臺灣博碩士論文加值系統

治療細菌感染時抗菌藥物的普遍使用，往往伴隨細菌抗藥性的增加及多重抗藥菌株頻繁的出現，使得用藥治療益形困難。而細菌發展出多重抗藥性可能與細菌能獲得並傳遞抗藥基因有密切的關係。Integron是近年來被認為繼抗藥性質體與轉位子外，另一個與細菌獲得新抗藥基因及抗藥基因散佈有關的機制。許多不同的抗藥基因是位於一種mobile element─稱之為基因片匣內，integron可主導基因片匣以特定部位重組作用的方式嵌入或移出integron，造成integron所攜帶之抗藥基因的改變及抗藥基因的散佈，因此integron及基因片匣可能在臨床菌株抗藥性扮演著重要角色。
目前已發現的integron有四型，以class 1 integron在臨床感染菌株中最常見，本研究為探討臨床菌株中integron之存在及特性，以聚合連鎖反應增幅包含class 1 integron基因片匣的片段，來偵測自臨床檢體分離出的104株大腸桿菌class 1 integron的存在情形，PCR的結果有54株（佔52％）可增幅得到PCR 產物，增幅片段的大小在1000 bp∼3000 bp。進一步以南方雜交法分析class 1 integron在菌體內存在的位置，發現integron可位於質體上。增幅片段經純化後，利用核酸直接定序讀得增幅片段之基因序列，再與GenBank database內的已知基因進行相似性比對，確定class 1 integron內所帶基因片匣的種類、數目及組合方式。結果顯示class 1 integron所攜帶的基因片匣種類，除了一個功能尚未知的open reading frame，orfF以外，其餘均為抗藥性基因，包括四個trimethoprim抗藥基因：dfrIa、dfrV、dfr12和dfr17；aminoglycosides類藥物的抗藥性基因（aadA1a、aadA2、aadA4和aadB）；以及chloramphenicol抗藥基因cmlA；erythromycin抗藥基因ereA2與β-lactamase PSE-1基因blaP1。其中dfr17 和aadA4是新發現的基因片匣，dfr17為一新型之trimethoprim抗藥性DHFR基因，與已知的dfrVII有91％的相似性，可歸屬於最常見的family 1 DHFR中之一員。至於aadA4則可對spectinomycin和streptomycin產生抗藥性，與已知的aadA3基因有95％之相似性。在本實驗中，Integron一般攜帶的基因片匣數目為1∼3個，基因片匣的組合方式則以dfr12，orfF和aadA2的組合最常見，攜帶dfr12-orfF-aadA2這三個基因片匣的integron可在33株E. coli菌株中偵測到（佔class 1 integron陽性菌株的61％）。此外本研究亦發現單一菌株內可同時存在兩個class 1 integron，且它們分別攜帶不同的基因片匣。
進一步以基因選殖和感受性試驗可證實integron所攜帶之抗藥基因片匣會表現在菌株之抗藥性上，但抗藥基因片匣無法完全涵蓋菌株的所有抗藥性，表示菌株內尚有其他抗藥機制存在。質體接合反應顯示class 1 integron及抗藥基因片匣可隨質體一起轉移，表示integron及抗藥基因片匣可藉質體之水平轉移方式在菌株間散佈。脈動電泳分析則發現攜帶相同抗藥基因片匣的菌株並無同源性存在，可見抗藥基因片匣早已在不同菌株間散佈了。
本研究亦一併偵測class 2 integron的存在及所攜帶之基因片匣，以聚合連鎖反應增幅class 2 integrase基因intI2，結果在104株E. coli中，僅10株（佔10％）具有intI2基因，依攜帶基因片匣的組合方式可分為3群class 2 integron。其中Group 1之基因片匣組合為dfrIa-sat-aadA1a，與轉位子Tn7相同；Group 2則攜帶dfrIa與sat片匣，卻不具aadA1a片匣，這種class 2 integron片匣組合型式是文獻中未曾報告過的，顯然class 2 integron攜帶之抗藥基因片匣組合有了變化；另外Group 3 integron上則除了sat與aadA1a片匣以外，多嵌入了一段功能未知的open reading frame。
比較integron陽性菌株與integron陰性菌株抗藥性結果，發現不論抗藥基因是否位於integron內，具有integron的菌株呈現多重抗藥性的比例遠高於不具integron的菌株，顯示integron之存在與菌株多重抗藥性有密切的關係。
綜合本研究的結果可知，integron及抗藥基因片匣在臨床E. coli菌株中普遍存在，不僅可讓菌株表現出抗藥性，並可藉質體在菌株間傳遞以達到散佈之目的。而抗藥基因片匣的種類及其出現頻率則適度反映了常用藥物所給予的篩選壓力。此外本研究發現integron均攜帶了抗藥基因，同時也發現了新型之抗藥基因片匣及片匣組合的變化，顯示在藥物持續的選擇壓力存在下，不僅抗藥基因本身發生了變異，也促使片匣的移動，造成細菌抗藥性的變化，更可能導致攜帶抗藥基因片匣的integron移至也攜帶抗藥基因的質體或轉位子上，因而使帶有integron的菌株呈現較高比例的多重抗藥性。未來研究的方向應更深入了解抗藥基因片匣形成的途徑及其與integron間之作用機轉，還有它們與轉位子和質體的相關性，以找出有效控制抗藥基因散佈的方法。

Increasing use of various drugs for the treatment of infectious diseases has resulted in an increase of drug-resistant clinical isolates. Acquisition and dissemination of antibiotic-resistant genes has been a major factor in the development of multiple drug resistance among bacteria. Many of the known resistance genes are contained in mobile elements called gene cassettes. Cassettes can be integrated into or excised from their receptor elements, the integrons, via site-specific recombination catalyzed by an integron-encoded integrase. Because of the ability of integrons to acquire gene cassettes and convert them into functional genes, integrons and antibiotic-resistant gene cassettes may play an important role in the development and dissemination of multiple antibiotic-resistant bacterial strains.
