博碩士論文 104326011 詳細資訊




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姓名 鄧教毅(Jiao-Yi Deng)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 重金屬生物有效性對於抗生素抗性基因在農地土壤的分佈與持續之影響
(The role of metal bioavailability in the distribution and persistence of antibiotic resistance genes in farmland soil)
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摘要(中) 近年來隨著抗生素的濫用與不當使用,已使得環境和臨床案例中感染具多重抗藥性的病原菌之案例逐年升高,由於大部分的致病菌已知多是透過環境中的一般微生物而獲得抗性基因,因此環境中抗生素抗藥性的加速發展所引發的公共衛生問題逐漸令人憂心。然而,減少抗生素的使用卻發現無法有效遏止抗藥性的擴散,代表環境中可能存有其他能持續對細菌造成壓力的物質;這當中,重金屬被視為極可能是維持抗生素抗性基因(antibiotic resistance genes, ARGs)在環境流佈存續的最關鍵因子,但此種非傳統抗生素抗性發展的途徑(即透過金屬的共選擇機制而誘發抗生素抗性)截至目前為止仍未得到廣泛的研究。有鑒於此,為瞭解重金屬是否/如何影響抗生素抗性基因在本土土壤中的維持及傳播,本研究選擇桃園中壢工業區周圍受重金屬污染且具有濃度梯度的農地為場址,透過土壤採樣與序列萃取進行初步的相關性分析。調查結果發現:(1)桃園地區受污染的農地土壤中,銅主要分佈於鐵錳氧化態、有機態及殘餘態,鉻主要分佈於殘餘態,鋅、鎳和鉛主要分佈於鐵錳氧化態及殘餘態;(2)當利用最小共選擇濃度(minimum co-selective concentration, MCC)來評估共選擇的潛勢時,發現在重金屬濃度高於MCC的場址,其ARGs和Class 1 integron相對豐富度顯著高於低於MCC的場址;(3)不同的ARGs透過基因連鎖同時存在於Class 1 integron,意指若有重金屬的環境壓力時,ARGs經由水平基因轉移而散佈;(4)重金屬全量濃度和移動相得到相似的結果,但移動相的結果顯示銅變為具有更強的驅動水平基因轉移潛勢,金屬移動相可能驅動水平基因轉移透過Class 1 integron將ARGs在環境中散佈,暗示著移動相可能適合作為生物有效性的代表,且當有數種重金屬的存在時更有助於誘發共選擇;(5) Class 1 integron同時對重金屬與ARGs具有顯著相關,代表其可能擁有應對重金屬與抗生素的能力,在受重金屬影響的農地種植,作物上可能發現同時擁有重金屬抗性和抗生素抗性的菌株,因而進入食物鏈之中。
摘要(英) In recent years, cases of infection with multi-drug resistant pathogens in environmental and clinical settings have increased drastically, which presumably is attributed to the abuse and improper use of antibiotics. Given that most of the pathogens are known to acquire resistance genes from environmental microbes, concern of rapid development and accumulation of the antibiotic resistome in the environment has raised. However, reduction in the use of antibiotics has shown to be unable to effectively hamper the spread of antibiotic resistance, implying that there may have been other substances in the environment that continue to exert resistance pressure on bacteria. Of them, heavy metals are considered the critical factors that highly likely promote the maintenance of environmental antibiotic resistance genes (ARGs). Yet, this non-traditional route of antibiotic resistance development (i.e., heavy metal driven co-selection of antibiotic resistance) has not been extensively studied. Therefore, in this study rice paddies irrigated with water from a creek flowing through the Jhongli Industrial Park and have been contaminated with heavy metals were selected as research sites to understand whether and/or how heavy metals would affect the maintenance and spread of ARGs in the farmland of Taiwan. Soil samples were collected and analyzed for the levels of heavy metals, ARGs and integrons. Results show that (i) the Fe-Mn oxide phase was a key in constraining metals in the soil of the studied sites; (ii) in addition to being corelated among one another, ARGs collectively had positive associations with class 1 integron, implying that ARGs might be co-selected via a gene cassette in which different ARGs co-existed with class 1 integraon; (iii) when the concentration of a heavy metal was above the minimum co-selective concentration (MCC) suggested by the literature, relative abundances of ARGs and Class 1 integron significantly increased, suggesting the MCC can be used as an initial assessment for the potential of metal-driven co-selection of antibiotic resistance; (iv) although the total concentration and mobile fraction (MF) of heavy metals possessed similar results, the mobile fraction results indicated that copper has become a stronger driving force for potential of horizontal gene transfer, implying that the MF may be a suitable indicator of metal bioavailability; (v) Class 1 integron had significant correlations with heavy metals and ARGs, indicating that it may have the ability to cope with heavy metals and thus ARGs may enter the food chain because metal resistance and antibiotic resistance may be found on crops when the farmland soil gets contaminated by heavy metals.
關鍵字(中) ★ 土壤重金屬
★ 抗生素抗性基因
★ 共選擇
★ 水平基因轉移
★ 序列萃取
關鍵字(英) ★ Heavy metal in soil
★ Antibiotic resistance gene (ARG)
★ Co-selection
★ Horizontal gene transfer
★ Sequence extraction
論文目次 摘 要 I
ABSTRACT III
誌謝 V
目 錄 VII
圖 目 錄 XI
表 目 錄 XIII
第一章 前言 1
1.1 研究背景 1
1.2 研究目的 3
第二章 文獻回顧 5
2.1 抗生素 5
2.1.1 抗生素使用現況 5
2.1.2 抗生素作用機制與抗性機制 6
2.2 重金屬 13
2.2.1 重金屬的生物毒性及其來源 13
2.2.2 金屬抗性機制 14
2.2.3 農田的重金屬污染 15
2.3 共選擇 17
2.3.1 共選擇機制 17
2.3.2 重金屬誘發抗生素抗性的共選擇 18
2.3.3 水平基因轉移 19
2.4 金屬的生物有效性 20
第三章 材料與方法 23
3.1 試劑與儀器 24
3.1.1 實驗藥品與分生試劑 24
3.1.2 實驗儀器 25
3.2 土壤現地採樣與前處理 26
3.2.1 研究場址介紹 26
3.2.2 現地採樣 27
3.2.3 土壤樣品前處理 28
3.3 土壤基本特性分析 28
3.3.1 土壤pH值測定 28
3.3.2 土壤水分含量 29
3.3.3 土壤中陽離子交換容量 29
3.3.4 土壤有機質含量 29
3.3.5 土壤質地分析 30
3.4 土壤重金屬分析 31
3.4.1 金屬全量分析 31
3.4.2 序列萃取試驗 31
3.5 分子生物試驗 33
3.5.1 土壤DNA萃取 33
3.5.2 抗生素抗性基因之標準品製備 33
3.5.3 土壤之抗生素抗性基因定量 39
3.6 統計分析 41
第四章 結果與討論 43
4.1 土壤物化性質 43
4.2 重金屬結合型態之分佈 46
4.3 抗生素抗性基因之分佈 50
4.4 ARGs、intI1與環境變量的相關性 56
4.4.1 重金屬誘發抗生素抗性的共選擇風險 56
4.4.2 ARGs、intI1與環境變量(金屬全量濃度與土壤性質)的相關性 56
4.4.2 ARGs、intI1與環境變量(金屬移動相與土壤性質)的相關性 60
4.5 ARGs、intI1及重金屬的環境意義 64
第五章 結論與建議 65
5.1 結論 65
5.2 建議 66
參考文獻 67
附 錄 87
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指導教授 林居慶(Chu-Ching Lin) 審核日期 2018-11-28
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