RamA, a transcriptional regulator conferring florfenicol resistance inLeclercia adecarboxylataR25

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-derivedLeclercia adecarboxylatastrain 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. TheramA-deleted variant (LA-R25 Delta ramA) decreased the level of resistance against florfenicol and several other antibiotics, while aramA-complemented strain (pUCP24-prom-ramA/LA-R25 Delta ramA) restored the drug resistance. The whole-genome sequencing revealed that there were five RND efflux pump genes (mdtABC,acrAB,acrD,acrEF, andacrAB-like) encoded over the chromosome, andramAlocated upstream of theacrAB-like genes. The results of this work suggest thatramAconfers resistance to florfenicol and other structurally unrelated antibiotics, presumably by regulating the RND efflux pump genes inL. adecarboxylataR25.