Jean-Michel Bolla

Sandrine Alibert-Franco

Jadwiga Handzlik

Jacqueline Chevalier

Abdallah Mahamoud

Gérard Boyer

Katarzyna Kiec-Kononowicz

Jean-Marie Pagès
email: jean-marie.pages@medecine.univ-mrs.fr

Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria ´

Keywords:

In Gram-negative bacteria, the envelope is a sophisticated barrier protecting the cell against exter-nal toxic compounds. Membrane transporters, e.g., porins or efflux pumps, are main filters regulat-ing the internal accumulation of various hydrophilic molecules. Regarding bacterial susceptibility towards antibacterial agents, membrane permeability is part of the early bacterial defense. The bacterium manages the translocation process, influx and efflux, to control the intracellular concentration of various molecules. Antibiotics and biocides are substrates of these mechanisms and the continuing emergence of multidrug resistant isolates is a growing worldwide health concern. Different strategies could be proposed to bypass the bacterial membrane barrier, comprising influx and efflux mechanisms, in order to restore the activity of antibiotics against resistant bacteria.

Introduction

After several decades of continued success of antibiotic therapy against bacterial infections, we are now facing a worrying prospect: the accelerated evolution of antibiotic resistance of important human pathogens [START_REF] Blot | Measuring the impact of multidrug resistance in nosocomial infection[END_REF][START_REF] Chopra | Treatment of healthcare-associated infections caused by Gram-negative bacteria: a consensus statement[END_REF][START_REF] Falagas | Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era?[END_REF][START_REF] Gandhi | Managing antimicrobial resistance in intensive care units[END_REF][START_REF] Rice | The clinical consequences of antimicrobial resistance[END_REF]. This bacterial adaptation to antibiotic use is directly involved in the current increase of morbidity and mortality caused by infection diseases. Gram-negative bacteria that correspond to a prominent part of drug resistant pathogens, display a complex envelope with an outer (OM) and an inner (IM) membrane delimiting a periplasmic space [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. This cellular organization results in the presence of various protein channels involved in the transport, uptake or efflux, of a large vari-ety of compounds, nutrients or toxic molecules (sugars, drugs, small peptides, chemicals) (Fig. 1). Among these various channels, porins and efflux transporters contribute to the balance of intracellular drug accumulation and are involved in the bacterial antibiotic susceptibility [START_REF] Nikaido | Multidrug resistance in bacteria[END_REF][START_REF] Pagès | The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria[END_REF].

Hydrophobic compounds, such as aminoglycosides, macrolides, rifamycins and other chemicals, can permeate through the OM bilayer by the self-promoting pathway [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. Regarding hydrophilic molecules, such as b-lactams and fluoroquinolones, the core region of lipopolysaccharide (LPS) impairs their diffusion and porins are the major way to enter the cell [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF][START_REF] Pagès | The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria[END_REF]. Consequently, LPS alterations can provide an efficient protection against the first class whereas porin modifications can protect bacteria against the second ones. The permeability of porins to b-lactam antibiotics has been demonstrated by various means and several porin mutants have been described in resistant clinical isolates [START_REF] Pagès | The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria[END_REF].

The over-expression of efflux pumps is involved in the Multi-Drug Resistance (MDR) phenotype associated with other more specific resistance mechanisms including target mutation and enzymatic modification of the antibiotic [START_REF] Alekshun | Molecular mechanisms of antibacterial multidrug resistance[END_REF][START_REF] Bolla | Membrane permeability and regulation of drug ''influx and efflux'' in enterobacterial pathogens[END_REF]. In addition to efthesis have been reported in various enterobacterial resistant isolates including Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, etc [START_REF] Pagès | The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria[END_REF]. In these strains, a complex regulation cascade is involved in the control of membrane transporters, porins and efflux pumps.

This regulation cascade involved the mar and ram genes that organize a tight and coordinated control of the expression of porins and efflux pumps in E. aerogenes, K. pneumoniae and Salmonella enteritidis [START_REF] Bolla | Membrane permeability and regulation of drug ''influx and efflux'' in enterobacterial pathogens[END_REF]. Various chemicals, antibiotics, biocides, and other molecules can trigger this regulation cascade [START_REF] Alekshun | Molecular mechanisms of antibacterial multidrug resistance[END_REF][START_REF] Bolla | Membrane permeability and regulation of drug ''influx and efflux'' in enterobacterial pathogens[END_REF]. The main clinical concern of efflux pump expression is related to their broad polyselectivity and their ability to expel a large collection of molecules [START_REF] Li | Efflux-mediated drug resistance in bacteria: an update[END_REF][START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Piddock | Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria[END_REF][START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF]. In Gram-negative bacteria, the archetype of drug characterized in E. coli and Pseudomonas aeruginosa (Fig. 1), Ac-rAB-TolC/MexAB-OprM efflux pump is the main multitransporter system reported in enterobacterial clinical isolates [START_REF] Nikaido | Multidrug resistance in bacteria[END_REF][START_REF] Li | Efflux-mediated drug resistance in bacteria: an update[END_REF][START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Piddock | Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria[END_REF][START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF]. Ac-rAB-TolC homologous efflux complexes have also been reported in many other pathogens [START_REF] Li | Efflux-mediated drug resistance in bacteria: an update[END_REF][START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Piddock | Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria[END_REF][START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF]. The poly-specificity of transporters confers a ''general resistance mechanism'' contributing to the first line of bacterial defense that can favor the acquisition of modifications [START_REF] Bolla | Membrane permeability and regulation of drug ''influx and efflux'' in enterobacterial pathogens[END_REF][START_REF] Li | Efflux-mediated drug resistance in bacteria: an update[END_REF]. It has been demonstrated that the expression of Salmonella and Campylobacter efflux pump is an important prerequisite for the selection of fluoroquinolone resistant strains exhibiting target mutations [START_REF] Bolla | Membrane permeability and regulation of drug ''influx and efflux'' in enterobacterial pathogens[END_REF][START_REF] Pagès | Efflux pumps of gram-negative bacteria, a new target for new molecules[END_REF]. The mechanics of efflux pump have been investigated and dynamic-functional models have been recently proposed [START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Yao | Drug export and allosteric coupling in a multidrug transporter revealed by molecular simulations[END_REF][START_REF] Schulz | Transitions between closed and open conformations of TolC: the effects of ions in simulations[END_REF][START_REF] Symmons | The assembled structure of a complete tripartite bacterial multidrug efflux pump[END_REF][START_REF] Takatsuka | Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli[END_REF]. In addition, recent data indicate that some drug transporters, e.g., MdfA and AcrAB-TolC, can cooperate or can act sequentially to efficiently expel antibiotics outside the bacterium [START_REF] Tal | A coordinated network of transporters with overlapping specificities provides a robust survival strategy[END_REF].

These two transport mechanisms, influx (porin and selfpromoting pathway through LPS) and efflux (efflux pumps), that can be genetically regulated by a same genetic cascade involving global regulators and specific local regulators, can cooperate to strongly decrease the intracellular concentration of antibacterial agents.

These processes conjointly contribute to the membrane barrier, or mechanical barrier, that constitutes the first line of bacterial defense flux pump over-expression, a severe down regulation of porin syn-against antibacterial agents comprising antibiotics, biocides (disinfectants, antiseptics, preservatives), bacteriocins, detergents, surfactants, defensins, bile salts and also dyes and other chemicals. Consequently, in order to bypass the membrane barrier existing in resistant isolates, we need to develop various strategies to increase the diffusion of antibiotics through bacterial membranes (target the influx), or/and, circumvent the pump mechanism to preserve a high intracellular concentration of antibiotics (target the efflux).

Increasing the antibiotic influx

During last decade, in order to restore the efficacy of b-lactams or other antibiotics in multidrug resistant strains, several ways efflux pump is the AcrAB-TolC/MexAB-OprM [START_REF] Eicher | Structural and functional aspects of the multidrug efflux pump AcrB[END_REF][START_REF] Murakami | Multidrug efflux transporter, AcrBthe pumping mechanism[END_REF][START_REF] Nikaido | Mechanisms of RND multidrug efflux pumps[END_REF][START_REF] Alibert-Franco | Efflux mechanism, an attractive target to combat multidrug resistant Plasmodium falciparum and Pseudomonas aeruginosa[END_REF][START_REF] Pos | Drug transport mechanism of the AcrB efflux pump[END_REF][START_REF] Yao | Drug export and allosteric coupling in a multidrug transporter revealed by molecular simulations[END_REF]. Firstly have been investigated. In addition to explore new compounds able to inhibit cephalosporinase and other enzymatic activities [START_REF] Bush | Updated functional classification of betalactamases[END_REF][START_REF] Drawz | Three decades of b-lactamase inhibitors[END_REF], an alternate possibility is to facilitate the diffusion of antibiotics through the bacterial envelope in order to increase their intracellular concentration (see Fig. 1, arrows a and b). To bypass the barrier due to porin alterations (decreased expression or altered channel) and LPS modifications, chemical facilitators and chemosensitizers, have been proposed such as detergents, surfacother resistance mechanisms such as target mutations or drug tants, chaotropic agents, polymyxines, antimicrobial peptides [START_REF] Hurdle | Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections[END_REF][START_REF] Vooturi | Synthetic membrane-targeted antibiotics[END_REF]. Some of them, e.g., polycationic cyclic lipopeptides and cationic antimicrobial peptides have been assayed in combination with usual antibiotics to combat resistant clinical strains [for recent reviews see [START_REF] Falagas | Resistance to polymyxins: mechanisms, frequency and treatment options[END_REF][START_REF] Landman | Polymyxins revisited[END_REF][START_REF] Vaara | Polymyxins and their novel derivatives[END_REF]. It is important to note that only very few results have been published regarding the molecular basis of this kind of combination, the synergy efficacy, the concentration ratio, the kinetics, and the combination parameters.

