多重抗藥性 Pseudomonas aeruginosa 菌株中 class 1 integron基因體分子多樣性之研究

Molecular diversity of class 1 integrons in multidrug-resistant isolates of Pseudomonas aeruginosa

蕭可妤(Ke-Yu Hsiao)

指導教授 ： 彭健芳

高雄醫學大學/健康科學院/醫學檢驗生物技術學研究所/碩士(2011年)

摘要

Pseudomonas aeruginosa 是重要的院內感染病原菌，被認為是細菌抗藥性的一種表現。本研究的目的是描述多重抗藥性 P. aeruginosa 菌株中，class 1 integron 基因體中基因卡匣的多樣性。從2009年1月至2010年12月期間，從南台灣地區某一教學醫院收集臨床上所分離的多重抗藥性 P. aeruginosa 菌株。收集方式以不同病患與不同檢體類別而且不重複為原則。首先利用 PCR 技術檢測 P. aeruginosa 菌株class 1、2、3 integron 基因體的盛行率。針對 class 1 integron 基因體陽性的菌株，進一步利用引子組 5’-CS 和 3’-CS 來增幅 class 1 integron 變異區的基因卡匣，將其 PCR 產物作兩端定序，採用 primer walking 定序的策略。分析 class 1 integron 基因體中基因卡匣及其抗藥基因序列組合特性。共收集735株多重抗藥性P. aeruginosa。其中，有203株 (27.6%) P. aeruginosa 菌株具有 class 1 integron 基因體。在這203株 P. aeruginosa 菌株中，只有68株 (33.5%) 其 class 1 integron 基因體變異區中帶有基因卡匣。經過 BLAST (Basic Local Alignment Search Tool) 分析，共有22種不同型式的基因卡匣排列組合。在本實驗中，所有的 P. aeruginosa 菌株中均沒有 class 2 和 3 integron 基因體的存在。所得到的基因卡匣中，aac(6’)-Ⅱ-catB2-aadA2 (GU934609) 為最常見的基因卡匣序列。其次是catB3-blaOXA-10-aadA15 (HM467196) 和 dfrB1 (In46; HQ317920)。具臨床意義的結果是， P. aeruginosa 菌株同時帶有 catB3-blaOXA-10-aadA15 和 aac(6’)-Ⅱ-catB2-aadA2 兩種基因卡匣序列。同時，利用 BLAST 系統比對的結果，發現一些新穎的基因卡匣序列包括: aac(6’)-Ⅱ-aadA2 (GU966684)、aac(6’)-Ⅱ-catB2 (HM366563)、aacC1-orfA-orfB-aadA1 (HM366564)、cm1A-aadA1 (HM446156)、aacA4-catB8-aadA1 (HM989924)、aadB-orfF1-aadA11 (HQ202807)、In216 dfrB4a-aacA4-aacA4-aadA1 (HQ317921)和aacA4-aadA15 (HQ705763)。值得注意的是，在dfrB4a-aacA4-aacA4-aadA 基因卡匣排列中，一個新穎的 dfrB4a 基因與dfrB4基因的差別是在第 26 胺基酸白胺酸 (Leucine) 變成色胺酸 (tryptophan)。同時也發現 catB3-blaOXA -10 -aadA15 基因卡匣序列中，aadA15 基因與其他報告不同處，是在第31個密碼子有一個無症狀變異 (silent mutation)。另外，對 carbapenem 藥物抗性的 P. aeruginosa菌株，其metallo-β-lactamase (MBL) 基因，只有1株 (7%)是存在在 class 1 integron基因體的基因卡匣序列中為 blaVIM-3-orf2a-aacA4-aadB-aacA4，另外 6 株 (93%) 是存在 class 1 integron 基因體的週邊基因。由於 class 1 integron 基因體中基因卡匣排列的多樣性，說明綠膿桿菌有多種抗藥性的能力。其中包括兩個新穎的，而且首度發表的 class 1 integron In46 及 class 1 integron In216基因體。

關鍵字

多重抗藥性綠膿桿菌 ； class 1 integron 基因體 ； 基因卡匣

並列摘要

Pseudomonas aeruginosa is an important nosocomial pathogen and considered to be a phenomenon of bacterial resistance. The aim of this study was to characterize molecular diversity of gene cassettes in class 1 integron from multidrug-resistant (MDR) P. aeruginosa isolates. In this study, isolates of MDR P. aeruginosa obtained from different clinical specimens in hospitalized patients admitted at a teaching hospital in southern Taiwan between January 2009 and December 2010. The presence of class 1, 2 and 3 integrons was investigated using PCR technology. The gene cassettes inserted in the variable region of class 1 integron were amplified using universal 5’-CS and 3’-CS primer pairs. The PCR amplicons were directly sequenced on both strands by the primer walking sequence strategy. Of 735 isolates of MDR P. aeruginosa, 205 (27.9%) carried detectable class 1 integrons. Sixty-eight (33.1 %) out of 205 isolates of P. aeruginosa harbored gene cassettes in class 1 integron variable region. Total of twenty-two different types of gene cassette arrays were found in class 1 integron-positive isolates. There are no class 2 and 3 integrons detected in these P. aeruginosa isolates tested. In our data, gene cassette array of aac(6’)-Ⅱ-catB2-aadA2 (GU934609) was the most frequent detected, followed by catB3-blaOXA-10-aadA15 (HM467196) and In46 dfrB1(HQ317920). Of note, bacterial isolates carrying two gene cassette arrays of catB3-blaOXA-10-aadA15 and aac(6’)-Ⅱ-catB2-aadA2 simultaneously, was commonly found in our isolates tested. BLAST analysis of DNA sequence showed other novel gene cassette arrays in P. aeruginosa isolates, including aac(6’)-Ⅱ-aadA2 (GU966684), aac(6’)-Ⅱ-catB2 (HM366563), aacC1-orfA-orfB-aadA1 (HM366564), cm1A-aadA1 (HM446156), aacA4-catB8-aadA1 (HM989924), aadB-orfF1-aadA11 (HQ202807), In126 dfrB4a-aacA4-aacA4-aadA1 (HQ317921) and aacA4-aadA15 (HQ705763). Interestingly, a novel dfrB4a gene detected in gene cassette array of dfrB4a-aacA4-aacA4-aadA1 was identified. The novel dfrB4a gene showed a Leu26Trp variant that was different from the dfrB4 gene. We also firstly find a silent mutation in codon 31 (GTC31GTA) of the aadA15 gene in the gene cassette array of catB3-blaOXA -10-aadA15. Moreover, the metallo-β-lactamase gene encoding carbapenem resistance in P. aeruginosa isolates were found to be carried not only on gene cassettes in class 1 integron (7%) such as blaVIM-3-orf2a-aacA4-aadB-aacA4 but also in the neighborhood regions of the integron genetic structure surroundings (93%). In conclusion, molecular diversity of gene cassettes presenting in class 1 integron may contribute to multidrug resistance for P. aeruginosa isolates. They are including two novel class 1 integrons of In46 and In216 that are the first reported.

並列關鍵字

multidrug-resistant Pseudomonas aeruginosa ； class 1 integron ； gene cassette

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