台灣Acinetobacter baumannii臨床分離菌株攜帶Metailo-B-Lactamase基因及Integrons分子 特性之探討
林傑裕、劉淑瑛，邱政洵
E-mail: 9314525@mail.dyu.edu.tw
摘 要
Acinetobacter baumannii 為醫院中造成院內感染常見的菌 株，由於臨床上抗生素的使用頻繁，形成了多重抗藥菌株，造成 治療上的困難。根據台大醫院對此多重抗藥菌株研究的統計數據 顯示，從1998 年篩選到的0％到2000 年的6.5％，有逐年 增加的 趨勢，可見此菌株抗藥性之快速傳播及其嚴重性。故本研究的目 的，就是希望從分子學上了解此菌株是攜帶何種 抗藥基因和其散 播的主要機制。 在醫院臨床中受A. baumannii 感染後所使用之治療藥物以β- 內醯胺類之抗生素
“imipenem”為主，故本研究主要探討對於 imipenem 具抗藥性的A. baumannii。研究之菌株來源，是由嘉義 長庚醫院所 分離到188 株之A. baumannii，再以紙綻擴散感受性試 驗檢測其抗藥性，從中篩選到2 株具imipenem 抗藥性的菌株 (P-78 和P-210)，再加上由林口長庚醫院所篩選到6 株imipenem 抗藥菌株 (AB-394、AB-1756、AB-1757、AB-1758、AB-1759 和 AB-1760)，共8 株進行其imipenem 抗藥性比對和研究分析。並從 菌株中再挑出只對imipenem 不具抗藥性的菌株 (P-21 和P-23) 和 對所有抗生素都不具抗藥性的菌株 (P-2) 進行分析，以了解其抗 藥基因散播的情形。 為了解抗藥菌株是否存在 常見對imipenem 抗藥之metallo-β -lactamase (例如blaIMP、blaVIM 與cfiA) 的基因，因此由已知的 blaIMP、blaVIM 與cfiA 序列設計出適當引子，再以PCR 的方法確 認出其中的AB-394 和P-78 兩株菌株具有blaIMP-1 的抗藥基因。相 對地，P-210
、AB-1756、AB-1757、AB-1758、AB-1759 和AB-1760 則不具有blaIMP、blaVIM 或cfiA 其中任一種之抗藥基因。此外，為 了進一步研究抗藥基因的位置，以快速分離質體DNA (Kado and Liu) 之方法，分離出這8 株抗藥菌株之質體與染色體，並 轉漬至 尼龍膜 (nylon membrane) 上，再以blaIMP-1 序列為探針做南方雜 交法實驗。結果發現AB-394 所攜帶的抗藥基因位 於大質體 (>100 kb) 上，P-78 所攜帶的抗藥基因則位於約95-kb 的質體上。而 P-210、AB-1756、AB-1757、AB-1758
、AB-1759 和AB-1760 則 不帶有質體，因此若攜帶抗藥基因則很可能位於染色體中。同時 進一步分析菌株中是否帶有散播 抗藥基因之“integon”結構，根據 臨床中最常被發現之integon I 序列設計引子，再以PCR 的方法確 認出P-21、P-23、P-78
、P-210、AB-394、AB-1756、AB-1757、 AB-1758、AB-1759 和AB-1760，共10 株菌株具有散播抗藥基因 之integron I 之結 構。將PCR 產物經純化解序比對後發現，AB-394 與P-78 具有相同之integron I 之結構及序列，並且攜帶有imipenem 抗藥 之blaIMP-1 基因。但在抗藥菌株P-210、AB-1756、AB-1757、 AB-1758、AB-1759 和AB-1760 之integron I 結構中未發現對 imipenem 抗藥之基因。至於這些菌株攜帶何種抗藥基因，或是其 它的抗藥機制，則需進一步研究。同時也藉由脈衝式膠 體電泳 (pulsed-field gel electrophoresis, PFGE) 之分子分型方法，分析其 抗藥菌株與非抗藥菌株之間的遺傳差異與親源性。
