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

Shinsuke Toba,Takanori Kumagai,Daichi Morita,Yusuke Minato,Yuma Kondo,Shu Minagawa,Teruo Kuroda,Yasuyuki Matoba,Tomofusa Tsuchiya,Naomasa Gotoh,Kanami Hoshikawa,Shiho Komaki,Wakano Ogawa

doi:10.1038/s41598-019-41237-7

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.

Highlights

Introduction of sdeS inKAM43 or KAM43/pSOS2 didn’t render the increase for any tested antimicrobial agents (Table 2), but in KAM32, increase of Minimum inhibitory concentrations (MICs) for novobiocin, sodium dodecyl sulphate (SDS), deoxycholate was observed (Table 1)
To clarify whether S. marcescens resistance-nodulation-cell division superfamily (RND) efflux systems utilize TolC when expressed in E. coli, we introduced S. marcescens RND efflux system genes into E.coli KAM43, a tolC-deficient strain, and tested antimicrobial susceptibilities by measuring MICs
Previous studies suggested that RND efflux systems play a major role in multidrug resistance in S. marcescens[10,11,12,13,14]

Summary

Introduction

Introduction of sdeS inKAM43 or KAM43/pSOS2 didn’t render the increase for any tested antimicrobial agents (Table 2), but in KAM32, increase of MICs for novobiocin, SDS, deoxycholate was observed (Table 1). The sdeXY deletion mutant of S. marcescens is susceptible to a broad range of antimicrobial agents. Since the present results indicated that SdeXY has the broadest substrate specificity among the characterized S. marcescens efflux pumps[10], we constructed a sdeXY mutant strain from S. marcescens. We attempted to construct the deletion strain from Db10, but were unsuccessful for an unknown reason. We used another strain KS24, a derivative of the clinically isolated strain of S. marcescens SM3938. The sdeXY mutant strain showed the decreased energy-dependent efflux of ethidium (Fig. 4). These results indicated that SdeXY is a major RND efflux pump that confers intrinsic resistance to S. marcescens against multiple antimicrobial agents

Objectives

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Results

Conclusion