Polymyxins as chemosensitizers

Membranotropic compounds such as polymyxin B and polymyxin E (colistin) perturb the integrity of the bacterial membranes and can increase the enterobacterial susceptibility to various compounds [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF]. In addition, they can act as opsonins to enhance Steps of antibiotic transport through the membranes of Gram-negative bacteria. To be active against bacterium, an antibiotic needs a critical concentration threshold to inhibit the corresponding target. Using the concentration gradient, the influx is controlled by several elements: the antibiotic can permeate through the OM via porins (a, for hydrophilic molecules) or through the OM (b, for hydrophobic drugs) [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. (c) Represents the diffusion through the periplasmic space in which enzymatic inactivation may occur [START_REF] Bush | Updated functional classification of betalactamases[END_REF][START_REF] Bush | New b-lactam antibiotics and b-lactamase inhibitors[END_REF]. Finally, the drug will pass the cytoplasmic/IM (d). Regarding the efflux, the drug may be recognized and transported by active pump (e) or (f). The pump (e.g., AcrB) has to recruit the OM pore TolC (g), in addition it may conjointly acts with other pumps (e.g., MFS family). The success of a drug is given by the kinetics of all individual steps [START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Amaral | Efflux pumps of Gram-negative bacteria: genetic responses to stress and the modulation of their activity by pH, inhibitors and phenothaizines[END_REF].

destruction by phagocytic cells. These molecules are pentacationic cyclic lipodecapeptides that present bactericidal activity by acting on the outer membrane of Gram-negative bacteria [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF][START_REF] Mogi | Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics[END_REF]. Their action is limited to Gram-negative bacteria and polymyxin used was almost halted between 1970 and 1980 as systemic therapy due to their severe nephrotoxicity. The molecular mechanism for permeabilization by polymyxin B is thought to involve the competition for the binding of divalent cations that normally cross-bridge neighboring LPS molecules. The displacement of these stabilizing interactions leads to enhance lateral diffusion of LPS [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF][START_REF] Soon | Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions[END_REF]. The resulting destabilization of the LPS layer allows the penetration of polymyxin B into the periplasm, providing essentially a ''selfpromoted uptake pathway'' for polymyxin B to reach its target, the cytoplasmic membrane. Then, the fatty acid tail on polymyxin B allows it to permeabilize the IM, thus leading to its antibacterial action. Polymyxin B nonapeptide (PMBN) lacks the fatty acid chain, and is less bactericidal and less toxic for eukaryotic cells, but the fact that it sensitizes cells to hydrophobic antibiotics demonstrates that it retains membrane permeabilizing properties [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF]. Now, faced with the continuous increase of infectious diseases involving MDR bacteria and with the scarcity of new active antibiotics, polymyxin group represents a last resort for clinicians [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF][START_REF] Giamarellou | Multidrug-resistant Gram-negative infections: what are the treatment options?[END_REF][START_REF] Nation | Colistin in the 21st century[END_REF]. Regarding their use in monotherapy, it must be noted that polymyxin resistance has been reported for clinical isolates although the resistant strains showed a noticeable decrease of fitness [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF][START_REF] Urban | Polymyxin-resistant clinical isolates of Escherichia coli[END_REF]. A possible alternate way is to combine low concentration (sub-inhibitory) of polymyxin with another clinical antibiotic to synergize its activity. Interesting results have been reported and the combination of meropenem and colistin showed increased synergic killing of Acinetobacter baumannii and P. aeruginosa [START_REF] Pankuch | Activity of meropenem with and without ciprofloxacin and colistin against Pseudomonas aeruginosa and Acinetobacter baumannii[END_REF]. Similar results have been reported with a combination of high-dose tigecycline and colistin [START_REF] Humphries | Successful treatment of pan-resistant Klebsiella pneumoniae pneumonia and bacteraemia with a combination of high-dose tigecycline and colistin[END_REF]. A synergistic combination of colistin with rifampin [START_REF] Aoki | Efficacy of colistin combination therapy in a mouse model of pneumonia caused by multidrug-resistant Pseudomonas aeruginosa[END_REF] and vancomycin [START_REF] Gordon | Potent synergy and sustained bactericidal activity of a vancomycin/colistin combination versus multi-drug resistant strains of Acinetobacter baumannii[END_REF] has been reported. Moreover, significant bactericidal activity was obtained in carbapenem-resistant K. pneumoniae, A. baumannii, P. aeruginosa, and E. coli isolates using combinations of polymyxin B, doripenem, and rifampin [START_REF] Urban | In vitro double and triple bactericidal activities of doripenem, polymyxin B, and rifampin against Klebsiella pneumoniae, and Escherichia coli[END_REF]. Combination of polymyxins with these antibacterials may support a strategy for treatment of patients infected with such bacteria exhibiting resistant phenotype. The effect and of b-lactam molecules or other antibiotics. Polymyxin B and colistin have been also successfully used in combination with tigecycline and gentamicin against Gram-negative resistant isolates [START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection[END_REF]. Consequently they are mentioned on the treatment of resistant bacterial infections and with association with particular antibiotics [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF]. Interestingly, it has been recently demonstrated that PMBN and polymyxin B can potentialize the activity of a new family of antibacterial agents, peptide deformylase inhibitor, against Gram-negative bacteria such as E. coli, E. aerogenes, K. pneumoniae, and P. aeruginosa [START_REF] Mamelli | New antibiotic molecules: bypassing the deformylase inhibitors[END_REF]. The increase of bacterial susceptibility is associated with the increase of the entry of new antibacterial agents obtained by the addition of sub-inhibitory concentration of polymyxin [START_REF] Mamelli | New antibiotic molecules: bypassing the deformylase inhibitors[END_REF].

Less toxic polymyxin derivatives would be highly welcome, such as the polymyxin B non-apeptide, to be used in combination with usual antibiotics [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF]. Interestingly, the group of M. Vaara has produced and characterized many polymyxin derivatives showing a significant decrease in toxicity but exhibiting chemosensitizer properties. Using sub-inhibitory concentrations, some compounds are able to increase the activity of rifampin, clarithromycin, vancomycin to E. coli, K. pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, and A. baumannii [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF][START_REF] Vaara | Novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents[END_REF]. In addition, a compound is effective in combination with erythromycin in an infection animal model [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF]. Furthermore, it is important to mention that the use of polymyxin compounds in combination with another antibiotic may reduce the risk of resistance emergence [START_REF] Vaara | Polymyxins and their novel derivatives[END_REF].

Interestingly, some natural compounds can act as membrane permeabilizers. The terpenic compound, eugenol is the major component of clove oil from Eugenia aromatica and was reported to act primarily by disrupting the cytoplasmic membrane [START_REF] Gill | Disruption of Escherichia coli; Listeria monocytogenes and Lactobacillus sakei cellular membranes by plant oil aromatics[END_REF]. The combination study of eugenol and ten antibiotics against Gramnegative bacteria, E. coli, P. aeruginosa, E. aerogenes, Salmonella typhimurium and Proteus vulgaris indicated that the minimal inhibitory concentration (MIC) of the antibiotics was 5-1000-fold lower than when used alone, indicating that the combination was synergistic [START_REF] Hemaiswarya | Synergistic interaction of eugenol with antibiotics against Gram negative bacteria[END_REF]. The enhancement of nitrocefin uptake associated with a sensitization of the microorganisms to lysis by lysozymes or detergents in the presence of eugenol demonstrated its effect on the bacterial membrane.

Destabilization of LPS barrier

An other way to circumvent the membrane barrier is to destabilize the LPS layer by using chaotropic agents or detergents that consequently facilitate the diffusion of hydrophilic compounds through the membrane lipid bilayer. Treatment by Tris/EDTA leads to massive release of LPS in the medium, and it is believed that the reduced amount of LPS in the OM leaflet is compensated by glycerophospholipids, essentially creating patches of phospholipid bilayer, which are much more permeable to lipophilic compounds [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. A similar situation may also be found in deep rough mutants where there is a decrease in OM protein incorporation associated with the expression of defective LPS molecules generating an unbalanced ratio of LPS/phospholipids/proteins in the OM. The achieved susceptibilities become similar to those of deep rough mutants [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. It has been demonstrated that LPS mutants are more susceptible to antibiotics. However, a major adverse effect in the use of chaotropic agent (EDTA) is associated with their noticeable toxicity on biological membrane and consequently, their use is limited to laboratory assays.

For laboratory assays including diagnostic assays, evaluation of resistance, detection of involved mechanisms and epidemiology surveys, several compounds have been used in combination with antibiotics. EDTA could be used to promote the entry of various antibiotics in resistant strains exhibiting some LPS alterations or kinetics reported may suggest that polymyxin may favor the entry to reduce the enzymatic activity against specific antibiotics [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF][START_REF] Pournaras | Inhibitor-based methods for the detection of KPC carbapenemase-producing Enterobacteriaceae in[END_REF]. EDTA can increase the susceptibility of bacteria producing metallo-b-lactamases towards b-lactams [START_REF] Bush | New b-lactam antibiotics and b-lactamase inhibitors[END_REF]. Some detergents, like Triton or SDS, deoxycholate, could be used to permeabilize the OM and in addition facilitate the entry of various antibacterial agents and subsequently increase the susceptibility [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. Regarding the use of detergents or biocides in combination with antibiotics, the situation is especially complex and depends on the specific use (skin treatment, local decontamination, medical devices or catheters treatment).

