新质粒介导喹诺酮耐药基因qnrB24的基因分析

Analysis of plasmid mediated quinolone resistance gene qnrB24

目的 对新的质粒介导喹诺酮耐药基因（qnrB24）进行相关研究和分析。方法 对新发现的qnrB24基因阳性菌株进行转移接合实验,了解qnrB24对喹诺酮类药物的耐药性,碱裂解法提取接合子质粒,Southern blot明确质粒大小。采用热不对称交错聚合酶链反应（PCR）技术研究基因5′端以及3′端未知侧翼碱基序列。结果 这个突变基因命名为qnrB24（Genbank登录号HM192542）。药敏试验结果显示携带qnrB24基因接合子对环丙沙星的最小抑菌浓度（MIC）值为0.125μg/mL,左氧氟沙星MIC值为0.190μg/mL。接合子对常见氟喹诺酮类抗菌药物的敏感性较大肠杆菌J53受体菌下降约8~11倍,接合子耐药水平低于临床分离菌株。Southern杂交显示该基因位于约60.0Kb质粒上。热不对称交错PCR结果显示,qnrB24基因5′端为假定的转座酶。结论 qnrB24通过质粒介导引起细菌对喹诺酮类药物的耐药性上升,容易发展成高水平耐药,因此需要监测其在细菌中的流行性。

Objective To detect and analyze the function of plasmid mediated quinolone resistance gene qnrB24.Methods The positive strains of qnrB24 gene detection were conducted transfer joint experiment,to explore the drug resistant of these strains to quinolonne drug,zygote plasmid was extracted by alkaline lysis method,and the size was detected by southern blot.The unknown base sequence on the wing of gene 5′end and 3′end were detected by thermal asymmetric interlaced polymerase chain reaction（PCR）technology.Results Susceptibility results showed that the minimum inhibitory concentration（MIC）of the transconjugant（carrying qnrB24gene）against ciprofloxacin,levofloxacin were 0.125,0.190μg/mL respectively.The sensitivity of the transconjugant was lower than the recipient starin,but the drug resistance was lower than the bacterial isolated in clinic.Southern hybridization of plasmid DNA from transconjugant of qnrB24 revealed that the gene was located on about 60 Kb plasmid.The quinolone resistance of qnrB24 could be transferred by conjugation.There was putative transposase adjust to 5′flank of qnrB24 gene.Conclusion The qnrB24 gene is discovered firstly.Gene qnrB24 could improve the drug resistance to fluoroquinolones,it′s necessary to monitor the epidemic characteristic of qnrB24 gene.