臺灣博碩士論文加值系統

近年來臨床上抗藥性細菌大幅出現，尤其是對第三代頭孢菌素 (cephalosporins) 抗藥性腸內菌越來越多。篩選對第三代頭孢菌素抗藥之菌株，自林口長庚醫院受感染病人的檢體中分離出Salmonella enterica serotype Choleraesuis (SC72) 和Salmonella enterica serotype Anatum (SA323)，都攜帶ceftriaxone抗藥性之質體 (分別為pSC72-1及pSA323-1)，這是台灣地區罕見具ceftriaxone抗藥性之沙門氏菌。針對兩質體進行聚合酶鏈鎖反應及序列分析之研究，發現兩質體都帶有blaCTX-M-3抗藥基因，且抗藥基因是由transposon所攜帶。兩質體分別和Salmonella enterica serotype Typhimurium (LBNP4417) 及E. coli (HB101) 進行接合實驗，顯示有接合能力 (conjugative)。進一步分析質體間之差異，pSC72-1大小約為74 kb，而pSA323-1大小約為82 kb，分別利用三種不同限制酶，以單一或混合兩種限制酶做切割，呈現限制酶多樣性，再以限制酶切割片段之差異性構築其物理圖譜。由南方墨點雜合法結果顯示兩質體除了攜帶同種抗藥基因之外，其相關性不高。
由於臨床上分離出攜帶blaCTX-M-3抗藥基因的菌株多為E. coli 和Klebsiella pneumoniae，從林口長庚醫院受感染病人的檢體中分離出4株E. coli及5株K. pneumoniae，由聚合酶鏈鎖反應、序列分析及南方墨點雜合法的結果顯示其抗藥基因blaCTX-M-3位於質體上，也是由transposon所攜帶。利用接合實驗分離出具有接合能力的質體pKP104-1和pKP116-1，以限制酶做切割，兩質體之限制酶片段一致，和pSC72-1、pSA323-1的限制酶片段比較，亦呈現限制酶多樣性。由pKP104-1和pKP116-1限制酶切割的結果顯示攜帶blaCTX-M-3抗藥基因之質體已經在K. pneumoniae菌株之間傳播。由已知nikA基因序列設計引子，進行聚合酶鏈鎖反應及序列分析，其結果顯示除了HB101/323-1之質體 (pSA323-1) 外，其他臨床分離菌株質體的接合系統 (conjugation system) 都一樣。
根據我們的了解，此研究應是台灣地區第一個構築S. Choleraesuis 和S. Anatum抗藥質體之物理圖譜。希望藉由此研究進一步了解其抗藥性及流行病學上的意義，並控制由 S. Choleraesuis 和S. Anatum所引起的感染。

More and more drug-resistant clinical isolates have emerged in recent years, especially significant numbers of Enterobacteriaceae showed resistance to the third generation cephalosporins. Through screening over hundreds of clinical isolates from Chang Gung Memorial Hospital, we found two isolates, Salmonella enterica serotype Choleraesuis, SC72, and Salmonella enterica serotype Anatum, SA323, both of which carried ceftriaxone-resistant plasmids, designated as pSC72-1 and pSA323-1, respectively. It is unusual for Salmonella clinical isolates from Taiwan to exhibit ceftriaxone-resistance. Further PCR and sequencing analysis revealed that both plasmids harbored blaCTX-M-3 gene conferring the resistance phenotype. Both plasmids, pSC72-1 and pSA323-1, were proved to be conjugative through conjugation experiments, which were carried out using Salmonella enterica serotype Typhimurium (LBNP4417) and Escherichia coli ( HB101 ) as recipients. The differences between two plasmids were investigated further. The sizes of pSC72-1 and pSA323-1 was 74-kb and 82-kb, respectively. The physical maps of both plasmids were also constructed from overlapping the restriction fragments digested with various combinations of restriction enzymes. Results from restriction fragment profiles and Southern hybridization suggested that these two plasmids are not closely related, although both carried the same drug resistance gene blaCTX-M-3.
