【作者】 魏勇；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2011， 博士

【摘要】 养殖粪污中细菌等微生物的种类、数量、存活时间及其耐药性等直接关系到生态环境保护、人与动物的健康。养殖粪污作为丰富的耐药基因库,在细菌及其耐药性的传递、保有、转归等多个环节具有重要作用,而养殖粪污耐药基因的传递与转归也并不总是依赖活细菌或可培养细菌；猪场粪污中含有丰富的可培养及不可培养细菌类群,其细菌类群随环境综合因素复杂变化而变化,因而其细菌及其耐药性研究较困难；已有研究以细菌培养及其药敏筛选方法为主,难以准确获取猪场粪污中的细菌及其耐药基因信息,因此开展规模化猪场粪污总DNA指纹图谱及其耐药性研究有重要意义。本试验研究针对规模化养猪生产中以沼气为纽带的循环利用系统的粪污处理主流模式,以规模化猪场粪污总DNA为主要研究对象,采用PCR-DGGE、multi-PCR等现代分子生物学及生物信息学分析方法开展研究,取得了以下结果：1 PCR-DGGE用于猪场粪污细菌群落分析的方法优化针对猪场粪污总DNA样品,以变性梯度凝胶电泳(PCR-DGGE)方法为研究对象,对基于16S rDNA V3区的PCR-DGGE电泳的最佳变性剂梯度范围、电泳时间、染色时间进行了优化。研究结果表明：最佳变性剂梯度范围为35%-60%,电泳时间为12h, SYBR? Green Fluorescent Dye染料的染色时间是30min,优化后的PCR-DGGE确保了实验的准确性、灵敏度和可重复性。运用此优化后的PCR-DGGE技术对5个猪粪样品及5份粪污样品的细菌群落进行了研究,获得了清晰的多样性分离条带,表明优化后的PCR-DGGE方法适合于猪场粪污的细菌多样性研究。该研究首次优化了用于猪场粪污细菌多样性分析的DGGE检测方法,为深入研究养殖粪污治理的菌群多样性相关研究奠定了基础。2猪场粪污细菌总DNA的细菌多样性PCR-DGGE分析取得嘉陵江上游连续分布的5个猪场的出粪口(A)→沼气池(B)→氧化塘(C)→地表水(D)→地下水(E)依次5个主要粪污处理环节的25份粪污样品,采用优化的DGGE方法进行了细菌多样性分析。结果表明：1号猪场1 E条带数最多、为17条,1A与1E菌群差异性较大、为91.5%,1E与1D菌群差异性较小为55.2%；2号猪场2E条带数最多、约9条,2A与2B菌群差异性较大、为85.0%,2A与2E菌群差异性较小、为48.5%；3号猪场3A条带数最多、约12条,3A与3D菌群差异性较大、为75.7%,3A与3B菌群差异性较小、为42.6%；4号猪场4A条带数最多、约7条,4A与4E菌群差异性较大、为85.5%,4A与4B菌群差异性较小、为47.6%；5号猪场5A条带数最多、约7条,5A与5E菌群差异性较大、为76.4%,5A与5C菌群差异性较小、为68.9%。经DGGE优势条带回收及序列鉴定,不可培养细菌是粪污样品中的优势菌。该研究首次采用DGGE方法对猪场粪污菌群多样性进行了研究,证实规模化猪场的粪污中细菌多样性在沿河连续分布猪场之间及依次处理环节间菌相无明显关联,为猪场控制抗生素残留、粪污治理的技术标准化及养殖适度规模的量化提供了科学资料。3猪场粪污细菌总DNA中菌群16S rDNA文库的构建对5个猪场的依次5个主要粪污处理环节的25份粪污样品,通过PCR扩增全长16S rDNA,获得了106条有效克隆,序列平均长度约为1400bp,构建了粪污菌群16S rDNA文库。通过生物信息学分析,依据97%的相似性原则。结果表明,粪污菌群16S rDNA文库中包括葡萄球菌(Staphylococcus)、大肠杆菌(E. coli.)、气单胞菌(A. viscosus)、亚硝化球菌(Nitrosococcus)等可培养细菌,以及大量不可培养细菌构成的优势菌群；近60%的克隆序列与Genbank中已知序列的同源性低于97%,表明其可能是新的或未识别的菌种；所构文库的覆盖率约为73.6%。本研究首次构建了猪场粪污的细菌菌群16S rDNA文库,为深入研究猪场粪污微生物的结构组成及粪污处理菌种的筛选与开发利用提供了科学素材。4猪场粪污细菌总DNA中的4类抗生素耐药基因调查针对25份粪污样品,采用细菌对氯霉素类药物耐药基因三重PCR检测试剂盒、细菌对氨基糖苷类药物耐药基因四重PCR检测试剂盒、细菌对磺胺类药物耐药基因三重PCR检测试剂盒及动物源细菌对四环素类药物耐药基因三重PCR检测试剂盒对猪场粪污总DNA进行规模化猪场常用抗菌素的耐药基因检测。结果表明：耐氯霉素类药物的cmlA、flor及cat1基因检出率分别为32%、60%和40%,耐四环素类药物的tet A, tet M及tet C基因检出率分别为60%、60%和56%,耐磺胺类药物的Sul 1、sul2及sul 3基因检出率分别为80%、76%和32%,耐氨基糖苷类药物的aph (3’)-IIa、aac (6’)- Ib, aac (3)-IIa及ant (3")-Ia基因检出率分别为58%、96%、64%与96%。在猪场粪污灌溉利用之前的粪污处理环节中,氯霉素类、四环素类、磺胺类及氨基糖苷类抗菌素耐药基因检出率100%。而氯霉素类、四环素类药物耐药基因在经过灌溉农田后出现了显著消除、地下水中无检出,磺胺类和氨基糖苷类抗菌素耐药基因在经过灌溉农田后出现了未被显著消除、地下水中检出率90%以上。本试验首次对猪场粪污细菌总DNA进行了抗生素耐药基因的multi-PCR检测,提示猪场粪污中氯霉素类、四环素类、磺胺类及氨基糖苷类抗菌素耐药基因普遍存在,灌溉农田利用可能是抗菌素耐药基因消除重要的方式之一。5猪场粪污中大肠杆菌和葡萄球菌的分离鉴定及其耐药性研究针对25份粪污样品,进行了粪污样品中的大肠杆菌与葡萄球菌的分离鉴定、对18种常用抗生素耐药表型K-B法筛选及4类抗生素的耐药基因型multi-PCR检测。结果表明：猪场粪污中大肠杆菌分离率为44%,对常用抗菌素以多重耐药为主,对氯霉素类、四环素类、磺胺类、氨基糖苷类等常用抗菌素耐药基因携带率>94%;葡萄球菌的分离率64%,高于大肠杆菌,规模化养猪生产中常用抗菌素也以多重耐药为主,氯霉素类、四环素类、磺胺类、氨基糖苷类等常用抗菌素耐药基因携带率高,均超过90%。本研究结果与其他环境样品的大肠杆菌分离率及耐药性研究结果基本一致。这提示在猪场粪污中细菌耐药表型及耐药基因型的存在都较普遍,需要注重抗生素的合理使用。首次证实了葡萄球菌在猪场粪污中的分离率及耐药基因携带率都高,因此提示葡萄球菌可能作为猪场粪污治理工艺及耐药基因行为研究的一个重要生物指示器。通过对猪场粪污样品的上述研究,探索了猪场粪污中细菌群落及其耐药性的存在、传播、转归及对人畜健康的潜在危害等,为猪场粪污致病菌、耐药基因控制及粪污治理工艺标准化积累了重要科学资料。更多还原

