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题名: 集约化养猪场废弃物处理和排放过程中抗生素迁移 及相关抗性基因的变化研究
作者: 王健
学位类别: 博士
答辩日期: 2015-11
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 强志民
关键词: 抗生素,抗生素抗性基因,集约化养猪场,迁移,堆肥 ; Antibiotics, antibiotic resistance genes (ARGs), concentrated swine feedlot, migration, composting
其他题名: Migration of antibiotics and variation of corresponding resistance genes from concentrated swine feedlots along waste treatment and discharge paths
学位专业: 环境工程
中文摘要:       基于预防疾病和促进生长的目的,抗生素在养殖业中大量使用。我国是养猪第一大国,每年所产生的大量养猪粪便和废水中含有未被生物体消化利用的抗生素。当这些养殖废弃物通过施肥、灌溉和排放等方式扩散到环境中时,其中残留的抗生素会给周围环境细菌带来选择性压力,诱导其抗性的增强及抗生素抗性基因(ARGs)的增殖,增加了环境病原菌获得抗生素抗性的机会,给抗生素的临床治疗带来很大困难,严重威胁人类健康。ARGs 作为一种新型污染物,其在我国养猪业中的污染现状及迁移规律,目前尚缺乏相关的系统性研究。
      本文通过固相萃取(SPE)及超高效液相色谱质谱联机(UPLC-MS/MS)方法测定了养猪粪便和废水、施肥和未施肥土壤、受纳水体及沉积物等相关样本中养殖业常用的四环素类(TCs)和磺胺类(SAs)抗生素的浓度;使用聚合酶链式反应(PCR)和定量PCR 方法定性、定量检测了样本中与TCs 和SAs 相关的ARGs 的相对丰度和分布;使用变性梯度凝胶电泳(DGGE)和高通量测序方法分析了样本中细菌群落结构的变化。在此基础上,揭示了ARGs 在养猪场中的迁移规律,阐明了多种养猪废弃物处理工艺对ARGs 的影响,取得以下主要结论:
      (1) 以北京市某典型集约化养猪场为研究对象,考察了TCs、SAs 两类抗生素及其相关ARGs 在废弃物排放或处理过程中的扩散规律,探讨了影响ARGs沿程扩散和传播的相关因素,结果表明,TCs 和SAs 的浓度及相关ARGs 的相对丰度总体上沿着粪便的处理和废水的排放途径逐渐降低。粪便和废水中TCs 的最高检出浓度分别为166.7 mg kg-1 和388.7 μg L-1,而SAs 的最高检出浓度仅分别为98.4 μg kg-1 和7.6 μg L-1,所检测ARGs 的相对丰度处于2.74×10-6−1.19 范围内。抗生素的检出浓度、ARGs 的检出率和相对丰度冬季高于夏季。粪便处理途径中抗生素和ARGs 的消减程度大于废水排放路径。核糖体保护机制(RPP)四环素类抗基因(TRGs)的丰度水平在上游粪便、废水样本中均占优势,而在下游下降明显,而外排泵(EFP)TRGs 和磺胺类抗性基因(SRGs)表现则较稳定。粪便样本中,4 种RPP TRGs 的相对丰度之和与TCs 的总浓度呈现显著正相关;水体样本中,SRGs 的相对丰度与SAs 的总浓度呈现强正相关。水塘沉积物是ARGs 随废水排放的一个主要“汇”。整合酶基因与部分ARGs 显著相关,表明整合子密切参与ARGs 的传播。在所调查的各类样本中,COD、氨氮、总氮、总磷等污染物浓度与部分ARGs 显著相关;在粪便和土壤样本中,含水率同部分ARGs 显著相关,说明环境因子同样影响ARGs 在环境中的传播。
      (2) 在北京市典型养猪场的研究基础上,开展了山东省九家集约化养猪场中TCs 和SAs 两类抗生素及相关ARGs 的大规模区域性调研,发现总体上两类抗生素在养猪粪便和废水中的检出浓度高于北京猪场,表明山东省养猪场抗生素的使用剂量相对较高。同样,土壤中抗生素的总检出浓度也高于北京猪场,这是由于山东省养猪场多数缺乏粪便处理设施,致使土壤易受污染。受纳水体沉积物中的抗生素浓度变化与上游养猪废水中相似,表明其受废水排放影响显著。九家养猪场相关样本中ARGs 的变化规律与北京猪场类似,呈现沿程逐渐降低的趋势。土壤和水体沉积物中,ARGs 的相对丰度在夏季均高于冬季,这可能与夏季排水量大导致猪场污染物易向外扩散有关。进一步考察了不同养猪废弃物处理工艺对ARGs 的影响,发现发酵床对ARGs 的去除效果最好,而化粪池和静置风干效果最差,革兰氏阴性菌可能是养猪废弃物处理工艺中ARGs 的主要宿主菌群。
      (3) 基于北京和山东多家养猪场的研究结果,重点考察了猪粪高温堆肥过程中土霉素(OTC)和磺胺二甲嘧啶(SMN)的降解及相应ARGs 的演变。结果表明,经过32 天的高温堆肥,OTC 和SMN 降解率分别达到了89.9%和100%,SMN 的降解主要依赖于非生物作用,OTC 的降解则是生物和非生物共同作用的结果。堆体中添加的抗生素没有阻碍粪便的腐熟,但诱导了其中相关ARGs 的增殖。在整个堆肥过程中,RPP TRGs 基本保持稳定,而EFP TRGs、tetX 和SRGs的相对丰度在堆肥中温期显著提高。相关性分析表明,高温和高浓度抗生素残留对RPP TRGs 在堆肥过程中的驻留有利,而其他TRGs 和SRGs 则更适于较低温度和较低抗生素残留的环境。整合酶基因与多种机制ARGs(EFP TRGs、tetX 和SRGs)存在显著正相关,说明整合子介导的基因水平转移对堆肥过程中ARGs的扩散起到重要作用。
英文摘要:       Antibiotics are extensively used in animal feeding industries for disease prevention and growth promotion. China is the largest country for pork production in the world. Accordingly, large amounts of swine manure and wastewater containing unmetabolized antibiotics are produced every year. When the swine wastes are spread to the environment via fertilization, irrigation or even direct discharge, the residual antibiotics contained therein may exert selection pressure on environmental microbes, thus raising their resistant levels and promoting the proliferation of antibiotic resistance genes (ARGs). As a result, environmental pathogens will have an opportunity to acquire the resistance, which severely hampers the clinical application of antibiotics and threatens the public health. As a group of emerging contaminants, the present pollution status, as well as the migration rule of ARGs in the swine