Ceragenins are a family of bile acid derivatives that have been modified to yield an amphiphilic morphology similar to that of endogenous antimicrobial peptides [START_REF] Epand | Depolarization, bacterial membrane composition, and the antimicrobial action of ceragenins[END_REF]. With relatively low concentrations of ceragenin, a permeabilization of the OM was observed and a synergistic effect of ceragenin and erythromycin in combination was also observed [START_REF] Saha | Enhancement of the efficacy of ´ pathogens[END_REF]. In addition, a synergistic effect is observed with combination of ceragenins and host defense molecules on resistant P. aeruginosa isolates [START_REF] Leszczynska | Potential of ceragenin CSA-13 and its mixture with pluronic F-127 as treatment of topical bacterial infections[END_REF]. Recently, squalamine, a natural aminosterol, has been studied for its antimicrobial activity and this molecule is able to destabilize the OM of various Gram-negative bacteria [START_REF] Salmi | Squalamine: an appropriate strategy against the emergence of multidrug resistant Gram-negative bacteria?[END_REF][START_REF] Alhanout | New insights into the antibacterial mechanism of action of squalamine[END_REF]. In combination with usual antibiotics, squalamine can restore susceptibility to various antibiotic classes including b-lactams, fluoroquinolones, macrolides, phenicols, cyclines to resistant isolates that are devoid of porins [START_REF] Lavigne | Squalamine, an original chemosensitizer to combat antibiotic-resistant Gram-negative bacteria[END_REF]. This is interesting in the case of cefepime or ciprofloxacin, two antibiotics that required porins to penetrate the OM [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF][START_REF] Pagès | The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria[END_REF]. Neamine derivatives represent also a possibility to permeabilize bacterial strated that some neamine compounds are able to induced a decrease of the thickness of P. aeruginosa envelope in parallel with membrane depolarization. In addition, a binding to LPS and the induction of membrane permeabilisation is reported with some specific neamine derivatives [START_REF] Ouberai | The Pseudomonas aeruginosa membranes: A target for a new amphiphilic aminoglycoside derivative?[END_REF].

In conclusion, these data suggest than this class of amphiphilic derivatives could be used for the development of a direct antibacterial agent, or alternatively for the production of original adjuvants that rejuvenate old antibiotic activity on MDR bacteria [START_REF] Epand | Ceragenins (cationic steroid compounds), a novel class of antimicrobial agents[END_REF][START_REF] Findlay | Cationic amphiphiles, a new generation of antimicrobials inspired by the natural antimicrobial peptide scaffold[END_REF].

Nanoparticles and antibiotics

At this moment, new complexes using nanoparticle technology have been produced and comprise several antibacterial agents associated with nanoparticle surfaces. This new presentation of antibacterials is attractive for efficiently combating resistance associated with biofilm structures or bacterial isolates exhibiting a modified envelope that contributes to antibiotic resistance. Now, precise informations are needed to clearly identify the benefit and possible use of such approach. A serious concern associated with biomedical and surgical devices and transplants, grafts, and other invasive treatments, is that of bacterial infections and the devices colonized by bacteria may cause infection or mortality. To prevent and to combat such infections, an effective strategy is to develop original surface modification processes that provide antibacterial abilities to biomedical devices [START_REF] Cao | Silver nanoparticles-modified films versus biomedical device-associated infections[END_REF].

Nano silver particles comprise nano-sized structures formed from silver atoms. It is proposed that silver ions interact with main bacterial components to produce the bactericidal effect: the peptidoglycan and the membrane, DNA and proteins and especially enzymes involved in vital cellular processes such as the electron transport chain [for a review see 60]. Taking into account these destabilizing effects generated in the bacterial envelope, it may be possible to propose a combination of such new medical devices associated with usual antibiotics for the treatment of specific infectious diseases caused by resistant bacteria [START_REF] Abeylath | Drug delivery approaches to overcome bacterial resistance to beta-lactam antibiotics[END_REF][START_REF] Pissuwan | Functionalised gold nanoparticles for controlling pathogenic bacteria[END_REF].

Blocking the efflux

Because of the clear involvement of the resistance nodulation cell division (RND) transporters in the increased frequency of MDR clinical bacteria, efflux pumps are now considered as an attractive target for the development of a combinational therapy using antibiotic/efflux pump inhibitor (EPI) as adjuvant of usual antibiotics [START_REF] Drawz | Three decades of b-lactamase inhibitors[END_REF]. The deciphering of structure and function, e.g., structure-activity relationship (SAR) of the RND efflux pump and its individual components is required for their rational design [START_REF] Drawz | Three decades of b-lactamase inhibitors[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. All details of interactions and processes that functionally govern and regulate the behaviour of the RND family can be useful for the development of an effective new mode of therapy. The design of active molecules capable of restoring antibiotic susceptibility in MDR pathogens is a promising alternative taking into account the scarcity of new molecules available and active against Gram-negative bacteria. These compounds must have no intrinsic antibacterial effect but induce an increase of intracellular antibiotic concentration. Some data provide some clues for the future development of distinct strategies targeting efflux pumps: alteration of pump gene expression, inhibition of membrane assembly of pump component, blocking OM exit duct, collapsing the energy driven source [START_REF] Pagès | Efflux pumps of gram-negative bacteria, a new target for new molecules[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF][START_REF] Falagas | Potential of old-generation antibiotics to address current need for new antibiotics[END_REF][START_REF] Yeaman | Mechanisms of antimicrobial peptide action and resistance[END_REF]. At the moment, efflux pump activity blockers are the main group that are described and tested on Gram-negative bacteria from both, natural and synthetic sources.

Although several families of chemical compounds that display EPIs activity have been identified, the EPIs-library is still not sufficient for a complete characterization of various aspects of multi-membrane. In a recent publication, Ouberai et al., clearly demon-drug resistance in Gram-negative bacteria. Many lines of evidence indicate that in case of tripartite pumps, AcrAB-TolC or MexAB-OprM, the RND protein (AcrB or MexB) is responsible for selection of a substrate to be extruded. Thus, lot of efforts are concentrated on search for compounds which tend to affine to these RND-proteins and, in this way, they could inhibit their efflux action. The 3D-structure of AcrB has been identified experimentally in several variants [START_REF] Drew | The structure of the efflux pump AcrB in complex with bile acid[END_REF][START_REF] Murakami | Crystal structure of bacterial multidrug efflux transporter AcrB[END_REF][START_REF] Yu | Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump[END_REF][START_REF] Yu | A perisplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study[END_REF] for last eight years. Twenty five structures of AcrB have been submitted in Protein Data Bank (PDB) since 2002. In case of MexB, the first experimental structure has been available in PDB since 2009 [START_REF] Sennhauser | Crystal Structure of the Multidrug Exporter MexB from Pseudomonas aeruginosa[END_REF]. It gives a great perspective to develop knowledge about the efflux mechanism and substrates binding pockets to discover new groups of efflux pump inhibitors. Nevertheless, a number of described potent EPIs is rather low, particularly, the number of inhibitors that were found basing on docking studies to the binding sites identified for the tripartite pumps within both mutagenic-and computer aided modelling studies. A natural non-selective character of the bacterial efflux proteins enables pathogens to abroad substrates with wide varieties of spatial and physicochemical properties [START_REF] Seeger | The AcrB efflux pump: conformational cycling and peristalsis lead to multidrug resistance[END_REF]. Structure-activity relationship studies performed for families of known EPIs (Table 1), gave ''step by step'' new information about electron-and spatial properties of inhibitors that could be used to create pharmacophore models describing features likely necessary to interact with protein pump binding sites. The pharmacophore models could be useful for design of novel compounds with expected higher EPIproperties. Molecular modelling based pharmacophore model of Nakayama et al. [START_REF] Nakayama | MexAB-OprM membrane barrier of gram negative bacteria increases the activity of peptide specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 3: Optimization of potency in the pyridopyrimidine series through the application of a pharmacophore model[END_REF] allowed to identify favourable features for inhibitors of MexAB-OprM in P. aeruginosa in the group of pyridopyrimidine derivatives. According to this model, four hydrophobic sites, including two sites in close neighbourhood and two peripheral sites, as well as an acidic ionisable fragment are desirable for EPI properties. The model of Nakayama allowed finding new active EPIs among pyridopyrimidine derivatives. Nevertheless, other active EPIs (Table 1) do not fit in the model as their number and position of hydrophobic sites are different, and acidic ionisable centre appears rather occasionally. A structure-activity relationship analysis which can be performed across all families of described EPIs (Table 1) gives information about structural requirements that may be desirable for interactions with efflux system of Gram-negative bacteria. The presence of various linear-or cyclic amines as well as aromatic moieties, often in neighbourhood (fused aromatic rings of naphthyl or quinoline side chains), can be noted in all representative EPIs. Amide fragments appear in several groups of EPIs, in which hydrogen bond acceptor on carbonyl oxygen may interact with amino acid residues of a binding pocket of protein pumps. Among the synthetic products with anti-efflux properties, the following chemical families stand out.

Synthetic products as efflux pump activity blockers

Peptidomimetics

In the case of synthetic compounds, the first recognized EPIs belong to peptidomimetic group [START_REF] Renau | Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin[END_REF], which are active against P. aeruginosa strains that overexpress the MexAB-OprM efflux pump. These compounds are the first described molecules that are able to block fluoroquinolone efflux using levofloxacin as marker [START_REF] Lomovskaya | Waltzing transporters and 'the dance macabre' between humans and bacteria[END_REF]. Phe-Arg-b-naphthylamide (PAbN) was the first EPIpeptidomimetic, selected as a pioneer lead compound for further chemical modifications. PAbN-like peptidomimetics effectively inhibit the quinolone efflux mechanism in P. aeruginosa strain expressing MexAB-OprM, particularly levofloxacin efflux. This group of EPIs induces a competition-like process during the recognition/transport steps of antibiotic molecule [START_REF] Pagès | Efflux pumps of gram-negative bacteria, a new target for new molecules[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. EPI activity of PAbN was also confirmed in other Gram-negative bacteria, including E. coli, E. aerogenes, K. pneumoniae and S. enterica [START_REF] Pagès | Efflux pumps of gram-negative bacteria, a new target for new molecules[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF][START_REF] Lomovskaya | Practical applications and feasibility of efflux pump inhibitors in the clinica vision for applied use[END_REF].

The mechanism proceeds using the competitive nature of PAbN with antibiotic substrates of the efflux pump system; that is while the pump preferentially pumps out PAbN, the antibiotic remains in the cell increasing its amount until the concentration required for its activity on the target is sufficient. The final effect of PAbN is to reduce resistance or completely reverse resistance to a given antibiotic to which the bacterium was initially resistant. Results of various microbiological assays suggested that PAbN may occupy an affinity site in the large substrate-binding pocket which differs from the site occupied by a given antibiotic in respect to the involved amino acid residues [START_REF] Murakami | Crystal structures of a multidrug transporter reveal a functionally rotating mechanism[END_REF].

