以蛋白質體學技術研究鮑氏不動桿菌之多黏桿菌素抗藥性

Proteomic Analysis of Polymyxin-Resistance Acinetobacter baumannii

葉昱辰(Yu-Chen Yeh)

指導教授 ： 張紹光

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10701

摘要

鮑氏不動桿菌 (Acinetobacter baumannii) 是一種分布廣泛的伺機性革蘭氏陰性菌，為人體皮膚上的常在菌叢（normal flora），目前也已在多種動物及相關的產品發現其存在。近年來，多重抗藥性鮑氏不動桿菌 (multidrug- resistant A. baumannii，MDRAB) 隨著抗菌劑的廣泛使用而出現，目前已成為醫療照護相關感染（healthcare associated infections，HAIs）中，難以控制細菌之一。由於MDRAB的出現，以及在藥理學上的重新認識，colistin再次的被用於臨床醫療，但抗藥性菌株也漸漸的開始出現。本研究自2010年9月至2011年3月間，自台灣北、中、南三區的傳統市場和超級市場進行檢體的逢機採樣，共216件。並以改良式Leeds Acinetobacter Medium（MLAM）培養基及馬康奇培養基進行初步分離。再以聚合酶鏈鎖反應增幅16S-23S核糖核酸基因內轉錄區，並搭配序列分析進行比對鑑定。結果共分離28/216（13％）株鮑氏不動桿菌，而傳統市場及超級市場的分離率分別為27/108（25%）及1/108（1％），北、中、南三區的分離率分別為6/36（17％）、10/36（28％）及12/36（33％），以及自台北地區醫院收集72株非重複之鮑氏不動桿菌。本研究亦進行抗菌劑感受性試驗及利用脈衝式電泳（pulsed-field gel electrophoresis，PFGE）調查兩來源菌株的抗藥性及親緣性。由結果得知，市售雞肉來源的菌株對cefazolin外藥品都保持一定的感受性（0∼36％），而人類臨床菌株則有較高的抗藥性（72∼100％），僅對colistin（7％）與ampicillin/sulbactam（10％）保持較佳的感受性。親緣性分析的結果發現，市售雞肉與人類臨床來源的菌株並不共有相同的基因型，故兩來源間應鮮少發生菌株及抗藥性的交流。本研究另外以蛋白質體學方法比較鮑氏不動桿菌標準菌株ATCC 19003及人工誘導抗colistin菌株19003 CR間蛋白質體的差異，結果發現誘導colistin的抗藥性產生，並不會造成菌株基因型別上的變化。藉由二維蛋白質電泳與質譜儀鑑定差異性蛋白質的身分，並以即時定量聚合酶鏈鎖反應（real-time PCR，qPCR）測定這些蛋白質的表現量，結果發現外膜蛋白Omp38在標準菌株有較高的表現量；sensor protein QseC、kdsA蛋白和aacD蛋白則是在抗colistin菌株有較高的表現量，推測colistin抗藥性的產生與細胞膜的合成相關，並受到二元訊號轉導系統調控，但還需更進一步研究其發生的機制。

關鍵字

鮑氏不動桿菌 ； 抗藥性 ； 多黏桿菌素 ； 親緣性分析 ； 蛋白質體學

並列摘要

Acinetobacter baumannii are widely distributed opportunistic Gram-negative bacteria, and also a part of normal bacteria flora of human skin. It can be found in a variety of animals and related product. In recent years, multidrug-resistant A. baumannii ( MDRAB) were emerged, caused by extensive use of antimicrobial agents, and become a part of difficult to control bacterial in healthcare associated infections (HAIs). Due to the emergence of MDRAB, and advances in pharmacology of colistin, colistin is used for clinical again. But colistin-resistant strains began to appear when it is used. In this study, two hundred and sixteen fresh raw chicken meat samples from the traditional markets and supermarkets of northern, medium and southern areas of Taiwan were collected during Sep. 2010 to Mar. 2011. Modified Leeds Acinetobacter medium (MLAM) and MacConkey agar was used as the isolation and selective medium. The samples were identified by amplify the 16S-23S internal transcribed spacers (ITS) region by polymerase chain reaction and sequence method. The total ratio of isolation was 13% (28/216). The ratio of isolation for traditional markets and supermarkets were 25% (27/108) and 1% (1/108), respectively. The prevalence of the northern, middle and southern regions was 17% (6/36), 28% (10/36) and 33% (12/36), respectively. A total of 72 isolates were consecutively collected from regional hospital in Taipei. In this study, antimicrobial susceptibility testing and pulsed field gel-electrophoresis (PFGE) are used to analysis antimicrobial resistance and phylogenic between two origin strains. The results indicated that strains from fresh raw chicken meat samples are sensitive to most tested antimicrobials (0-36%) except cefazolin. Most human clinical origin strains are highly resistant to all tested antimicrobials (72-100%); only colistin (7%) and ampicillin/sulbactam (10%) have good susceptibility. Phylogenetic analysis didn’t show any genetic relativity between chicken isolates and human isolates. The results showed isolates from two different sources were genetic and antimicrobial resistance independent. In this study, also use proteomic tool to compare the proteome differences between reference strain ATCC 19003 and artificial induced colistin-resistant strain 19003 CR. The results showed there was no genetic changes caused by induce reference strain to colistin-resistant strain by using PFGE analysis. Two-dimensional electrophoresis and MALDI-TOF analysis were used to identify the differentially expression proteins and real-time PCR was used to confirm the expression levels of these proteins. The results showed that the expression levels of outer membrane protein 38 (Omp38) increased in reference strain; sensor protein QseC, kdsA protein and aacD protein increased in artificial induced colistin-resistant strain, alternatively. It is speculate that A. baumannii may obtain their resistance against colistin through membrane biosynthesis, and it may be regulated by two component regulatory systems, but still needs further studies to verify its mechanism.

並列關鍵字

Acinetobacter baumannii ； antimicrobial resistance ； colistin ； phylogenetic analysis ； proteomics

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