水产动物源气单胞菌喹诺酮类耐药与PMQR基因、QRDR突变相关性分析

Correlation between PMQR genes/QRDR mutations and quinolones resistance in Aeromonas from aquatic animals

关键词:  气单胞菌  PMQR基因  QRDR突变  喹诺酮类  耐药

Key words:  Aeromonas  PMQR  QRDR mutation  quinolones  resistance

摘要:

为了解PMQR基因、QRDR突变与气单胞菌喹诺酮类耐药相关性，以116株水产源气单胞菌为研究对象，采用PCR法检测PMQR基因（qnrA、qnrB、qnrS、qepA、aac（6'）-Ib-cr）并分析QRDR靶基因gyrA、parC突变情况，用微量二倍稀释法测定萘啶酸（NAL）、环丙沙星（CIP）、恩诺沙星（ENR）的最小抑菌浓度（MIC）值。结果表明：116株气单胞菌中，有18株（15.52%）菌株携带PMQR基因，其中8株（6.90%）携带qnrS2基因，9株（7.76%）携带aac（6'）-Ib-cr基因，1株同时携带qnrS2、aac（6'）-Ib-cr基因，未检测到qnrA，qnrB和qepA基因；56株（48.28%）菌株发生QRDR靶位点突变，主要突变方式为GyrA：Ser⁸³Ile和GyrA：Ser⁸³Ile+ParC：Ser⁸⁷Ile；对NAL、CIP及ENR耐药率分别为47.41%、12.07%及11.21%；7株仅携带qnrS2基因菌株未发生QRDR突变，对喹诺酮类药物敏感性略下降，NAL、CIP及ENR的MIC值分别小于4.00、0.50和1.00 mg/L；10株携带aac（6'）-Ib-cr基因的菌株均发生QRDR突变并对NAL表现耐药，MIC值均> 128.00 mg/L，其中8株对CIP和ENR表现耐药，MIC值均≥ 4.00 mg/L；发生gyrA单突变或gyrA和parC双突变菌株均对NAL表现耐药，MIC值均≥ 64.00 mg/L，CIP和ENR的MIC值有的< 4.00 mg/L，有的≥ 4.00 mg/L。综上，QRDR靶基因突变可影响水产动物源气单胞菌对萘啶酸的药物敏感性，而气单胞菌对氟喹诺酮类药物耐药可能与PMQR和QRDR协同作用有关。

Abstract:

To analyze the correlation of plasmid-mediated quinolone resistance (PMQR) genes/quinolone resistance determining region (QRDR) mutations with quinolones resistance in Aeromonas,116 Aeromonas isolates were taken as study object.PMQR genes including qnrA,qnrB,qnrS,qepA and aac(6')-Ib-cr in Aeromonas were detected by PCR.The target genes of gyrA and parC were also amplified by PCR to analyze the mutations in QRDR.The minimal inhibitory concentrations (MICs) of all 116 isolates against nalidixic acid (NAL),enrofloxacin (ENR) and ciprofloxacin (CIP)of quinolones were evaluated by micro broth 2-fold dilution.The results showed that:Eighteen (15.52%) isolates were detected carrying PMQR gene.Among those strains,8 (6.90%) and 9 (7.76%) strains carried qnrS2 gene and aac(6')-Ib-cr gene,respectively.One strain carried both of these two genes;qnrA,qnrB and qepA genes were not detected in this study;Mutations in the QRDRs of gyrA and parC were found in 56 strains (48.28%) of all isolates with GyrA:Ser⁸³Ile and GyrA:Ser⁸³Ile + ParC:Ser⁸⁷Ile combination being the most common mutations.The resistance rates of 116 Aeromonas isolates to NAL,CIP and ENR were 47.41%,12.07% and 11.21%,respectively;With the exception of one strain carrying qnrS2 gene alone was resistant to CIP,7 strains were susceptible to the 3 drugs of quinolones examined;No mutations in QRDR.The MICs of NAL,CIP and ENR were less than 4.00,0.50 and 1.00 mg/L,respectively;Ten aac(6')-Ib-cr positive strains were found mutations in QRDR and resistant to NAL (MIC>128.00 mg/L), and eight strains were resistant to CIP and ENR (MIC≥4.00 mg/L).Strains with either gyrA single mutation or gyrA + parC double mutation were resistant to NAL (MIC≥64.00 mg/L).The MICs of CIP and ENR for the strains with QRDR mutation,some strains were less than 4.00 mg/L,and there were more than 4.00 mg/L.In conclusion,QRDR target gene mutation may contribute to the susceptibility decrease of NAL,and the fluoroquinolone resistance in Aeromonas might be attributed to the mutual cooperation between PMQR and QRDR mutations.