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系統識別號 U0026-0812200912043152
論文名稱(中文) 台灣地區大腸桿菌超廣效乙內醯胺 抗藥菌株之乙內醯胺酶之分析
論文名稱(英文) Analysis of β-lactamases in Extended-Spectrum Cephalosporin-Resistant Escherichia coli in Taiwan
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 94
學期 2
出版年 95
研究生(中文) 陳宏謨
研究生(英文) Hung-Mo Chen
學號 chenhm
學位類別 碩士
語文別 中文
論文頁數 89頁
口試委員 口試委員-顏經洲
口試委員-柯文謙
指導教授-吳俊忠
中文關鍵字 大腸桿菌、超廣效性乙內醯胺酶、乙內醯胺酶 
英文關鍵字 Escherichia coli、extended-spectrum β-lactamase 
學科別分類
中文摘要 本研究從2003 年 3 月到 8 月收集 7 家醫學中心共 291 株大腸桿菌 (Escherichia coli) 臨床菌株,這些菌株對第三代頭孢子菌素抗生素具有較高的抗性。利用最小抑制濃度及紙錠擴散確認法等方法分析這些菌株中總共有 60.5% (176/291) 可產生超廣效性乙內醯胺酶。利用聚合酶連鎖反應及最小抑制濃度分析這些菌株中有 43.6% 擁有 plasmid-mediated AmpC 酵素。根據等電點電泳與聚合酶連鎖反應結果發現每一測試菌株中可能同時含有2或3種乙內醯胺酶對廣效性頭孢子菌素具有抗性。 291株大腸桿菌分離株中具 TEM, CTX-M, SHV ESBLs及CMY-2相關性酵素基因分別佔 81.7, 54.3, 9.3及43.6%。利用核酸序列定序發現 blaCTX-M 基因存在 5 種subtypes, blaCTX-M-3、blaCTX-M-9 、 blaCTX-M-14、 blaCTX-M-15、 blaCTX-M-19 等基因,其中以blaCTX-M-3 和 blaCTX-M-14 最為常見。而blaCTX-M-19 基因則首次在台灣被發現。從不同醫院隨機選取具 blaCTX-M-3 或 blaCTX-M-14基因菌株,以 隨機放大基因多型性圖譜分析可分別將其分為 14 種blaCTX-M-3 與 16 種 blaCTX-M-14 types。 根據此結果發現, 在不同醫院的分離株中 blaCTX-M-3 或 blaCTX-M-14 基因存在不同的基因圖譜。以同種接合實驗及質體分析發現該具有乙內醯胺酶質體可在相同種細菌間傳遞,且相似的抗藥性質體也可在不同醫院間傳播。


英文摘要 A total of 291 Escherichia coli isolates were collected from seven medical centers between March and August 2003. These isolates showed highly resistant to third generation of cephalosporins. The extended-spectrum β-lactamases (ESBLs) production was detected in 60.5% (176/291) of E. coli isolates by minimum inhibitory concentration (MIC) and disk diffusion confirmatory tests, and 43.6% of the E. coli isolates exhibited plasmid-mediated AmpC enzymes by PCR and MIC analysis. According the results of isoelectric focusing electrophoresis and PCR showed these isolates harbored two or three β-lactamases involved in resistance to extended-spectrum cephalosporins. In 291 isolates of E. coli, TEM (81.7%), CTX-M (54.3%), SHV ESBLs (9.3%), and CMY-2-related enzymes (43.6%) were detected. Nucleotide sequencing of blaCTX-M genes revealed the presence of 5 subtypes, which were blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, and blaCTX-M-19 genes. Among them,blaCTX-M-3 and blaCTX-M-14 were predominant in E. coli. The blaCTX-M-19 genes were firstly identified in Taiwan. The blaCTX-M-3- or blaCTX-M-14-positive strains were randomly selected from different hospitals. Fourteen types of blaCTX-M-3 and 16 types of blaCTX-M-14 were classified by randomly amplified polymorphic DNA (RAPD) analysis from these isolates. The results exhibited blaCTX-M-3 or blaCTX-M-14 genetic diversity among the isolates from different hospitals. The transconjugation experiments and plasmid analysis suggested these bla–positive plasmids could transfer to same species and interhospital spreading.


