耐药整合子在鳗鲡病原菌、正常菌群和养殖环境细菌中的传播

Integron, a mobile DNA element that can capture, and carry genes based upon which integrase gene they contain, particularly those responsible for antibiotic resistance. At present, the integron has become a hot research of drug-resistance of pathogens associated with human being or animal. In contrast, the investigation of integron about aquatic animal pathogens is more scarce and inadequate. In this project, transfer of resistance integrons in pathogens and normal flora of eel and enviromental bacteria will be investigated. Firstly, a large number of preserved pathogenic strains isolated from diseased eels will be used to analyzed the prevalence and distribution of integrons, and the result is helpful to reveal the relationship between the resistance patterns and gene cassette arrays of integrons. Secondly, new drug-resistant strains of pathogens, normal flora in the same diseased eel and enviromental bacteria in the outbreak site were screened and isolated. The resistance spectrums and integrons of the three flora of bacteria will be compared, and the spread way of resistance integrons between different flora of bacteria will be discussed. Furthermore, the effect of bacterial species, integrase, antimicrobial agents on the integron gene transfer will be studied by conjugation experiments. So the conditions for spread of integrons can be explored. After above transmission research of resistance integron, a mathematic model based on the monitoring of integron to predict the probability of antibiotic resistance will be built. It will provide an important reference for decision in drug-resistance risk assessment and control of disease in aquaculture areas. To carry out this project, it will not only clarify the transmission and mechanisms of bacterial resistance, but also introduce new idea and method for the discussion on drug-resistance of pathogen of aquatic animals in China.

整合子是一个能够通过自身编码的整合酶获取胞外游离基因或基因片段并使之表达的遗传元件系统。目前，整合子已成为人、兽病原菌耐药性研究的热点。而相比而言，水产动物病原菌在整合子方面的研究就显得较为匮乏和不足。为此，本项目利用已分离保藏的大量病原菌菌株，全面分析耐药整合子在鳗鲡病原菌中的种属分布和流行特征；再利用新分离的鳗鲡病原菌和同宿主正常菌群以及水体菌群的耐药菌株，深入分析不同菌群的耐药相关性；进而利用接合实验，多方面探索耐药基因转移的条件；最终在整合子传播规律的研究基础上，建立病原菌耐药性预测的数学模型，为水产动物源耐药性风险评估和病害防控提供决策依据。开展本项目的研究将有助于阐明细菌耐药性的传播途径和机制，同时也为我国水产动物耐药性的研究引入新的思路和方法。

本项目针对耐药整合子在水产领域研究的不足，开展鳗鲡病原菌耐药整合子的种属分布特征和水平传播规律等方面的研究。首先，对97株已知种属的鳗鲡病原菌进行了耐药性分析和整合子的检测分析，结果显示这些菌株对PEN、AMP、AMX、SMZ、TMP、T/S、CHL、RIF等8种药物的耐药率达到50%以上，其中对PEN、AMP、AMX和RIF等4种药物的耐药率甚至达到69%以上；对3种以及3种以上药物具有抗性的耐药菌株高达95.4%，其中有1株对18种药物具有抗性。在这些病原菌中有77株可PCR扩增得到1型整合子可变区，含34种基因盒32种阵列，基因盒aadA2和阵列dfrA12-aadA2出现的频率最高。其中，还发现了9种新型基因盒阵列。.继而，对94株新分离的鳗鲡病原菌、正常菌群以及水体菌群的耐药菌株，分析耐药性与整合子的分布特征与规律。结果显示，93.5%的菌株对3种（含）以上的抗菌药物具有耐药性。对AMX的耐药率高达90.7%，对TET、RIF以及磺胺类和酰胺醇类药物类的耐药率在60%~80%之间，对CTX、NEO以及喹诺酮类的耐药性弱（低于20%）。多重耐药指数显示各生态样品耐药程度较为严重，尤以水样为最。各菌属中，柠檬酸杆菌属（0.58）和克雷伯菌属（0.61）的多重耐药指数最高，而不动杆菌属（0.21）则相对较低。整合子检测结果显示，有20株扩增出共17种基因盒9种基因盒阵列，基因盒阵列dfrA12-orfF-aadA2出现的频率也是最高的（11/20）。 .进一步对耐药性的消除与接合进行研究，柠檬酸杆菌T4传代实验和影印实验结果显示，紫外照射3min和无抗生素培养条件下，传代菌株中具有抗性的菌株比例迅速下降，到80代时比例已低于10%。在37 ℃高温和低浓度抗生素条件下，传代菌株耐药性基本无消除。耐药基因检测结果则显示，耐药基因strA/B、aphA1和blaTEM在消除子中始终未丢失，而tetD、floR、dfrA12和整合子在消除子中均同时丢失。上述含整合子的耐药菌株与大肠杆菌E.coil NK进行接合实验，获得16株接合子，进一步研究发现适当浓度的抗生素有利于提高接合效率。下一步我们将对所获得的一些超广谱（耐16种以上）耐药菌株进行基因组或质粒的高通量测序，进一步探讨细菌多重耐药性形成和传播的机制。