多重耐药杂合型IncHI2质粒特征及抗生素压力对其形成的影响研究

Widespread antibiotic resistance poses an enormous threat for human and animal health, and this problem has been exacerbated with the emergence of multidrug resistance plasmids conferring resistance to most classes of antimicrobials. Recently, multi-resistance hybrid plasmids with resulting from IncHI2 plasmids cointegrated with other plasmids has increasingly emerged in Enterobacteriaceae. Up to now, studies mainly focused on characterization of the complete sequences of these hybrid plasmids, but few paid attention to the factors causing the formation of these hybrid plasmids, and their persistence and dissemination in Enterobacteriaceae. In our previous study, multi-resistance hybrid IncHI2 plasmids were found, thus, on the basis of these investigations, the main proposal in this study is aimed to further screen for positive plasmids from Enterobacteriaceae of animals origins, and by combining molecular biology techniques, high-throughput sequencing, comparative genomics and bioinformatics analysis, to study the traits and complete sequences of these hybrid plasmids, to clarify their multi-drug resistance, genetic stability, the bacterial host range and fitness burden conferred to new host strains; to explore the modes involved in formation of hybrid plasmids; to evaluate the effect of antibiotic selective pressure on the cointegration between different plasmids co-resident within the same cell; at last, to elucidate the factors causing the formation of the multi-resistance hybrid IncHI2 plasmids and determine their maintenance and dissemination in Enterobacteriaceae. This research would provide a scientific guideline on slowing down the development of resistance in multiresistance Enterobacteriaceae isolates and exploiting a novel strategy for prevention and control of antibiotic resistance.

抗生素耐药性的广泛传播对人和动物健康构成了巨大威胁，而介导对多种抗菌药物耐药的多重耐药质粒的出现，使得这一问题更加严重。近来，多重耐药杂合型IncHI2质粒在肠杆菌中不断出现，相关研究主要集中于质粒全序列特征分析，而很少关注影响其杂合形成的因素及其是否能够在肠杆菌中稳定存在、广泛传播。申请人在前期研究中，已检出多重耐药杂合型IncHI2质粒，因此本研究拟进一步在动物源肠杆菌中筛选阳性质粒，通过分子生物学技术、高通量测序、比较基因组学以及生物信息学分析，研究杂合质粒的特性及全序列特征，旨在探明其多重耐药性、遗传稳定性、宿主细菌范围及适应性代价，明确杂合质粒的形成模式，评估抗生素压力对同一宿主细菌内不同质粒间发生杂合的影响，从而阐明影响多重耐药杂合型IncHI2质粒的形成因素及其在肠杆菌中的维持与传播能力，为减缓多重耐药肠杆菌耐药性的发展及探索新的耐药控制策略提供科学指导。

抗生素耐药性的广泛传播对人和动物健康构成了巨大威胁，而介导对多种抗菌药物耐药的多重耐药质粒的出现，使得这一问题更加严重。本项目围绕多重耐药IncHI2型杂合质粒的序列特征分析、质粒的生物学特性以及抗生素压力对同一宿主细菌内不同质粒间发生杂合的影响开展研究。明确了大肠杆菌、沙门菌和肺炎克雷伯菌中IncHI2型杂合质粒的流行分布特征，并筛选出51个IncHI2型杂合质粒，其携带多个耐药基因包括一些临床重要耐药基因，甚至还携带重金属抗性基因、毒力因子等。明确了IncHI2型杂合质粒的序列结构特征并发现了6种杂合方式，其中以IncHI2/N型为主。发现了杂合质粒的IncHI2型质粒骨架区大部分都比较完整，但来源于沙门菌的接合转移区I丢失现象明显，杂合质粒的其他复制子型质粒区域的整合位点多发生在多重耐药区。发现了整合区域的大小不等，包括接合转移相关基因、质粒维持相关基因、耐药基因、毒力因子等，且整合区域的两端常常存在插入序列，主要为IS26，提示插入元件可能介导IncHI2型质粒与其它不同复制子型质粒之间发生杂合。明确了IncHI2型杂合质粒的宿主范围，其可在多种肠杆菌科细菌中发生接合转移，尤其是大肠杆菌和沙门菌，但接合频率相对较低。发现了携带IncHI2型杂合质粒降低了沙门菌和弗氏柠檬酸杆菌的竞争能力以及大肠杆菌的生长能力。明确了IncHI2/N杂合质粒中N型杂合区域的整合有利于IncHI2型杂合质粒的生长和竞争能力。通过体外实验证实了抗生素压力选择促进了IncHI2型质粒与IncFII质粒之间发生杂合。研究结果阐明了多重耐药杂合型IncHI2质粒的形成因素及其在肠杆菌中的维持与传播能力，为减缓多重耐药肠杆菌耐药性的发展及探索新的耐药控制策略提供科学指导。