臨床所分離出來具廣效性乙內醯胺酶特性的大腸桿菌具有與此特性有關的質體

Clinically isolated Escherichia coli with the extended-spectrum beta-lactamases (ESBL) property have plasmids related to its ESBL

傅麗玲(LI-LING FU)

指導教授 ： 蔡榮宗

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2015年)

摘要

1983年之後，陸續發現廣效性乙內醯胺酶(Extended-spectrum β-lactamases; ESBL)的蹤跡，它是一種突變的酵素，可經由質體為媒介在不同菌種間傳播，且大部分的質體size都大於100 kb。這些酵素經由一或多個胺基酸的改變就能水解更多頭孢菌素類(cephalosporin)抗生素，使得抗生素失效，這樣的問題造成感染管制上的一大隱憂。
因此我們想要探究，由臨床上所分離出來具ESBL特性的腸內菌，是否含有與ESBL相關的質體，使得細菌產生抗藥性。從實驗的結果得知，野生株的大腸桿菌(no. 18)與實驗室轉型後的大腸桿菌(no. 18-1)都可得到同樣一條大size 質體DNA，且再經由藥敏試驗測試，皆具ESBL結果，因此可以肯定的是細菌帶有ESBL特性可能與此大size DNA片段有直接的關係。另外，為了確認大尺寸質體的真實性，我們使用限制酶EcoRI或BamHI切割從野生株18號及轉型的18-1菌株中抽到的質體，結果發現皆可切成幾條固定大小的DNA片段，因此確認此DNA非染色體，因為染色體會切割成連續條帶狀(smear)。綜合實驗結果，此臨床挑選出來具有ESBL特性的大腸桿菌(no. 18)是因帶有一個大尺寸的質體，而使細菌產生抗藥性。因此，經由本研究我們建立了一個從帶有ESBL特性的大腸桿菌中純化大尺寸質體的方法，並將運用這個方法來研究臨床菌株的ESBL特性。

關鍵字

廣效性乙內醯胺酶

並列摘要

After 1983, Extended-spectrum β-lactamases (ESBL) were discovered. They are distributed between bacteria via a plasmid with a mutant form of this enzyme. Most of the plasmids with these genes have a size greater than 100 kb. By changing one or more amino acid of these enzymes through mutations, they will be able to hydrolyze more cephalosporin antibiotics, resulting a major worry on infection control.
Therefore, we want to explore whether the clinically isolated intestinal bacteria with ESBL characteristics have ESBL-containing plasmids, allowing them to become resistant. From the experimental results, we found that clinically isolated wild-type E. coli (no.18) with ESBL has many plasmids with different size. One transformant (no. 18-1) generated from transforming DH5α with plasmid mixture from no.18 E. coli has a large size plasmid longer than 100 kb. We compared the result of sensitivity test on no. 18 and no. 18-1 and found that they are similar, confirming that this large size plasmid was related to its ESBL property. In addition, to confirm the authenticity of this plasmid, we use the restriction enzyme EcoRI or BamHI to digest this plasmid and found that it could be cut many times by this two enzymes to generate several distinct DNA fragments. This property is impossible to happen in chromosome, because the chromosome will become smear by doing this. Taking together, our results indicated that no. 18 wild-type E. coli with ESBL property may exhibited its antibiotic-resistance from this large size plasmid. Hence, we have established a procedure to find out ESBL-related plasmid and will employ this method to explore the ESBL property from clinically isolated bacteria.

並列關鍵字

ESBL

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