Derivatives molecules from PAbN-EPIs have been prepared in order to improve the stability in biological fluids, the activity and the pharmacokinetic properties of the EPI [START_REF] Seeger | The AcrB efflux pump: conformational cycling and peristalsis lead to multidrug resistance[END_REF]. One of them, MC-04,124, is more stable than the original molecule and exhibits reduced toxicity [START_REF] Renau | Addressing the stability of Ccapped dipeptide efflux pump inhibitors that potentiate the erythromycin in multiple antibiotic-resistant gram-negative bacterial activity of levofloxacin in Pseudomonas aeruginosa[END_REF][START_REF] Watkins | The relationship between physicochemical properties; in vitro activity and pharmacokinetic profiles of analogues of diamine-containing efflux pump inhibitors[END_REF]. These EPIs are useful to identify the presence of active efflux pump in P. aeruginosa clinical isolates [START_REF] Kriengkauykiat | Use of an efflux pump inhibitor to determine the prevalence of efflux pump-One 3, e2765. mediated fluoroquinolone resistance and multidrug resistance in Pseudomonas aeruginosa[END_REF].

From this original family of diamine EPIs several compounds have been described [START_REF] Yoshida | MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa; Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001; a potential preclinical candidate[END_REF]. At this moment, these PAbN-derived EPIs are the more studied and developed family of anti-efflux compounds.

One of them is under early phase preclinical development by Mpex Pharmaceuticals. This company is now developing a fixedcombination drug product consisting of an existing antibiotic and an EPI for systemic treatment of serious hospital-acquired infections due to MDR Gram-negative bacterial pathogens such as P. aeruginosa.

From quinoline to quinazoline derivatives as AcrB blockers

A screening test bases on clinical isolates of E. aerogenes has allowed the identification of quinoline, quinazoline derivatives, two new classes of efflux pump blockers [START_REF] Chevalier | Quinazoline ´ ´ ´ ´ ´ ´ derivatives are efficient chemosensitizers of antibiotic activity in Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa resistant strains[END_REF][START_REF] Mahamoud | Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy[END_REF][START_REF] Mahamoud | An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates[END_REF]. The compound structures have been selected according to their structural similarity with quinolones, a main efflux substrate. The activity of these compounds has been evaluated on a collection of MDR clinical strains expressing various resistance mechanisms to different antibiotics including efflux pumps, altered membrane organisation, target mutations and enzymatic barriers [START_REF] Bolla | Membrane permeability and regulation of drug ''influx and efflux'' in enterobacterial pathogens[END_REF]. In each class, some derivatives (Table 1) noticeably increase the susceptibility to the quinosubstrates of efflux pumps of E. aerogenes [START_REF] Pagès | Efflux pumps of gram-negative bacteria, a new target for new molecules[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF][START_REF] Chevalier | Quinazoline ´ ´ ´ ´ ´ ´ derivatives are efficient chemosensitizers of antibiotic activity in Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa resistant strains[END_REF][START_REF] Mahamoud | Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy[END_REF][START_REF] Mahamoud | An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates[END_REF]. In addition, they also increased the intracellular accumulation of radiolabelled norfloxacin or chloramphenicol [START_REF] Chevalier | Quinazoline ´ ´ ´ ´ ´ ´ derivatives are efficient chemosensitizers of antibiotic activity in Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa resistant strains[END_REF][START_REF] Mahamoud | Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy[END_REF][START_REF] Mahamoud | An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates[END_REF]. The difference in the restoration level obtained with the various derivatives and the antibiotics tested may reflect: (i) a variation in the respective location of ligands inside the AcrB cavity such that binding of a compound interferes with the extrusion of the antibiotic; (ii) a competition between the antibiotic and derivative for the same pump active site; (iii) the compounds differ with respect to the degree of affinity for a specific internal site; and (iv) binding of the compound reduces the affinity of the antibiotic to a different site.

The presence of different sites located inside the AcrB pump has been reported [START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Yu | A perisplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study[END_REF]. With the recent model illustrating functioning of the AcrB pump [START_REF] Murakami | Multidrug efflux transporter, AcrBthe pumping mechanism[END_REF][START_REF] Pos | Drug transport mechanism of the AcrB efflux pump[END_REF], it is possible that the same amino acid residues participated at different drug binding sites located in the pump tunnel. Moreover, several recent data on the E. coli AcrB efflux mechanism indicate that some amino acid residues are involved in the transport process to a varying extent during specific steps such as recognition, binding, transport and release [START_REF] Nikaido | Multidrug resistance in bacteria[END_REF][START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Yu | A perisplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study[END_REF][START_REF] Wehmeier | Site-directed mutagenesis reveals amino acid residues in the Escherichia coli RND efflux pump AcrAB that confer macrolide resistance[END_REF]. This can explain the divergence observed in the competitive capacity of the compounds with drugs of different families (chloramphenicol, quinolones, ethidium bromide) for binding sites within the pump type (AcrB, MexB) as well as the level of expressed efflux pump activity in the tested strains [START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. Analyses of structure-activity relationships regarding homologous derivatives allowed the identification of pharmacophoric profile in each class of compounds.

Arylpiperazines

The screening of a limited library indicated that several arylpiperazines are able to block RND-type efflux pumps in E. coli [START_REF] Bohnert | Selected arylpiperazines are capable of reversing multidrug resistance in Escherichia coli overexpressing RND efflux pumps[END_REF]. The blocking activities of these compounds are modulated by the spacer length between the benzene ring and the piperazine ring as well as halogenic substitutions at the benzene ring. Among piperazine compounds, NMP (Table 1) was the most active unsubstituted arylpiperazines that increases the intracellular concentration of diverse substrates such as fluoroquinolones and fluorescent dyes [START_REF] Kern | Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Escherichia coli[END_REF][START_REF] Schumacher | Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli[END_REF]. NMP was active in several bacterial species including A. baumannii, in different Enterobacteriaceae, but less efficient in P. aeruginosa [START_REF] Kern | Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Escherichia coli[END_REF][START_REF] Schumacher | Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli[END_REF][START_REF] Pannek | Multidrug efflux inhibition in Acinetobacter baumannii: comparison between 1-(1-naphthylmethyl)-piperazine and phenyl-argininebeta-naphthylamide[END_REF][START_REF] Schumacher | Intracellular accumulation of linezolid in Escherichia coli, Citrobacter freundii and Enterobacter aerogenes: role of enhanced efflux pump activity and inactivation[END_REF][START_REF] Hannula | Effect of putative efflux pump inhibitors and inducers on the antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli[END_REF]. The mechanism of action of NMP and related compounds has not been elucidated and because of the primary activity (''serotonin agonist'' properties), they are likely to be too toxic for clinical use. Nevertheless, arylpiperazine could be an interesting pharmacophoric group for other molecular structures as hydantoins [START_REF] Kiec-Kononowicz | Antimycobacterial activity of 5-arylidene derivatives of hydantoin[END_REF][START_REF] Szymanska | Antimycobacterial activity of 5-arylidene aromatic derivatives of hydantoin[END_REF][START_REF] Szymanska | Antimicrobial activity of 5-arylidene aromatic derivatives of hydantoin Part 2 #[END_REF][94] which are promising agents to combat bacterial resistance (Handzlik et al., unpublished data).

Phenothiazines

Phenothiazines are an other class of EPIs, or indirect effectors of antibiotic potency; derivatives such as chlorpromazine or thioridazine, have been proved to sensitize resistant bacteria to the antibiotic to which it was initially resistant [START_REF] Martins | Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gram-negative infections: mechanisms for their direct and indirect activities[END_REF].

For example the activity of antimicrobials against Burkholderia pseudomallei and Salmonella enterica has been increased using phenothiazines [START_REF] Bailey | RamA confers multidrug resistance in Salmonella enterica via increased expression of acrB; which is inhibited by chlorpromazine[END_REF]. Moreover, in the presence of chlorpromazine, the efflux of ethidium bromide was decreased in the wild type parental strain as well as with thioridazine, those activity was recognized in Mycobacterium avium and smegmatis [START_REF] Rodrigues | Thioridazine and chlorpromazine inhibition of ethidium bromide efflux in Mycobacterium avium and Mycobacterium smegmatis[END_REF].

Because the primary mode of action of phenothiazines is to inhibit enzyme activities involved in the generation of metabolic energy the question of whether a similar mechanism was involved in the EPI activity of this group of agents, was of importance [START_REF] Amaral | Promising therapy of XDR-TB/MDR-TB with thioridazine an inhibitor of bacterial efflux pumps[END_REF]. The effect of phenothiazine on efflux activity was demonstrated by measuring the accumulation and extrusion of ethidium bromide [START_REF] Viveiros | New methods for the identification of efflux mediated MDR bacteria; genetic assessment of regulators and efflux pump constituents; characterization of efflux systems and screening for inhibitors of efflux pumps[END_REF]. lone, phenicol and cycline antibiotics, all of which being

The activity of phenothiazines on the strain over-expressing efflux pump AcrAB E. coli has been reproduced with other Gramnegative bacteria [START_REF] Viveiros | New methods for the identification of efflux mediated MDR bacteria; genetic assessment of regulators and efflux pump constituents; characterization of efflux systems and screening for inhibitors of efflux pumps[END_REF]. From these preliminary data, the study on phenothiazines will be attractive for developing new physiologically relevant EPIs [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF].

Natural products as a source of bacterial resistance modulators

Plants have exceptional ability to produce cytotoxic agents, and there is an ecological rationale that antimicrobial natural products should be present or synthesized de novo in plants following microbial attack to protect themselves from pathogenic microbes [START_REF] Stavri | Bacterial efflux pump inhibitors from natural sources[END_REF]. The scarcity of infective diseases in wild plants is in itself an indication of the successful defense mechanisms developed by them.

