铜绿假单胞菌整合子及其基因盒与耐药性的相关性研究

Study on Relationship between Pseudomonas aeruginosa Integrons and Antibiotic-resistance

目的调查致呼吸系统感染的铜绿假单胞菌中整合子的存在情况,研究整合子中基因盒与耐药表型的关系。方法测定97株铜绿假单胞菌对抗菌药物的敏感性;应用简并引物PCR方法扩增整合子5’保守区的Ⅰ类、Ⅱ类和Ⅲ类整合酶基因,对阳性产物用限制性内切酶HinfⅠ作限制片段长度多态性(RFLP)分析,并进行整合子分类;对整合酶阳性标本进行整合子可变区的扩增,并对大小相同的整合子进行酶切分析;对纯化后的整合子进行DNA测序。结果 20株铜绿假单胞菌检测出Ⅰ类整合子,未检出Ⅱ类和Ⅲ类整合子,其中17株含有基因盒,共有3种类型:979,1 833和2 337bp,测序结果显示有多种耐药基因。Ⅰ类整合子阳性的菌株对抗菌药物的耐药率普遍比整合子阴性的菌株高。结论铜绿假单胞菌的Ⅰ类整合子普遍携带耐药基因盒,参与了多重耐药性的形成。

Objective To analyze the relationship between gene cassettes and drug-resistant phenotype according to the distribution integrons in Pseudomonas aeruginosa isolated from respiratory infection and arrangement of gene cassette in integron.Methods Antibiotic susceptibility test of 97 strains of Pseudomonas aeruginosa were performed by using bioMerieux VITEK 2.Integrons were detected by PCR using degenerate primers targeted to 5＇conserved regions of class Ⅰ,Ⅱ and Ⅲ integrase genes,The class of the integron was determined by analyzing integrase PCR products by restriction fragment length polymorphism（RFLP） following digestion using Hinf I restriction enzyme.The variable region of integron was amplified by long fragment PCR,and the PCR products were restricted with Hinf I.Three representative products were purified and sequenced.Results Integrons were found in 20 Pseudomonas aeruginosa,which were all classⅠ integrons as PCR-RFLP analysis,no class Ⅱand Ⅲ integrons.Seventeen of the 20 strains contained three kinds of gene cassettes： 979,1 833 and 2 337 bp.These cassettes included many kinds of resistance genes.The rate of drug resistance in isolates of integron positive was generally higher than that of in the integron negative strains.Conclusions Resistance gene cassettes were generally found in class Ⅰ integrons of Pseudomonas aeruginosa and involved in the formation of multi-drug resistance.