Riemerella anatipestifer is a Gram-negative bacterium, which is an important pathogen infecting ducks and resistant to various antibiotics. The efflux pump is an important resistance mechanism of Gram-negative bacteria, but little research has been done in R. anatipestifer. In this study, the drug resistance mediated by RIA_1614 gene of R. anatipestifer RA-GD strain was studied, because the gene was presumed to be an efflux pump component of ABC. Firstly, the deletion strain RA-GD△RIA_1614 and complemented strain RA-GD△RIA_1614 pCPRA::RIA_1614 were constructed. Then, MICs of various antimicrobial agents to parent and deletion strains and the tolerance of the strains to organic solvents were detected to screen the substrates for RIA_1614 gene. Moreover, the transcription levels of RIA_1614 gene in the parent and the complemented strains exposed to the substrates were detected by quantitative real-time RT-PCR. Furthermore, the efflux abilities of parent, deletion and complemented strains to substrates were determined by antibiotic accumulation test. In addition, in vitro competition ability and virulence of the strains were also detected. The results showed that the deletion strain was more sensitive to aminoglycosides and organic solvents than parental strain RA-GD. When RA-GD and complemented strain were exposed to sub-repression levels of aminoglycosides and organic solvents, the transcription levels of RIA_1614 gene were significantly up-regulated. Sodium o-vanadate inhibitor assay confirmed that RIA_1614 protein contributed to amikacin and streptomycin resistance and organic solvent tolerance. Streptomycin accumulation test showed that the RIA_1614 protein was able to export streptomycin, and the addition of ATPase inhibitor sodium o-vanadate increased the accumulation of streptomycin, indicating that RIA_1614 protein was an ATP-dependent efflux transporter. Growth and competition experiments revealed that RIA_1614 protein had no significant effect on growth of RA-GD, but decreased in vitro competition ability of the strain. Furthermore, pathogenicity tests showed that RIA_1614 protein involved in the virulence of the strain. Based on the results and amino acid sequence analysis, it was determined that RIA_1614 protein was a member of ABC efflux pumps, and the protein was named RanB.

厌氧里氏杆菌是革兰氏阴性细菌，是一种重要的病原菌，可感染鸭并对多种抗生素具有抗性。外排泵是革兰氏阴性细菌的重要抵抗机制，但是在拟南芥中的研究很少。在这项研究中，研究了由R. anatipestifer RA-GD菌株的RIA_1614基因介导的耐药性，因为该基因被认为是ABC的外排泵成分。首先，缺失菌株RA-GD△ RIA_1614和互补菌株RA-GD△ RIA_1614 pCPRA :: RIA_1614被建造。然后，检测各种抗微生物剂对亲本和缺失菌株的MIC，以及该菌株对有机溶剂的耐受性，以筛选用于RIA_1614基因的底物。此外，通过定量实时RT-PCR检测亲本和暴露于底物的互补菌株中RIA_1614基因的转录水平。此外，通过抗生素积累测试确定了亲本，缺失和互补菌株对底物的外排能力。另外，体外还检测了菌株的竞争能力和毒力。结果表明，与亲本菌株RA-GD相比，该缺失菌株对氨基糖苷类和有机溶剂的敏感性更高。当RA-GD和互补菌株暴露于氨基糖苷和有机溶剂的亚阻水平时，RIA_1614基因的转录水平显着上调。邻钒酸钠抑制剂测定证实RIA_1614蛋白有助于阿米卡星和链霉素的耐药性以及对有机溶剂的耐受性。链霉素的积累试验表明，RIA_1614蛋白能够输出链霉素，并添加了ATPase抑制剂钠o-钒酸盐增加链霉素的积累，表明RIA_1614蛋白是ATP依赖性外排转运蛋白。生长和竞争实验表明，RIA_1614蛋白对RA-GD的生长无明显影响，但降低了该菌株的体外竞争能力。此外，致病性测试表明，RIA_1614蛋白与菌株的毒力有关。根据结果​​和氨基酸序列分析，确定RIA_1614蛋白是ABC外排泵的成员，并且将该蛋白命名为RanB。