虾源大肠杆菌耐药可移动遗传元件 II 型整合子的适应度代价研究

Integrons are mobile genetic elements that play an important role in the spread of bacterial antibiotic resistance genes and in the emergence of multidrug-resistant bacteria. Class I and II integrons were frequently detected in many aquatic products in recent years. Class II integron becomes increasing concerned because it has different structures from class I integron and with unknown dissemination mechanism. However, the fitness cost of class II integron in the host bacteria is unclear, which severely restricts the understanding of its dissemination mechanism. Thus, in our study, the class II integron-carrying Escherichia coli isolates obtained from intestinal tracts of Penaeus vannamei from seawater and freshwater aquacultures and markets were used for materials. The aim of our study was thus to assess fitness cost of important components in class 2 integron in E. coli by the methods of construction of genetically engineered bacteria, competition experiments and flow cytometry. In addition, the risk of dissemination of class 2 integrons in the aquaculture and market circulation links, as well as under micro and trace antibiotic conditions will be assessed. It will further reveal the integron-mediated horizontal dissemination mechanism of antibiotic resistance genes and to provide theoretical basis for risk assessment and prevention and control technology of antibiotic resistance of foodborne pathogenic bacteria.

整合子作为一种可移动遗传元件，在细菌耐药基因的水平传播和多重耐药菌的出现中起着重要作用。近年来，日常食用的水产品中I型和II型整合子屡有检出。其中，II型整合子因其有别于I型整合子的特殊结构和仍未清晰的传播机制而日益受到关注。然而，II型整合子在相关宿主菌中的适应度代价不明，严重制约了对其传播机制的理解。因此，本项目将以前期从海水、淡水养殖场和市售南美白对虾肠道中筛选到的携带II型整合子的耐药大肠杆菌为材料，通过基因工程菌构建、菌株间的竞争试验和流式细胞术等方法研究II型整合子中各重要元件在大肠杆菌中的适应度代价，在此基础上评估其在水产养殖、水产品市场流通等环节以及含微量乃至痕量抗生素等条件下的传播和扩散风险，进一步揭示整合子介导的耐药基因水平传播机制，为食源性致病菌耐药性的风险评估和防控技术提供理论依据。

本研究围绕食源性病原微生物耐药基因传播扩散这一全球共同关注的食品安全和人类健康热点问题，以本项目组前期分离到的携带II型整合子的虾源耐药大肠杆菌为研究对象，旨在阐明II型整合子中各重要元件的适应度代价，以及携带II型整合子的耐药大肠杆菌在不同环境中的适应度代价，以期为进一步揭示整合子介导的耐药基因水平传播机制提供理论依据。本研究首先以筛选到的典型II型整合子intI2*-dfrA1-sat2-aadA1序列为模板，利用基因重组技术构建含不同整合酶、不同Pc启动子和不同基因盒的II型整合子大肠杆菌试验菌株13株以及不含整合子的对照菌株1株。其次，细菌体外竞争试验和流式细胞术分析可得，II型整合子的结构成本随着整合酶催化能力的增强而增加；同时，其结构成本随着Pc启动子增强而增加，其中启动子Pc2A引入的代价比Pc2B稍大；此外，II型整合子结构成本随着阵列中耐药基因盒数量的增加而增加，单个耐药基因盒的适应度代价均较小；最后，我们发现典型的II型整合子在大肠杆菌中通常是低成本结构，但在“从农田到餐桌”的环境中，尤其是在贫营养水平条件下，仍有一定的生物成本。然而，抗生素亚抑制浓度条件有利于携带II型整合子的耐药大肠杆菌的富集与传播。本项目的完成为进一步揭示整合子介导的耐药基因水平传播机制奠定研究基础，为食源性致病菌耐药性的风险评估和防控技术提供理论依据。