In this study, the presence of class 1 integrons in clinical isolates of Escherichia coli was detected by PCR. Of 104 E. coli isolates, 54 (52%) carried detectable class 1 integrons, with inserted DNA regions ranging in size from 1000 bp to 3000 bp. These integrons were located on plasmids, as demonstrated by Southern hybridization. DNA sequencing was used to identify the genetic content of the integron-variable regions. Different class 1 integrons contained various numbers, kinds and combinations of gene cassettes within their variable regions. The gene cassettes found included those encoding resistance to trimethoprim (dfrIa, dfrV, dfr12 and dfr17), aminoglycosides (aadA1a, aadA2, aadA4 and aadB), chloramphenicol (cmlA), erythromycin (ereA2) and -lactams (blaP1). Two new gene cassettes, dfr17 and aadA4, inserted in a class 1 integron are reported in this study. The dfr17 cassette encodes trimethoprim resistance and has 91% identity with the dfrVII dihydrofolate reductase gene. The aadA4 cassette confers resistance to spectinomycin and streptomycin and shows 95% identity with the aadA3 gene. An integron carrying three inserted cassettes, dfr12-orfF-aadA2, was present in 61% (33/54) the isolates with class 1 integrons (33/54).
Antimicrobial susceptibility tests and further cloning and expression of the amplicons containing the cassette regions demonstrated the gene cassettes could express their phenotypes. The conjugation experiment revealed that the transfer of class 1 integrons and gene cassettes could be plasmid-mediated. Furthermore, isolates carrying identical class 1 integrons were found to have genetic backgrounds unrelated as detected by the PFGE, suggesting the horizontal integron transfer.
Class 2 integrons were also sought in these 104 E. coli isolates by PCR with intI2-specific primers. Ten isolates (10%) were intI2-positive and three different groups of class 2 integrons were identified further. The group 1 class 2 integron carried the same cassettes (dfrIa, sat and aadA1a) as transposon Tn7. The group 2 integron carrying dfrIa and sat cassettes but without aadA1a has never been reported, indicating a novel combination of gene cassettes within class 2 integrons. Besides sat and aadA1a, the group 3 integron carried an additional open reading frame encoding an unknown product.
The present study reveals that integrons are widespread in clinical E. coli isolates and their location on plasmids could contribute to the horizontal dissemination of the antibiotic resistance gene cassettes in integrons. The types, combinations and frequency of the gene cassettes in integrons may reflect the specific selective pressures to which the isolates were exposed. This information could provide useful surveillance data for relation to antibiotic usage information. This study also demonstrated that all of the integrons contained antibiotic-resistant gene cassettes and the presence of new cassettes and novel combinations of gene cassettes. This indicates that continued antibiotic selective pressure does not only favor the incorporation of gene cassettes in integrons but also select new resistance gene variants and different combinations of cassettes. Moreover, such selection pressure may also promote the association of integrons carrying more than one antibiotic-resistant gene cassette with other genetic elements containing resistance determinants, such as plasmids and transposons, making multiple drug resistance more common in integron-positive isolates than integron-negative isolates as found in this study. Further studies on understanding the process of cassette genesis and its interaction with integrons, relating to transposons and plasmids could provide new insight about resistance spread control.

