對carbapenem感受型不一致之綠膿桿菌分離株其抗藥機轉之探討

The Resistance Mechanisms of Discordant Carbapenem Susceptibility in Pseudomonas aeruginosa Isolates

李靜宜(Ching-yi Li)

指導教授 ： 羅雪霞

中臺科技大學/健康科學院/生命科學研究所/碩士(2008年)

摘要

本研究採用自台中榮民總醫院由2004年8月至2006年6月間收集之66株對carbapenem類藥物感受型不一致之Pseudomonas aeruginosa分離株進行實驗，嘗試瞭解其carbapenem抗藥機轉。首先以PFGE進行菌株親源性的分析，並篩選其中是否有metalloβ-lactamase生產菌；接著以Western blot進行膜蛋白OprD表現之偵測並進行oprD基因定序，並以real time RT-PCR分析ampC, oprD, mexA, mexE及mexX等基因之轉錄。
這66分離株中以PFGE分型，主要區分為五大群 (A-E) ，而無法分群的有26株菌 (cluster O) 。顯示這些分離株具有相當的歧異性。在oprD基因定序時發現，有12株屬於cluster C的分離株，其oprD皆被相同的帶有Thauera sp B4 transposase基因的插入序列在核?酸916與917之間插入，暗示此菌株在院內水平散佈的可能。在收集的菌株中，Imipenem (IMP) 呈非感受性的分離株數量遠大於對meropenem (MER) 非感受性分離株，分別為92.4 %及47.0 % ，所有的菌株均未發現metalloβ-lactamase，有95.5 %菌株oprD基因具有變異。MER非感受性菌株中，有25.6 %菌株之mexA或mexX並無過度轉錄；對MER感受性菌株中，有48.6 %之mexA或mexX卻呈過度轉錄。mexA及mexX的過度轉錄與否似乎不足以完全解釋菌株對MER抗藥性的產生。在61株對IMP呈非感受性菌株中有86.9 %菌株因為oprD基因缺失，導致無法製造出OprD蛋白質，6.55 % OprD具有胺基酸的變異，另有6.55 %菌株胺基酸雖無變異但卻有基因轉錄下降或伴隨ampC過度轉錄，顯示IMP的抗藥性產生主要與OprD異常有關。
本研究結果顯示，對carbapenem感受型不一致P. aeruginosa分離株對IMP呈抗藥性機轉主要是因為oprD基因變異，對MER呈抗藥性卻不一定會伴隨mexA與mexX基因過度轉錄。

關鍵字

綠膿桿菌 ； carbapenem ； oprD ； efflux pump system

並列摘要

In this study, sixty-six Pseudomonas aeruginosa isolates with discordant imipenem (IMP) and meropenem (MER) susceptibility were colletcted from Taichung Veterans General Hospital during August 2004 to June 2006. The possible carbapenem resistance mechanisms among these isolates were explored.
PFGE typing was performed to clarify bacteria isolates’ relatedness. Metallo β-lactamase producers were screened and confirmed. The presence of OprD was examined by Western blotting, then oprD gene was sequenced. Gene transcription were analyzed by real time RT-PCR for ampC, oprD, mexA, mexE and mexX genes, respectively.
PFGE method typed 66 isolates into five major clusters (A to E), however, 26 isolates showed unique patterns and thus was designated as cluster O. The result revealed that these isolates were quite genetically diverse. While oprD gene was sequenced, twelve isolates belonging to cluster C had the same oprD gene insertion by insertion sequence containing Thauera sp B4 transposase gene between nucleotide 916 and 917. It was suggested that these isolates were horizontal spread in the hospital. The population of IMP non-susceptible isolates was greater than that of MER non-susceptible isolates (92.4 % compared with 47.0 %). No metallo β-lactamase producer was found. Most of isolates (95.5%) showed oprD gene mutation. Among MER non-susceptible isolates, 25.6 % had no mexA or mexX over-transcription. Meanwhile, among MER susceptible isolates, 48.6 % showed mexA or mexX over-transcribed. mexA and mexX over-transcription could not fully explain MER resistance mechanisms among these isolates. Among IMP non-susceptible isolates, 86.9% had oprD gene mutation, which led to OprD protein dysfountion, 6.55 % of isolates had amino acid variation in OprD protein. The remaining 6.55 % had no oprD gene mutation, but decreased oprD transcription or increased ampC transcription was observed. The main IMP resistance mechanism among P. aeruginosa isolated was OprD abnormality.
The result of this study show that, in discarsdant carbapenem susceptibility P. aeruginosa isolates, IMP resistance mechanisms are mainly due to oprD gene mutation, while MER resistance mechanisms may not necessarily be accompanied with over-transcription of mexA or mexX gene.

並列關鍵字

Pseudomonas aeruginosa ； carbapenem ； oprD ； efflux pump system

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