[Objective] This study investigated the resistance mechanisms of Escherichia coli from giant pandas against pradofloxacin.[Method] Mutants resistant to pradofloxacin,marbofloxacin and ciprofloxacin were selected by stepwise exposure of E.coli to increasing concentrations of pradofloxacin,marbofloxacin and ciprofloxacin.The sequences of quinolone resistance determining region (QRDR) of gyrA and parC were analyzed.The activities of three efflux pumps were evaluated by efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβA) and quantitative real-time reverse transcriptase PCR (qRT-PCR).[Result] The resistant mutants were obtained after one or two rounds selection by three fluoroquinolones.It was easy to select resistant mutants by ciprofloxacin,followed by marbofloxcain and pradofloxacin.Pradofloxacin firstly targeted the gyrA subunit of DNA gyrase,and the number of mutations in target genes correlated with MICs.Four ciprofloxacin and mabofloxacin associated mutants had a point mutation at position 119 (Ala 119 Glu) in gyrA except two mutations at position 83 (Ser 83 Leu) and position 87 (Asp 87 Asn).Overexpression of acrB,emrE and mdfA genes was identified in all resistant mutants compared with parental isolates,and their activities increased with the increase of MICs.[Conclusion] Pradofloxacin resistance in giant panda E.coli was mainly caused by point mutations and overexpression of efflux pump gene.%[目的]探究大熊猫源大肠埃希菌对新型动物专用氟喹诺酮类药物普多沙星的耐药机制.[方法]以从大熊猫体液分离的对氟喹诺酮类药物敏感的大肠埃希菌株为研究对象,采用环丙沙星、麻保沙星和普多沙星通过体外逐渐递增药物浓度的方法诱导其成为耐药菌株.采用PCR扩增和基因测序的方法,分析诱导耐药菌株的靶位基因gyrA和parC耐药决定区(QRDR)的基因突变情况.采用外排泵抑制剂苯丙氨酸-精氨酸-β-萘酰胺(PAβA)和实时荧光定量PCR方法探讨外排泵在体外耐药中的作用.[结果]采用体外浓度递增法可诱导大熊猫大肠埃希菌对3种氟喹诺酮类药物产生耐药性,其中环丙沙星最易产生耐药性,其次是麻保沙星,普多沙星相对较慢.普多沙星诱导的耐药菌首先在gyrA基因发生点突变,随着MIC的升高,靶基因突变位点也逐渐增多.环丙沙星和麻保沙星诱导的4株耐药菌的gyrA基因出现Ala 119 Glu突变,而该突变在普多沙星所诱导的突变体中未发现.acrB,emrE和mdfA 3种外排泵基因在所有诱导的耐药菌株中均出现不同程度的过量表达,特别是在环丙沙星和麻保沙星诱导突变体中的表达明显高于在普多沙星诱导突变体中的表达.3种受试外排泵基因的表达均随MIC的升高而增加.[结论]普多沙星引起的大熊猫致病性大肠埃希菌多重耐药性,主要由靶基因的点突变和外排泵基因的过量表达所导致.
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