Since most clinical isolates which carried blaCTX-M-3 were either E. coli or Klebsiella pneumoniae, four E. coli isolates and five K. pneumoniae isolates, all ceftriaxone-resistant, were selected from Linkou Chang Gung Memorial Hospital. In order to see if these isolates possess the same drug resistance constructs, PCR assays, DNA sequencing, and Southern blot experiments were performed. The results showed that the plasmids of these E. coli and K. pneumoniae isolates harbored identical blaCTX-M-3 downstream of tnpA. Two of the conjugative plasmids pKP104-1 and pKP116-1, were further digested with various restriction enzymes and revealed the same pattern of restriction fragment profiles. The results suggested that the dissemination of ceftriaxone-resistant plasmids carrying blaCTX-M-3 among clinical isolates of K. pneumoniae. Primers specific for nikA were designed to characterize these plasmids. All plasmids except pSA323-1 contained the same conjugation system.
To our knowledge, this is the first physical map of ceftriaxone-resistant plasmid from clinical isolates of S. Choleraesuis or S. Anatum in Taiwan. This pioneer research work would help to the understanding of epidemiology and the control of infections caused by S. Choleraesuis and S. Anatum.

目錄
封面內頁
簽名頁
授權書 ………………………………………..…………………. iii
中文摘要 …………………………………..………………….. iv
英文摘要 ………………………………..…………………….. vi
誌謝 …………………………………...……………………… viii
目錄 ………………………………...………………………… ix
圖目錄 ………………………………...………………………… xi
表目錄 ………………………………...………………………… xiii
第一章 緒論 ………………………………………………….. 1
1.1 沙門氏菌 ( Salmonella ) 簡介 ..................................... 1
1.2 沙門氏菌之抗藥性............................................................ 2
1.3 頭孢菌素 (cephalosporin) 簡介 ..................................... 3
1.4 β-內醯胺酶 (β-lactamases) 簡介.................................... 4
1.4.1 超廣效性β-內醯胺酶 (extended-spectrum β-lactamases)
....................................................................................... 5
1.5 研究動機及目的 ............................................................... 6
第二章 實驗材料及方法 ........................................................ 7
2.1 抗藥基因分析 .............................................................. 7
2.1.1 菌株篩選及培養 ....................................................... 7
2.1.2 洋菜膠電泳 (agarose gel electrophoresis) ............... 7
2.1.3 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR)... 8
2.1.4 聚合酶鏈鎖反應產物之純化及回收......................... 9
2.2 質體分析 ...................................................................... 9
2.2.1 細菌質體快速檢驗 .................................................. 9
2.2.2 接合實驗 (conjugation) .............................................. 10
2.2.3 大量抽取質體DNA ................................................. 10
2.2.4 限制酶切割 (Restriction enzyme digestion) ......... 12
2.2.5 DNA片段大小之分析 .......................................... 12
2.2.6 DNA片段分離及純化 .......................................... 12
2.2.7 質體DNA之物理圖譜 .......................................... 13
2.2.8 南方墨點雜合法 (Southern hybridization) ............... 13
第三章 結果與討論................................................................... 16
