高密度聚乙烯表面之生物膜藉由銅暴露所引起的抗生素抗性共選擇

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蔡睿澤(Ruei-Tze Tsai) 查詢紙本館藏

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論文名稱

高密度聚乙烯表面之生物膜藉由銅暴露所引起的抗生素抗性共選擇
(Co-selection of antibiotic resistance induced by copper exposure in biofilms grown on HDPE slides)

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摘要(中)

由於塑膠製品非常耐用且價格低廉，很快地成為生活中實用的物品。然而，隨著塑料產量的增加，大量廢棄塑膠開始在自然棲地堆積。一旦塑膠進入環境，風化後產生的凹坑使表面粗糙，疏水性降低，細菌便會在表面定殖並形成生物膜。生物膜中的細菌可以通過水平基因轉移(HGT)來共享和傳播抗生素抗性基因(ARG)。此外，塑膠顆粒上的生物膜對於大多數重金屬的累積相當顯著，例如廣泛用於工業製程和畜牧業的銅離子，因此ARGs在塑膠碎片上生物膜中的傳播可能因共選擇而受到銅的影響。本研究選擇高密度聚乙烯(HDPE)薄膜作為生物膜生成的載體，並在HDPE表面形成生物膜後將HDPE薄膜暴露在含有不同銅離子濃度的緩衝溶液中，最後從HDPE薄膜表面收集生物膜並定量生物膜中的相關基因(包括β-內醯胺類、四環素類、大環內酯類、磺胺類ARGs、銅抗性基因(CRGs)、第1類整合子整合酶基因intI1和16S rRNA基因)和銅濃度，以探究抗性基因的生成程度是否由胞外銅物種組成所調控。結果顯示與對照組相比，低濃度銅、低濃度銅加EDTA、和高濃度銅加EDTA在第2天時pcoA相對豐度增加；與第2天相比，低濃度銅、低濃度銅加EDTA、和高濃度銅加EDTA在第5天的胞內銅濃度降低，說明胞內外生物可利用銅離子的濃度梯度可作為帶有pco操縱子的移動基因元件(MGE)水平基因轉移的驅動力。除此之外，blaCTX-M和pcoA之間的相關性高於其他ARGs和pcoA之間的相關性，基於ARGs和CRGs可同時存在於質粒等MGE，此相關性暗示blaCTX-M的傳播可能受到銅的影響；不僅如此，sul1的傳播也可能因第1類整合子而受到銅的影響，因與對照組相比，低濃度銅在第2天的sul1和intI1相對豐度同時增加。本研究的結果為HDPE生物膜中生物可利用銅離子濃度梯度藉由共選擇驅動抗生素耐藥性，提供了一定程度的證據與見解。

摘要(英)

Because plastic products are superbly durable and cheap, they quickly become practical items in life. Yet, massive increases in plastic production have resulted in plenty of waste plastics accumulated in natural habitats. Once the plastic enters the environment, the pits generated after weathering make the surface rough and reduce the hydrophobicity, which enables bacteria to colonize the surface and form biofilms. Bacteria in biofilms can share and spread antibiotic resistance genes (ARGs) through horizontal gene transfer (HGT). In addition, a biofilm on plastic particles can significantly enrich most heavy metals, including copper (Cu) that is widely used in industrial processes and animal husbandry. Hence, the spread of ARGs in biofilms on plastic debris may be affected by Cu through co-selection. In this study high-density polyethylene (HDPE) films were selected for biofilm formation; once biofilms formed on HDPE, HDPE films were exposed to buffer solution containing varying levels of Cu ions; biofilms were then collected from surfaces of HDPE films, and the related genes (including the β-lactam, tetracycline, macrolide, sulfonamide ARGs, Cu resistance genes (CRGs), class 1 integron-integrase gene intI1 and 16S rRNA gene) and Cu concentrations in biofilm were quantified to probe whether the generation of resistance genes would be regulated by extracellular Cu speciation. Results show that compared with controls, pcoA relative abundance on day 2 was increased in LC, LC + EDTA and HC + EDTA. Intracellular Cu concentrations on day 5 were decreased in LC, LC + EDTA and HC + EDTA compared with day 2, indicating concentration gradients of bioavailable Cu ions between intracellular and extracellular regions can be the driving force of HGT related to mobile genetic element (MGE) carrying pco operon. Furthermore, the correlation between blaCTX-M and pcoA was higher than that between other ARGs and pcoA, suggesting the spread of blaCTX-M could be affected by Cu due to co-existing of ARGs and CRGs in MGEs like plasmid. The spread of sul1 also could be affected by Cu through class 1 integron because sul1 and intI1 relative abundance on day 2 were simultaneously increased in LC compared with controls. This study provides preliminary evidence and insights into co-selection of antibiotic resistance driven by concentration gradients of bioavailable Cu ions in HDPE biofilm.

關鍵字(中)

★ 共選擇
★ 抗生素抗藥性
★ 生物可利用銅離子
★ 高密度聚乙烯薄膜
★ 生物膜

關鍵字(英)

★ Co-selection
★ Antibiotic resistance
★ Bioavailable cupric ions
★ HDPE films
★ Biofilms

論文目次

摘要 I
Abstract II
誌謝 III
Table of Contents IV
List of Figures V
List of Tables VIII
Chapter 1 Introduction 1
1.1.1 Microplastics in natural habitats 1
1.1.2 Biofilm formation on microplastics 2
1.1.3 Horizontal gene transfer in biofilm 3
1.1.4 Metal accumulation on biofilm-developed microplastics 4
1.1.5 Heavy metal-driven co-selection of antibiotic resistance 4
1.2 Research objective 5
Chapter 2 Materials and methods 6
2.1 Microcosm preparation 6
2.2 Biofilm detachment 6
2.3 Copper exposure 7
2.4 Biofilm collection 8
2.5 Copper analysis 8
2.6 Sample DNA extraction 8
2.7 Quantification of genes 9
2.8 Statistical analysis 11
Chapter 3 Results and discussions 12
3.1 Biofilm detachment tests 12
3.2 CRG clones and standard curves 15
3.3 Copper in HDPE biofilms 18
3.4 CRGs in HDPE biofilms 23
3.5 ARGs in HDPE biofilms 27
3.6 Relationships between CRGs, ARGs and copper 33
3.7 Environmental implications 40
Chapter 4 Conclusions 42
Chapter 5 Future works 43
References 44
Appendices 50
口試委員口試問題及回答 59

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指導教授

林居慶

審核日期

2022-9-6

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