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Influence of Anti-FloR Antibody on Florfenicol Accumulation in Florfenicol-Resistant Escherichia coli and Enzyme-Linked Immunosorbent Assay for Detection of Florfenicol-Resistant E. coli Isolates

机译:抗FloR抗体对耐氟苯尼考的​​大肠杆菌中氟苯尼考积累的影响和酶联免疫吸附法检测耐氟苯尼考的​​大肠杆菌分离物

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摘要

To detect florfenicol-resistant Escherichia coli isolates by enzyme-linked immunosorbent assay (ELISA), anti-FloR1 antibodies were produced in mice using a recombinant glutathione S-transferase (GST)-FloR1 protein, which was expressed in a prokaryote expression system, as the antigen. The specificity of the murine anti-GST-FloR1 antibody and its influence on florfenicol accumulation in florfenicol-resistant isolates were investigated using Western blotting and high-performance liquid chromatography, respectively. Western blotting using the anti-FloR1 antibody showed specific binding of the antibody to the florfenicol-resistant FloR protein. Preincubation of florfenicol-resistant strains with the antibody significantly increased the intracellular accumulation of florfenicol and enhanced the bacterial susceptibility to florfenicol, suggesting that antibody binding to the FloR protein inhibited the activity of the efflux protein conferred by the floR gene. Analyses of florfenicol-resistant and -sensitive isolates by ELISA using the anti-FloR1 antibody showed good correlation between FloR protein expression and the floR genotype. The anti-FloR1 antibody-based ELISA is a useful tool for the detection of florfenicol-resistant bacteria harboring the floR gene.
机译:为了通过酶联免疫吸附测定(ELISA)检测耐氟苯尼考的​​大肠杆菌分离株,使用重组谷胱甘肽S-转移酶(GST)-FloR1蛋白在小鼠中产生抗FloR1抗体,该蛋白在原核表达系统中表达为抗原。分别使用蛋白质印迹和高效液相色谱法研究了鼠抗GST-FloR1抗体的特异性及其对氟苯尼考耐药菌株中氟苯尼考积累的影响。使用抗FloR1抗体的蛋白质印迹显示该抗体与耐氟苯尼考的​​FloR蛋白具有特异性结合。用该抗体对氟苯尼考抗性菌株进行预培养显着增加了氟苯尼考的​​细胞内积累并增强了细菌对氟苯尼考的​​敏感性,这表明与FloR蛋白结合的抗体抑制了由floR基因赋予的外排蛋白的活性。使用抗FloR1抗体通过ELISA对氟苯尼考耐药和敏感的分离株进行分析表明,FloR蛋白表达与floR基因型之间具有良好的相关性。基于抗FloR1抗体的ELISA是检测含有floR基因的耐氟苯尼考的​​细菌的有用工具。

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