新质粒介导喹诺酮类耐药基因qnrB24的克隆表达

Cloning and expression of qnrB24:a new gene conferring plasmid-mediated quinolone resistance

目的研究新质粒介导喹诺酮耐药基因qnrB24的耐药特性。方法 PCR扩增qnrB24基因全编码区,对PCR产物进行测序分析,将qnrB24与克隆载体PHSG398双酶切连接,转化入E.coil JM109受体菌中表达。用E试验纸条法对携带qnrB24的临床菌株、受体菌和克隆表达株进行常用喹诺酮类抗菌药物敏感性试验。结果 qnrB24与qnrB10同源性为98.7%,3个碱基位点发生同义突变,分别为139位C→A,257位C→T,670位A→G,导致氨基酸改变为47位亮氯酸→蛋氨酸,86位丙氨酸→缬氨酸,224位异亮氨酸→缬氨酸。qnrB24基因表达株对常见氟喹诺酮类抗菌药物的敏感性较受体菌下降为原来的近1/8,但耐药水平低于临床分离菌株1/32～1/128。结论本研究首次发现qnrB24基因,qnrB24基因可以轻度增加氟喹诺酮类药物的耐药性。

Objective To study a new gene, qnrB24, which confers susceptibility to fluoroquinolones. Methods The qnrB24 was sequenced and compared with those included in the GenBank database. The PCR amplicons of qnrB24 and vector PHSG39g gene were digested with BamHI and EcoR, and then ligated to similarly restricted vector PHSG398, and cloned into E. coil JM109. The minimum inhibitory concentrations （MICs） of eiprofloxacin and levofloxacin were determined by E-test strips ac- cording to the manufacturers instructions. Results Sequence analysis revealed that the qnrB24 gene had a maximal identity of 98.7% to the qnr1310 gene. Three nucleotide changes were observed. The C--A at nucleotide position 139 resulted in Leu-Met. The C-T at position 257 produced a Ala--Val change. The A---G at position 670 produced a Ile--Val change. Suscepti- bility of the transformant containing qnrB24 gene against common fluoroquinoiones was about 1/8 of the recipient strain, hut the MIC value was about 1/32 to 1/128 of the clinical isolate. Conclusions This may be the first report of qnrB24 gene. qnrB24 gene may slightly raise the resistance to fluoroquinolones.