基于淤长型海涂土壤耐药组演变探索耐药基因的“源库关系”

In the previous study, it was found that the diversities of cultivable antibiotic-resistant bacteria decreased, whereas the detection rates of four kinds of tetracycline resistance genes increased vertically along the coastline. Based on the facts that marine is of severe environment, facile transfer of mobile genetic elements, and abundant antibiotic-producing microbes versus soil, a hypothesis that marine may be the main source and soil may be the main reservoir of ARGs is advanced. Soil samples with different formation time in Jiangsu coastal mud flat offer us an ideal sampling sites for this study. In this proposal, a strategy spatial sequence instead of time successional sequence is adopted. From Sheyang, Yancheng City and Rudong, Nantong City, serial samples from sediment submerged in seawater to agricultural soil perpendicular to the coastline of the Yellow Sea will be sampled. Metagenomic libraries will be constructed, and antibiotic resistant clones will be selected for Illumina sequencing. Thus, the successional characteristics of soil antibiotic resistomes from 0 to 100 years will be uncovered to test the hypothesis. Besides, in order to uncover the influences of human activities on antibiotic resistome, each antibiotic resistome of soil samples derived from different areas with distinct reclamation modes will be determined as mentioned above. Meanwhile, bacterial 16S rRNA gene will be sequenced by Illumina high-throughput sequencing approach in order to reveal bacterial diversities and communities of the samples. Then, the assembled resistance-conferring DNA fragments will be accurately predicted the source phylum by using RAIphy model. The correlation and network between soil ARG content and bacterial composition will be clarified. Moreover, antibiotics contents, physical and chemical properties of soils et al. will be determined for better understanding the responses of succession of antibiotic resistomes to environmental factors. This proposed study will probably offer us new knowledge regarding the source of ARGs, and offer us novel indices for assessing current and future conditions of the Great Coastal Development from the viewpoint of public health.

前期研究发现海洋垂直沿线土壤可培养耐药菌多样性下降，但耐药基因检出率却上升的现象，结合海洋较土壤具有产抗生素微生物种类数量丰富、可移动遗传元件易于转移等特点，我们提出“海洋可能是耐药基因源，土壤是耐药基因库”假设。江苏淤长型海涂规律性演化土壤为本研究提供了样品来源。拟用“空间代替时间”策略，对盐城射阳和南通如东海岸线垂直带土壤系列取样，构建宏基因组文库，筛选耐药克隆，用Illumina测序技术探究不同成土时间土壤耐药组的变化规律，推断耐药基因的来源及扩散机制，验证项目假说；对海岸线平行带不同围垦利用方式区域土壤取样，基于宏基因组技术揭示耐药基因特性对不同人为活动的响应及机制；对各样本16S rRNA基因进行高通量测序，分析其群落结构和多样性，模型预测揭示耐药基因组和细菌群落之间的相关性和网络关系。本研究可就耐药基因来源问题提供新的认知，也为滩涂围垦开发等政府决策提供基于公共卫生角度的依据。

本项目根据前期研究发现，提出“海洋可能是耐药基因源，土壤是耐药基因库”假设。江苏淤长型海涂规律性演化土壤为本研究提供了样品来源。我们用“空间代替时间”策略，对盐城射阳无明显人为活动（国家自然保护区核心区）的海岸线垂直带土壤系列取样，构建宏基因组文库，用Illumina测序技术探究不同成土时间土壤耐药组的变化规律，推断抗性基因的来源及扩散机制。主要发现有：①在非根际和根际土壤分别检测到196种亚型和192种亚型ARGs；②69年来，ARGs的多样性和丰度保持稳定，其原因可能是碱性土壤环境而非抗生素诱导所致；相比之下，成土86年土壤ARGs的增加可能是候鸟活动频繁造成的ARGs扩散；③多药抗性基因是最丰富的类型，并且为所有土壤样本共享。进一步研究表明，土壤样品随成土时间不能被明确区分，表明沿海泥滩中ARGs演替过程缓慢；④方差分解分析表明，ARGs特征由细菌群落、微生物和环境因素的综合效应共同驱动；⑤研究还暗示了成土86年的区域存在由候鸟介导的耐药致病菌，这一点需要在未来予以特别关注。研究指出海洋是周边土壤环境中ARGs和MGEs的重要来源。科学意义：本研究为领域内学者探索环境及人为活动对土壤ARGs丰度的影响提供了基准和背景数据；同时为海洋中ARGs向土壤扩散这一论点提供了证据。项目执行期间，以第一/通讯作者身份发表标注项目号高质量学术论文10篇，授权发明专利1件，完成了项目既定目标和任务。