industry of China are lacking in systematic research
      In this study, solid phase extraction (SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) were employed to determine the concentrations of tetracyclines (TCs) and sulfonamides (SAs), two commonly-used classes of antibiotics in animal feeding industries, in various related samples including swine manure and wastewater, fertilized and unfertilized soils, and receiving water and sediment. Polymerase chain reaction (PCR) and real-time PCR were adopted to detect and quantify the tetracycline resistance genes (TRGs) and sulfonamide resistance gens (SRGs), and denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing were adopted to analyze the variation of bacterial community structure in these samples. On this basis, we revealed the
migration rule of ARGs in the swine industry and clarified the impacts of various swine waste treatment processes on the ARGs. The main conclusions can be drawn as follows:
      (1) Taking a typical concentrated swine feedlot located in Beijing as example, we investigated the dissemination of TCs, SAs, and their corresponding ARGs along the swine waste discharge and treatment paths as well as the impacts of various influential factors. Results show that both the concentrations of TCs and SAs and the relative abundances of the corresponding ARGs generally decreased along the manure treatment and wastewater discharge paths. In the manure and wastewater samples, the highest detected concentrations of total TCs reached 166.7 mg kg-1 and 388.7 μg L-1, respectively; while those of SAs were only 98.4 μg kg-1 and 7.6 μg L-1, respectively. The relative abundances of target ARGs ranged from 2.74×10-6 to 1.19. The detected antibiotic concentrations and the detection frequencies and relative abundances of ARGs were higher in winter than in summer. The reduction of both antibiotics and ARGs was more rapid along the manure treatment path than along the wastewater discharge path. TRGs encoding ribosomal protection proteins (RPP) exhibited a dominant abundance level in the upstream manure and wastewater samples, but decreased remarkably in the downstream samples. In comparison, TRGs encoding efflux pumps (EFP) and SRGs remained relatively stable. In the manure samples, the total relative abundance of RPP TRGs was significantly correlated with the total
concentration of TCs; meanwhile, in the wastewater samples, the total relative abundance of SRGs was strongly correlated with the total concentration of SAs. Lagoon sediment acted as a major sink of ARGs along the wastewater discharge path. Some ARGs were significantly correlated with the integrase genes, indicating that the integrons were intimately involved in the dissemination of ARGs. In all tested samples, COD, ammonia, total nitrogen, and total phosphorus were significantly correlated with certain ARGs; and in the manure and soil samples, the moisture content was significantly correlated with certain ARGs. This indicates that environmental factors could also affect the dissemination of ARGs.
