多重耐药革兰阴性杆菌I类整合子结构与功能分析

目的探索I类整合子在多重耐药的革兰阴性杆菌中的功能作用，并对其基因结构进行研究，为阐明革兰阴性杆菌的耐药机制和抑制耐药性的产生提供理论依据。方法选择温岭市妇幼保健院2009年7月～2011年10月的多重耐药革兰阴性杆菌80株，其中铜绿假单胞菌40株，鲍曼不动杆菌40株，使用K—β纸片法和双纸片协同实验对细菌的耐药性和超广谱β-内酰胺酶（ELSBs）的产生情况进行考察。对耐药且产ELSBs的细菌进行筛选．使用聚合酶链式反应（PCR）扩增实验、电泳法、整合酶Souhen杂交实验对耐药细菌体内的I类整合子存在情况、整合子基因结构、基因位置进行考察。结果药敏结果显示，40株铜绿假单胞菌中多重耐药且产ELSBs的菌株为27株，40株鲍曼不动杆菌中多重耐药且产ELSBs的菌株为28株。对多耐药且产ELSBs的菌株通过PCR扩增实验进行的I类整合子存在情况的考察结果显示，40株鲍曼不动杆菌和40株铜绿假单胞菌中I类整合子的阳性率分别为57．5％和55．O％。I类整合子的保守区和可变区PCR扩增与测序分析结果显示，保守区内含有qaeEal-F和sul1-B这两种特征性基因片段，可变区内以长度为0．15、1．00、2．00、2．30kb的基因片段出现频率最高．0．15kb含有保守区内基因结果，其它基因片段依次为aadA、aaeA4、catB8这3种基因盒。结论I类整合子与革兰氏阴性杆菌耐药性的产生有着密切的关系，qaeEal-F和sull一B为I类整合子基因保守区的特征性片段。两者同时出现则可确证该细菌体内含有I类整合子。I类整合子基因可变区内含有aadA、aaeA4、catB8三种出现频率最高的基因盒且长短为0．15kb的基因片段含有保守区的基因结构，这3种基因盒和保守区基因片段的存在是导致细菌间耐药性平行转移的重要因素。

Analysis about the structure and function of I integron of Gram-negative bacilli with multi-drug resistance

Objective To explore the functional role of class I integrons in multidrug-resistant for Gram-negative bacteria, and to study on the genetic structure to provide some theoretical basis for elucidating the mechanism of resis- tance of gram-negative bacteria and inhibiting the development of drug resistance. Methods 80 cases of multi-resis- tant gram-negative bacteria in Maternal and Child Health Hospital of Wenling City from July 2009 to October 2011 were selected, 40 cases of pseudomonas aeruginosa and 40 cases of acinetobacter baumannii were contained, K-B disk and double-disk synergy test were used to evaluate the drug-resistance and extended-spectrum 13-1actamase （ELSBs） of gram-negative bacteria. The bacteria with drug-resistance and ELSBs were screened, polymerase chain reaction （PCR）, electrophoresis, electrophoresis and integrated enzyme Sourthen hybridization experiments were used to evaluate the presence of class I integron, integron gene structure and gene position. Results 27 strains bacteria with mul- tidrug-resistant and ELSBs were found in 40 cases of pseudomonas aeruginosa, 28 strains bacteria with multidrug-re- sistant and ELSBs were found in 40 cases of acinetobacter baumannii according to the results of susceptibility. The positive rate of class I integron in 40 cases of pseudomonas aeruginosa and 40 cases of acinetobacter baumannii were 57.5% and 55.0% according to the results of PCR amplification experiment. Characteristic gene fragment of qaeE A 1-F and sul 1-B gene sequence were found in conserved region, the highest frequency of gene segment were found with length of 0.15, 1.00, 2.00, 2.30 kb in variable region and other fragment was gene cassette such as aadA, aaeA4, catB8 according to the results of PCR amplification for conservative and variable regions. Conclusion Class I inte- gron has a close relationship with the muhidrug-resistant of gram-negative bacteria, resistant generation qaeE A 1-F and sull-B are the characteristic fragments of the gene conserved region of class I integrons, both of their appear- ance of the two fragment are the evidence of the bacteria contains a class I integron. Class I integron gene variable region contains three kinds of gene cassettes such as aadA, aaeA4 catB8, and the 0.15 kb gene fragment contains the gene structure of class integron gene conserved region, this three kinds of gene cassette and the presence of the conserved region gene fragments play an improtant role for the parallel transfer of multidrug-resistance of gram-negative bacteria.