土壤耐药性及抗生素对土壤氮素影响研究

发布时间：2018-07-11 18:38

本文选题：耐药基因 + 土壤耐药菌　；参考：《浙江大学》2015年硕士论文

【摘要】：本文以不同环境土壤抗生素的耐药性及抗生素污染对土壤氮素转化的影响为目的,综合运用传统的微生物生态学方法以及分子生物学研究手段,并借鉴临床医学方法,研究茶园、养殖场、医院和森林等四种环境中土壤抗生素抗性基因的存在及丰度特征,筛选高抗生素耐药菌,探明耐药菌中抗性基因的遗传位置;同时研究外源抗生素添加对土壤氮转化及相关微生物的响应,初步研究了土壤环境条件与土壤耐药性及养分转化之间的内在联系。主要研究结果如下:1.四类土壤环境中,养殖场土壤中抗生素抗性基因(AGRs)含量较高,是耐药基因的一个重要存储库,而森林土壤受到人为影响最小,各基因拷贝数较低。茶园土壤中耐药基因虽然绝对拷贝数较低,但是相对表达量较高。可见,耐药基因的积累受人类活动的影响。四类土壤环境中均检测到了一类整合子,含量达到4.75× 105-1.39× 107 copies/g;二类整合子在茶园、养殖场、猪粪中存在,但拷贝数低于一类整合子。说明整合子广泛存在于土壤环境中,耐药基因在环境中有较高的基因转移水平。2.不同环境土壤存在不同程度的耐青霉素和四环素细菌,其中四类土壤均能筛选出超级青霉素耐药菌,耐受浓度高达1000 mg/L。所分离纯化的20株耐药菌属于Pseudomonas sp.或Variovorax sp.。多重耐药表明每个菌株至少对两种抗生素存在抗性,但是所有菌株均对四环素类抗生素敏感。基因检测表明所有分离的高耐药菌不仅含有青霉素抗性基因blaTEM,而且含有编码四环素外排泵基因tet(C),说明基因的存在并不一定能够表达,基因存在只能说明这种基因具有表达的潜能。质粒去除后,有6株耐药菌消除了对大环内脂类抗生素的抗性,说明这些菌株的部分耐药基因是存在于质粒上的,易于横向转移。3.外源添加抗生素处理初期能使土壤中硝态氮含量增加,后慢慢平衡。土壤NH4+-N的含量在培养9 d显著下降,后趋于平衡;土壤在高浓度抗生素处理下,AOA呈平缓下降的趋势,而低浓度抗生素处理下AOA呈先抑制再激活后平缓下降趋势,总体培养前期与氨氮硝氮变化相呼应。因此,抗生素污染压力下AOA可能对土壤的硝化起主要贡献。
[Abstract]:Aiming at the effects of antibiotic resistance and antibiotic contamination on soil nitrogen conversion in different environments, the traditional methods of microbial ecology and molecular biology were used in this paper, and the clinical medical methods were used for reference. To study the existence and abundance of antibiotic resistance genes in tea garden, farm, hospital and forest, to screen high antibiotic resistant bacteria and to find out the genetic position of resistance genes in drug resistant bacteria. At the same time, the responses of exogenous antibiotics to soil nitrogen transformation and related microorganisms were studied, and the internal relationship between soil environmental conditions, soil resistance and nutrient transformation was studied. The main results are as follows: 1. In the four kinds of soil environment, the content of antibiotic resistance gene (AGRs) in farm soil is high, and it is an important repository of drug resistance gene, but forest soil is least affected by human being, and the copy number of each gene is low. Although the absolute copy number of drug resistance gene in tea garden soil was lower, the relative expression of drug resistance gene was higher. It can be seen that the accumulation of drug resistance genes is affected by human activities. A class of integron was detected in four kinds of soil environment, and the content of integron was 4.75 脳 105-1.39 脳 107 copies-g.The second class integron was found in tea garden, breeding farm and pig manure, but the copy number was lower than that of one class integron. The results showed that integron existed widely in soil environment, and drug resistance gene had higher gene transfer level. Penicillin-and tetracycline-resistant bacteria were found in different soil environments. Four kinds of soils were able to screen super penicillin resistant bacteria and the tolerance concentration was up to 1000 mg 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) ~ (-1). Twenty strains of drug-resistant bacteria isolated and purified belong to Pseudomonas sp. Or Variovorax sp. Multidrug resistance indicated that each strain was resistant to at least two antibiotics, but all strains were sensitive to tetracycline antibiotics. Gene analysis showed that all highly resistant strains contained not only the penicillin resistance gene blaTEM, but also the tetracycline efflux pump gene tet (C), indicating that the presence of the gene may not be able to express it. The presence of a gene only indicates that the gene has the potential to express. After the removal of plasmids, 6 strains of drug-resistant bacteria eliminated the resistance to macrocyclic lipids, indicating that some of the resistant genes of these strains existed on the plasmids and were easy to be transfered horizontally. Exogenous antibiotics could increase nitrate content in soil at the initial stage and then balance slowly. The content of NH4-N in soil decreased significantly after 9 days of culture, and then tended to balance, and AOA decreased slowly under high concentration of antibiotics, while that of AOA in low concentration of antibiotics decreased at first, then activated, and then decreased slightly in the treatment of low concentration of antibiotics, while in the treatment of high concentration of antibiotics, the content of AOA decreased slowly. The change of ammonia, nitrite and nitrogen in the early stage of the whole culture coincided with the change of ammonia and nitrate. Therefore, AOA may play a major role in soil nitrification under antibiotic pollution.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X53;R124

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