      (2) Based on the results obtained from the typical concentrated swine feedlot in Beijing, a large-scale regional surveillance was carried out on the pollution of TCs, SAs and corresponding ARGs in nine concentrated swine feedlots located in Shandong Province. Results show that in general, higher levels of target antibiotics were detected in the manure and wastewater samples from the nine feedlots than those from the Beijing feedlot, suggesting that higher antibiotic doses were applied in the swine industry of Shandong Province. Similarly, the detected antibiotic concentrations in the soil samples near the nine feedlots were also higher than those near the Beijing feedlot. This was due to a serious lack of manure treatment facilities in Shandong Province, which rendered the soils more vulnerable to antibiotic pollution. The antibiotic concentrations in the sediment of receiving waters showed a similar variation pattern to those in the upstream wastewater samples from the nine feedlots, indicating a strong impact of wastewater discharge on the receiving waters. The ARGs tested in the nine feedlots gradually decreased along the waste treatment and
discharge paths, which agrees with the result obtained in the Beijing feedlot. In the soil and sediment samples, more abundant ARGs were detected in summer than in winter, probably because the pollutants were easier to disseminate when more wastewater was discharged in summer. In addition, the effects of different swine waste treatment processes on the ARGs were examined. The microbial fermentation bed process showed the most effective removal of ARGs , whereas the septic tank and static drying processes were the least effective. Gram-negative bacteria appeared to be the main hosts of ARGs in the swine waste treatment processes tested.
      (3) According to the results from the swine feedlots in Beijing and Shandong, the removal of oxytetracycline (OTC) and sulfamethazine (SMN) as well as the evolution of their corresponding ARGs were emphatically studied in a simulated thermophilic composting process with swine manure. Results show that the removal efficiencies of OTC and SMN reached 89.9% and 100% after 32 days of composting, respectively. Abiotic process was mainly responsible for the degradation of SMN, while both abiotic and biotic processes contributed to the degradation of OTC. Antibiotics added in the composting piles did not retard the compost maturation but induced the proliferation of certain ARGs. RPP TRGs remained quite stable throughout the composting process, while the relative abundances of other TRGs and SRGs increased obviously in the mesophilic stage. Correlation analysis revealed that the RPP TRGs were more adaptive to a relatively higher temperature and antibiotic concentration, while other TRGs and SRGs favored a lower temperature and antibiotic concentration. Integrase genes showed a significant and positive correlation with most of the studied ARG types (i.e., EFP TRGs, tetX, and SRGs), implying that the integrons played an important role in the dissemination of ARGs during the manure composting.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36973
Appears in Collections:环境水质学国家重点实验室_学位论文

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Recommended Citation:
王健. 集约化养猪场废弃物处理和排放过程中抗生素迁移 及相关抗性基因的变化研究[D]. 北京. 中国科学院研究生院. 2015.
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