临床分离肺炎克雷伯菌Ⅰ类整合子的结构与功能

doi:10.3724/SP.J.1005.2014.0603

遗传 ›› 2014, Vol. 36 ›› Issue (6): 603-610.doi: 10.3724/SP.J.1005.2014.0603

临床分离肺炎克雷伯菌Ⅰ类整合子的结构与功能

张跃进^{1, 3}, 常清利¹, 汪倩¹, 卢俊婉^{1, 2}, 王欢¹, 李佩珍¹, 应俊¹, 包其郁¹, 胡云良¹

1. 温州医科大学检验医学与生命科学学院, 温州 325000;
2. 丽水学院医学院, 丽水 323000;
3. 温州医科大学护理学院, 温州 325000

收稿日期:2013-11-18 修回日期:2014-01-21 出版日期:2014-06-20 发布日期:2014-05-28
通讯作者: 胡云良,硕士,教授,研究方向：生化与分子生物学。E-mail:huyunliang66@163.com E-mail:zhangyuejin1111@126.com
作者简介:张跃进,实验师,大学本科,研究方向：基础医学。Tel:0577-86699168;E-mail:zhangyuejin1111@126.com
基金资助:
浙江省科技计划项目(编号：2008C23074)与卫生部科研基金(编号：WKJ2012-2-032)资助

Structure and function of class 1 integron in clinical isolates of Klebsiella pneumoniae

Yuejin Zhang^{1, 3}, Qingli Chang¹, Qian Wang¹, Junwan Lu^{1, 2}, Huan Wang¹, Peizhen Li¹, Jun Ying¹, Qiyu Bao¹, Yunliang Hu¹

1. School of Laboratory and Life Sciences, Wenzhou Medical University, Wenzhou 325000, China;
2. School of Medicine, Lishui College, Lishui 323000, China;
3. School of Nursing, Wenzhou Medical University, Wenzhou 325000, China

Received:2013-11-18 Revised:2014-01-21 Online:2014-06-20 Published:2014-05-28

摘要/Abstract

摘要：

为了探索细菌多重耐药性的产生和播散的分子机制, 文章对2002～2007年间179株临床分离的肺炎克雷伯菌进行耐药性、I类整合子可变区基因盒结构以及基因盒携带的耐药性基因进行分段克隆和耐药性功能测定。结果显示：65.9%(118/179)的肺炎克雷伯菌表现出对至少两种以上的抗生素(主要为β-内酰胺类、氨基糖苷类和喹诺酮类抗菌药物)的耐药性; 36.3%(65/179)的菌株检出单条或者双条I类整合子基因盒条带; 对整合子阳性组与阴性组的耐药率进行比较发现, 除氨基糖苷类、喹诺酮类和复方新诺明等药物的耐药性存在显著性差异(P<0.01)外, 其余药物的差异不显著; 共发现15种耐药基因构成形式的整合子基因盒, 其中以dfrA17-aadA5最为多见, 实验证明整合子可由接合转移耐药性质粒携带; 对整合子基因盒(dhfr17-orfF-aadA2)分段克隆的耐药性功能研究发现, 3个克隆重组子(pET28a-dhfr17、pET28a-dhfr17-orfF和pET28a-dhfr17-orfF-aadA2)对复方新诺明的抗性(MIC值)均为256 µg/mL, 重组子pET28a-dhfr17-orfF与重组子pET28a-dhfr17对链霉素的抗性无明显区别, 和受体菌一样MIC值均为8 µg/mL, 而pET28a-dhfr17-orfF-aadA2对链霉素的抗性则明显提高, MIC值为256 µg/mL。结果表明, I类整合子在肺炎克雷伯菌中较常见, 携带氨基糖苷类和甲氧苄啶类的耐药基因盒在数量上占优势, 且整合子携带的耐药基因具有耐药性功能, 位于可水平转移耐药性质粒的耐药性基因相关的整合子对病原菌耐药性播散具有重要意义。目的基因

关键词: I类整合子, 基因盒, 克隆, 耐药性

Abstract:

To investigate molecular mechanism of multi-resistance of Klebsiella pneumoniae and its spreading, 179 strains isolated from different clinical samples in the period of 2002-2007 with serious resistance to 14 anti-bacterial agents were examined. Among them, 118 (65.9%) were resistant to at least two anti-bacterial agents; 36.3% (65/179) were found to contain class 1 integrons. There was a significant difference for resistance rate between the integron positive and the negative groups, especially for antimicrobial agents of aminoglycosides, quinolones and sulfonamides (P<0.01). Gene cassette structures of the class 1 integrons in these bacteria were analyzed and their resistance genes were further cloned and tested for antibiotic resistance activities. Fifteen gene cassettes were identified with dfrA17-aadA5 being the most popular form. Three recombinant plasmids pET28a-dhfr17, pET28a-dhfr17-orfF and pET28a-dhfr17-orfF-aadA2 were cloned from a gene cassette of dhfr17-orfF-aadA2. When introduced into a recipient E. coli strain BL21, all of them rendered resistance to co-trimoxazole, with minimum inhibitory concentration (MIC) value up to 256 µg/µL. The E. coli BL21 carrying pET28a- dhfr17 or pET28a-dhfr17-orfF had the same MIC value of 8 µg/µL to streptomycin as the recipient strain without plasmid. However, the E. coli carrying pET28a-dhfr17-orfF-aadA2 was resistant to streptomycin with MIC level up to 256 µg/µL. In conclusion, class 1 integrons were regularly identified in Klebsiella pneumoniae. They mainly carry resistance genes against antimicrobial agents of aminoglycosides and sulfonamide. Transferable plasmid carrying integrons with resistance genes may play an important role in resistance spreading among bacterial species.

Key words: class 1 integron, gene cassette, cloning, antibiotic resistance

张跃进, 常清利, 汪倩, 卢俊婉, 王欢, 李佩珍, 应俊, 包其郁, 胡云良. 临床分离肺炎克雷伯菌Ⅰ类整合子的结构与功能[J]. 遗传, 2014, 36(6): 603-610.

Yuejin Zhang, Qingli Chang, Qian Wang, Junwan Lu, Huan Wang, Peizhen Li, Jun Ying, Qiyu Bao, Yunliang Hu. Structure and function of class 1 integron in clinical isolates of Klebsiella pneumoniae[J]. HEREDITAS(Beijing), 2014, 36(6): 603-610.

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临床分离肺炎克雷伯菌Ⅰ类整合子的结构与功能

Structure and function of class 1 integron in clinical isolates of Klebsiella pneumoniae

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