論文目次 目 錄
中文摘要…………………………………………………………………......I
英文摘要…………………………………………………………….. .…….II
誌謝………………………………………………………...……..……......III
目錄………………………………………………………...……..…….......Ⅳ
表目錄……………………………………………………………................Ⅵ
圖目錄………………………………………………………………......…..Ⅷ
符號及縮寫………………………………………………………….... ...... IX
第一章、緒論
I. 大腸桿菌簡介………………………………………………....……..….1
II. 抗生素(Antibiotics) ……………………………………………........…2
III.細菌之抗藥機制……………………………………..…..……..…..…11
IV. 乙內醯胺酶(-lactamases)………………………..……………… .….16
V. 產生 ESBL 之相關因 ……………………………………...…...……19
VI. 世界及台灣 ESBL-producing菌株發生之概況 …………...……….24
VII.研究目的.………………………………..……....…….….…...…......25
第二章、材料與方法
I. 本研究各實驗所使用之藥品、溶液配方及儀器…………………. ..26
II. 大腸桿菌菌株來源、培養與保存………………….……………..…26
III.大腸桿菌之抗藥性測試…………………………..…..…………….. 26
IV. 大腸桿菌染色體 DNA 之抽……………………………………….28
V. 大腸桿菌質體 DNA 之抽取…………………………….…………. 29
VI. 聚合酶連鎖反應(Polymerase chain reaction, PCR)…….......…...29
VII. DNA 序列解序 (DNA Sequencing)………………………. …...…30
VIII. 洋菜膠體電泳分析………………………………… .. ……….......30
IX. 等電點蛋白膠體電泳 (IEF) 分析…………………….………........31
X. Randomly amplified polymorphic DNA (RAPD)分析……….............31
XI. 接合作用 (Conjugation) ...........................................32
XII. 影像處理.................................................32
第三章、結果
I. MIC susceptibility test 及 double disk confirmatory test ...33
II. Non-ESBL 與 ESBL-producing 不同beta-lactamase 基因的分佈與分型34
III. 菌株分型..........................................................36
IV. 接合實驗 (Conjugation)測試大腸桿菌菌株所攜帶transferred plasmid 之分型..37
第四章 討論………………………………………………………………...39
第五章 結論……………………………………………………….……......45
參考文獻……………………………………………………...……...……...47
表……………………………………………………...……………..............63
圖……………………………………………………...………………..........75
附錄……………………………………………………...………………......82
表 目 錄
Table 1 Specific PCR primer sets used in PCR analysis…………..….....63
Table 2 Distribution of ESBL-producing types among 291 Escherichia coli
isolates collected from seven medical centers in Taiwan.... ..64
Table 3 The antimicrobial susceptibility assay of 291 Escherichia coli
isolates in Taiwan……………………..65
Table 4 Characteraction of antimicrobial susceptibility of 291 Escherichia
coli isolates in Taiwan……... ………..…….66
Table 5 Classification of pI types by IEF analysis among 291 Escherichia coli
isolates collected from seven medical centers in Taiwan…………67
Table 6 Distribution of blaTEM gene among 291 Escherichia coli isolates
collected from seven medical centers in Taiwan…………………68
Table 7 Distribution of blaSHV-related gene among 291 Escherichia coli
isolates collected from seven medical centers in Taiwan…………69
Table 8 blaCTX subtypes detected in randomly selected E. coli (n=128)
isolates collected from seven medical centers in Taiwan ………70
Table 9 Distribution of AmpC enzymes among 291 Escherichia coli isolates
collected from seven medical centers in Taiwan ………………71
Table 10 Distribution of blaCTX-M-14 RAPD typing patterns from 20 Escherichia
coli tested isolates……………………….72
Table 11 Distribution of blaCTX-M-3 RAPD typing patterns from 16 Escherichia
coli tested isolates …………………………….73
Table 12 Distribution of bla CMY-2 RAPD typing patterns from 15 Escherichia
coli tested isolates…………………………..…74
圖 目 錄
Fig. 1. (A) Double-disk synergy tests
(B) Disk diffusion confirmatory tests……… ……… 75
Fig. 2. Classification of pI types by isoelectric focusing analysis (IEF)76
Fig. 3. Distribution of cefotaxime (A) and ceftazidime
(B)MICs of 291 Escherichia coli ………………………………..….77
Fig. 4. The predicted size of antimicrobial resistant genes was
determined by PCR.……………………..……......……78
Fig. 5. The PCR NheI restriction pattern of SHV genes in Escherichia coli..79
Fig. 6. RAPD patterns of 15 CMY-2 like (A)16 CTX-M-3-producing (B)20 CTX-M-
14-producing (C)and Escherichia coli isolates randomly selected from
different hospitals. ..............................80
Fig. 7. EcoRI restriction patterns of conjugative plasmids from 13 CMY-2 like
(A), and 18 CTX-M-14-producing (B) Escherichia coli isolates ……81
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