結果顯示多重 抗藥菌株AB-1758、AB-1759、AB-1760 和P-23 可能來自同一親 源，而P-21 和P-210 顯然具相同的親源，此 結果也顯示近期從林 口長庚醫院所篩選到的臨床多重抗藥菌株，很可能源自同一親源 (same clone)。 目前對於台灣臨床菌 株A. baumannii 之抗藥基因及integron 的探討才剛起步，本研究之結果將有助於台灣臨床A. baumannii 感染之控制及流行病 學之了解。
關鍵詞 : Acinetobacter baumannii，抗藥基因，integron，PFGE 目錄
封面內頁 簽名頁 授權書---iii 中文摘要---v 英文摘要---viii 誌 謝---xi 目錄---xii 圖目錄---xvi 表目
錄---xviii 第一章 前言---1 1.1Acinetobacter baumannii簡介---1 1.2抗生素之簡介 ---2 1.3細菌對抗抗生素之策略---3 1.4β-內醯胺?---4 1.5Metallo-
β-lactamse---6 1.6抗藥基因之傳播---7 1.7integron介紹 ---8 1.7.1 integron之構
造---9 1.7.2 integron之種類---9 1.7.3 基因片匣的構造---11 1.7.4 integron與基因片匣之表現----11 1.7.5 integron與基因片匣在抗藥性基因散佈之角色----12 1.8 脈衝式膠體電泳(Pulsed-field gel electrophoresis,PFGE)---13 1.9 研究動 機及目的---14 第二章 材料與方法---16 2.1 菌株來源---16 2.2 E-test最低抑 菌抗生素濃度---16 2.3 偵測imipenem抗藥基因及integron之步驟----17 2.3.1 引子(primer)設計---17 2.3.2
Polymerase chain reaction;PCR---18 2.3.3 電泳分析及PCR產物分子量計算---20 2.3.4 blaimp及integron在菌體位置之 分析---20 2.3.5 PCR產物之純化---21 2.3.6 序列資料分析---22 2.4 快速抽取質體DNA(Kado and Liu)---22 2.5 南方雜交法(Southern hybridization)---23 2.5.1 薄膜轉漬(Membrane transfer)---23 2.5.2 製作探針---24 2.5.3 雜交(Hybridization)與顯影---25 2.6 脈衝式膠體電泳(PFGE)---27 第三章 實驗結果---29 3.1 E-test最低抑菌抗
生素濃度之結果分析---29 3.2 Imipenem抗藥基因之PCR結果分析---29 3.2.1 blaimp之imipenem抗藥基因---29 3.2.2 blavim之imipenem抗藥基因---30 3.2.3 cfiA之imipenem抗藥基因----31 3.3 Integron之PCR結果分析---31 3.3.1 Integron之PCR 結果---31 3.3.2 Integron PCR產物定序分析比對---34 3.4 Kado and Liu之方法分離質體---33 3.5 抗藥基因位置之分析 結果---34 3.6 脈衝式膠體電泳(PFGE)之分子分型---34 第四章 討論---36 4.1 E-test 最低抑菌抗生素濃度---36 4.2
Imipenem抗藥基因之PCR 結果討論----36 4.3 抗藥基因位置分析---38 4.4 Integron之PCR結果討論---39 4.5 脈衝式膠體 電泳(PFGE)之分子分型----41 第五章 結論---43 參考資料---77 附錄---86 附錄一 菌株感受性試 驗---86 a、紙錠擴散感受性試驗---87 b、微量稀釋感受性試驗---88 附錄二 Metallo-β-lactamase之各blaIMP 比較表---89 附錄三 Metallo-β-lactamase之各blaVIM比較表---90 附錄四 AB-394、p-78與P-210之菌株integron之比 較---91
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