臺灣博碩士論文加值系統

本研究是針對乙內醯胺或非乙內醯胺類藥物呈抗藥性之Enterobacter cloacae (E. cloacae)的菌株，進行超廣效性乙內醯胺酶盛行率之探討。從台灣中部大型醫院收集的100株E. cloacae經double disc試驗、E-試驗及聚合酶連鎖反應發現有31（31%）株菌攜帶有超廣效性乙內醯胺酶。此31株菌對乙內醯胺類藥物具抗藥性，但對imipenem則為敏感性。31株攜帶有超廣效性乙內醯胺酶菌株之質體，以聚合酶連鎖反應和等電點聚焦電泳顯示，23株（74%）具SHV的基因，23株（74%）攜帶有CTX-M之基因，同時帶有CTX-M及SHV之基因的則有16株（52%），並於27株菌（84%）偵測出攜帶有TEM的基因。實驗中也發現E35具DHA-1乙內醯胺酶，且31株菌的質體上皆有AmpC（MIR-1或ACT的基因）存在。從PCR 反應發現E71的SHV附近具有嵌入子，因此進行選殖的實驗，以pOK12為載體選殖到E71 質體上4.5 kb BamHI片段具cephalosporinase高表現量，構築的質體命名為pE71-1。此4.5 kb BamHI片段定序後發現帶有四個完整的基因intI1、tnpA、blaSHV-12、orf2以及部分的orf3基因。在blaSHV-12基因部分相似於blaSHV-1基因，於緊鄰blaSHV-12上游發現有IS26 element插入於接近-10的區域，而blaSHV-12基因更上游的位置也發現有第一型嵌入子的integrase完整基因存在。這樣的結果顯示此4.5 kb含blaSHV-12的基因片匣為ㄧ新的嵌入子。本研究也發現一些菌株的抗藥基因可藉由接合作用（Conjugation）進行散播。聚合酶連鎖反應的實驗中19株菌被偵測出有第一型嵌入子且攜帶aadA2基因。最後於脈衝式膠體電泳的實驗中，將此31株帶有超廣效性乙內醯胺酶菌株之E. cloacae分為18種不同的分子型，由此結果得知菌株間同源性不高。

The prevalences of extended-spectrum beta-lactamases (ESBLs) and their encoding bla genes were investigated in isolates of Enterobacter cloacae (E. cloacae) that were resistant to beta-lactam and/or non-beta-lactam antibiotics. Of the 100 E. cloacae isolates investigated, all of which came from patients in a major hospital in middle Taiwan, 31 (31%) were found to have ESBLs, their production of beta-lactamase being confirmed by the double disc methods, E-test and PCR. All 31 ESBL isolates were resistant to beta-lactams and sensitive to imipenem. The results of isoelectric focusing analysis and the PCR-based amplification of the bla gene in plasmid DNA from the 31 ESBL isolates indicated that 23(74%) of the isolates carried the bla(SHV) gene, and 23(74%) carried the bla(CTX-M) gene, 16 (52%) of the isolates carried both the bla(CTX-M) and bla(SHV) genes, 27 (84%) carried the bla(TEM) gene. We also found the AmpC -lactamase DHA-1 in strain E35, and AmpC (MIR-1 or ACT) gene to be present on the plasmid in all 31 isolates. From PCR amplification showed integron near the bla(SHV-12) gene in strain E71. Shotgun cloning experiments with restriction enzyme-digested plasmid DNA from E. cloacae E71, which expresses high levels of cephalosporinase, into the pOK12 cloning vector gave a recombinant plasmid, pE71-1 with 4.5 kb BamHI fragment, which encoded four entire gene: intI1, tnpA, blaSHV-12. orf2 and part of orf3. The immediate upstream sequence of the bla(SHV-12) gene showed little similarity to the part of the prototype bla(SHV-1) gene due to the insertion of an IS26 element next to the -10 region, and further upstream by a full gene encoding integrase gene of class 1 integron. This finding indicate that the 4.5 kb cassette array contains bla(SHV-12) is a new integron. The resistance gene dissemination by conjugation also found in some strains in this study. Base on PCR the class 1 integron with aadA2 gene in 19 strains were also detected. Finally, Pulse Field Gel Electrophoresis (PFGE) showed 18 different genotypes in the 31 E. cloacae strains that revealed all isolates of harboring ESBLs were genetically unrelated.

表目錄............................................................Ⅰ
圖目錄............................................................Ⅱ
英文摘要..........................................................Ⅲ
中文摘要..........................................................Ⅴ
第一章 緒論........................................................1
第一節 前言................................................1
第二節 Enterobacter cloacae的介紹...........................1
第三節 乙內醯胺酶的分類命名及歷史回顧.........................2
第四節 乙內醯胺酶相關的調控因子..............................4
第五節 乙內醯胺酶相關的散播因子..............................6
第六節 超廣效乙內醯胺酶的分佈................................8
第七節 超廣效乙內醯胺酶菌株的篩選與鑑定.......................10
第八節 研究動機.............................................11
第二章 材料和方法...................................................13
Ⅰ.實驗材料................................................13
第一節 培養基...............................................13
第二節 試劑與緩衝溶液........................................14
第三節 實驗之菌種及質體......................................15
Ⅱ.實驗方法.................................................16
第一節 菌株來源.............................................16
第二節 抗生素敏感性試驗......................................17
第三節 質體DNA的分析........................................19
第四節 等電點聚焦電泳 (Isoelectric focusing; IEF)............20
第五節 聚合酶連鎖反應 (Polymerase chain reaction; PCR).......22
第六節 細菌接合試驗 (Conjugation)............................23
第七節 DNA選殖 (Cloning analysis)...........................23
第八節 南方雜交分析 (Southern hybridization).................25
第九節 脈衝式電泳試驗 (Pulsed-Field Gel Electrophoresis,PFGE).28
第三章 結果.........................................................32
第一節 檢體收集和抗生素敏感性試驗.............................32
第二節 超廣效性乙內醯胺酶菌株初步鑑定..........................32
第三節 超廣效性乙內醯胺酶型別鑑定.............................35
第四節 其它乙內醯胺酶分析....................................37
第五節 抗藥基因散播因子之分析.................................38
第六節 脈衝式膠體電泳分子分型.................................42
第四章 討論.........................................................44
圖表................................................................52
參考文獻............................................................69

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