抗生素重复污染土壤中抗性基因多样性与基因点突变研究

Most of the used antibiotics are excreted from animal body by feces and urine as the parent compound or metabolites in livestock and poultry breeding. These antibiotics-contaminated animal manures were repeatedly applied into agricultural soil as organic fertilizer, resulting in "persistent" residues of antibiotics, and became a long-term selection pressure for soil microorganism and then substantive antibiotics resistance genes (ARGs) occurred by adaptive mutation. Therefore, there is an increasing concern on the diversity, mutation, and ecological risk of ARGs in agricultural soil. In this project, tetracyclines (chlortetracycline) and quinolones (ciprofloxacin) were selected as the representative antibiotics due to their extensive and substantive applications as feed additives in livestock and poultry breeding. According to their actural residual levels in agricultural soil, the soil was treated with chlortetracycline and ciprofloxacin alone and in combination by two treatment means (only antibiotics used, both antibiotics and manure used), respectively. The objectives of this project were: 1) to investigate the diversity, abundance, and mobility of ARGs in soil; 2) to measure the activity of ARGs in soil; 3) to quantify ARGs in soil; 4) to isolate and identify antibiotics-resistant bacteria; and 5) to conduct ARGs cloning and detection of point mutation and expression level. This project will elucidate the diversity, abundance, mobility, activity, and fluorescent quantitation of ARGs after a long-term treatment of single antibiotic and coupled antibiotics in agricultural soil, and reveal point mutation site, mutation rate, mutation frequency, and expression level of ARGs based on antibiotics-resistant bacteria, which is valuable for ecological risk assessment of ARGs in agricultural soil.

畜禽养殖中抗生素大多以母体或代谢产物经动物粪便和尿液排出体外，这些畜禽粪便作为有机肥长期、频繁施入农田造成抗生素"持久"性残留，形成对土壤微生物的长期选择压，从而易发生适应性突变产生大量抗性基因。抗性基因多样性、基因突变规律及其生态风险性是当前研究的热点。本项目选择常用四环素类抗生素金霉素和喹诺酮类抗生素环丙沙星，参照其在农业土壤中的实际残留水平，以单一和复合形式并采用直接添加和与有机肥共施添加两种方式重复处理土壤，研究：1）土壤中抗性基因多样性、丰度与移动性；2）土壤中抗性基因活性；3）土壤中抗性基因的定量；4）土壤中抗性菌的分离与鉴定；5）抗性基因克隆、点突变检测与活性检测。研究可望阐明单一和复合抗生素长期污染土壤中抗性基因多样性、丰度、移动性、活性及其含量水平，并结合分离鉴定的抗性菌，重点揭示抗性基因点突变位点、突变率、突变频率及其表达水平，可为抗性基因生态风险性评价提供基础数据。

含抗生素残留的畜禽粪便作为有机肥频繁大量施入农业土壤造成抗生素“持久”性残留，对土壤微生物施加长期选择压，从而易发生适应性突变产生大量抗性基因，因此，抗性基因多样性及其突变研究具有重要的科学和现实意义。本项目选择常用四环素类抗生素金霉素和喹诺酮类抗生素环丙沙星，以单一和复合形式并与有机肥共施添加方式多次重复处理土壤，主要研究了抗生素在重复处理土壤中的降解与积累性、土壤生物学功能变化及其土壤微生物群落诱导抗性，重点开展了土壤中抗性基因多样性、丰度、活性以及突变位点多样性及其突变频率、抗性基因荧光定量以及抗性菌分离与鉴定及其抗性谱与抗性水平等研究。主要研究结果包括：1）抗生素重复处理能改变其在土壤中的降解速率，随着处理次数的增加金霉素降解半衰期逐渐减小，而环丙沙星逐步产生积累；2）抗生素重复处理能改变土壤微生物功能多样性和土壤酶活性，抗生素诱导土壤微生物群落抗性显著大于有机肥处理，并且随着处理次数的增加而显著上升；3）结合宏基因组和宏转录组分析表明抗生素重复处理不仅改变了土壤微生物群落结构，而且抗性基因丰度、活性、突变位点多样性及其突变频率显著上升，网络分析揭示了Actinobacteria和Firmucutes是四环素类抗生素抗性基因主要潜在宿主；4）荧光定量PCR分析表明抗生素重复处理土壤中金霉素和环丙沙星（交叉）抗性基因相对丰度逐步上升；5）抗生素重复处理土壤中抗性菌多样性及其抗性水平显著大于有机肥处理，并且随着处理频率逐渐上升。研究结果在阐明单一和复合抗生素长期重复污染土壤中的降解与积累特征及其土壤生物学功能变化及其微生物群落诱导抗性的基础上，重点揭示了土壤中抗性基因多样性、丰度、活性以及抗性基因突变位点多样性及其突变频率，相关成果已在Environ Sci Technol、J Hazard Mater、Sci Total Environ等国际期刊发表标注SCI论文7篇，另有3篇SCI论文待发表。研究结果为抗生素长期污染土壤中抗性基因生态风险性评价提供了科学依据。