基于质粒介导的禽源弯曲菌多重耐药传播的分子机制研究

Campylobacter is one of the most common foodborne pathogens worldwide, and is a leading cause of diarrhea illness and acute gastroenteritis in humans. Poultry are recognized as a source and reservoir of Campylobacter. Along with the antimicrobial use in the food animals, multidrug resistant Campylobacter isolated from poultry emerge constantly, and can be transmitted from poultry to humans through the food chain. It may severely threaten food safety and human public health. Plasmid is considered as the efficient mobile genetic elements and can mediate multidrug resistant gene, which contributed to the rapid dissemination of these resistance genes and has become a serious clinical problem. Based on the investigation of the current situation of antimicrobial resistance of Campylobacter in poultry from partial areas of China, the aims of this project are I. to investigate the distribution of antibiotic-resistant plasmids types in Campylobacter and plasmid-mediated resistant genes by application of molecular biology and genome sequencing, plasmid elimination; II. to reveal the multidrug resistance transmission mechanisms were confirmed by using various methods including plasmid conjugation, plasmid profiling, the S1-PFGE ( PFGE with S1 nuclease digestion), southern hybridization and plasmid fitness cost. This project will be helpful to obtain the molecular epidemiology of resistance Campylobacter and its evolutional trend. It can clarify the transmission mechanism of plasmid-mediated the multidrug resistance and provide a reliable evidence for reducing and controlling the spreading of multidrug resistant Campylobacter.

弯曲菌(Campylobacter)是重要的食源性致病菌，能够引起人的腹泻性肠炎。家禽是人弯曲菌病最重要贮存宿主和传染源，随着抗生素在畜牧生产上的大量使用，多重耐药弯曲菌不断出现，并有可能通过食物链传递给人类，给食品安全和人类健康构成潜在威胁。基因水平转移重要元件-质粒可携带多重耐药基因，且易于传播扩散，临床危害极大。本项目前期已开展我国部分地区禽源弯曲菌耐药性调查，在此基础上，以耐药质粒为切入点，应用分子生物学、基因组测序、质粒消除等相关技术，系统分析弯曲菌质粒类型及其介导的多重耐药基因；通过质粒接合转移、质粒酶切图谱、S1-PFGE、核酸杂交和质粒适应性等技术揭示质粒介导弯曲菌多重耐药基因传播的分子机制。通过本项目研究，探明我国部分地区禽源弯曲菌多重耐药流行现状和趋势，阐明质粒在弯曲菌多重耐药中所起作用及传播机制，为降低和控制耐药弯曲菌的产生和扩散提供可靠的理论依据。

弯曲菌是一种重要的食源性病原菌。多重耐药弯曲菌的出现和传播，给公共卫生安全带来了严重危险。本项目以弯曲菌为研究对象，通过分离规模化养殖场、市场以及屠宰场的弯曲菌，借助耐药性检测、分子分型、全基因组序列分析等手段，探明禽源弯曲菌的流行分布和耐药性演变规律，从而解析弯曲菌的多重耐药机制。结果表明：（1）分离鉴定286株弯曲菌，其中空肠弯曲菌204株，结肠弯曲菌82株，空肠弯曲菌仍然是禽源弯曲菌中的优势菌种。弯曲菌分离株的药敏结果显示多重耐药现象严重，MLST分析结果显示ST-464和ST-828克隆复合体为优势克隆复合体，遗传进化分析表明菌株之间具有丰富的遗传多样性；（2）采用多重PCR和全基因组测序技术均未能检测到质粒介导的耐药，55株弯曲菌分离株共筛选出6大类14种耐药基因，其中庆大霉素、卡那霉素、链霉素和四环素的耐药基因与耐药表型具有相关性；（3）弯曲菌Ⅰ型整合子的流行率为18.6%，部分携带有完整的Ⅰ型整合子结构，经SDS化学处理消除整合子耐药基因盒可降低菌株的耐药率；（4）研究发现氨基糖苷类耐药基因在分离菌中普遍存在，本项目以弯曲菌中携带率高的aph(2")-If为研究对象，发现aph(2")-If基因能通过自然转化的方式进行水平传播，对其转化子测序发现了一个包括aph(2")-If和ISCco2 family transposase的基因岛，转座子介导了aph(2")-If的传播；（5）探究了酰胺醇类/恶唑烷酮类耐药基因optrA和cfrC在弯曲菌中的流行情况，率先在鸡源空肠弯曲菌中检测到optrA耐药基因的存在，对含有optrA耐药基因的菌株进行遗传进化分析表明其在弯曲菌中存在克隆传播。本项目研究结果有助于准确掌握我国动物源弯曲菌的流行、耐药特征及耐药机制，为开展动物源弯曲菌的有效防控提供理论依据。