Numerous data are already available on Gram-positive resistance modulators in crude plant extracts or isolated from natural products [START_REF] Gibbons | Phytochemicals for bacterial resistancestrengths; weaknesses and opportunities[END_REF] with the lead example of the 5 0 -methoxyhydnocarpin produced by berberry plants that inhibits efflux of staphylococcals [START_REF] Stermitz | Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin; a multidrug pump inhibitor[END_REF] thus allowing the active hydrophobic alkaloid berberine to reach its target [START_REF] Lewis | Prospects for plant-derived antibacterials[END_REF]. In Gram-negative bacteria, despite the limited results available, some very interesting tracks are open [START_REF] Stavri | Bacterial efflux pump inhibitors from natural sources[END_REF].

Ethanol extract from Mentha avensis, an herbaceous plant that occurs in the whole of South America, was shown to strongly decrease resistance to gentamicin of an E. coli clinical isolate [START_REF] Coutinho | Enhancement of the antibiotic activity against a multiresistant Escherichia coli by Mentha arvensis L. and chlorpromazine[END_REF]. The same authors reported that extracts from Turnera ulmifolia could be used to increase the susceptibility of E. coli to aminoglycosides probably by altering efflux pump activity [START_REF] Coutinho | Increasing of the aminoglicosyde antibiotic activity against a multidrug-resistant E. coli by Turnera ulmifolia L. and chlorpromazine[END_REF]. However, in both cases the active compound was not identified from the extracts.

Lemongrass oil (Cymbopogon citratus) or its main component, citral, were tested alone or in combination with streptomycin and kanamycin on Salmonella species. A synergy was observed for one strain of S. typhimurium in each combination between natural products and kanamycin or streptomycin. The stronger effect was observed with the combination of the essential oil and kanamycin [START_REF] Shin | Anti-Salmonella activity of lemongrass oil alone and in combination with antibiotics[END_REF]. Interestingly, citral derivatives have been showed to efficiently block NorA pump [START_REF] Thota | Citral derived amides as potent bacterial NorA efflux pump inhibitors[END_REF].

Three molecules isolated from the hop flower, lupulone (also known as beta-acid), humulone and xanthohumol were tested in combination with antibiotics on various Gram-negative bacteria. The disc diffusion assay was used to measure susceptibility to polymyxin B and zones of inhibition were compared from regular plates, to plates containing molecules at a sub-inhibitory concentration. Results indicated that in the presence of lupulone, inhibition zones around the polymyxin B discs increased for Proteus mirabilis, Serratia marcescens and P. vulgaris; the latter showing the most significant response, the inhibition zone increasing from 0.05 cm to 0.8 cm in the presence of the product [START_REF] Natarajan | Positive antibacterial co-action between hop (Humulus lupulus) constituents and selected antibiotics[END_REF]. Authors hypothesized that the changes in envelope permeability, which may favor the penetration or reduce the efflux, caused by the hop compounds help in overcoming the resistance associated with bacterial membrane.

The essential oil of the Corsican Helichrysum italicum (HI) exhibited a strong activity against MDR E. aerogenes clinical isolates. Moreover, the essential oil was also efficient in modulation of bacterial resistance of E. coli, P. aeruginosa and A. baumanii strains. Efflux pump mutants analysis showed that HI could modulate the AcrAB efflux mechanism and other resistance mechanisms that are produced in MDR Gram-negative bacteria. Composition determination of HI indicated that the activity described comes at least in part from geraniol, which was not described to date as modulator of antibiotic resistance [START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug[END_REF].

Conclusion

Decrease of membrane permeability associated with overexpression of efflux pumps largely contribute to the MDR phenotype. Down regulation of porins synthesis is described in a large collection of resistant clinical isolates associated or not with an alteration of OM organization e.g., modification of LPS structure.

It is now admitted that the membrane-associated mechanisms of resistance represent a key step for clinical isolates and it is urgent to define new ways to circumvent these bacterial mechanisms. The re-use of polymyxins reported by several teams illustrates a possible strategy to combat MDR bacteria. With our increasing knowledge of antibiotic translocation through bacterial membrane, e.g., influx and efflux processes, in clinical isolates and the molecular details of transporter-ligand interactions, we can consider new possibilities in order to restore intracellular antibiotic concentrations. Attempts to develop original agents to combat this membrane barrier mechanism used by bacteria include the research and development of inhibitors of multidrug efflux pumps and of entry facilitators.

The variety and redundancy of membrane transporters involved in the efflux pump activity, the bacterial membrane organization and toxicity issues have hampered this. However, the recent studies focused on the specificity and mechanics (structure-activity relationships) of drug efflux may lead to the development of perhaps more effective inhibitors that exhibit a reduced toxicity associated to an efficacy in restoring the antibiotic accumulation inside the bacterium. These new agents may be able to rejuvenate the activity of ''old antibiotics'' against MDR bacteria. A functional, pharmaco-chemical and structural analysis of pump-substrate interactions will be engaged to decipher the molecular bases of interaction of the pump with specific ligands allowing us to develop the rational synthesis of efflux inhibitors.

Regarding molecules able to facilitate the entry of antibiotics, several main aspects must be considered, toxicity on membrane and adverse side effects on eukaryotic cells, two-step entry in Gram-negative bacteria, OM and IM, for antibiotics that must reach a cytoplasmic target (proteins, nucleic acids). The natural compounds, polycationic cyclic lipopeptides and cationic antimicrobial peptides and the new derivatives issued from polymyxin class are quite attractive but required intensive studies to determine activity, stability and toxicity. A major issue must address the pharmacokinetic and pharmacodynamic aspects of these membranotropic molecules as a therapeutic agent and their precise conditions of use.

Until now, most adjuvants or chemosensitzers have been identified through usual random screening of synthetic compounds or natural-product libraries. With the improved resolution of the 3D structures of membrane components and bacterial transporters, the dynamic simulations and molecular modelling (SAR, QSAR, HQSAR), and the recent approaches about the kinetics of transport (influx and efflux), it will be possible to design the pharmacophoric groups involved in the drug translocation. However, this original way requires studies about the penetration and membrane diffusion and also toxicity of such complex compounds.

Whatever the selected ways, favoring the entry or blocking the efflux, a functional pharmaco-chemical and biological-structural analysis of adjuvant-antibiotic interactions must be engaged to determine the best efficient combination in order to combat specific MDR bacteria and prevent the emergence of new resistances against the adjuvant action.

Regarding the future of these adjuvants in combination with antibiotics, the development and current use of b-lactamase inhibitors such as clavulanate or tazobactam is an encouraging and fruitful clinical example.

  Fig.1. Steps of antibiotic transport through the membranes of Gram-negative bacteria. To be active against bacterium, an antibiotic needs a critical concentration threshold to inhibit the corresponding target. Using the concentration gradient, the influx is controlled by several elements: the antibiotic can permeate through the OM via porins (a, for hydrophilic molecules) or through the OM (b, for hydrophobic drugs)[START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. (c) Represents the diffusion through the periplasmic space in which enzymatic inactivation may occur[START_REF] Bush | Updated functional classification of betalactamases[END_REF][START_REF] Bush | New b-lactam antibiotics and b-lactamase inhibitors[END_REF]. Finally, the drug will pass the cytoplasmic/IM (d). Regarding the efflux, the drug may be recognized and transported by active pump (e) or (f). The pump (e.g., AcrB) has to recruit the OM pore TolC (g), in addition it may conjointly acts with other pumps (e.g., MFS family). The success of a drug is given by the kinetics of all individual steps[START_REF] Nikaido | Structure and mechanism of rnd-type multidrug efflux pumps (E.J. Toone[END_REF][START_REF] Amaral | Efflux pumps of Gram-negative bacteria: genetic responses to stress and the modulation of their activity by pH, inhibitors and phenothaizines[END_REF].

Table 1

 1 Most active efflux pump blockers, with antibiotics and bacterial species in which their activity has been demonstrated.

	Compound		Molecular structure	Bacterial species	Antibiotic	Reference
	Synthetic products	Peptidomimetics							P. aeruginosa, E. coli, E.	Quinolones	[63,73-
									aerogenes, K.	80]
									pneumoniae, S. enterica
			H2N	H N	H N MC-04,124 O N H O	N
			H2N	O	H N	O	N H	NH
								H N	NH2
						PAN
		Quinolines/			HN	N	E. aerogenes	Quinolones	[21,63,81-
		quinazolines							Phenicols	83]
			O2N	N BG 814			Cyclines Ethidium bromide
				HN			N
					N				O
				N
				BG 1167
		Arylpiperazines		N	NH			E. coli, Acinetobacter baumanii	Fluoroquinolones	[85-90]
			NMP
			N						N
		Phenothiazines		N		Cl		N	S	B. pseudomallei, S.	Ethidium bromide	[95-99]
				S					S	enterica, M. avium, M.	Aminoglycosides, Levofloxacin
			Chlorpromazine			Thioridazine	smegmatis, E. coli
	Natural products	Lupulone,		OH O			OH O	P. mirabilis, S.	Polymyxin B	[108]
		Humulone ,	HO			O			HO	O	marcescens, P. vulgaris
		Xanthohumol							OH
				Lupulone			Humulone
							OH O
						HO			O
							Xanthohumol	OH
						OH
		Eugenol							E. coli, P. aeruginosa, E.	b-Lactams, Erythromycin, Norfloxacin,	[48]
						O			aerogenes, S.	Chloramphenicol, Polymyxin B,
		Geraniol	HO						typhimurium, P. vulgaris E. aerogenes, E. coli, P.	Tetracycline, Vancomycin, Rifampycin Chloramphenicol, Norfloxacin	[109]
									aeruginosa, A. baumanii

Acknowledgements

We gratefully thank Anne Favard for the preparation of this manuscript. This study was supported by the Service de Santé des Armées, by the Université de la Méditerranée, COST Action BM0701 (ATENS) and by PHC Polonium (22531ZG).


