臺灣博碩士論文加值系統

摘 要
超廣效乙醯胺酶 (extended-spectrum β-lactamases，簡稱ESBLs) 基因可藉由質體自由地在菌種間傳播，使細菌獲得額外的抗藥性，而導致具有多重抗藥性的菌株產生。blaTEM和blaSHV是最常見的ESBLs基因，由於基因不斷地發生多點突變，使得blaTEM和blaSHV衍生出的ESBLs基因數目龐大，且數量持續地擴大，因而引發嚴重的抗藥性問題。為了深入探討多重抗藥菌株之抗藥分子機制，本論文針對經常發生多重抗藥性的Klebsiella pneumoniae和Escherichia coli，以抗微生物藥劑敏感性試驗，分析台灣北區臨床多重抗藥菌之抗藥表型。實驗結果發現，台灣北區多重抗藥的E. coli 和K. p.菌有70%為ESBLs菌株，而所有ESBLs菌株對β-lactam類抗生素 (ampicillin、piperacillin、cefazolin、cefuroxime、cefotaxime及aztreonam) 具有抗藥性，但對同屬於β-lactam類的cephamycin (cefoxitin及flomoxef) 卻較敏感。我們也研究blaTEM和blaSHV的基因型及分佈情形，以進一步瞭解其與菌株之抗藥表型間的關聯性。實驗結果發現，蒐集所得之TEM和SHV基因具有多處點突變，且TEM-type以blaTEM-1b (57%) 和新發現的blaTEM-A (28%) 為主，而SHV-type則以blaSHV-12 (63%) 居多。根據實驗發現TEM和SHV基因型與抗藥表型之關聯，我們也利用基因晶片可以檢測單點突變 (single nucleotide mutation) 的優點，設計檢驗TEM-和SHV-type基因型之基因晶片，並調整至最佳化。由於基因晶片具有快速、大量，及同時檢測多種抗藥性菌株之優點，因此適用於臨床醫療檢測，未來希望可以補現有醫療檢測系統之不足，選用正確抗生素，避免細菌產生抗藥性之問題。

ABSTRACT
Extended-spectrum β-lactamases (ESBLs) which predominantly encoded by plasmids can spread among different bacterial strains through conjugative dissemination of ESBL-encoding plasmids. With this, bacteria acquire additional resistance and generate multidrug resistance. ESBLs are predominantly derived from plasmid-mediated TEM- or SHV-type β-lactamases through one or more point mutations that lead to a large number of TEM or SHV derivatives causing serious drug resistance issue. To explore the molecular mechanism concerning multidrug resistance, we adopt an antimicrobial sensitivity testing method to analyze the resistance phenotypes that expressed from multidrug resistant isolates collected in northern Taiwan. We found that 70% of these isolates were ESBLs strains and all of them were resistant to β-lactam antibiotics (ampicillin, piperacillin, cefazolin, cefuroxime, cefotaxime and aztreonam), but most of them were sensitive to another kind of β-lactam antibiotics─cephamycin (cefoxitin and flomoxef). We also analyzed TEM and SHV genotypes and their distribution in order to understand the correlations between resistance genotypes and phenotypes. We found that the TEM and SHV isolates collected in this study contain multiple point mutations and most of the TEM isolates belong to the TEM-1b (57%) and another TEM-A (28%) types, however, most of SHV-12 isolates were the SHV-type (63%). According to the available TEM and SHV genotypes, we designed a gene chip that can detect most of the genotypes of TEM and SHV-ESBLs in clinical isolates, because gene chip has the advantage of detecting single nucleotide mutation. For the purpose of clinical diagnostics, we optimized the hybridization and washing conditions, and also probe design. Gene chips are suitable for use in clinical diagnosis because of the advantage of detecting many resistance strains at the same time. It is likely, in the future, that clinical diagnostic gene chip will be able to make up a deficiency of existing diagnostic system and help us to choice correct antibiotics, then avoid bacteria producing drug resistance.

目 錄
名詞縮寫…………………………………………………………………I
中文摘要…………………………………………………………………II
英文摘要…………………………………………………………………III
壹、 緒論……………………………………………………………1
貳、 材料與方法
一、 細菌菌株(Bacteria strains)………………………………16
二、 培養基(Culture media)………………………………………17
三、 抗微生物藥劑敏感性試驗(Antimicrobial sensitivity tests)……17
四、 DNA萃取(DNA extraction)……………………………………17
五、 ESBL基因篩檢(ESBL gene screening)………………………18
六、 DNA定序(DNA sequencing)……………………………………19
七、 探針設計(Probe design)………………………………………19
八、 探針固定(Immobilization)……………………………………21
九、 標的物製備(Target preparation)……………………………21
十、 晶片雜合反應(Hybridization)………………………………23
十一、 雜合訊號讀取及量化(Detection & Quantitation)…………24
參、 結果
一、多重抗藥菌株之研究…………………………………………………26
( 1 ) .多重抗藥菌經鑑定可分為ESBLs和non-ESBLs菌株………………26
( 2 ) .多重抗藥菌株之抗微生物藥劑敏感性試驗分析…………………26
( 3 ) .ESBL基因及AmpC基因之篩選與基因型鑑定………………………28
( 4 ) .分析ESBLs基因型分佈與菌株抗藥表型之關聯性………………32
二、建立TEM-和SHV-type基因型之基因晶片……………………………34
( 1 ) .設計TEM-和SHV-type基因型晶片及最佳化測試………………35
( 2 ) .設定基因晶片的臨床檢測標準…………………………………40
( 3 ) . 運用基因晶片檢驗TEM-和SHV-type基因型及菌種鑑定………41
肆、 討論……………………………………………………………44
伍、 參考文獻………………………………………………………61
陸、 附圖及表………………………………………………………69

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