)-producing isolates, the multidrug resistant V. parahaemolyticus has become a serious threat to public healthy and food safety. The IncA/C-type plasmids are known to strongly associate with the dissemination of ESBLs genes and aquatic environment. In the present project, through molecular and bioinformatic studies, we aim to elucidate the molecular mechanism of acquisition and evolution of ESBLs genes mediated by IncA/C-type plasmids from the foodborne V. parahaemolyticus. From our previous studies, we have identified more than thirty strains carrying those plasmids from a collection of over three hundred isolates of foodborne resistant V. parahaemolyticus. In the present work, the plasmid-mediated resistance will be further identified through screening the resistance genes by PCR, conjugal transferring or electro-transformation of the plasmids into Escherichia coli, and testing their antimicrobial drug susceptibility profiles. Then the complete nucleotide sequences of three to eight IncA/C plasmids will be determined by high throughput sequencing. The genetic structures of the new resistance plasmids, resistance gene clusters and mobile elements will also be characterized in details. The promoter structure, activity of key resistance genes, and the enzymatic activity, and the protein crystal structure of the key resistance determinants will be further investigated. Through the phenotypic, molecular and bioinformatic data of two to three new types of resistance plasmids and three to five mobile elements, we are anticipating to gain deeper knowledge about the molecular mechanism of IncA/C-type plasmid-mediated ESBLs genes acquisition and evolution. This study should be able to improve the quality and safety of aquatic raw materials, provide further theoretical understandings, and benefit human health and food safety.

副溶血弧菌是影响水产原料品质与安全的重要微生物，也是引起食源性疾病的重要病原菌。近几年来，IncA/C质粒携带超广谱β-内酰胺酶（ESBLs）基因介导的耐药副溶血弧菌严重威胁人们的健康与食品安全。本项目拟通过分子生物学和生物信息学等手段揭示IncA/C质粒介导ESBLs基因在副溶血弧菌中获得及进化的分子机制。课题组对已分离的300余株副溶血弧菌耐药株进行耐药基因筛查、质粒接合转移、耐药谱分析等获得30余株候选菌株。本研究拟从这些菌株中选出3-8个IncA/C质粒进行高通量测序，通过对耐药质粒、耐药基因座位、移动元件的遗传结构分析，结合耐药表型、遗传背景和酶学特征，最终得到2-3个目标耐药质粒，再对其携带的3-5个移动元件进行精细生物信息学分析，阐明IncA/C质粒介导ESBLs基因获得及进化的分子机制。本研究可为提高水产原料品质与安全提供科学参考，对保障民众健康和食品安全具有重要理论意义。

副溶血弧菌等食源性致病性细菌是影响水产原料品质与安全的重要微生物，也是引起食源性疾病的重要病原菌，尤其是质粒携带超广谱β-内酰胺酶（ESBLs）基因介导的耐药菌严重威胁人们的健康与食品安全。本研究主要完成了以下工作：（1）调查了辽宁省大连、丹东、葫芦岛、锦州、营口五座沿海城市的部分海产品受耐药菌污染情况。（2）对分离的所有菌株进行了耐药谱、耐药基因分析；（3）对耐药谱较广、耐药基因种类较多的部分菌株进行了高通量测序，并进行了深入的分子生物信息学分析，阐明质粒在介导细菌ESBLs基因获得及进化的分子机制研究；（4）以研究中分离的多株耐药菌为宿主菌分离噬菌体，开展了噬菌体清除效果评价与绿色环保生物清除剂研发方面的研究。研究结果表明，在辽宁省沿海五市采集的样品受耐药菌污染的阳性率为67.9%，分离的菌株中耐药菌的比例达70%以上。样品的耐药菌污染率与季节显著相关，不同城市采集的样品污染率不尽相同，不同样品种类受耐药菌污染情况也存在差异。9类12种常用抗菌药物敏感性分析表明，菌株对头孢唑啉和复方新诺明耐药性最高，对多粘菌素耐药性最低。部分菌株携带有多达12种耐药基因，且耐药基因在移动元件的作用下可随质粒的接合转移在不同菌株中进行水平传播，导致耐药基因的播散，使部分菌株多重耐药现象更为严重。作为有前景的抗菌药物替代品、控制水产品中致病性细菌的绿色环保的生物清除剂，可开发噬菌体制剂，控制、清除水产品中的致病性细菌及其耐药菌。本项目研究的细菌获得耐药的机制及研究清除耐药菌的方法可以为提高水产原料品质与安全提供科学参考，对保障民众健康和食品安全具有重要理论意义和实际应用价值。