Abdallah Mahamoud

Jacqueline Chevalier

Sandrine Alibert-Franco

Winfried V Kern

Jean-Marie Pages

Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy `

Keywords: antibiotic resistance, drug efflux pumps, efflux pump inhibitors

After several decades of continuously successful antibiotic therapy against bacterial infections, we are now facing a worrying prospect: the accelerated evolution of antibiotic resistance to important human pathogens and the scarcity of new anti-infective drug families under development. Efflux is a general mechanism responsible for bacterial resistance to antibiotics. This active drug transport is involved in low intrinsic susceptibility, cross-resistance to chemically unrelated classes of molecules, and selection/acquisition of additional mechanisms of resistance. Thus, inhibition of bacterial efflux mechanisms appears to be a promising target in order to (i) increase the intracellular concentration of antibiotics that are expelled by efflux pumps, (ii) restore the drug susceptibility of resistant clinical strains, and (iii) reduce the capability for acquired additional resistance. Structurally unrelated classes of efflux pump inhibitors (EPIs) have been described and tested in the last decade, including some analogues of antibiotic substrates and new chemical molecules. Among the current collection of EPIs, only a few compounds have been studied taking into account the structure-activity relationships and the spectrum of activity in terms of antibiotics, pumps and bacteria. While large efforts have characterized an increasing number of bacterial efflux pumps and generated several potentially active EPIs, they have not elucidated the molecular basis of efflux transport and inhibition. Recent studies of pumpsubstrate complexes, the 3D resolution of the efflux pumps, the synthesis of novel compounds and molecular dynamic studies may generate new clues to decipher and select novel targets inside the efflux mechanisms and, finally, may result in a clinically useful molecule.

Efflux pumps and antibiotic resistance

Bacterial resistance to antibiotics such as b-lactams, aminoglycosides and quinolones can be summarized as different, interacting steps based on the discovery of novel molecules belonging to various antibiotic classes, their clinical use, and the characterization of emerging resistance mechanisms. A consequence of these tight relationships and increasing antibiotic resistance is the urgent requirement to develop novel molecules that are insensitive to resistance processes in order to combat resistant pathogens. The accelerating increase of bacterial resistance and resultant problematic therapy are directly responsible for the current increase in morbidity and mortality associated with bacterial infections.

Among the various mechanisms involved in bacterial resistance, the balance of membrane permeability which controls the traffic (in and out) of various molecules, plays a key role in the influx and efflux of antibiotics, thereby limiting their intracellular concentration. Multidrug resistance efflux pumps have now been described in both Gram-positive and Gram-negative bacterial pathogens. The poly-specificity of efflux transporters confers a 'general resistance mechanism' that can reinforce the effect, and/or favour the acquisition, of other mechanisms of antibiotic resistance such as mutations of the antibiotic targets or modification of the drugs. 1 -5 Gram-negative bacteria become, in general, more readily resistant to antibiotics owing to the sophisticated architecture of their cell envelope, including the outer and inner membranes which delineate the periplasmic space. A toxic compound can be picked up in the periplasm and expelled directly to the external medium, strongly reducing the number of molecules reaching their cytoplasmic targets. 1 -7 Conjointly to these, the external barrier to diffusion, the outer *Corresponding author. Tel.: þ33-4-91-32-45-87; Fax: þ33-4-91-32-46-06; E-mail: Jean-Marie.PAGES@medecine.univ-mrs.fr membrane, acts as an additional protective step which strongly limits the passive penetration of hydrophilic, charged and hydrophobic molecules. [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF][START_REF] Pages | Role of bacterial porins in antibiotic susceptibility of Gram-negative bacteria[END_REF] Moreover, because this envelope architecture is responsible for additional levels of resistance to some antibiotic classes (e.g. b-lactams, fluoroquinolones), Gram-negative bacteria are a major cause of antibiotic-resistant infections.

The complex envelope of Gram-negative bacteria contains a variety of protein channels involved in the transport (influx or efflux) of a large variety of nutrients (sugars, amino acids, salts, metals, etc) or noxious compounds (metabolites, drugs, biocides, detergents, etc.). Among these transporters are distinct energydependent efflux pumps that recognize toxic agents such as antibiotics and extrude ( pump out) the agent from the periplasm/cytoplasm to the exterior (environment) of the cell, thereby reducing the intracellular accumulation of the agent. Overexpression of one or more of these efflux pumps prevents the intracellular accumulation of the agent to thresholds necessary for its inhibitory activity. 1 -6 This efflux pump overproduction is generally accompanied by an increase in resistance to two or more structurally unrelated antibiotics [multidrugresistance (MDR)] and significantly contributes to the emergence and spread of MDR pathogens. The mechanisms of influx and efflux cooperate in the control of intracellular concentration by various efficient regulation cascades.

Phylogenetically, bacterial antibiotic efflux pumps belong to five superfamilies (see http://www.biology.ucsd.edu/~msaier/ transport/ for classification, and reviews [START_REF] Li | Efflux-mediated drug resistance in bacteria[END_REF][START_REF] Paulsen | Multidrug efflux pumps and resistance: regulation and evolution[END_REF][START_REF] Saier | Tracing pathways of transport protein evolution[END_REF] ), namely: (i) ABC (ATP-binding cassette), which are primary active transporters energized by ATP hydrolysis; (ii) SMR [small multidrug resistance subfamily of the DMT (drug/metabolite transporters) superfamily]; (iii) MATE [multi-antimicrobial extrusion subfamily of the MOP (multidrug/oligosaccharidyl-lipid/polysaccharide flippases) superfamily]; (iv) MFS (major facilitator superfamily); and (v) RND (resistance/nodulation/division superfamily), which are all secondary active transporters driven by ion gradients. The MFS and RND pumps are the most abundant: the MFS pumps are found in both Gram-positive and Gram-negative bacteria, and are characterized by a relative narrow spectrum, recognizing usually one or sometimes a few antibiotic classes; the RND pumps are found exclusively in Gram-negative bacteria and display an extremely wide spectrum of substrates (polyselectivity), including not only several classes of antibiotics, but also antiseptic compounds, dyes, or detergents. 1 -7 These pumps have been discussed in detail (regulation and expression, topology, presence in bacterial species, main substrates, etc.) in recent reviews. 1 -7,12 -16 In this article we will focus on efflux pump inhibitors (EPIs) that are active against resistant Gram-negative bacteria.

Circumventing the efflux mechanism: an emerging strategy to face an emerging problem It comes as no surprise that efflux mechanisms responsible for the decreased effectiveness of common antibiotics also account for the resistance to new, recently described antimicrobial agents such as a peptide deformylase inhibitor, [START_REF] Dean | Role of the AcrAB-TolC efflux pump in determining susceptibility of Haemophilus influenzae to the novel peptide deformylase inhibitor LBM415[END_REF] plectasin [START_REF] Mygind | Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus[END_REF] and platensimycin. [START_REF] Wang | Platensimycin is a selective FabF inhibitor with potent antibiotic properties[END_REF] This strongly supports the need for research and development into compounds that are able to circumvent or block efflux pumps and to restore/preserve antibacterial potency of older as well as newer antibiotics. 20 -25 Moreover, it has been recently demonstrated that the expression of AcrAB-TolC pump is an important prerequisite for the selection of ciprofloxacinresistant mutants. [START_REF] Ricci | Ciprofloxacin-resistant Salmonella enterica serovar Typhimurium strains are difficult to select in the absence of AcrB and TolC[END_REF] Similar observations have been reported concerning the role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter strains. [START_REF] Yan | Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure[END_REF] Response to drug efflux mechanisms: a present challenge In order to address the problem of efflux pumps and their consequences on decreasing the intracellular active concentration of antibiotics, it is necessary to search for and develop new molecules to circumvent efflux activity. Different strategies to reach this objective are:

(1) By-passing efflux activity: improving the molecular design of old antibiotics to reduce their efflux. The profile of antibiotics will be changed: † by modifying the structure of a molecule in order to decrease its affinity for the affinity sites located inside the efflux pump; † by introducing new side chains in order to block/impair the transport. (2) Direct action on the permeability of the bacterial cell envelope: decreasing the efficacy of the membrane barrier.

The permeability of the cell envelope will be modified by: † a direct modification, via a detergent-like effect, ensuring an improved uptake that yields increased intracellular concentration of the antibiotic; † a channel-blocker that induces a 'traffic jam' in the outer membrane channel (TolC) restoring a high intracellular drug concentration. (3) Blocking the efflux capacity of bacterial cell: alteration of pump function. The reversal of resistance will be obtained by using: † competitive inhibition: competition between an agent that uses the same substrate (ligand) site inside the pump and the antibiotic(s); † non-competitive inhibition: poly-selective binding to pump components producing steric hindrance within the cavities of the pump; † energy wasting, direct/specific via a antiporter site or indirect/general via a collapse of energy driven mechanisms of the bacterial cell envelope.

Concerning strategy (1), no novel drug has been designed according to the recent criteria obtained from the determination of pump structures. However, the recent advances in bacterial genomics, enabling resolution of the 3D structure of efflux pumps, and the combinatorial chemistry will provide new means, tools and concepts for the improvement of existing antibiotics.

