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.

Department of Clinical Laboratory Zhejiang Hospital Hangzhou 310013 Zhejiang China

Medical Research Center Taizhou Hospital of Zhejiang Province Taizhou 317000 Zhejiang China

School of Laboratory Medicine and Life Sciences Institute of Biomedical Informatics Wenzhou Medical University Wenzhou 325035 Zhejiang China

The 5th Affiliated Hospital Wenzhou Medical University Lishui 323000 Zhejiang China

Vocational and Technical College Lishui University Lishui 323000 China

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