封面
目
中文摘要
英文摘要
緒言
一、前言
二、Integron與基因片匣之發現
三、Integron之基本特徵及種類
(一)Integron構造之基本特徵
(二)Integron的種類
四、基因片匣的構造及片匣的重組部份
(一)基因片匣的構造
(二)基因片匣的重組部份-59base element
五、Integron做為表現載體與基因片匣之表現
六、Integron與基因片匣之特定部份重組作用
七、Integron與基因片匣在細茵抗藥性扮演之角色
八、本論文研究目的
材料與方法
一、實驗茵株月來源與鑑定
二、茵株之藥物感受性試驗
三、Class 1 intergron 之偵測(Detection for the presence of class 1 integrons)
1.引言的設計(primer desige)
2.模板DNA之製備(Preparation of template DNA for PCR)
3.聚合□鏈反應
4.瓊脂醣明膠電泳(Agarose gel electrophoresis)
5.PCR產物分子量之計算
四、Class 1 integron 於茵株內存在位置之分析
(一)質體DNA之抽取
(二)瓊脂醣明膠電泳
(三)南方雜交分析 (Southern hybrizization)
1.DNA的轉印(Blotting)
2.非輻射性dna探針標記
3.前雜交(Prehybridization)
4.雜交(Hybridization)
5.雜交後清洗(Post-hybridzation washes)
6.偵測以DIG標記之已雜交探針(Detection of DIG-labeled, hybridized probe)
7.自動放附顯影(Autoradiography)
五、Class 1 integron 轉移之分析─接合試驗 (conjugation )
六、Class 1 integron 攜帶之基因片匣之分析 (Identification of genecassettes inserted in calss 1 integrons)
(一)PCR產物的純化
(1)單一PCR產物之純化
(2)非單一之PCR產物純化
(二)核酸自動定序分析
1.Cycle Sequencing 及反應產物之純化
2.自動定序分析儀之開機與電泳用玻璃之準備
3.電泳凝膠的製作
4.電泳前預跑(Pre-run gel)
5.電泳前之檢體處理
6.電泳及訊息之接收與分析
(三)基因序列之電腦分析
七、抗藥基因片匣之抗藥性表現分析
(一)增輻片段之選殖(Amplicon cloning)
1.增輻片段與載體(vector)之連接作用(ligation)
2.X-gal plate之製備
3.勝任細胞(competent cell)之製備
4.重組質體(recombinant plasmid)之轉形作用
5.轉形茵株之篩選
(二)摧帶增輻片段之轉形茵株抗藥性表現之分析
八、摧帶抗藥基因片匣茵株之脈動電泳分析
(一)樣品製備 (Sample preparation)
(二)限制□的消化與DNA的脈動電泳(Restriction enzymedigestion and PFGE)
九、Class 2 integron 及其所攜帶之基因片匣之分析
十、統計分析
結果
一、Class 1 integron 於E.coli 茵株中之存在情形
二、Class 1 integron 於茵株內的存在位置
三、Class 1 integron 所攜帶之基因片匣
(一)基因片匣特徵之識別
(二)基因片匣之種類
(三)基因片匣之組合排列
(四)基因片之出現頻率
(五)新型抗藥基因片匣之發現
1. 1600bp之增輻片段攜帶新型抗藥基因片匣
2. dfr 17 cassette
3. aadA14 cassette
四、抗藥基因片匣之表現及與茵株抗藥性之相關性
(一)抗藥基因片匣之表現
(二)E.coli茵株之抗藥性
五、Class 1 integron之轉移
六、攜帶抗藥基因片匣株之脈動電泳分析
七、Class 2 integron 所攜帶之基因片匣
八、Integron陽性茵株與integron 陽性茵株抗藥性之比較
討論
一、Class 1 integron 之存在情形
二、Class 1 integron 攜帶之抗藥基因片匣
三、Integron 的轉移與抗藥基因片匣的散佈
四、抗藥基因片匣與茵株抗藥性之相關性
五、攜帶之抗藥基因片匣
六、Integron存在對茵株抗藥性的影響
七、結論
圖
表
附圖
附表
參考文獻
與本論文相關期刊發表

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