生鲜鸡肉中抗生素耐药基因池污染特征及整合子/质粒介导的耐药基因在共生菌中的HGT迁移机制研究

The rapid emergence and spread of antibiotic resistant (AR) bacteria threatens public health. Horizontal gene transmission (HGT) mediated by conjugative plasmids or integrons is considered as an important mechanism for the spreading of AR genes. However, most of the research are proceed in pathogens and conditional pathogens up to now, data on AR characterization of commensal bacteria which might play an important role in AR transmission is very limited. Therefore, the present project focuses on the mechanisms of HGT of AR genes mediated by integrons or plasmids in commensal bacteria in commercial fresh chicken products. AR genes including tet, sul, qnr, aac, dfr and bla as well as conserved sequences of mobile elements including int, traA and trbC are screened by quantitative PCR, and the composition of AR bacteria are analyzed by 16s rDNA PCR to investigate the characterization of AR gene pools in fresh poultry products. Then the screening techniques for integrons/plasmids-AR genes systems are established to get the plasmids carrying AR genes, whose migration potential to heterogenous bacterial are analyzed, and the effects of donor strain / recipient strain of AR genes as well as the sequence characteristics of mobile elements on the HGT of AR gene are investigated to propose the mechanisms of HGT of resistance genes mediated by integrons or plasmids in commensal bacteria.

抗生素耐药（AR）菌的快速出现和传播严重威胁人类健康。由质粒或整合子等介导的水平基因转移（HGT）被证实是AR基因迁移扩散的重要机制之一。但现有研究多在（条件性）致病菌中展开，关于在AR迁移中可能扮演重要角色的共生菌的耐药数据十分有限。因此，本项目以市售生鲜鸡肉产品中的共生菌为研究对象，展开整合子/质粒介导的AR基因在共生菌中的迁移机制研究。拟用定量PCR技术筛查AR基因（tet、sul、qnr、aac、dfr、bla等）和移动元件保守序列（intI和traA/trbC）的组成和含量，用16s rDNA PCR技术分析耐药菌的种属组成，初步探明鸡肉产品中AR基因池的污染特征。再建立整合子/质粒-AR基因盒系统的筛查技术，筛得携带AR基因的质粒，考察其向异源菌迁移的潜势，并分析AR基因在共生菌中进行HGT的影响因素，提出整合子/质粒介导的AR基因在共生菌中的迁移机制。

抗生素耐药菌的快速出现和传播严重威胁人类健康。由质粒或整合子等介导的水平基因转移被证实是耐药基因迁移扩散的重要机制之一，但现有研究多在（条件性）致病菌中展开，而在耐药迁移中可能扮演重要角色的共生菌的耐药数据十分有限。因此，本项目以市售生鲜鸡肉产品中的共生菌为研究对象，展开市售生鲜鸡肉产品表面细菌耐药基因池的组成、含量和寄主菌等特征的分析，筛查并获取携带耐药基因的整质粒，最终研究质粒-耐药基因系统迁移潜势及影响因素。结果表明，鸡肉产品表面细菌中包含了巨大的耐药基因池：27种耐药基因检测到24种，且四环素耐药基因tetE和tetM以及磺胺耐药基因sul2和dfrA1的绝对含量高达106拷贝/克肉样；对磺胺甲恶唑/甲氧苄氨嘧啶（Sul/Triis）、四环素（Tetis）、强力霉素（Doxis）、头孢噻肟（Ctxis）、红霉素（Ermis）和环丙沙星（Cipis）不敏感的细菌在108份鸡肉样品中的检出率分别为97.22%、100%、96.30%、100%、100% 和98.15%，抗性菌的含量分别为（1.00×103 ~1.00×108）CFU/g 、（1.00×103 ~1.51×108）CFU/g、（5.01×10~7.76×106）CFU/g、（3.47×103 ~1.51×108）CFU/g、（5.01×102 ~1.70×108）CFU/g、（5.01×10 ~7.59×107）CFU/g。分离到的耐药菌的多重耐药现象普遍，耐药基因的寄主菌包括埃希氏菌、葡萄球菌、肠球菌、奇异变形杆菌、不动杆菌、鞘氨醇杆菌和土地杆菌。目前已获得6个携带耐药基因的质粒，其中有2个质粒（葡萄球菌来源，质粒携带sul2基因；肠球菌来源，质粒携带tetG基因）能迁移到大肠杆菌DH5-α中。本项目获得的数据和分离到的携带耐药基因的质粒将为农产品表面耐药基因的迁移机制的揭示提供参考。