堆肥过程中土霉素含量及四环素类抗性基因tet（C）多样性变化规律

发布时间：2018-03-10 13:28

  本文选题：抗生素抗性基因　切入点：tet(C)　出处：《黑龙江八一农垦大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着畜禽养殖业的快速发展,越来越多的抗生素类药物以药剂或饲料添加剂的形式被广泛的应用于畜禽养殖业中。这些抗生素并不能完全被畜禽吸收,绝大多数的抗生素类药物会以母体化合物或者代谢物的形式随着畜禽粪便排出体外,从而造成了抗生素在畜禽粪便中的积累,进而诱导产生了大量的耐药菌。这些耐药菌含有的抗生素类抗性基因(Antibiotic Resistance Genes,ARGs)不仅可以在不同环境介质中传播扩散,而且还能持久性残留,比抗生素本身对环境的危害要大。堆肥是降解和消除ARGs的有效方法,但ARGs在堆肥过程中的变化分布却不清楚。本研究通过牛粪和树叶堆肥的方式,分析堆肥过程中土霉素(OTC)的含量及四环素类抗性基因tet(C)多样性的变化规律,为阐明ARGs在堆肥过程中的消减机制提供数据支撑,也为减少抗生素的残留及控制ARGs的传播提供理论依据。本研究以牛粪和树叶为主要堆肥原料,经检测牛粪中不含土霉素。试验设置两个处理,即处理组(TG)和空白对照(CK)。其中TG的土霉素添加量为60 mg·kg-1,CK不添加土霉素。在堆肥的的0 d、7 d、14 d、28 d、63 d取样并测其理化参数和土霉素的含量。结果显示,TG的土霉素含量在第7 d下降到初始含量的一半为32 mg·kg-1,到堆肥结束,土霉素含量为2 mg·kg-1。TG和CK温度50℃以上分别持续16 d和18 d,最高温度分别为62.0℃和61.5℃。TG和CK的pH在整个堆肥过程中均保持在7.5以上。堆肥结束时,两处理含水率变化差异性显著(p0.05),TG和CK的含水率由初始的70.0%分别下降到28.4%和34.8%。在整个堆肥过程中,TG和CK全碳含量逐渐降低,到堆肥结束,TG和CK全碳含量由初始的36.86%分别下降到26.34%和26.48%,差异性不显著(p0.05)。TG和CK全氮含量均先升高后降低,然后逐渐升高,到堆肥结束,TG和CK全氮含量由初始的1.59%分别升高到1.86%和2.0%。通过分子克隆技术研究四环素类抗性菌的多样性变化,结果表明,TG和CK两种堆肥过程中,四环素类抗性菌的多样性和种类均有差异。CK的微生物类群主要包括Uncultured bacterium、Salmonella enterica和Enterococcus faecalis,随着堆肥的进行CK处理四环素类抗性菌多样性逐渐减少。从堆肥第14 d开始,CK的堆体中只能检测到一种四环素类抗性菌。TG的微生物类群主要包括Uncultured bacterium、Salmonella enterica、Enterococcus faecalis、Uncultured Geobacter sp.和Lactococcus garvieae,在堆肥初期,TG的四环素类抗性菌的多样性增加,但是到堆肥结束时仅检测到Uncultured Geobacter sp。利用冗余分析(RDA)研究两处理堆体中四环素类抗性菌的菌群结构与环境因子的相关性。RDA二维排序图显示,TG和CK的四环素类抗性菌的菌群结构差异性较大。相关分析未发现对四环素类抗性菌的菌群结构变化具有显著性影响的环境因子(p0.05)。通过SPSS16.0对土霉素含量与四环素类抗性菌的多样性进行显著性分析可知,土霉素对四环素类抗性菌多样性变化具有显著性影响(p0.05)。
[Abstract]:With the rapid development of livestock and poultry breeding, more and more antibiotic drugs are widely used in animal husbandry in the form of medicament or feed additive. These antibiotics can not be completely absorbed by livestock and poultry. Most antibiotics are excreted in the form of parent compounds or metabolites, resulting in the accumulation of antibiotics in animal manure. In turn, a large number of drug-resistant bacteria were induced. The antibiotic resistance gene, Antibiotic Resistance Genes-ARGs, not only spread in different environmental media, but also persistent residues. Composting is an effective way to degrade and eliminate ARGs, but the distribution of ARGs in composting process is not clear. The content of oxytetracycline (OTC) and the diversity of tetracycline resistance gene (tetC3) during composting were analyzed, which provided data support for elucidating the mechanism of ARGs subtractive during composting. In this study, cattle dung and leaves were used as the main composting materials, the oxen manure was detected without oxytetracycline, and two treatments were set up in the experiment, which were used to reduce the residue of antibiotics and to control the spread of ARGs. TG) of the treatment group and CK of the blank control group, in which the amount of oxytetracycline was 60mg 路kg ~ (-1) CK without adding oxytetracycline. The physicochemical parameters and the content of oxytetracycline were measured at 0 d ~ 7 d ~ 14 d ~ (14) d ~ (28) d ~ (63) d of compost. The results showed that the content of oxytetracycline in TG was determined. On the 7th day, half of the initial content decreased to 32 mg 路kg ~ (-1), and the compost reached the end of the compost. The content of oxytetracycline was 2 mg 路kg-1.TG and CK temperature was above 50 鈩，

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