海水养殖动物源弧菌喹诺酮类药物耐药表型与基因型分析

采用琼脂稀释法测定121株海水养殖源弧菌对喹诺酮类药物(恩诺沙星、诺氟沙星和环丙沙星)的敏感性,利用PCR方法检测其质粒介导的喹诺酮类耐药基因(qnrVC、qnrS、qnrA和qnrB)、外排泵耐药基因(oqxA、oqxB、acrR、marR和soxR)和整合子(Int1、SXT和ISCR1),同时研究4种外排泵抑制剂(甲基吡咯烷酮,NMP;利血平,RSP;羰基氰氯苯腙,CCCP;苯丙氨酸-精氨酸-β萘酰胺,PAβN)对弧菌恩诺沙星、诺氟沙星和环丙沙星最小抑菌浓度的影响。在121株弧菌中,恩诺沙星耐药菌株31株(25.6%),诺氟沙星耐药株21株(17.4%),环丙沙星耐药株22株(18.2%);4种质粒介导喹诺酮类耐药基因仅检测到qnrA(2株)和qnrVC(30株),5种喹诺酮类外排泵基因仅在溶藻弧菌352菌株中检测到oqxB;121株弧菌中检测到39株菌携带Int1、42株菌携带SXT、44株菌携带ISCR1;在NMP、RSP、CCCP和PAβN作用下,分别有22株、25株、31株和7株弧菌对恩诺沙星的敏感性下降,分别有6株、5株、9株和4株弧菌对诺氟沙星的敏感性下降,分别有17株、13株、5株和14株弧菌对环丙沙星的敏感性下降。研究结果表明,弧菌对喹诺酮类药物耐药表型与基因型存在较大的不一致性,外排泵抑制剂对弧菌喹诺酮类药物的耐药性具有显著影响,预示弧菌对喹诺酮类药物耐药存在着新的机制。

Analysis of phenotype and genetype in quinolone resistance in Vibrio from mairculture

The minimal inhibitory concentrations of Enrofloxacin, Norfloxacin and Ciprofloxacin of 121 Vibrio isolated from maricultural animals were determined using the agar dilution method. PCR amplication method was used to detect the plasmid-mediated quinolones resistance genes(qnrVC, qnrS, qnrA, qnrB), efflux pump genes(oqxA, oqxB, acrR, marR, soxR)and integron-related genes(Int1, SXT, ISCR1).To investigate the effect of efflux pump on quinolones resistance, the minimal inhibitory concentrations of Enrofloxacin, Norfloxacin, Ciprofloxacin with/without efflux pump inhibitors were analyzed. To investigate the effects of four efflux pumps(NMP, RSP, CCCP, PAβN) on quinolones resistance, the minimal inhibitory concentrations of Enrofloxacin, Norfloxacin, Ciprofloxacin with/without efflux pump inhibitors were analyzed. In the total 121 Vibrio strains, 31 strains,21 strains and 22 strains were resistant to Enrofloxacin, Norfloxacin and Ciprofloxacin, respectively. As shown, qnrVC gene was detected positive in 30 strains of Vibrio isolates. but only 2 positive strains for qnrA gene and 1 positive oqxB strains. Furthermore, Int1, SXT, and ISCR1 were found in 39, 42, 44 strains of the 121 Vibrio isolates tested, respectively. Under the action of NMP, RSP, CCCP and PAβN, the sensitivity of 22, 25, 31, 7 strains of Vibrio isolates to Enrofloxacin decreased respectively. The sensitivity of 6, 5, 9, 4 strains to Norfloxacin decreased respectively, with 17, 13, 5, 14 strains of Vibrio isolates decreased sensitivity to Ciprofloxacin. There was a large difference between the genotype of drug resistance and genetype of resistance genes. Antibiotic susceptibility testing suggested that efflux pump inhibitors were effective on the treatment for the quinolones resistance of Vibrio isolates, indicating that there was a new mechanism for the quinolones resistance of Vibrio.