碳青霉烯耐药基因的溯源及传播机制研究

In recent years, with the increasing frequencies of multi-drug-resistant bacteria, especially Carbapenem-Resistant Enterobacteriaceae(CRE), the antibiotic-resistant bacteria has posed the greatest risk to global health. Recently, CRE is ranked in priority 1: urgent threat according to global priority list of antibiotic- resistant bacteria published by WHO. Based on the fact that the carbapenem resistance gene has been detected in the bacteria of animals and environmental origin, it is necessary to study its origin and transmission at the human-animals-environment interface. In this proposal, a large number of samples from human, animals and environment in the farms located at key region for aquaculture in China were collected and investigated by multidisciplinary methods such as metagenomics, bioinformatics and molecular epidemiology. Furthermore, the resistome, the transmission mechanism of resistance gene, and the relationship of antibiotic with pig’s gut microbiome were also carried out. The exploration the epidemiological characteristics, possible sources and mechanisms of carbapenem resistance genes and the clarification of basic rules on its epidemic and spread quickly, as well as the association of the use of β-lactam antibiotics and its prevalence in veterinary medicine, will provide a scientific evidence for the antibiotic usage in veterinary medicine and supply new control strategies and approaches for restricting the spread of high-risk resistant bacteria and resistance genes and slowing down the development of resistance.

近年来，细菌耐药性问题十分突出，多重耐药菌尤其是产碳青霉烯酶革兰氏阴性菌的不断出现，已经成为威胁人类健康和公共卫生的重大隐患，WHO已将耐碳青霉烯类肠杆菌科列为优先1级（危急）的重要耐药菌。基于当前已经在动物和环境源细菌中检出碳青霉烯耐药基因的存在，有必要对其来源及在不同界面的传播规律开展深入研究。本项目拟综合运用宏基因组学、生物信息学和分子流行病学等多学科方法，采集我国重点养殖地区养殖场的人-动物-环境多个界面中的大量样品，从耐药基因组、耐药基因传播机制、抗菌药-肠道菌群-耐药菌之间的相互作用关系，深入探究碳青霉烯耐药基因流行特征、可能来源及传播机制，明确具有重大风险的碳青霉烯耐药基因是否在我国养殖场流行扩散？解析兽医临床使用抗生素与碳青霉烯耐药基因流行的关联，从而为制定抗生素使用策略提供科学依据，也为控制重要耐药基因以及耐药菌的扩散传播和减缓耐药性发展探索新的控制策略和途径。

近年来，多重耐药菌尤其是产碳青霉烯酶革兰氏阴性菌的不断出现，已经成为威胁公共卫生的重大隐患。本项目围绕开发产碳青霉烯酶菌株的检测方法，探究水禽、候鸟、宠物源重要碳青霉烯耐药基因的分子传播特征，挖掘水禽及野生动物及其生活环境中新型β-内酰胺类耐药基因及碳青霉烯耐药基因的传播载体，以及探究碳青霉烯类耐药基因/耐药菌定植和扩散的影响因素等方面开展研究。建立了CIMB.S和运用AI模型的快速表型检测产碳青霉烯酶菌株的方法，在鸭场、鹅场、候鸟栖息地、宠物诊所等不同生态位的动物-环境-人中碳青霉烯耐药基因blaNDM阳性肠杆菌科细菌的污染情况以及传播特征，发现了来源于候鸟的产碳青霉稀酶的新型肺炎克雷伯菌亚种，明确了鸭源、鹅源blaNDM阳性大肠杆菌与人源菌株具有密切的亲缘关系，以及宠物和医护人员之间可能存在着blaNDM阳性大肠杆菌的克隆传播；发现了多个新型blaNDM阳性质粒，阐明了blaNDM主要以IncX3质粒介导的水平传播在不同生态位动物-环境-人之间扩散。证实了水禽养殖环境和野生动物栖息地是β-内酰胺类基因的重要储库，发现了9个具有水解β-内酰胺类抗生素的新型β-内酰胺酶，并一定程度上解析了其进化和起源；在菌群水平揭示了鹅场小生境中重要碳青霉烯耐药基因blaNDM的分布特征及传播的载体，并证实了大肠杆菌是其主要的宿主菌。明确了亚抑菌浓度氨苄西林和阿莫西林/克拉维酸一定程度上可促进blaNDM-5-IncX3质粒在不同肠杆菌科细菌之间的水平传播；明确了不同日龄猪粪中CPKP生长和定植规律，揭示了猪的常驻肠杆菌科细菌在营养限制条件下能够抑制产碳青霉烯酶肺炎克雷伯菌CPKP在猪肠道的定植。研究结果为制定抗生素使用策略提供科学依据，也为控制重要耐药基因/耐药菌的扩散和减缓耐药性发展提供新的策略和途径。