大腸桿菌中第一類Integron結構分析

Structural characterization of class 1 integrons in clinical isolates of Escherichia coli

林季榆(Ji-Yu Lin)

指導教授 ： 張仲羽

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

Integron和基因片匣是細菌獲得抗藥基因及基因在細菌間散佈的一種水平轉移形式，在細菌多重抗藥性的形成和抗藥基因的變化上扮演著重要的角色。目前在臨床菌株中所發現之integron以class 1 integron最普遍，它可藉由質體轉移與轉位子跳躍達到在細菌間散佈抗藥基因甚至於integron本身結構的目的，然而對於integron本身散佈能力，以及散佈能力與結構之間的相關性則尚未完全了解。
文獻曾報告在某些class 1 integron 的上下游兩端發現有特異性的反轉重複序列（inverted repeat，5’端稱為IRi，3’端稱為IRt）及位在基因片匣下游和轉位功能相關的tni基因群存在，顯示intgron和轉位子間可能有相關性。故本研究主要目的在分析大腸桿菌中class 1 integron結構，以了解臨床菌株class 1 integron結構變異和演化情形。
實驗菌株為1993年及2002年所收集自臨床檢體分離的88株E. coli菌株，利用聚合酶連鎖反應（polymerase chain reaction，PCR）分析E. coli菌株之class 1 integron兩端的反轉重複序列IRi及IRt，並增幅3’端保留片段上的sul1基因下游至IRt的序列，再進行定序分析、基因比對和限制酶片段多型性分析，以了解class 1 integron的結構。
結果發現在88株E. coli中，IRi的存在率約90%，而IRt的存在率約62.5%。而且class 1 integron 5’的IRi端序列較無變化，但3’的IRt端的序列結構則有較大的變異。在已分析完成的57株E. coli class 1 integron sul1基因下游結構中，與轉位能力相關的tni基因群以tniA和tniB有較高的保留度，但多數的tniA和tniB序列有短少，而tniQ和tniR則未發現。另外有IS26、IS6100、IS1326和IS1353四種插入序列嵌入，其中以IS26存在最為普遍。此外在sul1基因下游發現有抗藥基因存在，包括與macrolide抗藥性相關的基因mphR(A)、mrx和mphA；trimethoprim抗藥基因dfr19和streptomycin抗藥基因strA、strB。
綜合本研究結果顯示，Class 1 integron本身具有轉位子構造特徵，在5’端的IRi序列有較高的保留性，而IRt序列保留度較低且sul1基因下游的變化也較大，可見class 1 integron的3’端序列結構仍然在持續演變中，這也可能影響integron之散佈。

關鍵字

大腸桿菌 ； 第一類Integron ； 結構分析 ； 轉位子

並列摘要

Integrons and gene cassettes are known as a horizontal transfer mechanism of acquisition and dissemination of resistance genes among bacteria. They play important roles in the development and variation of multiple antimicrobial resistances of bacteria. Most integrons from clinical isolates belong to class 1 and their location on plasmids and transposons may contribute to the spread of antibiotic resistance genes and even integrons themselves. However, it has not yet been elucidated that the movement ability of integrons themselves and the relations between the movement and structures of integrons.
Several studies have reported that some class 1 integrons are bounded by 25-bp inverted repeats, designated IRi and IRt, at the 5’ and 3’ ends, respectively. Furthermore, tni genes required for transpositions were present downstream the gene cassettes. This suggests integrons may be related to transposons and move by transposition. This study was to characterize the structures of class 1 integrons in clinical isolates of Escherichia coli to understand the structural variety and evolutionary lineage of class 1 integrons.
In this study, inverted repeats IRi and IRt, at the outer boundaries of class 1 integrons, were detected in 88 clinical isolates of E. coli collected in 1993 and 2002 by polymerase chain reaction. Then, the fragments of class 1 integrons between the sul1 gene and the IRt were amplified, followed by sequencing, sequence analysis and restriction fragment length polymorphism to confirm the structures of class 1 integrons.
Of the 88 E. coli isolates, the inverted repeats IRi and IRt were present in 90% and 62.5 % of the isolates, respectively. The sequences neighboring the IRi end of class 1 integrons were more conserved than those neighboring the IRt end. The structures of class 1 integrons downstream the sul1 gene have been characterized in 57 E. coli isolates. Among the four transposition genes (tni A, B, Q, R) contained in a complete tni module, tniA and tniB frequently existed, but the other two genes tniQ and tniR were not found. However, most of the tniA and tniB genes had sequence deletions. Four types of insertion sequences were found associated with class 1 integrons, including IS26, IS6100, IS1326 and IS1353, IS26 were the most prevalent among them. Besides, several resistance genes not contained in cassettes were present downstream the sul1, including a macrolide resistance operon consisting of the genes mphR(A), mrx and mphA, dfr19 for trimethoprim resistance and strA-strB for streptomycin resistance.
The present study reveals that class 1 integrons carry the characteristic features of transposons. In addition, the structural variations of class 1 integrons are still ongoing, especially at 3’ end, thus may affect the mobility of integrons.

並列關鍵字

structural characterization ； class 1 integrons ； transposon ； Escherichia coli

參考文獻

1. Yu, H.S., J.C. Lee, H.Y. Kang, D.W. Ro, J.Y. Chung, Y.S. Jeong, S.H. Tae, C.H. Choi, E.Y. Lee, S.Y. Seol, Y.C. Lee, and D.T. Cho, Changes in gene cassettes of class 1 integrons among Escherichia coli isolates from urine specimens collected in Korea during the last two decades. J Clin Microbiol, 2003. 41; 5429-33.

2. Normark, B.H. and S. Normark, Evolution and spread of antibiotic resistance. J Intern Med, 2002. 252; 91-106.

3. Boyle-Vavra, S., M. Challapalli, and R.S. Daum, Resistance to autolysis in vancomycin-selected Staphylococcus aureus isolates precedes vancomycin-intermediate resistance. Antimicrob Agents Chemother, 2003. 47; 2036-9.

4. Hall, R.M., Mobile gene cassettes and integrons: moving antibiotic resistance genes in gram-negative bacteria. Ciba Found Symp, 1997. 207; 192-202.

5. Cohen, S.N., Transposable genetic elements and plasmid evolution. Nature, 1976. 263; 731-8.