【作者】 刘燕；

【导师】 路璐；

【作者基本信息】 西华师范大学 ， 环境科学， 2021， 硕士

【摘要】 抗生素抗性基因（Antibiotic resistance genes,ARGs）作为一种新型环境污染物,对不同环境的生态系统安全和人类健康产生严重的威胁。近期的研究中ARGs已在不同环境中被大量检测出且产生了富集污染。然而,不同环境中ARGs分布规律的综合比较分析,及其传播方式仍处于研究初期。因此,本研究针对城市黑臭河流、养猪场周边农田土壤、受人为干扰少的贡嘎山以及网络交易的粪肥,采用高通量实时荧光定量PCR（High-Throughput Real-time fluorescence quantitative PCR,HT-q PCR）的方法,对283对ARGs和12对可移动遗传元件（Mobile Genetic Elements,MGEs）遗传标记物进行检测,探讨不同环境介质中ARGs和MGEs的多样性和丰度以及其分布特征,并耦合生物理化指标,揭示ARGs与各环境因子的相关性。其研究结果如下:（1）在城市黑臭河流中,水样中共检测到199种不同的ARGs和12种MGEs,底泥中共检测到160种ARGs和9种MGEs。在城市河流水样中黑臭区域的ARGs和MGEs的亚基种类和相对丰度均显著高于河流上游的农村对照区域（P<0.05）,表明人类活动引起的恶劣水质可能会加剧ARGs和MGEs的富集和污染。在底泥中没有显著差异。冗余分析表示,NH₄⁺-N、p H以及Fe离子是影响河流水体中ARGs分布和组成的显著环境因子,底泥中并未发现显著影响其ARGs分布的理化因素。（2）在养猪场周边土壤样品中总共检测到198种ARGs和12种MGEs。养猪场排污口、堆肥氧化池边、施用粪肥的土壤中ARGs和MGEs的亚基种类和丰度均显著高于未施肥的对照土壤（P<0.05）,表明养猪场对周围农田土壤有显著的ARGs贡献。此外,冗余分析表明p H和NH₄⁺-N分别解释了不同样品间ARGs差异的61%和28%,但并无显著的相关性（P>0.05）。（3）在贡嘎山2948-3651米海拔土壤样品中总共检测到132种ARGs和10种MGEs。在贡嘎山各海拔梯度的土壤样品中ARGs和MGEs的种类组成和丰度有显著差异（P<0.05）,然而其并未随着海拔高度变化而变化。冗余分析表明NO₃^--N、NH₄⁺-N、含水量、海拔和TOC分别解释了贡嘎山各海拔梯度样品中ARGs分布差异的33%、29%、20%、9%和5%,但均没有显著的相关性（P>0.05）。（4）在网络交易平台上购买的四种粪肥中共检测出165种ARGs和10种MGEs,检测到ARGs种类为:羊粪肥（138种）＞鸡粪肥（98种）＞鸡羊混合肥=牛粪肥（89种）。不同粪肥的ARGs丰度和组成差异显著,且都有其独有的ARGs亚型。同时,四种粪肥的总ARGs与总MGEs的相对丰度呈极显著相关（P＜0.01）,表明ARGs的水平转移可能加剧粪肥环境中ARGs的迁移和传播风险。（5）河流水样、底泥样品、农田土壤样品、高山土壤和粪肥样品之间检测到的ARGs和MGEs种类数和相对丰度均有显著差异（P<0.05）。ARGs种类数的总体变化如下:农田土壤>河流水样>河流底泥>粪肥>高山土壤;ARGs的相对丰度变化为:粪肥>河流底泥>农田土壤>河流水样>高山土壤。综上所述,不同环境中ARGs的分布特征有显著差异,ARGs的多样性和丰度随着人为干扰强度的增加而增加。本研究为评估其不同环境中微生物生态安全提供了理论依据,也为探究不同环境介质中ARGs的传播机制、以及评估ARGs污染水平提供重要的数据基础。更多还原

【Abstract】 Antibiotic resistance genes（ARGs）,as a new type of environmental pollutant,pose a serious threat to environmental.In recent studies,ARGs have been extensively detected and enriched in different environments.However,the comprehensive comparative analysis of the distribution of ARGs in different environments,and the ARGs transmission mechanism are largely unexploredl.Therefore,the diversity and abundance of ARGs and MGEs in different environmental samples were determined using High-Throughput Real-time q PCR（HT-q PCR）.283 ARGs and 12 MGEs genetic markers were used in the HT-q PCR measurement.Biophysical and chemical indicators were also measured to reveals the correlation between ARGs and various environmental factors.The research results were as follows:（1）The diversity of ARGs in the urban black-odorous river was determined.A total of 199 ARGs and 12 MGEs were detected in water samples.And a total of 160ARGs and 9 MGEs were detected in sediment samples.The relative abundances of ARGs and MGEs in the urban water samples were significantly higher than those in the rural control areas in the upper reaches of the river（P<0.05）,indicating that human activities may aggravate ARGs and MGEs enrichment and pollution.However,there was no significant difference in the sediment samples between the two areas.Redundant analysis indicated that NH₄⁺-N,p H,and Fe were significant environmental factors that affecting the distribution and composition of ARGs in river water.However,no physical and chemical factors significantly affect the distribution of ARGs in sediment samples.（2）A total of 198 ARGs and 12 MGEs were detected in the soil samples near to the pig farm.The abundances and number of ARGs and MGEs in the soil of sewage outlet,compost oxidation pond side and manure application were significantly higher than those in the unfertilized control soil（P<0.05）,indicating that the pig farms had significant contribution of ARGs to the surrounding farmland soil.Moreover,redundancy analysis showed that p H and NH₄⁺-N accounted for 61%and 28%of ARGs differences among different samples,respectively,but there was no significant correlation（P>0.05）.（3）A total of 132 ARGs and 10 MGEs were detected in soil samples at an altitude of 2948-3651 meters in the Gongga Mountain.There were significant differences in the composition and abundance of ARGs and MGEs in soil samples at different altitude gradients in the Gongga Mountain（P<0.05）.The ARGs and MGEs abundance did not correlate with altitude.Redundancy analysis showed that NO₃^--N,NH₄⁺-N,water content,altitude and TOC explained 33%,29%,20%,9%and 5%of the differences in the distribution of ARGs in different altitudes of Gongga Mountain,respectively,but there was no significant correlation（P>0.05）.（4）A total of 165 ARGs and 10MGEs were detected in the four types of manure that were purchased in the online shops.The number of detected ARGs types were as follows:sheep manure,chicken manure,and chicken and sheep mixed manure=earthworm manure.The abundant and composition of ARGs subtypes significantly differed among the four manures,each manure had its own unique ARGs subtypes.The relative abundant of total ARGs significantly and positively correlated with the total MGEs（P＜0.01）,indicating that the horizontal transfer of resistance genes may exacerbate the migration and spread of ARGs.（5）The number and relative abundance of ARGs and MGEs in the river water and sediment samples,farmland soil samples,alpine soil and manure samples were significantly different（P＜0.05）.The number of detected ARGs varied as follows:farmland soil,water samples,sediment samples,manure samples and alpine soil.The relative abundance of ARGs varied as follows:manure samples,sediment samples,farmland soil,water samples and alpine soil.In summary,the results show that the distribution characteristics of ARGs in different environments were significantly different,and the enrichment and abundance of were greatly affected by human activities.This study provides a theoretical basis for evaluating the ecological safety of microorganisms in various environments,and also provision important data basis for exploring the transmission mechanism of ARGs in different environmental media.更多还原