Riemerella anatipestifer (R. anatipestifer) is a multidrug-resistant bacterium and an important pathogen responsible for major economic losses in the duck industry. Our previous study revealed that the efflux pump is an important resistance mechanism of R. anatipestifer. Bioinformatics analysis indicated that the GE296_RS02355 gene (denoted here as RanQ), a putative small multidrug resistance (SMR)-type efflux pump, is highly conserved in R. anatipestifer strains and important for the multidrug resistance. In the present study, we characterized the GE296_RS02355 gene in R. anatipestifer strain LZ-01. First, the deletion strain RA-LZ01ΔGE296_RS02355 and complemented strain RA-LZ01cΔGE296_RS02355 were constructed. When compared with that of the wild-type (WT) strain RA-LZ01, the mutant strain ΔRanQ showed no significant influence on bacterial growth, virulence, invasion and adhesion, morphology biofilm formation ability, and glucose metabolism. In addition, the ΔRanQ mutant strain did not alter the drug resistance phenotype of the WT strain RA-LZ01 and displayed enhanced sensitivity toward structurally related quaternary ammonium compounds, such as benzalkonium chloride and methyl viologen, which show high efflux specificity and selectivity. This study may help elucidate the unprecedented biological functions of the SMR-type efflux pump in R. anatipestifer. Thus, if this determinant is horizontally transferred, it could cause the spread of quaternary ammonium compound resistance among bacterial species.

Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last‐resort defense against lethal infections with carbapenem‐resistant pathogens. In addition to the plasmid‐borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A‐modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296_RS09715 gene‐encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%–33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR‐like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)‐recognizable cavities. Comparative analyses of colistin resistance in RA‐LZ01 and RA‐LZ01ΔRaEptA showed the level of colistin has fallen from 96 μg mL−1 down to 24 ~ 32 μg mL−1. Site‐directed mutagenesis assay of the PE‐binding cavity and expression of the mutants reveals that K309‐rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point‐mutation of P309K is necessary for EptA‐mediated lipid A modification. Moreover, the virulence of RA‐LZ01ΔRaEptA was attenuated compared with RA‐LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram‐negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience.