Comprehensive analysis of resistance-nodulation-cell division superfamily (RND) efflux pumps from Serratia marcescens, Db10

Abstract

We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.

Figure 1

Putative S. marcescens RND efflux systems in Db10. White arrow; gene for the periplasmic membrane fusion protein, Black arrow; gene for the inner membrane protein, Gray arrow; gene for the outer membrane protein.

Figure 2

Unrooted phylogenetic tree of the inner membrane protein of RND efflux pumps. The phylogenetic tree was obtained using CLUSTALW (https://clustalw.ddbj.nig.ac.jp). E. coli: AcrB, AcrD, AcrF, MdtB, MdtC, MdtF, CusA^,,,. V. parahaemolyticus: VmeB, VmeD, VmeF, VmeI, VmeK, VmeM, VmeO, VmeQ, VmeS, VmeW, VmeV, VmeZ^–. V. cholerae: VexB, VexD, VexF, VexH, VexK, VexM^,. S. marcescens: SdeB, SdeD, SdeE, SdeY, SdeH, SdeJ, SdeO, SdeQ, SdeS, SM39_1914, SM39_1958^–. A baumannii: AdeB, AdeE, AdeG, AdeJ^,,,. P aeruginosa: MexD, MexF, MexI, MexK, MexN, MexQ, MexW, MexY, MuxB, MuxC, TriC^,,,,,,–. K pneumoniae: AcrB(K.P.), KexD, OqxB, KexC(KPN_RS15040), KexF(KPN_RS19875), KexH(KPN_RS21805), KexK(KPN_RS11560), KexM(KPN_RS25535), KexS(KPN_RS04245), KexU(KPN_RS03035), KexW(KPN_RS13595), KexX(KPN_RS13600)^,,.

Figure 3

Rhodamine 6 G efflux assay. Energy-starved cells of E. coli KAM32 strains that express S. marcescens RND efflux systems were prepared as described in the Materials and Methods. Energy-starved cells were resuspended in PBS containing 5 mM MgSO₄ and 1 µM rhodamine 6 G. At the time point indicated by the arrow, 20 mM potassium lactate (K-Lactate) was added to energize cells. Intracellular rhodamine 6 G levels were monitored continuously by measuring the fluorescence of rhodamine 6 G at excitation and emission wavelengths of 529 and 553 nm, respectively. IJ; E. coli KAM32/pURS6 (sdeIJ), PQ; E. coli KAM32/pURS8 (sdePQ-omsA), XY; E. coli KAM32/pURS2 (sdeXY), GH; E. coli KAM32/pURS5 (sdeGH), AB; E. coli KAM32/pURS3 (sdeAB), control; E. coli KAM32/pUC19.

Figure 4

Ethidium efflux activity in S. marcescens cells. The cells of the S. marcescens KS24 strain (A) and its KS24∆sdeXY (B) were prepared as described in the Materials and Methods. Ethidium bromide was added to cell suspensions at a final concentration of 10 μM at the time point indicated by the first downward arrow. Intracellular ethidium levels were monitored continuously by measuring the fluorescence of ethidium at excitation and emission wavelengths of 500 and 580 nm, respectively. At the second downward arrow, CCCP was added to the suspensions at a final concentration of 100 µM. Assays were performed at 37 °C.