建立台灣地區流行性嗜血桿菌分子指紋圖譜資料庫及其抗藥性相關之研究

data establishment of molecular fingerprinting of haemophilus influenzae in taiwan and related mechanism of antibiotic resistance

張容榕(jung-jung chang)

指導教授 ： 吳瑞裕

臺北醫學大學/醫學院/醫學科學研究所/碩士(2006年)

摘要

流行性嗜血桿菌（Haemophilus influenzae）感染症為台灣的法定疾病，目前流行性嗜血桿菌感染症所使用的抗生素為ampicillin及chloramphenicol，但是將近一半以上會產生抗藥性，且帶有β-lactamase的抗藥性基因，有些菌株甚至對多種抗生素產生抗藥性。與歐美國家相較之下，台灣地區嗜血桿菌的抗藥性問題更加嚴重。在抗生素濫用、缺乏管理的情形下，其具抗藥性之比例已有50% ~ 60%以上，因此相關抗藥性的機轉研究是刻不容緩的。 本論文主要是要探討流行性嗜血桿菌在台灣地區的感染情況與抗藥性在各菌株間分布的情況，以及建立台灣地區流行性嗜血桿菌的分子分型指紋圖譜資料庫，以供臨床及用藥之參考。我們收集到2003年台灣地區全國各大醫學中心嗜血桿菌的感染樣本。首先做菌種培養確定為嗜血桿菌，以及MIC的檢測，以供後續的資料研究。我們利用數種分子生物學的技術建立嗜血桿菌的分子指紋圖譜，包括ERIC-PCR、REP-PCR與16S rDNA - RFLP來建立嗜血桿菌菌種資料庫。我們亦針對嗜血桿菌可能帶有之質體DNA，設計引子來檢測帶有抗藥性基因，利用核酸定序進行分析抗藥性基因之種類與分布，最後再與醫院嗜血桿菌MIC試驗的資料做整體之統計分析。不論是REP PCR、ERIC PCR或者是16S rDNA的分子指紋圖譜鑑定方法，都可以有效的分型流行性嗜血桿菌。ERIC / REP PCR的優點為簡單操作、省時間與金錢，且準確度很高，鑑別菌種的多樣性比16S rDNA大。REP PCR所產生的pattern不論起全國或各醫院觀察都比ERIC有較多多樣性，但是ERIC / REP PCR兩者所分類的族群ERIC比REP多。所以以流行性嗜血桿菌的分子指紋圖譜來看，我們會建議採用ERIC PCR的方法，快速又便利，且花費最低，且菌種的多樣性多。台灣地區的流行性嗜血桿菌的抗藥性問題嚴重，其中以ampicillin最為嚴重，有63％的細菌具有抗生素抗藥性，在我們檢測的八種抗生素中，發現流行性嗜血桿菌不僅僅對一種抗生素產生抗藥性，具有兩種抗藥性。嗜血桿菌內普遍存在質體，以兩種形式存在，excised或是integrated types。嗜血桿菌抗藥性產生的原因主要是細菌內的具有質體不論是excised或是integrated types的都與抗生素的抗藥性有高度的相關性。

關鍵字

指紋圖譜 ； 抗生素 ； 相關性 ； 抗藥性 ； 準確度

並列摘要

Haemophilus influenzae is the one of most important bacterial pathogen of pediatric infection. This species is a pathogen causing variety of clinical manifestations such as meningitis, septicemia, epiglottitis, cellulities, septic arthritis, pneumonia, otitis media, sinustitis, and other respiratory tract infections in children. According it having the capsular or not, H. influenzae can be divided into two groups. H. influenzae with capsular have six serotypes, a through f, on the basis of structurally and serologically distinct capsular polysaccharides. All over the world the majority of severe infections related to this species are caused by H. influenzae type b (Hib). Hib is the leading cause of bacterial meningitis in children less than 5 years old of the world, including Taiwan（about 41%）. Ampicillin (200-400mg/Kg/day) is a representative drug for treatment of Hib. Up to 30% of the pathogens, however had developed drug-resistance of ampicillin in United States. Beta-lactamase production was found in 50% to 60% of H. influenzae in Taiwan. Among amoxicillin-resistant H. influenzae isolates, beta-lactamase nonproducers were rare (<2%). A continuing upsurge of H. influenzae isolates resistant to macrolide (30%) and to trimethoprim sulfamethoxazole (50%) during the last decade has turned out to be popular. Since resistance to several drugs continues to emerge among the H. influenzae worldwide. Continued surveillance of this pathogen group appears to be necessary. For above the reasons, we hope to establish a database of H. influenzae which have variable subtypes and its resistance mechanism that is useful and can be applied to clinical therapy. In our study, we compared 384 H. influenzae isolates from Taiwan by enterobacterial repetitive intergenic consensus (ERIC), repetitive extragenic palindromic (REP) polymerase chain reaction (PCR), RFLP-16S rDNA ribotyping and MIC test. Furthermore, we have used variable PCR method to analysis the plasmids of H. influenzae and its resistance mechanism.

參考文獻

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