【Abstract】 Presently, the biological and physical variable of microflora in pig manure is not clear yet. In this study, denaturing gradient gel electrophoresis was optimized in the running parameters including level electrophoresis, electrophoresis time and dying time, respectively. The optimized DGGE method was subsequently performed to analyze the diversity of the manure samples from 5 main running scenorios of 5 pig farms distributed along Jialing River. And then, bacterial 16S rDNA clone library of corresponding manures was established. Moreover, antibiotics resistance genes in microbial community DNA extracted from manure samples were amplified by using multiplex PCR detection kit of 4 different antibiotics resistance genes in bacteria isolated from animals. What’s more, it’s also concerned with isolation of antibiotics resistance detection of Staphylococcus and E.coli. from the manure samples.This research will benefit further study on wide microbial diversity, manure management practices, inestimable number of combinations of potential pathogen fates in environment, and environmental factors that influence the presence, persistence, survival, and transport of pathogens.1. Optimization of denaturing gradient gel electrophoresis for bacterial communities in manure from pig farm Ploymerase chain reaction and denaturing gredient gel electrophoresis (PCR-DGGE) technique was used to reveal the bacterial community composition and diversity associated with manure from pig farm. In order to improve the accuracy, sensitivity and repeatability of DGGE based on 16S rDNA V3 region, the running parameters of DGGE were optimized by level electrophoresis, electrophoresis time and dying time, respectively. The results showed that the best range of denaturing gradients was 35-60% in 8% polyacrylamide gels, running time was 12 hours, and the dying time was 30 min using SYBR Green Fluorescent Dye. Furthermore, bacterial communities from 5 excrement samples and 5 slurry samples were also performed by using these optimized reaction conditions. It was found that 10 manure samples exhibited effective separation and rich microbial diversity. This revealed that different manure samples result in the distinct changes of microbial diversity and microbial community structure. PCR-DGGE is proven to be a powerful tool for describing the bacterial flora in manure from pig farms.2. Study on the bacterial abundance of manure sample in pig farms using DGGEDenaturing gradient gel electrophoresis (DGGE) technology was used in studying the diversity of manure samples from 5 main running scenario of 5 pig farm continuously distributed along Jialing River. The detection result showed that the abundance of manure samples was not very rich, while the DGGE bands of manure samples were around 10 respectively. And as for the bacteria diversities and community, there is no positive relationship among the same scenario of difference pig farms, the successive scenario in the same pig farm, and the pig farm distributing along the same river. Furthermore, after recovery and identification the bands from DGGE profiles, the results revealed that above 70% recovery band were Staphylococcus, Escherichia coli and uncultured microorganisms. Therefore, it could be concluded that Staphylococcus, Escherichia coli and uncultured microorganisms were the majority of bacterial species in manure from pig farms.3. Generation and characterization of 16S rDNA library of the manure form pig farmsBacterial 16S rDNA clone library of manure form pig farms was constructed in this research. From cloned PCR products,200 randomly-selected clones containing inserts were sequenced. After removing the