填埋场抗生素抗性基因传播扩散规律及其微生物驱动机制

Differ from regular environmental chemical pollutants, the antibiotic resistance genes could be replicated and amplified in the bacteria. Meanwhile, they could be spread with mobile genetic elements. In addition, both amplification and spread could be happened without selective stress of antibiotics. Thus, the pollution of antibiotic resistance genes in the environment pose a significant risk to human health. Compared with other environmental media, there are relatively few studies on the contamination of antibiotic resistance genes in landfills, a reservoir of multiply kinds of pollutants. The rules of spread of antibiotics resistance genes in landfills is still unclear. In addition, its microbial driving mechanism, as one of the research hotpots for antibiotic resistance gene, is in urgent need of breakthrough. Based on this, the project will select typical landfills to clarify systematically the characteristics of antibiotic resistance genes in landfills. Then will focus on the co-selection under the stress of heavy metals and hazardous and noxious organic pollutants to analyze the rules of spread of antibiotic resistance genes in complex environment. Last but not least will take horizontal gene transfer and quorum sensing as the main line to explore the microbial mechanism on driving the spread of antibiotic resistance genes in environment. The implementation of the project can not only be helpful to understand the deep-seated mechanism of antibiotic resistance gene contamination, but also provide a forward-looking scientific basis for the control of the antibiotic resistance gene contamination, which is of great significance to the overall promotion of ecological health and environmental security in our country.

与常规环境化学污染物不同，抗生素抗性基因不仅可以通过微生物载体进行复制倍增，还可以通过各类可移动元件在微生物载体间进行传播扩散，且复制传播扩散可在无抗生素选择压力下发生，给人类健康带来了巨大风险。相较于其他环境介质，作为众多污染物聚集地的垃圾填埋场，非抗生素污染对抗生素抗性基因传播扩散影响尚不清晰；此外，抗性基因污染的微生物驱动机制仍是该领域的研究重点和难点，亟需突破。基于此，本项目拟选择典型生活垃圾填埋场，系统研究抗生素抗性基因的污染特征；结合生活垃圾填埋场复杂的环境因素，聚焦重金属和有毒有机污染物胁迫下的共选择，解析抗性基因的传播扩散规律；关注生活垃圾填埋场关键的抗性菌类群，以基因水平转移和群体感应为切入，探究抗性基因传播扩散的微生物驱动机制。项目实施有助于理解抗生素抗性基因污染的深层次机制，可为抗性基因污染管控提供前瞻性科学依据，对全面推动我国生态健康及环境安全具有重要意义。

垃圾填埋场是城市固体废物的最终处置场所，也是抗生素和抗性基因（ARGs）重要的汇与源。但垃圾填埋场中抗生素及ARGs的污染研究相对较少，ARGs的传播扩散规律、影响因素及微生物驱动机制尚不清晰。基于此，本项目通过对垃圾填埋场实地取样，结合理化检测、高通量定量PCR、宏基因测序及生信分析，对填埋场中抗生素抗性基因的污染水平、分布特征、影响因素和驱动机制等进行了深入研究。首先，对浙江省10个不同城市的生活垃圾填埋场取样，发现氟喹诺酮类和四环素类抗生素在垃圾填埋场检出浓度最高，分别为1406.85 μg/kg和1101.04 μg/kg。垃圾填埋场中ARGs普遍存在，在嘉兴，温州和丽水垃圾填埋场中qnrD，qnrS，mexF，ermA，ermB，mefA，sul1，sul2，tetM，tetX 和tetW污染程度较高，其丰度均高于10-4（ARGs/16S rRNA），且抗生素和抗性基因在这些填埋场中的生态风险都比较高。其次，实验对填埋场理化指标进行测定，并通过方差分解分析和冗余分析识别了填埋场ARGs传播的驱动因素，发现移动遗传元件（MGEs）是影响垃圾填埋场中ARGs丰度的主要因素，贡献率为50.6％。环境因素、抗生素和重金属对ARGs的贡献度仅分别占13.8％、6.8％和2.1％。此外，固体垃圾中ARGs受填埋龄的影响，丰度随填埋龄的增加而降低。最后，实验通过宏基因组测序分析了不同填埋龄垃圾的ARGs、毒力因子基因、群体感应相关基因和病原菌的分布特征，并甄别出关键类群。抗性基因、毒力因子基因和病原菌丰度均呈现随填埋龄增加而升高的趋势。在所有垃圾样品中多重耐药基因是主要的ARGs类型，占所有ARGs的33~44%，其次是MLS耐药基因和四环素耐药基因。L.monocytogenes、S. enterica、S. pneumoniae、A.baumannii、E. coli和X. campestris同时具有多种ARGs和VFs基因，可被列为垃圾填埋场中的高风险人类致病菌。此外群体感应相关基因与抗生素抗性基因之间关系密切，可能通过影响微生物菌群的分布驱动ARGs的扩散传播。研究内容丰富了对复杂环境系统下抗生素抗性基因污染水平的认识，加深了对抗生素抗性基因污染机制的深层次理解，可为抗性基因污染的管控提供前瞻性科学依据，对全面推动我国生态健康以及环境安全具有重要意义。