Abstract
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.
Highlights
IntroductionMore than ten florfenicol resistance genes have been reported
Cong Cheng and Yuanyuan Ying contributed to this work.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Florfenicol, a derivative of chloramphenicol with better antibacterial activity and fewer adverse effects, has been widely used in veterinary medicine (Schwarz and Chaslus-Dancla 2001; Schwarz et al 2004)
We found that a ramA gene encoded on the chromosome of L. adecarboxylata R25 was involved in resistance to florfenicol and several other antibiotics
Summary
More than ten florfenicol resistance genes have been reported. These genes belong to four molecular categories: the major facilitator superfamily (MFS, including floR, floRv, flost, fexA, fexB, and pexA) (Alessiani et al 2014; Braibant et al 2005; He et al 2015; Kehrenberg and Schwarz 2004; Lang et al 2010; Liu et al 2012); the rRNA methyltransferase family [cfr, cfr(B), and cfr(C)] (Hansen and Vester 2015; Schwarz et al 2000; Tang et al 2017); the ATP-binding cassette (ABC) family (optrA) (Wang et al 2015b); and a chloramphenicol acetate esterase-encoding gene, estDL136 (Tao et al 2012). No efflux pump from RND family-related genes has been reported to be associated with florfenicol resistance
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