Abstract
Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identified. With respect to the substrate specificity of B739_0873, the antibiotic susceptibility testing showed that the B739_0873 knockout strain was more sensitive to aminoglycosides and detergents than the wild-type strain. The transcription of B739_0873 was up-regulated when R. anatipestifer CH-1 was exposed to sub-inhibitory levels of these substrates. From the gentamicin accumulation assay, we concluded that B739_0873 was coupled to the proton motive force to pump out gentamicin. Furthermore, site-directed mutagenesis demonstrated that Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 were the crucial function sites of B739_0873 in terms of its ability to extrude aminoglycosides and detergents. Finally, we provided evidence that B739_0873 is co-transcribed with B739_0872, and that both B739_0872 and B739_0873 are required for aminoglycoside and detergent resistance. In view of these results, we designate B739_0873 as RaeB (Riemerella anatipestifer efflux).
Highlights
Riemerella anatipestifer is a Gram-negative bacterium that belongs to the Flavobacteriaceae family (Segers et al, 1993)
resistance-nodulation-cell division (RND) efflux pumps are widespread among Gram-negative bacteria and play an important role in producing multidrug resistance (Nikaido, 2011)
Genome sequence analysis suggested that the raeB gene in R. anatipestifer CH-1 encodes a putative RND efflux pump, and we investigated the biological function of RaeB in R. anatipestifer CH-1, as well as its energy source and functional site
Summary
Riemerella anatipestifer is a Gram-negative bacterium that belongs to the Flavobacteriaceae family (Segers et al, 1993). It infects ducks, geese, turkeys, chickens, and other birds, leads to contagious septicemia, and causes large economic losses in the duck industry (Ryll et al, 2001; Hess et al, 2013). Antibiotics are still the major preventative and therapeutic approach against R. anatipestifer infection in poultry. The widespread use of antibiotics to treat R. anatipestifer infection has resulted in multidrug resistance in R. anatipestifer. It is necessary to understand the multidrug resistance mechanisms of R. anatipestifer to find a way to prevent and treat R. anatipestifer infection
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