Decreasing the efficacy of the membrane barrier: permeabilizing compounds

Concerning permeabilizing agents, one molecule has presented some attractive properties, the polymyxin B nonapeptide. [START_REF] Tsubery | Structure-function studies of polymyxin B nonapeptide: implications to sensitization of gram-negative bacteria[END_REF] This molecule and neopeptide antibiotics, which exhibit a structure close to the polymyxin class with a reduced direct bactericidal activity, are able to destabilize the membrane allowing the enhanced penetration of antibiotics into the bacterium. [START_REF] Tsubery | Structure-function studies of polymyxin B nonapeptide: implications to sensitization of gram-negative bacteria[END_REF][START_REF] Tsubery | Neopeptide antibiotics that function as opsonins and membrane-permeabilizing agents for gramnegative bacteria[END_REF] The cyclic moiety of the molecule binds to the Gram-negative lipopolysaccharide and permeabilizes the outer membrane to hydrophobic antibiotics as well as to other bactericidal agents. [START_REF] Tsubery | Neopeptide antibiotics that function as opsonins and membrane-permeabilizing agents for gramnegative bacteria[END_REF] The use of these molecules is limited by the same adverse effects produced by polymyxin-type antibiotics such as acute nephrotoxicity, although some of the most recent polymyxins are less toxic. [START_REF] Tsubery | Neopeptide antibiotics that function as opsonins and membrane-permeabilizing agents for gramnegative bacteria[END_REF] However, more investigations are necessary to elucidate the conditions necessary to restore the activity of conventional antibiotics against multidrug-resistant (MDR) bacteria. Because various detergents, such as SDS and Triton-X, which affect the integrity of the cell envelope, are also substrates of efflux mechanisms due to their lipophilic and amphipathic properties, [START_REF] Li | Efflux-mediated drug resistance in bacteria[END_REF] the use of such agents may be limited unless they can be modified in a manner that renders them non-substrates.

What about specifically blocking the TolC-or other similar family channel? Although this possibility seems to be attractive, especially with regard to their dual role in the secretion of virulence factors, [START_REF] Nishino | Virulence and drug resistance roles of multidrug efflux systems of Salmonella enterica serovar Typhimurium[END_REF] this approach has not been really investigated to the authors' knowledge. However, study of this interesting approach will be facilitated by the recent data concerning the functional organization and the structure of the AcrAB-TolC efflux pump. 31 -33 The efflux pump energy: targeting the driving force of the mechanism Compounds that seriously affect the energy level of the bacterial membrane such as carbonyl cyanide m-chlorophenylhydrazone (CCCP), are used in the laboratory to abolish totally the efflux of various molecules. [START_REF] Pages | Inhibitors of efflux pumps in Gram-negative bacteria[END_REF][START_REF] Mallea | Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes[END_REF][START_REF] Thanassi | Active efflux of bile salts by Escherichia coli[END_REF] These compounds reduce the viability of the bacterium and cause cell death via the dissipation of the proton-motive force of the membrane. Consequently, there is always the question of whether it is their effect on the efflux pump that is the cause of an increase in the penetration of the antibiotic, or whether it is due to the alteration of the cell envelope itself that results in the death of the bacterium. In addition, some of them, like CCCP, are recognized as highly noxious and cytotoxic and are also substrates of bacterial efflux pumps. Today, no molecule belonging to the energy-blocker family has been developed for clinical use or has been patented.

What about general inhibitors of efflux pumps such as reserpine and verapamil? These molecules were initially documented as inhibitors of vesicular monoamine transporters and blockers of transmembrane calcium entry (or calcium ion antagonists), respectively. Verapamil is an inhibitor of MDR pumps of cancer cells and parasites and also improves the activity of tobramycin. [START_REF] Li | Efflux-mediated drug resistance in bacteria[END_REF] Reserpine inhibits the activity of Bmr and NorA, two Gram-positive efflux pumps. [START_REF] Li | Efflux-mediated drug resistance in bacteria[END_REF] They alter the generation of the membrane proton-motive force required for the function of MDR efflux pumps. Although these molecules are able to inhibit the ABC transporters involved in the extrusion of antibiotics (i.e. tetracycline), the concentrations necessary to block bacterial efflux are neurotoxic. [START_REF] Li | Efflux-mediated drug resistance in bacteria[END_REF] Even though reserpine has been used as an anti-hypertensive drug, its concentration for this purpose is far lower than that employed for the inhibition of efflux. Verapamil and reserpine are routinely used to evaluate the activity of efflux pumps in Gram-positive bacteria. [START_REF] Li | Efflux-mediated drug resistance in bacteria[END_REF] Chemically synthesized derivatives and natural products such as 5-methoxyhydnocarpin or berberine have activities against efflux pumps. [START_REF] Lewis | In search of natural substrates and inhibitors of MDR pumps[END_REF][START_REF] Tegos | Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials[END_REF] Problems with their synthesis, purification, stability and solubility, in addition to their potential toxicity, have reduced interest in further development of these promising compounds as efflux pump inhibitors.

Alteration of pump function: the flux inhibitors

Peptidomimetics

From the study of efflux mechanisms acting on Gram-negative antibiotic-resistant strains of Pseudomonas aeruginosa, Microcide and Daiichi Pharmaceuticals have produced a large family of peptidomimetics that exhibit EPI properties (Table 1). Among this family, the first identified EPI from a screening based on the ability to restore levofloxacin susceptibility in various P. aeruginosa clinical strains, was MC-207 110 or phenylalanine arginyl b-naphthylamide (PAbN). In addition to its effect on levofloxacin activity, PAbN is also reported to restore the activity of various other antibiotic classes, including chloramphenicol and macrolides. 24,37 -39 The ability of this agent to restore antibiotic susceptibility of resistant bacteria is attributed to its inhibiting the efflux of one or more antibiotics, [START_REF] Lomovskaya | Practical applications and feasibility of efflux pump inhibitors in the clinic-a vision for applied use[END_REF] and therefore the molecule can be considered to exhibit a broad spectrum of efflux pump inhibition.

The analyses of the inhibitory mechanism have demonstrated that this EPI is a substrate of efflux pumps and may act as a competitive inhibitor. [START_REF] Lomovskaya | Practical applications and feasibility of efflux pump inhibitors in the clinic-a vision for applied use[END_REF] These studies, in addition to the recent work describing the co-crystallization of AcrB with various substrates, [START_REF] Yu | A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and sitedirected mutagenesis study[END_REF][START_REF] Yu | Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump[END_REF] support the idea that MC-207 110 may recognize and bind to the same affinity pocket used by the potentiated antibiotics (e.g. levofloxacin) located inside the AcrB cavity or inside the MexB cavity in P. aeruginosa. Alternatively, due to a close location of binding site, the EPI binding may also generate steric hindrance, impairing the antibiotic binding at its affinity site. In order to clarify these possibilities, affinity constants and kinetic parameters should be determined (see the concluding section 'A future for combating efflux pumps'). The pharmaceutical companies Essential Therapeutics and Daiichi Pharmaceutical have produced MC-207 110 derivatives exhibiting structural modifications in order to improve the biological stability of EPIs and to enhance the therapeutic and pharmacodynamic profiles (Table 1). Some of them [START_REF] Renau | Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin[END_REF][START_REF] Renau | Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa[END_REF][START_REF] Renau | Addressing the stability of C-capped dipeptide efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa[END_REF] result from the substitution of an amino acid unit (Orn in place of Arg), or use D-amino acid residues (D-Orn in place to L-Orn). Additional structural modifications have provided derivatives for in vivo evaluations. In parallel studies, structureactivity relationships have examined the role of the peptidic backbone present in this inhibitor family. 42 -46 In addition to these MC-207 110 derivative compounds, pyridopyrimidines have been developed as EPIs in P. aeruginosa. 47 -51 A major problem with members of this EPI family is their toxic properties, which prevents their clinical application. 23 -25 However, these products are widely used to experimentally determine and evaluate the efflux mechanisms of bacterial pathogens. In addition, they are used to measure the efflux activity and determine the level of inhibitor-sensitive efflux for specific antibiotics in various bacteria. [START_REF] Lomovskaya | Practical applications and feasibility of efflux pump inhibitors in the clinic-a vision for applied use[END_REF][START_REF] Kriengkauykiat | Use of an efflux pump inhibitor to determine the prevalence of efflux pump-mediated fluoroquinolone resistance and multidrug resistance in Pseudomonas aeruginosa[END_REF] Quinoline derivatives This novel class of compounds was discovered by using several screening procedures with Enterobacter aerogenes strains. [START_REF] Mahamoud | Quinolone derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes[END_REF][START_REF] Chevalier | New pyridoquinoline derivatives as potential inhibitors of the fluoroquinolone efflux pump in resistant Enterobacter aerogenes strains[END_REF] These compounds have been assayed for their activity against various MDR clinical strains overexpressing efflux pumps that expel different antibiotics (e.g. chloramphenicol, norfloxacin). Active quinoline derivatives have then been evaluated for their ability to restore the activity of various antibiotic families (e.g. quinolone, phenicol, cycline). 52 -57 Data from many sources confirm the potential of these EPI compounds, and several quinoline derivatives are now considered as broad-spectrum EPIs for rendering antibiotic-resistant E. aerogenes and Klebsiella pneumoniae susceptible to chloramphenicol, tetracycline and norfloxacin (Table 1). [START_REF] Chevalier | Inhibitors of antibiotic pneumoniae strains[END_REF] Moreover, the direct action of this family of molecules on the drug extrusion mechanism has been clearly demonstrated by measuring the intracellular concentrations of antibiotics (norfloxacin, chloramphenicol) after their addition to bacterial cultures; their ability to increase the accumulation of the antibiotic has been compared to ` that resulting from the addition of CCCP or PAbN to the culture. 52 -57 An interesting point to note is the relatively poor efficacy of these products against the MexAB-OprM efflux pump of P. aeruginosa (J. Chevalier, A. Mahamoud and J.-M. Pages, unpublished results) if compared with the substantial activity of PAbN to reverse drug resistance in this bacterium. This observation could be attributable to differences in the sequences of AcrB and MexB, which may induce some change in the functional organization of the efflux pump; these modifications may affect amino acid residues that are directly involved in the substrate affinity sites, e.g. in the transport of EPIs or antibiotic molecules in both E. aerogenes and P. aeruginosa. In addition, the original screening protocols are different, at least for the selected bacterial efflux target and for the antibiotic used as substrate (levofloxacin versus chloramphenicol), for the two EPI families (peptidomimetics or quinoline derivatives). [START_REF] Renau | Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin[END_REF][START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF][START_REF] Mahamoud | Quinolone derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes[END_REF] The analyses of structureactivity relationships have indicated that the alkyl side-chain linked to the heterocyclic moiety of alkylaminoquinolines plays a key role in EPI activity. [START_REF] Mahamoud | Quinolone derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes[END_REF] Alkoxy-and thioalkoxy-quinolines having piperidinoethyl chains restore susceptibility to chloramphenicol, with the degree of susceptibility increased depending upon the type of derivative-less so with oxo-derivatives than with amino substitutions in thioalkyl molecules. [START_REF] Mahamoud | Quinolone derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes[END_REF] In addition, the connecting heteroatom and the position of substituted groups on the ring seem also to be of importance. In this inhibitor class, additional studies are necessary to define the role of pharmacophoric groups and their reactivity with the affinity pockets reported in AcrB. [START_REF] Murakami | Crystal structures of a multidrug transporter reveal a functionally rotating mechanism[END_REF][START_REF] Seeger | Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism[END_REF][START_REF] Yu | Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump[END_REF] Alkylamino-, alkoxy-, thioalkoxy-, chloro-quinoline derivatives present two advantages: their similarity with the quinolone family, which greatly argues for an efficient pharmacokinetic profile and a negligible intrinsic activity, and no additional side-effect ( permeabilization or alteration) on the membrane. [START_REF] Mahamoud | Quinolone derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes[END_REF] However, toxicity assays and pharmacodynamic studies are still needed to determine the therapeutic potency of these compounds.

