臺灣博碩士論文加值系統

Integron與基因片匣（gene cassette）是與細菌獲得抗藥基因及抗藥基因散佈有關的機制，可能在細菌抗藥性扮演著重要角色。本研究主要探討大腸桿菌中與抗藥性相關之class 2 integron及其所攜帶的基因片匣。首先，利用聚合酶鏈鎖反應（PCR）增幅class 2 integron的integrase基因intI2，結果在193株大腸桿菌中，有30株（佔16％）可得到一預期的440 bp增幅片段，表示帶有class 2 integron。進一步利用PCR增幅class 2 integron上包含基因片匣的片段，來分析此30株大腸桿菌的class 2 integron攜帶的基因片匣，結果得到長約4 kb、3 kb及4.3 kb三種不同大小的增幅片段，將之區分為GroupⅠ、Ⅱ、Ⅲ三群。增幅片段經DNA定序及核酸限制酶切割分析，確定class 2 integron內所帶基因片匣的種類及組合型式。GroupⅠ（13株）約4 kb的增幅片段上抗藥基因片匣組合為dfrIa-sat-aadA1a，分別為trimethoprim、streptothricin及streptomycin的抗藥基因；GroupⅡ（16株）約3 kb的增幅片段其抗藥基因片匣組合為dfrIa-sat，卻不具aadA1a片匣，此種class 2 integron片匣組合型式是文獻中未曾報告過的；GroupⅢ（1株）約4.3 kb的增幅片段所具有之基因片匣除了sat與aadA1a片匣外，還多了一段目前仍不知功能的open reading frame。此外，藥物感受性試驗的結果發現E. coli菌株可表現出基因片匣所帶有的抗藥性，顯示class 2 integron及其攜帶之抗藥基因片匣與菌株的抗藥性有關。
為進一步探討class 2 integron與轉位子Tn7的關係，以位於Tn7左右兩端點處的引子分別與integron各個基因片匣的特異性引子進行PCR，其PCR產物經DNA定序及核酸限制酵素分析證實class 2 integron皆位於轉位子Tn7之上。同時，本研究發現其中有9株Group Ⅱintegron，它們所在的轉位子Tn7在轉位基因tnsE和tnsD間多嵌入了一段插入序列（insertion sequence）IS100，導致Tn7序列產生了改變，此亦為文獻中未曾報告的發現。
此外，曾有文獻報告指出在class 2 integron的intI2 基因部份含有一個內部終止密碼 ( internal stop codon ) －TAA，可能導致intI2基因無法產生有活性的integrase產物。因此，本研究也初步分析此30株含有class 2 integron的大腸桿菌其intI2基因序列， 發現確實皆有此內部終止密碼存在。
綜合本研究的結果顯示，菌株都能表現出基因片匣所攜帶的抗藥性，表示class 2 integron及所帶的抗藥基因片匣可造成菌株的抗藥性。另外，實驗中所發現的integron確實位在轉位子Tn7上，所以可能藉由轉位子在細菌間散佈。而insertion sequence的存在，則可能造成integron與轉位子構造甚至抗藥基因的變化。而本研究所發現的有別於已知的class 2 integron所具有的基因片匣組合型式，顯示class 2 integron攜帶的抗藥基因片匣組合有變化的可能。至於intI2基因所包含的內部終止密碼對integrase活性及片匣排列組合可能的影響，及integrase基因作用及基因片匣嵌入、移出integron的機轉將再進一步探討。

In this study, Escherichia coli isolates were investigated to demonstrate the prevalence and characteristics of class 2 integrons and the gene cassettes in these organisms. The presence of class 2 integrons in clinical isolates of E. coli was detected by PCR, which amplified a 440-bp fragment of the integrase gene, intI2. Of 193 E. coli isolates, 30 (16%) contained class 2 integrons. Class 2 integron cassette regions in these 30 isolates were amplified. Three kinds of amplicons with different sizes of 4 kb (13 isolates)、3 kb (16 isolates) and 4.3 kb (1 isolates) were obtained and defined as groupⅠ、Ⅱand Ⅲ, respectively. DNA sequencing and restriction enzyme analysis were performed to identify the gene cassettes inserted in the class 2 integrons. The groupⅠclass 2 integrons (amplicons of 4 kb) carried dfrIa, sat and aadAla cassettes, conferring resistance to trimethoprim, streptothricin and streptomycin, respectively. The groupⅡ (amplicons of 3 kb) carried dfrIa and sat cassettes, but without aadAla. This is a novel combination of gene cassettes within class 2 integrons and has never been reported. The group Ⅲ (amplicons of 4.3 kb) carried sat, aadAla and an additional open reading frame encoding an unknown product. Antimicrobial susceptibility tests showed that cassettes carrying resistance genes were expressed phenotypically. This indicated that gene cassettes are associated with drug resistance.
To understand the correlation between transposon Tn7 and class 2 integrons, PCRs were carried out to amplify the fragments with primers specific for each gene cassette and the termini of Tn7. Amplicons were obtained from each PCR reaction and then subjected to DNA sequencing and restriction enzyme analysis. The results indicated that class 2 integrons were located on transposon Tn7. Meanwhile, we also found that groupⅡintegrons in Tn7 from 9 isolates contained an insertion sequence IS100 between transposition genes tnsE and tnsD, which lead to the variation of Tn7. This has never been reported in the literature.
In addition, it has been mentioned that the intI2 gene of class 2 integrons contains an internal stop codon (TAA), which may inhibit the IntI2 integrase activity. Therefore we also examined the presence of this internal stop codon in the intI2 gene in these 30 E. coli isolates. DNA sequencing of intI2 gene verified the presence of the internal stop codon.
In conclusion, the present study confirmed that the gene cassettes in the class 2 integrons could contribute to drug resistance in bacteria. The location of class 2 integrons on transposon Tn7 allow their dissemination through transposition along with the drug-resistant gene cassettes. The existence of insertion sequence may lead to the structure variation of integrons or transposons. The novel combination of gene cassettes found in this study suggests the possibility of variation of gene cassettes carried by class 2 integrons. Furthermore, the effects of the internal stop codon in intI2 gene on the integrase activity and cassette combination need further elucidation to understand the mechanisms for cassette movement catalyzed by integrase.

中文摘要 1
英文摘要 4
緒論 7
一、前言 7
二、Integron與基因片匣之發現 8
三、Integron之結構與種類 10
（一）Integron之結構 10
（二）Integron之種類 10
四、基因片匣之結構與特異性重組部位 14
（一）基因片匣之結構 14
（二）基因片匣的重組部位－59-base element；attC site 16
五、特定部位重組作用 17
六、Integron表現抗藥基因片匣的情形 17
七、Integron及基因片匣與細菌抗藥性的關係 18
八、研究目的 18
材料與方法 20
一、實驗菌株之來源與鑑定 20
二、藥物感受性試驗 20
三、菌體DNA之抽取 21
四、Class 2 integron的偵測 23
（一）聚合酶鏈鎖反應（Polymerase chain reaction） 23
（二）瓊脂醣明膠電泳（Agarose gel electrophoresis） 25
（三）核酸限制酶切割試驗 26
五、Class 2 integron基因片匣的分析 27
（一）聚合酶鏈鎖反應 27
（二）PCR產物的純化 28
（三）核酸自動定序分析 29
（四）基因序列的電腦分析 30
1.基因序列相似性比對 30
2.限制酶切割位置分析 30
（五）限制酶切割試驗 31
六、Class 1 integron的偵測及其所帶之基因片匣分析 32
七、Class 2 integron與轉位子Tn7的關係 32
（一）聚合酶鏈鎖反應 32
1.增幅Tn7左右端至各個基因片匣的片段 32
2.增幅cassette region下游至Tn7左右端的片段 34
八、Class 2 integron之integrase基因intI2內部終止密碼（internal stop codon）的偵測 34
結果 36
一、Class 2 integron於E. coli菌株之存在情形 36
二、Class 2 integron攜帶之基因片匣 36
三、Class 1 integron於30株E. coli菌株之存在情形及其所攜帶之基因片匣 38
四、E. coli菌株之抗藥性 38
五、Class 2 integron與轉位子Tn7的關係 39
六、intI2基因內部終止密碼的偵測 41
討論 42
圖 49
表 60
附圖 66
附表 74
參考文獻 75

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