3.1 抗藥性分析 .................................................................... 16
3.2 質體分析 .................................................................... 18
3.3 E. coli和K. pneumoniae質體之分析 ...................... 22
3.4 抗藥質體之接合能力 ................................................ 25
第四章 結論 .......................................................................... 26
參考文獻 ............................................................................... . 52
附錄 ....................................................................................... 59
圖目錄
圖一、β-內醯胺環結構 ............................................................... 27
圖二、以SC72及SA323為DNA模板之聚合酶鏈鎖反應之
電泳圖 (1) 引子為CTX-F和CTX-R1，(2) 引子為
TPO-F和CTX-R1............................................................ 28
圖三、抗藥基因 bla CTX-M-3 之結構及引子設計之位置 ........... 29
圖四、以Kado and Liu法檢驗菌株所帶之質體 .................. 30
圖五、質體pSC72-1以BglⅡ、HindⅢ和ScaⅠ單一或混合
兩種限制酶酵素於37°C切割2.5h，再以50V、6.5h
進行電泳分析之電泳圖 ................................................... 31
圖六、質體pSA323-1以BamHⅠ、HindⅢ和BglⅡ單一或混
合兩種限制酶於37°C切割2.5h，再以50V、6.5h進
行電泳分析之電泳圖 ................................................... 32
圖七、(a) 質體pSC72-1，(b) 質體pSA323-1之物理圖譜.... 33
圖八、(1) 質體pSA323-1和質體pSC72-1以HindⅢ和BglⅡ
單一或混合兩種限制酶於37°C切割2.5h，再以50V、
6.5h進行電泳分析之電泳圖，(2) 將DNA轉印到轉漬
膜上，以CTX為引子之PCR產物為標定成雜合探針
的南方墨點雜合法之結果 ........................................... 34
圖九、(1) 質體pSA323-1和質體pSC72-1以HindⅢ和BglⅡ
單一或混合兩種限制酶於37°C切割2.5h，再以50V
、6.5h進行電泳分析之電泳圖，(2) 將DNA轉印到
轉漬膜上，以pSA323-1為探針的南方墨點雜合法之
結果................................................................................... 35
圖十、(1) 質體pSA323-1和質體pSC72-1以HindⅢ和BglⅡ
單一或混合兩種限制酶於37°C切割2.5h，再以50V
、6.5h進行電泳分析之電泳圖，(2) 將DNA轉印到轉
漬膜上，以pSC72-1為探針的南方墨點雜合法之結果
......................................................................................... 36
圖十一、以K. pneumoniae和E. coli為DNA模板之聚合酶
鏈鎖反應之電泳圖 (1) 引子為CTX-F和CTX-R1
，(2) 引子為TPO-F和CTX-R1 .............................. 37
圖十二、(1)以Kado and Liu法檢驗E. coli和K. pneumoniae
所帶質體之電泳圖，(2) 將DNA轉印到轉漬膜上，
以CTX為引子之聚合酶鏈鎖反應的產物標定成雜
合探針的南方點墨雜合法之結果 ............................... 38
圖十三、以Kado and Liu法檢驗菌株所帶之質體 .................. 39
圖十四、質體pKP104-1和質體pKP116-1以HindⅢ和BglⅡ
切割之電泳圖 ......................................................... 40
圖十五、以KP18、K P51、KP104、KP116、KP166、EC86、
EC365、EC548、EC626、HB101/323-1、和
LBNP4417/72-1為DNA模板之聚合酶鏈鎖反應之
電泳圖 .................................................................... 41
表目錄
表一、β-內醯胺酶之分類 ........................................................ 42
表二、所有的臨床菌株及實驗室菌株 .................................... 43
表三、聚合酶鏈鎖反應之反應混合液 (1)，反應條件 (2) ... 44
表四、本實驗中聚合酶鏈鎖反應所用的引子及其鹼基序列.. 45
表五、SC72和SA323之抗藥性 .......................................... 46
表六、質體pSC72-1以BglⅡ、HindⅢ和ScaⅠ單一或混合
兩種限制酶酵素切割之限制酶切割片段大小 .......... 47
表七、質體pSC72-1之 BglⅡ限制酶片段再經HindⅢ和
ScaⅠ切割之片段大小 .................................................. 48
表八、質體pSA323-1以BamHⅠ、HindⅢ和BglⅡ單一或
混合兩種限制酶酵素切割之限制酶切割片段大小 .... 49
表九、質體pSA323-1之 BamHⅠ限制酶片段再經HindⅢ
和BglⅡ切割之片段大小 ........................................... 50
表十、本實驗之E. coli和K. pneumoniae菌株之抗藥性 ... 51

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