Arylpiperidines and arylpiperazines

Several compounds belonging to these two families have been tested as potential EPIs (Table 1). Among arylpiperidines, some derivatives (dihalogens) are able to restore linezolid susceptibility and linezolid accumulation in Escherichia coli. 58 In addition, phenylpiperidine selective serotonin re-uptake inhibitors inhibited the function of two unique Staphylococcus aureus multidrug efflux pumps and also partially affected the activity of the AcrAB-TolC pump in E. coli. [START_REF] Kaatz | Phenylpiperidine selective serotonin reuptake inhibitors interfere with multidrug efflux pump activity in Staphylococcus aureus[END_REF] Concerning arylpiperazine, screening of a limited library of low-molecular-weight N-heterocyclic compounds in E. coli led to the discovery of several arylpiperazines with a potency to reverse multidrug resistance in cells overexpressing RND-type efflux pumps. [START_REF] Bohnert | Selected arylpiperazines are capable of reversing multidrug resistance in Escherichia coli overexpressing RND efflux pumps[END_REF] Elongation of the spacer between the benzene ring and the piperazine ring as well as halogenic substitutions at the benzene ring enhanced the potency of the compounds. Despite a significant sensitizing effect, some of these compounds were strikingly poor in their efficiency to increase the intracellular concentration of efflux pump substrates, suggesting alternative or additional mechanisms of action. A few anthrylpiperazines and naphthylpiperazines, notably N-methylpyrrolidone (NMP), were among the most potent unsubstituted arylpiperazines, with a minimal effective concentration and a dose-dependent ability to increase the intracellular concentration of such diverse substrates as fluoroquinolones, chloramphenicol, fluorescent dyes and linezolid. NMP was tested in several bacterial species including clinical isolates. Interestingly, the compound was also effective in Acinetobacter baumannii, in different Enterobacteriaceae except Serratia, but not in P. aeruginosa. 61 -63 Due to the serotonin agonist properties of arylpiperazines, these compounds are likely to be too toxic for use in man and animals. The mechanism of action of NMP and related compounds has not been elucidated. Unlike PAbN, the (low) intrinsic antibacterial activity of NMP and its cellular accumulation are not enhanced in cells with inactivated efflux pumps.

Other efflux pump inhibitors

Various antibiotic analogues have been developed in order to circumvent the efflux pumps and restore the antibiotic activity of efflux-mediated resistant strains. Among them are tetracycline analogues which were first described by S. B. Levy's team in 1993. Initially focused on S. aureus and tetracyclines, these molecules have been tested with other antibiotics and other bacterial pathogens. 64 -66 The problem with this class of modified antibiotics is its high similarity of structure to the true antibiotic and its residual activity on bacterial targets; an adverse consequence of these properties is the reinforcement of the selection of resistance mechanisms directed against the antibiotic.

In addition to the products described, several products have been patented, [START_REF] Van Bambeke | Inhibitors of bacterial efflux pumps as adjuvants in antibiotic treatments and diagnostic tools for detection of resistance by efflux[END_REF] some of them exhibiting interesting capabilities. However, their exact properties, activity, solubility, purification, stability, and toxicity remain to be clearly defined. [START_REF] Molnar | Inhibition of the transport funcefflux pump in resistant Enterobacter aerogenes and Klebsiella tion of membrane proteins by some substituted phenothiazines in E. coli and multidrug resistant tumor cells[END_REF][START_REF] Amaral | Antimicrobial activity of phenothiazines[END_REF] A future for combating efflux pumps It is now important to tackle a serious gap in our knowledge of efflux mechanisms. Although we have some information regarding the structure and activity of AcrAB-TolC or MexAB-OprM pumps, the archetype of drug transporters, and about some inhibitors, we know very little about the mechanical and dynamic aspects of their function. The resolution of the structure of co-crystals of AcrB with various substrates [START_REF] Yu | A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and sitedirected mutagenesis study[END_REF][START_REF] Yu | Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump[END_REF] and the recently proposed mechanisms including the 'peristaltic pump mechanism' and the 'functionally rotating ordered multidrug binding change mechanism' [START_REF] Yan | Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure[END_REF][START_REF] Murakami | Crystal structures of a multidrug transporter reveal a functionally rotating mechanism[END_REF] are very promising but not totally satisfactory for the definition of suitable physicochemical characteristics for a future efficient inhibitor. To date, the activity of putative EPIs is deduced from various measures using the susceptibilities towards different antibiotics in the absence and presence of the agent. Similarly, the degree of inhibition of antibiotic efflux is determined by the variation of intracellular concentrations induced by the addition of tested compounds and compared to the level obtained in the presence of a poison that collapses the membrane energy necessary for the drug expulsion. To quantify the effect of EPIs on an efflux pump, more effective assays are needed for determination of kinetic parameters and their relationships to the structure of the component of the efflux pump that is affected. [START_REF] Mahamoud | Quinolone derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes[END_REF] The definition of these parameters is necessary for intelligent design of new EPIs and their eventual use for the therapy of MDR bacterial infections. In addition, these data are important in order to choose between the development of a 'general inhibitor' targeting the transporter that expels various antibiotics in one bacterial species or a 'selective inhibitor' blocking the pumping out of one antibiotic family in various bacteria-or, an intermediate solution.

To date, no efflux pump inhibitor has been licensed for use in the treatment of bacterial infections in human or veterinary medicine, and it is clear that this gap in our antimicrobial armamentarium must stimulate research that leads to the development of new EPI molecules. [START_REF] Pages | Inhibitors of efflux pumps in Gram-negative bacteria[END_REF][START_REF] Lomovskaya | Practical applications and feasibility of efflux pump inhibitors in the clinic-a vision for applied use[END_REF] The major benefits derived from developing efficient efflux pump inhibitors will be the ability to re-use various antibiotics affected by the efflux pumps as well as the control of the emergence and the dissemination of MDR efflux strains. Until now, the majority of the known inhibitors have been obtained from screening libraries of synthetic compounds, from purification of natural compounds or 'naive' chemical modification of existing molecules. With the recent resolution of the 3D structure of drug transporters, the co-crystallization of AcrB with some substrates, and the analyses of the biological effects of several mutations in the pump, we are able to begin rational design of functional targets located inside the bacterial pumps. Consequently, the synthesis of efficient molecules with strong EPI capacity will be more readily achieved. In addition, the definition of binding sites located inside the pump will be used to improve the antibiotic profile in order to decrease the affinity for pump affinity sites and residues involved in the drug transport, e.g. by introducing groups favouring steric hindrance. This may support the rationale for a new generation of antibiotics that are 'resistant or less susceptible to efflux'.

With this new generation of designed antibacterial compounds that are able to restore the antibiotic susceptibility by selectively blocking membrane transport, novel uses of transport inhibitors will emerge in order to (i) compete with the diffusion of quorum sensing molecules, or (ii) block the secretion of virulence factors.

Table 1 .

 1 Known inhibitors of Gram-negative efflux pumps

	Type	Substrates	Bacteria	SAR Reference
	Phenothiazine	tetracyclines	Escherichia coli	2	67
	Phenylpiperidine	norfloxacin, tetracycline, ethidium bromide	E. coli	2	59
	Tetracycline analogue	tetracyclines	E. coli	2/þ	64,65
	Aminoglycoside analogue	tetracyclines, gentamicin	Haemophilus influenzae	2	25
	Fluoroquinolone analogue	fluoroquinolones, macrolides	E. coli, Pseudomonas aeruginosa	2	25
	Quinoline derivative	chloramphenicol, norfloxacin, tetracycline	Enterobacter aerogenes, Klebsiella pneumoniae þ	52
	Peptidomimetic	quinolones, chloramphenicol,	P. aeruginosa, E. aerogenes,	þ	24
		macrolides, carbenicillin, tetracycline	E. coli, K. pneumoniae,
			Acinetobacter baumannii,
			Campylobacter jejuni
	Pyridopyrimidine	fluoroquinolones, b-lactams	P. aeruginosa	þ	46-50
	Arylpiperidine	linezolid	E. coli	2/þ	58
	Arylpiperazine	fluoroquinolones, tetracyclines,	E. coli, Citrobacter freundii,	2/þ	60-63
		macrolides, linezolid, chloramphenicol	K. pneumoniae, E. aerogenes, A. baumannii

SAR, studies on structure-activity relationships; þ, available; 2/þ, partial data; 2, no data.

Acknowledgements

´

´

We thank the members of EA2197 UMR-MD for helpful discussions. This study was partly carried out within the frame of the European 'COST B16 Action' and from Eloi Collery prix -Academie Nationale de Medecine (J.-M. P.). W. V. K. is supported in part by a grant from the German Federal Ministry of Education and Research (BMBF 01KI9951).

Transparency declarations

None to declare.