施有机肥对土壤及生菜中耐药非致病菌及耐药基因的影响研究

发布时间：2018-08-26 21:19

【摘要】：由于抗生素的过量使用,养殖环境及禽畜粪便中存在大量抗生素残留及耐药菌和耐药基因。粪便被还田用于农业种植中,可能成为土壤中耐药菌及耐药基因的来源之一。细菌耐药及耐药基因已成为危害人类健康的环境及食品问题。粪肥和土壤作为耐药菌和耐药基因的储存环境和传播介质之一,其对耐药菌和耐药基因在环境中的传播有重要影响。蔬菜是人类的基本生活食品,其中的耐药菌及耐药基因会通过食物链影响人类的健康安全。而非致病菌是耐药基因的受体、供体和储存中间体,因其数量众多,占微生物生态系统中的大部分,在其中发生耐药基因的水平迁移(HGT)事件的频率可能更高,因此在耐药基因的迁移中扮演了重要角色。因此开展施用有机肥对土壤及蔬菜中耐药非致病菌及耐药基因的影响研究,以及其中耐药基因传播风险的研究十分必要,可为农业环境及农产品中抗生素耐药菌的控制策略的制定提供科学依据。本研究以土壤及生菜为研究对象,通过室内盆栽实验、菌落培养、平板计数的实验方法,研究分析施用有机肥对土壤及生菜中对头孢噻肟(CTX)、红霉素(ERM)、磺胺(SUL)、四环素(TET)、强力霉素(DOX)、环丙沙星(CIP)6种抗生素耐药性的非致病细菌数目和多重耐药表型的影响;通过基因克隆构建耐药基因库,耐药基因筛查和测序研究施有机肥对土壤及生菜中整合酶基因intI和9种耐药基因的丰度、组合类型,对含多重耐药基因菌种类型的影响;最后采用质粒提取及聚合酶链式反应(PCR)分析了耐药基因的潜在传播风险。主要结论如下:(1)施肥后明显增加了土壤中CTX~r、SUL~r、ERM~r、TET~r、CiP~r数目,DOX~r在对照组和施肥组中均未检出。施肥后15 d、30 d和45 d,施肥组CTX1·的数量均大于其他耐药非致病菌1-2个数量级,数量达到10~8CFU·g~(-1),DOX~r最少,数量均为10~5CFU·g~(-1)。除DOX~r外,随生菜的生长,施肥均增加土壤中所研究的其他5种耐药非致病菌(CTX~r、SUL~r、ERM~r、TET~r、CIP~r)的数目,但对其影响逐渐减少。施肥15d和30d时,对土壤中CTX~r、TET~r、CIP~r、DOX~r数目的影响显著,其中对CTX~r、CIP~r数目影响极显著,ERM~r在30d差异显著,施肥45 d时,即生菜采摘期时,施肥仅对土壤中CIP~r数目影响显著,对其他5种耐药非致病菌影响均不显著。施肥后土壤中多重耐药菌比例增加,不同时间点多重耐药菌比例均有不同程度的提高。(2)施肥会使生菜中的耐药非致病菌的数目增加。无论施肥与否,除DOX~r在生菜中均未检出外,生菜中其他5种耐药非致病菌普遍存在。随着种植时间的增长,施肥对土壤中SUL~r的影响逐渐增加,对CIP~r数目的影响先增加后减少,TET~r和CTX~r数目的影响逐渐减少,对ERM~r数目无明显影响。施肥对CTX~r数目在15 d时影响最大,对TET~r在15 d和30 d时影响显著,且15 d影响极显著,施肥对其余耐药非致病菌在15 d和30 d均无显著影响,施肥45 d时,即在生菜采摘期45 d时,施肥对其中的耐药非致病菌数目影响不显著。施有机肥对生菜中耐药菌的多重耐药性没有太大的影响,但是可以看出即使不施肥,生菜中也存在多重耐药菌。(3)intI在土壤和生菜中普遍存在,和施肥与否无关。施肥后增加了土壤和生菜中intI、tet和sul基因的检出率。土壤与生菜中不同tet基因类型变化均不同,sul基因中,检出率差异性大小均为sul1sul2。(4)施肥后,土壤中tet基因中tetC检出率明显增加,其次为tetG、tetB、tetE、tetA、tetM,tetL基本无变化。施肥对土壤中tet基因的影响在30 d最大,在45 d基本无影响。(5)施肥后,生菜中tet基因中tetG检出率明显增加,其次为tetM、tetB,施肥组tetL和tetA检出率小于对照组,tetC均未检出。随着时间的增长,施肥对生菜中tet基因的影响减弱甚至减少其中tet基因检出率,对生菜中sul基因的影响增强。(6)施肥可以增加土壤和生菜中含多重耐药基因的菌株和intI基因的检出率,可能一定程度上增加抗性基因的水平迁移风险。(7)施有机肥均可增加土壤和生菜中多重耐药菌株的菌种类型,且可以改变生菜多重耐药基因组合的类型和丰度。施肥土壤与对照土壤中的含多重耐药基因的菌种均主要为假单胞菌(Pseudomonas sp.)、短波单胞菌(Brevibacterium sp.)节杆菌(Arthrobacter sp.)。对照组与施肥组生菜中优势多重耐药菌株均为假单胞菌(Pseiudomonas sp.)和短波单胞菌(Brevibacterium sp.),施肥引入较多的菌株为节杆菌(Arthrobacter sp.)。(8)在所选取的多重耐药非致病菌的质粒上均检出intI基因及sul、tet耐药基因,其中土壤及生菜中intI在质粒上检出率100%。因此土壤及生菜中的耐药基因可能具有水平传播风险,还需要对这些质粒的特点进行进一步研究。
[Abstract]:Due to the overuse of antibiotics, a large number of antibiotic-resistant bacteria and genes exist in the farming environment and livestock excrement. Feces returned to farming may become one of the sources of drug-resistant bacteria and genes in soil. Bacterial resistance and genes of drug resistance have become environmental and food problems that endanger human health. Vegetables are the basic food for human life, in which drug-resistant bacteria and drug-resistant genes affect human health and safety through the food chain. Non-pathogenic bacteria are receptors for drug-resistant genes. Horizontal migration of drug-resistant genes (HGT) may occur more frequently in microbial ecosystems because of the large number of donor and storage intermediates, which play an important role in the migration of drug-resistant genes. It is necessary to study the influence of antibiotic resistance genes and the transmission risk of antibiotic resistance genes, which can provide scientific basis for the formulation of control strategies of antibiotic-resistant bacteria in agricultural environment and agricultural products. Effects of fertilizers on the number and multidrug resistance phenotypes of cefotaxime (CTX), erythromycin (ERM), sulfonamide (SUL), tetracycline (TET), doxycycline (DOX) and ciprofloxacin (CIP) resistant non-pathogenic bacteria in soils and lettuce; Resistance gene pool was constructed by gene cloning, resistance gene screening and sequencing. Finally, Plasmid Extraction and polymerase chain reaction (PCR) were used to analyze the