vector sequence and trimming the poor-quality sequences, we obtained 106 qualified sequences, with an average length of approximately 1400bp. Tentative annotations were performed by using BLASTX and results were manually validated. According to comparison analysis, Escherichia coli,Staphylococcus and Pseudomonas were the main species. The Homology analysis could be not more than 97% between 50% of 16S rDNA sequences and known DNA sequences in GenBank. It showed that there may be a new bacteria or species. The result indicated that diversiform bacteria maybe exist in manure form pig farms.4. Detection of antibiotics resistance genes in the microbial community DNA extracted from the manure of pig farmsThe antibiotics resistance genes were amplified in microbial community DNA extracted from 25 manure samples from 5 main running scenario of 5 pig farm distributed along Jialing R;ver by using multi-PCR detection kit of Choramphenicols resistance genes in animal original bacteria, the multi-PCR amplification kit of bacteria Aminoglycoside-resistance genes detection, the multi-PCR detection kit of Sulfonamides-resistance genes in bacteria isolated from animals and the multi-PCR detection kit of Tatracycline-resistance genes in bacteria isolated from animals. The result showed that the integrative detection rate of the antibiotics resistance genes from the sewage outlet, biogas pool and oxidation pond is 100%. The antibiotics resistance genes of Choramphenicols and Tatracyclines decreased remarkably after land application, and there was no antibiotics resistance genes detection in underground samples. On the other hand, the fertilization had poor effect on the elimination of the antibiotics resistance genes of Sulfonamides and Aminoglycosides, and the detection rate was above 90% yet in under ground water. It indicated that the resistance genes of often-used antibiotics spread universally, such as Choramphenicols, Tetracycline, Sulfonamides and Aminoglycosides. Furthermore, land application may be a most important scenario of reducing the antibiotics resistance genes.5. Isolation and antibiotics resistance gene detection of Staphylococcus and E.colL in the manure from pig farms This research was concerned with isolation of antibiotics resistance detection of Staphylococcus and E.coli. from 25 manure samples. The result indicated that the isolation rate of E.coli. was 44% in 25 manure samples, the integrative detection rate of drug-resistance genes of Choramphenicols, Tetracycline, Sulfonamides and Aminoglycosides was respectively 82%,100%,100% and 100%. The isolation rate of Staphylococcus was 64% in the manure of pig farms, the integrative detection rate of drug-resistance genes of Choramphenicols, Tetracycline, Sulfonamides and Aminoglycosides was 94%,94%,100% and 100% respectively. The research result indicated that Staphylococcus and E.coli. were commonly found in manure from pig farms, and they might be a important preserver of antibiotics resistance genes in the agricultural environment, and the reasonable application of antibiotics could be obtained special attention in the livestock production process. Moreover, Staphylococcus could be a candidate bacteria indicator similar to E.coli. for manure management of livestock production.更多还原