临床分离铜绿假单胞菌质粒介导的喹诺酮耐药机制研究

Detection of plasmid-mediated quinolone resistance genes in clinical isolates of Pseudomonas aeruginosa

目的了解临床分离的铜绿假单胞菌中PMQR基因及ESBLs的分布情况,并对PMQR阳性菌株中染色体介导的喹诺酮耐药机制进行分析。方法采用琼脂稀释法测定菌株对环丙沙星和左氧氟沙星的药物敏感性;PCR检测qnr A、qnr B、qnr C、qnr D、qnr S、aac(6’)-Ib-cr和qep A,对PMQR阳性菌株扩增blaTEM、blaSHV、blaCTX-M和blaPER,同时扩增测序分析染色体基因gyr A、gyr B、par C和par E的突变情况;接合转移试验验证PMQR与bla基因的转移性。结果 106株铜绿假单胞菌中,2株携带qnr S,1株携带qnr D,2株携带aac(6’)-Ib-cr。4株PMQR阳性菌株中有2株接合转移成功,qnr S1和blaTEM-1基因可以通过质粒共同转移。PMQR阳性菌株均在Gyr A亚基和/或Par C亚基存在氨基酸突变,其相应的接合子以及受体菌均未发现突变。结论临床分离的铜绿假单胞菌中存在qnr S、qnr D和aac(6’)-Ib-cr基因,PMQR与bla基因能共同转移,造成多重耐药的传播。

Objective To investigate the prevalence of plasmid-mediated quinolone resistance （PMQR） among clinical Pseudomonas aeruginosa, and the presence of extended spectrum β-lactamase （ESBL） genes and mutations in quinolone resistance-determining regions in PMQR-positive isolates. Methods Antimicrobial susceptibility test was performed using the agar dilution method. Isolates were screened for the presence of qnrA, qnrB, qnrC, qnrD, qnrS, aac（6′）-Ib-cr, qepA, blaTEM, blaSHV, blaCTX-M, and blapER. Mutations in gyrA, gyrB, parC, and parE genes were identified by DNA sequencing of their PCR products. The transfer of PMQR and bla genes was studied by performing conjugation experiments. Results Among the 106 isolates ofP. aeruginosa, two isolates harbored qnrS, one harbored qnrD, and two carried aac（6′）-Ib-cr. Quinolone resistance could be transferred from two of the four PMQR-positive donors, qnrS1 and blaTEM-1 genes can be transferred together. Mutations in GyrA and/or ParC were observed among the four PMQR-positive isolates. There were no mutations in the target genes among the transconjugans and the recipient. Conclusion Our study decribed the clinical isolates of P. aeruginosa carrying PMQR genes. The co-existence and co-transfer of PMQR and bla genes may lead to the emergence and spread of multidrug-resistant pathogens.