嗜水气单胞菌喹诺酮类药物抗性决定区基因片段的克隆与突变分析

Cloning and Sequence Mutation Analysis of Quinolone Resistance Determining Regions of Pathogenic Aeromonas hydrophil

将分离到的4株对环丙沙星、诺氟沙星和氧氟沙星等喹诺酮类药物耐药的嗜水气单胞菌、2株敏感菌,进行旋转酶基因A亚单位(gyrA)喹诺酮抗性决定区基因(QRDR)PCR扩增。引物序列A1:AGAGTTC CTATCTTGATTACG;A2:CTGTGATGTAGGTCATCAACT,在gyrA基因中的位置分别为53～73和620～640。PCR扩增后,将扩增产物克隆到pMD-18T载体,酶切鉴定后测序。用DNAstar软件分析比较其蛋白序列,发现4个氨基酸突变位点,分别是83位点的Ser-Ile(4株耐药菌),92位点的Leu-Met(4株耐药菌),174位点的Ile-Phe(3株耐药菌),202、203位点的Asn、Leu-Asp、Arg(3株耐药菌,另1株Leu未突变),83位点的突变与大肠杆菌等一致,122位点具有保守的Try,耐药菌突变后的氨基酸残基分子量均比对应增大。4株耐药菌、2株敏感菌的喹诺酮抗性决定区基因片段序列已登录GenBank,注册号AY039655,AY039656,AY138539,AY138540,AY138537,AY138538。

In order to invesgate the mechanism of quinolones drug resistance of Aeromonas hydrophil, the genome of four drug resistant strains, two susceptible strains isolated in Jilin Province were amplified by PCR. The primers, used included A1:AGAGTTCCTATCTTGATTACG;A2:CTGTGATGTAGGTCATCAACT, which were synthesized according to the gyr A gene sequence of A. salmonicida, which amplify the quinolones resistancedetermining region(QRDR) of the gyrA gene. PCR products were cloned into vector pMD18T, the recombinant vectors were transfered into the E.coli DH5αcompetent cells. After identification, the sequences of the amplified QRDRs were determined and analysised by DNA star software. Four mutation sites of amino acid sequence were found, which were serine 83toisoleucine, leucine 92tomethionine, isoleucine 174tophenylalanine and an adjacent site asparagines 202, leucine 203toasparatic acid, arginine. Among 4 mutation sites, only the mutation of site 83 was identical with E.coli and other drug resistant bacteria, the molecular weigh of the mutated amino acid residue was bigger than original residue. The QRDR sequences of four drug resistant strains and two susceptible strains were registered to Genebank with accession numbers being AY039655, AY039656, AY138539,AY138540,AY138537,AY138538.