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Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method

https://doi.org/10.1016/j.ijantimicag.2007.12.015Get rights and content

Abstract

Demonstration of efflux of ethidium bromide (EtBr) has been made for over 30 bacterial species, usually by showing enhanced efflux in multidrug-resistant strains that was then abolished by inactivating efflux pumps. Here we present a relatively simple automated method that employs EtBr as an efflux pump substrate for the demonstration of intrinsic efflux activity in Escherichia coli K-12 AG100. The method uses the Rotor-Gene 3000™ instrument for real-time fluorometric measurement of EtBr accumulation under conditions that limit energy (absence of glucose, low temperature) and of EtBr extrusion under optimum conditions. The method can be used for screening compound libraries for efflux inhibiting capacity.

Introduction

Overexpressed efflux pumps (EPs) as a cause of multidrug resistance (MDR) in bacterial clinical isolates have been demonstrated in all bacteria studied to date [1], [2]. Previously, we developed a simple ethidium bromide (EtBr)/agar-based method that allowed the identification of bacteria that overexpressed EP activity [2]. However, this method, as do others, suffers from the sensitivity required for the demonstration and characterisation of EP activity in clinically antibiotic-susceptible bacteria. EtBr, a common substrate of efflux pumps in Enterobacteriaceae [2], [3], at working concentrations was shown not to affect cell viability or cellular function, with properties of a very low signal outside the bacterial cell and the signal being amplified once inside the cell. This signal can be detected and quantified by time-course fluorescence spectroscopy, allowing correct assessment and quantification of the transport of this molecule across the living cell. Escherichia coli has been shown to have at least nine different major proton-dependent EP systems that bestow resistance to two or more antibiotics (defined as MDR) [4], [5]. They belong to one of three genetically and structurally defined families: the major facilitator superfamily (MFS) (emrD, mdfA, emrB); the resistance–nodulation–division (RND) family (acrB, acrF, acrD, yhiV); and the small multidrug resistance (SMR) family (emrE, tehA) [5]. The tripartite AcrAB-TolC system is the most well-studied MDR EP system, consisting of an inner membrane efflux transporter (AcrB), a linker protein (AcrA) and an outer membrane channel (TolC) [3], [4], [5], [6].

An automated method has been developed that demonstrates and quantifies intrinsic efflux activity of antibiotic-susceptible E. coli K-12 AG100 on a real-time basis. The method distinguishes conditions that result in the accumulation of EtBr from those that inhibit efflux itself and hence can be used routinely for the screening of efflux pump inhibitors (EPIs) as well as for the identification of overexpressed efflux activity of multidrug-resistant Gram-negative bacteria.

Section snippets

Bacteria

Wild-type E. coli K-12 AG100 strain (argE3 thi-1 rpsL xyl mtl Δ (gal-uvrB) supE44) [7] was kindly provided by Hiroshi Nikaido (University of California, Berkeley, CA).

Reagents

EtBr, potassium cyanide (KCN), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Phe-Arg-napthylamide (MC-207,110) (PAN), chlorpromazine (CPZ), thioridazine (TZ), glucose and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Química SA (Madrid, Spain). Luria–Bertani (LB) medium was purchased from Difco (Detroit, MI).

Results

The EP that is overexpressed in multidrug-resistant E. coli is mainly the tripartite AcrAB-TolC system [5], [6], [7], [8], [9]. Because the AcrAB EP also utilises EtBr as a substrate [6], [11], it is assumed, although not yet proven, that accumulation of EtBr and its extrusion, as assessed in the current study, is primarily the result of the activity of the AcrAB EP.

The MICs of the EPIs employed in this study have been published previously [8]: 60 mg/L for CPZ, 100 mg/L for TZ, 10 mg/L for CCCP

Discussion

It has been accepted that EtBr is a substrate extruded by bacteria [2], [3]. Because the addition of CCCP, an uncoupler of the proton-motive force (PMF), has been shown to cause an immediate increase in accumulation of EtBr [12], most studies assume that this accumulation is due to the inhibition of extrusion of EtBr [2], [3]. This assumption cannot be made because, as shown by the current study, simple killing of the bacterium results in the immediate maximum accumulation of EtBr.

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