New Roads Leading to Old Destinations: Efflux Pumps as Targets to Reverse Multidrug Resistance in Bacteria

Abstract

Multidrug resistance (MDR) has appeared in response to selective pressures resulting from the incorrect use of antibiotics and other antimicrobials. This inappropriate application and mismanagement of antibiotics have led to serious problems in the therapy of infectious diseases. Bacteria can develop resistance by various mechanisms and one of the most important factors resulting in MDR is efflux pump-mediated resistance. Because of the importance of the efflux-related multidrug resistance the development of new therapeutic approaches aiming to inhibit bacterial efflux pumps is a promising way to combat bacteria having over-expressed MDR efflux systems. The definition of an efflux pump inhibitor (EPI) includes the ability to render the bacterium increasingly more sensitive to a given antibiotic or even reverse the multidrug resistant phenotype. In the recent years numerous EPIs have been developed, although so far their clinical application has not yet been achieved due to their in vivo toxicity and side effects. In this review, we aim to give a short overview of efflux mediated resistance in bacteria, EPI compounds of plant and synthetic origin, and the possible methods to investigate and screen EPI compounds in bacterial systems.

Keywords: ABC-transporter; RND pump; efflux pump inhibitor (EPI); multidrug efflux pump; multidrug resistance; proton motive force.

Figure 1

Simplified structure of the AcrAB-TolC pump of a Gram-negative bacterium and proposed mechanism of action ((A): adapted from [101], (B): adapted from [160]). (A) The AcrAB–TolC efflux pump consists of two fusion proteins AcrA (made of six monomers) that anchors the AcrB transporter (made up of three monomers) to the plasma membrane. The latter is connected to the TolC channel (which consist of three monomers, therefore the stoichiometry of the respective pump proteins AcrA–AcrB–TolC is 3:6:3. providing a conduit to the exterior of the bacterium [161]. Noxious agents may enter the transporter from the periplasm or the cytoplasm [128]. The key enzyme is ATP synthase situated within the plasma membrane with one active site for ATP in the periplasm and one active site on the cytoplasmic side of the plasma membrane [162,163]. At pH 7 equilibrium favors the hydrolysis of ATP. At pH 6 equilibrium favors the synthesis of ATP. In order for the pump to work at pH 7, metabolic energy from glycolysis is required [164] for the creation of ATP which is hydrolyzed by the ATP synthase. The product hydronium ion (H₃O)⁺ present in water enters the AcrAB transporter which had recognized the noxious agent and bound it; this reduces the pH of internal part of the transporter thereby encouraging the dissociation of the agent (B) [165] which is then pushed out into the TolC channel by the movement of water and expelled to the milieu. Because the hydronium ions are attracted to components of the outer membrane (lysine, arginine, lipopolysaccharides), they contribute to the proton motive force (PMF) [166]. The AcrAB–TolC pump is now ready to recognize a noxious agent. When the bacterium is in medium of pH 5.5 there is no need for metabolic energy [158]. At this pH the concentration of hydronium ions is high and some are bound to the outer membrane resulting in a strong electrochemical gradient (PMF) that favours their movement into the periplasm via porins [167]. It should be noted that when the AcrAB efflux pump is over-expressed, the major porin C is down-regulated resulting in fewer porins [156]. This provides additional protection from noxious agents.

Figure 2

Accumulation methods to measure the activity of bacterial efflux pumps. (A) Accumulation of EB by Salmonella enterica serovar Enteritidis 104 at pH 5 in the presence of thioridazine (50 µg/mL) using real-time EB method. The flat curve represents the accumulation of the untreated strain [226]; (B) Cartwheel method to monitor efflux activity of control (NCTC 13349), clinical (104, 5408), and ciprofloxacin resistant (104-cip, 5408-cip) Salmonella enterica serovar Enteritidis strains using EB containing (2.5 mg/L) tryptic soy agar plate [177]; (C.1) Accumulation of EB (1 µg/mL) by E. coli AG 100 strain using flow cytometry [158]; (C.2) Accumulation of EB (1 µg/mL) by E. coli AG 100 strain in the presence of thioridazine (20 µg/mL) using flow cytometry [169].

Figure 3

Examples for natural EPI compounds. (1) Karavilagenin C; (2) uvaol; (3) jatropholone A and B; (4) ferruginol.

Figure 4

Examples of synthetic EPI compounds. (1) Thioridazine; (2) Phenylalanine-arginine β-naphthylamide (PAβN); (3) carbonyl cyanide 3-chlorophenylhydrazone (CCCP); (4) general structure of EPI hydantoins.