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题名: 猪场周围土壤中四环素类抗性基因丰度和多样性的研究
作者: 吴楠
学位类别: 硕士
答辩日期: 2010
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 朱永官
关键词: 抗生素 ; 抗性基因 ; antibiotics, resistance gene, soil, swine feedlot ; 土壤 ; 猪场
中文摘要:       近年来,致病菌耐药性的增加和扩散已成为全世界关注的一个健康问题,而人类医疗和畜禽养殖业抗生素的滥用正在加剧这一问题。四环素类抗生素是一类常见的抗生素,作为兽药和促生长剂被广泛用于猪场养殖业。由于其在畜禽养殖业中的大量使用,诱导了环境中四环素抗性微生物的产生,然而有关四环素类抗性基因在土壤中存在、迁移和扩散的研究还很少。 本研究采集了来自中国3个城市共9个猪场周围的农田土壤,对土壤中的四环素抗性进行了检测。利用普通PCR在土壤中共检测到15种四环素类抗性基因,包括7种编码泵外排蛋白的基因(tetA, tetC, tetE, tetG, tetK, tetL, tetA/P),7种编码核糖体保护蛋白的基因(tetM, tetO, tetQ, tetS, tetT, tetW, tetB/P),以及1种编码修饰或钝化四环素类分子的酶的基因(tetX)。 进一步建立了定量PCR程序对其中5种基因(tetM, tetO, tetQ, tetW, tetT)以及微生物总量16S rRNA基因进行了定量。不同土样中的4种四环素类抗性基因(tetM, tetO, tetQ, tetW)的相对丰度具有明显差别,数值范围从10-5到10-2不等。而每个土样中tetT基因的相对丰度(从10-6到10-4)都要低于其他4种基因(tetM, tetO, tetQ, tetW)的相对丰度。 利用LC-MS/MS共对土壤中5种典型四环素类抗生素及10种四环素类抗生素的降解产物进行了检测。土样中四环素类抗生素的浓度总和范围从5.4到377.8 μg•kg-1干土。相关性分析结果显示,土壤中的四环素类抗性基因水平(tetM, tetO, tetQ, tetW绝对基因拷贝数总和)与土壤中残留的四环素类抗生素的总量显著正相关(r2 = 0.45, P < 0.05);另外四环素抗性基因水平还与土壤微生物总量(r2 = 0.65, P < 0.01)以及土壤有机质(r2 = 0.46, P < 0.05)显著正相关。 通过建立克隆文库的方法,在猪场周围土壤中共发现了5种不同的tetM基因类型,大部分tetM基因型与各类菌种中的质粒、转座子等携带的tetM基因序列相同或相近,这些结合或者可移动元件有助于tetM基因在不同环境介质中各类微生物之间的传播和扩散,具有一定的生态及健康风险。
英文摘要:       The increasing prevalence of antibiotic-resistant pathogens has been recognized as a global public health issue, which is exacerbated by the overuse of antibiotics in human medicine and livestock systems. Tetracyclines are commonly used antibiotics in the swine industry for disease treatment and growth promotion. In recent years, the prevalence of tetracycline-resistant bacterial in environment are influenced by the wide use of tetracyclines in livestock production facilities. However, the survival, fate and dissemination of tetracycline resistance (tet) genes in soil are still poorly understood. In the present study, tetracycline resistance was determined in soils sampled from farmlands in the vicinity of nine representative swine farms located in three cities in China. Totally, 15 tetracycline resistance genes were commonly detected in soil samples using the Polymerase-chain-reaction (PCR), including seven efflux pump genes (tetA, tetC, tetE, tetG, tetK, tetL, tetA/P), seven ribosomal protection proteins (RPPs) genes (tetM, tetO, tetQ, tetS, tetT, tetW, tetB/P), and one enzymatic modification gene (tetX). The quantitative real-time PCR was further used to quantify five RPPs genes (tetM, O, Q, W, T) and 16S rRNA gene abundances. The relative abundances (target gene/16S rRNA genes) of tetM, tetO, tetQ, and tetW in soil showed significant variation over sampling sites, ranging from 10-5 to 10-2. However, tetT genes were lower in relative abundances (from 10-6 to 10-4) than other four genes at all sites where tetT genes were detected by using conventional PCR methods. The concentrations of total tetracyclines (5 typical tetracyclines and 10 of their degradation products) in these soils were measured using liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), and ranged from 5.4 to 377.8 μg·kg-1 dry soil. Bivariate correlation analysis confirmed that absolute tet gene copies (sum of tetM, O, Q, W genes) were strongly correlated with the concentrations of tetracycline residues (r2 = 0.45, P < 0.05), ambient bacterial 16S-rRNA gene copies in each soil sample (r2 = 0.65, P < 0.01) and organic matter in soil (r2 = 0.46, P < 0.05), respectively. Finally, the phylogenetic analysis on tetM combined with culture-independent molecular techniques, revealed at least five genotypes of tetM in nine soil samples. Most genotypes of tetM were confirmed to be associated with various conjugative transposons and plasmids, which may allow wider dissemination of the tetM gene among numerous bacteria in the environments.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35066
Appears in Collections:中澳联合土壤环境研究室_学位论文

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Recommended Citation:
吴楠. 猪场周围土壤中四环素类抗性基因丰度和多样性的研究[D]. 北京. 中国科学院研究生院. 2010.
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