嗜水气单胞菌对喹诺酮类药物耐药的分子机制

Molecular mechanisms of quinolone resistance in Aeromonas hydrophilia

【目的】调查从浙、苏、皖等地水产动物中分离的23株致病性嗜水气单胞菌的耐药谱并探索喹诺酮类抗生素耐药菌株的耐药分子机制。【方法】根据美国临床实验室标准化协会药敏判断标准(2011版)测定23株嗜水气单胞菌耐药谱并筛选喹诺酮类抗生素耐药株;对筛选的耐药菌株和体外诱导耐药菌株的gyrA和parC基因耐药决定区进行分析;用二倍稀释法测定加入多重耐药外排泵抑制剂碳酰氰基-对-氯苯腙前后耐药菌株对恩诺沙星最小抑菌浓度的变化,同时检测喹诺酮类药物相关的外排泵基因qepA、oqxA和mdfA;并检测质粒介导的喹诺酮耐药的qnr家族基因qnrA、qnrB、qnrC、qnrD和qnrS。【结果】所有菌株都存在对5种以上药物的耐药性;39.1%(9/23)的嗜水气单胞菌对喹诺酮类药物耐药,其中55.6%(5/9)对恩诺沙星耐药。耐恩诺沙星的5株菌株均携带qnrS,而不携带qnrA、qnrB、qnrC、qnrD基因,也不携带外排泵基因qepA、oqxA和mdfA。其中耐药菌株AH19同时存在gyrA与parC基因双突变、质粒介导的耐药基因qnrS和主动外排泵三种耐药机制,菌株AH4、AH7和AH20存在gyrA与parC基因双突变和质粒介导的耐药基因qnrS两种耐药机制,AH6存在gyrA基因突变和质粒介导的耐药基因qnrS机制,体外诱导耐药菌株ATCC7966-QR相对于原始菌株ATCC7966发生了gyrA与parC基因双突变。【结论】喹诺酮类药物作用靶位的改变和质粒介导的耐药基因qnrS的存在是本研究中涉及的嗜水气单胞菌对喹诺酮类药物耐药的主要作用机制,主动外排泵机制是个别菌株存在的耐药机制。

[ Object ] To study the resistance mechanisms to quinolones in Aeromonas hydrohila isolated from aquatic animals. [ Methods] The drug-resistant spectrum of 23 strains was determined. Quinolone-resistance determining regions of gyrA and parC genes in both screened and in-vitro induction drug-resistant strains were analyzed. Then the detection of quinolone drugs relative efflux pump genes qepA, oqxA and mdfA was performed. The qnrA, qnrB, qnrC, qnrD and qnrS genes were also analyzed at the same time. [ Results] All organisms were resistant to more than 5 drugs; 39.1% （9/23） of the isolates were quinolone resistant, of which 55.6% （5/9） were enrofloxacin resistant. All the enrofloxacin-resistant isolates harbored qnrS gene, but none of the enrofloxaein-resistant strains harbored qnrA, qnrB, qnrC , qnrD genes and the efflux pump genes of qepA, oqxA and mdfA. AH19 possessed the gyrA and parC genes double mutation, plasmid- mediated quinolone resistance gene qnrS and efflux pump, 3 drug resistance mechanisms simultaneously, while the two drug-resistant mechanisms of AH4, AH7 and AH20 were gyrA and parC genes double mutation and qnrS gene. GyrA gene mutation and qnrS gene occurred in AH6. Compared to the strain ATCC7966, the in-vitro induction drug-resistant strain ATCC7966-QR had both the gyrA and parC genes mutation. [ Conclusion] The mechanisms of resistance to quinolones in the A. hydrophila isolates of this study mainly depended on the existence of plasmid-mediated gene qnrS and the variation of the target site of quinolone drugs, whereas, the drug resistance mechanism relying on the efflux pump system only existed in individual strains.