Due to the inappropriate use of florfenicol in agricultural practice, florfenicol resistance has become increasingly serious. In this work, we studied the novel florfenicol resistance mechanism of an animal-derived Leclercia adecarboxylata strain R25 with high-level florfenicol resistance. A random genomic DNA library was constructed to screen the novel florfenicol resistance gene. Gene cloning, gene knockout, and complementation combined with the minimum inhibitory concentration (MIC) detection were conducted to determine the function of the resistance-related gene. Sequencing and bioinformatics methods were applied to analyze the structure of the resistance gene-related sequences. Finally, we obtained a regulatory gene of an RND (resistance-nodulation-cell division) system, ramA, that confers resistance to florfenicol and other antibiotics. The ramA-deleted variant (LA-R25ΔramA) decreased the level of resistance against florfenicol and several other antibiotics, while a ramA-complemented strain (pUCP24-prom-ramA/LA-R25ΔramA) restored the drug resistance. The whole-genome sequencing revealed that there were five RND efflux pump genes (mdtABC, acrAB, acrD, acrEF, and acrAB-like) encoded over the chromosome, and ramA located upstream of the acrAB-like genes. The results of this work suggest that ramA confers resistance to florfenicol and other structurally unrelated antibiotics, presumably by regulating the RND efflux pump genes in L. adecarboxylata R25.

由于在农业实践中不当使用氟苯尼考，氟苯尼考耐药性变得越来越严重。在这项工作中，我们研究了具有高水平的氟苯尼考耐药性的动物来源的莱克氏菌Adecarboxylata菌株R25对氟苯尼考的新型耐药机制。构建了一个随机基因组DNA文库以筛选新型氟苯尼考抗性基因。进行基因克隆，基因敲除和互补与最小抑制浓度（MIC）检测相结合，以确定抗性相关基因的功能。测序和生物信息学方法被用于分析抗性基因相关序列的结构。最后，我们获得了RND（抗性结节细胞分裂）系统的调控基因ramA赋予对氟苯尼考和其他抗生素的抗性。所述RAMA -deleted变体（LA-R25Δ RAMA）降低对氟苯尼考和其他几种抗生素抗性水平，而RAMA -complemented菌株（pUCP24-prom- RAMA / LA-R25Δ RAMA）恢复了耐药性。全基因组测序显示，在染色体上编码了五个RND外排泵基因（mdtABC，acrAB，acrD，acrEF和acrAB样），而ramA位于acrAB样基因的上游。这项工作的结果表明ramA可能通过调节阿德莫氏乳杆菌R25中的RND外排泵基因来赋予对氟苯尼考和其他与结构无关的抗生素的抗性。