potential transmission risk of drug-resistant genes. The main conclusions were as follows: (1) Fertilization significantly increased the number of CTX~r, SUL~r, ERM~r, TET~r and CiP~r in soil. The number of CTX1 ~ (-1) in the fertilizer group was higher than that in the other non-pathogenic bacteria (1-2 orders of magnitude) 15 days, 30 days and 45 days after fertilization. The number of CTX1 ~ (-1) in the fertilizer group was 10-8 CFU ~ (-1), and the number of DOX ~ (-1) was the smallest, all of which were 10-5 CFU (-1). Except DOX ~ (-1), the other five Non-drug-resistant non-pathogenic bacteria in the soil were increased with the growth of lettuce. The number of pathogenic bacteria (CTX~r, SUL~r, ERM~r, TET~r, CIP~r) decreased gradually, but the effect of Fertilization on the number of CTX~r, TET~r, CIP~r and DOX~r in soil was significant at 15 and 30 days of fertilization, especially on the number of CTX~r and CIP~r. ERM~r had significant difference at 30 days of fertilization, that is, at 45 days of lettuce picking stage, fertilization only had significant effect on the number of CIP~r in soil. (2) Fertilization could increase the number of drug-resistant non-pathogenic bacteria in lettuce. Except DOX~r, there were no other 5 species in lettuce except DOX~r. With the increase of planting time, the effect of Fertilization on the number of SUL~r in soil increases gradually, and then decreases. The effect of Fertilization on the number of TET~r and CTX~r decreases gradually, but has no significant effect on the number of ERM~r. Fertilization had no significant effect on the number of drug-resistant non-pathogenic bacteria on the 15th day and 30th day. Fertilization had no significant effect on the number of drug-resistant non-pathogenic bacteria on the 45th day, that is, on the 45th day of picking period. Multidrug-resistant bacteria also existed in lettuce. (3) InI was ubiquitous in soil and lettuce, and had nothing to do with fertilization. After fertilization, the detection rate of intI, tet and sul genes in soil and lettuce increased. The detection rate of tetC in lettuce was significantly increased, followed by tetG, tetB, tetE, tetA, tetM and tetL. Fertilization had the greatest effect on the Tet gene in soil at 30 days, but had no effect on it at 45 days. (5) After fertilization, the detection rate of tetG in lettuce was significantly increased, followed by tetM, tetB. The detection rate of tetL and tetA in fertilization group was lower than that in control group, and tetC was not detected. (6) Fertilization can increase the detection rate of strains and intI genes containing multiple resistance genes in soil and lettuce, which may increase the risk of horizontal migration of resistance genes to a certain extent. (2) Fertilization can increase the detection rate of strains and intI genes containing multiple resistance genes in lettuce. 7) Application of organic manure could increase the types of multi-drug resistant bacteria in soil and lettuce, and could change the type and abundance of multi-drug resistant genomic combination in lettuce. The dominant multidrug-resistant strains were Pseudomonas sp. and Brevibacterium sp. in the control group and the fertilization group. Arthrobacter sp. (8) InI gene and sul, tet resistance gene were detected in the plasmids of the selected multidrug-resistant non-pathogenic bacteria. The detection rate of intI in medium soil and lettuce was 100%. Therefore, the risk of horizontal transmission of drug-resistant genes in soil and lettuce may exist, and further study on the characteristics of these plasmids is needed.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S141

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