International audienceIn Gram-negative bacteria, the envelope is a sophisticated barrier protecting the cell against external toxic compounds. Membrane transporters, e.g., porins or efflux pumps, are main filters regulating 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

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Efflux pumps protect the bacterial cell by expelling toxic compounds before they reach intracellular targets. Because this mechanism actively contributes to the resistance of a given bacterium to more than one class of antibiotics, molecules that are able to block the relevant efflux pump are of potential significance to combat drug resistance caused by efflux pumps. Different quinoline derivatives including alkoxy, alkylamino, thioalkoxy and chloroquinolines have been previously reported to make Enterobacter aerogenes resistant isolates that over express the mechanism of efflux, noticeably more susceptible to structurally unrelated antibiotics. In addition, various quinoline derivatives significantly increase the intracellular concentration of chloramphenicol as reported with other inhibitors, thereby suggesting the inhibition of the drug transport by AcrAB-TolC pump, which is fully active in the clinicaly resistant isolates investigated. Here, we discuss the respective properties of this molecular family, taking into account the recent insights into the structural data of AcrB pump.

Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections. Several Gram-negative bacteria efflux pumps have been described. These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens. Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria. In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters. We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria. Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-[2'-(piperidino)ethyl]aminoquinoline acts as an inhibitor of the AcrAB-TolC efflux pump and restores a high level of intracellular drug concentration. Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes.

Antibiotic resistance mechanisms reported in Gram-negative bacteria are a worldwide health problem. The continuous dissemination of multi-drug resistant (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria the over-expression of efflux pumps expel structurally-unrelated antibiotics decreasing their intracellular concentration. It is necessary to clearly define the molecular and genetic bases of the efflux pump in order to combat this mechanism. The characterization of efflux pumps, from genetic to structural studies, allows the definition of a new, original antiresistance bullet, the efflux pump inhibitor (EPI). This new class of antibacterial molecules may act conjointly to the usual antibiotic in order to restore its activity. Several families of EPIs have been now reported and described. The use of these EPIs promotes a significant increase of susceptibility to one or more antibiotics in strains or clinical isolates which were initially resistant. These EPIs may target different efflux targets, (i) the expression of genes that induces MDR, the transporters that pump the antibiotic out of bacterium, (ii) the assembly of membrane transporter complex involved in drug efflux, (iii) the energy involved in this active transport, (iv) the inhibition of the flux of molecules inside the efflux channel by competition or blocking (via steric hindrances). With the recent thorough characterization of the efflux pump AcrB at its structural and physiological level including the identification of drug affinity sites and kinetic parameters for some antibiotics, it is now possible to rationally develop an improved new generation of EPIs.

In Enterobacter aerogenes and Klebsiella pneumoniae, efflux provides efficient extrusion of antibiotics and contributes to the multidrug resistance phenotype. One of the alkoxyquinoline derivatives studied here, 2,8-dimethyl-4-(2-pyrrolidinoethyl)-oxyquinoline, restores noticeable drug susceptibility to resistant clinical strains. Analyses of energy-dependent chloramphenicol efflux indicate that this compound inhibits the efflux pump mechanism and improves the activity of structurally unrelated antibiotics on multidrug-resistant E. aerogenes and K. pneumoniae isolates.Various multidrug resistance (MDR) phenotypes that confer active protection against environmental toxic compounds by efflux mechanisms have been described in Enterobacteriaceae (1,9,16,27,28). One of these drug ejection systems, the efflux detected in resistant gram-negative bacteria, depends on membrane energy and efficiently expels structurally unrelated antibiotic molecules across the bacterial envelope via a tripartite complex comprising an inner membrane pump, a periplasmic fusion protein, and an outer membrane channel (26,31).Enterobacter aerogenes and Klebsiella pneumoniae are frequently described in resistant nosocomial infections (2-4, 10, 12, 24). In these bacteria, the marRAB, acrAB-tolC, and ramA genes are involved in expression of the MDR phenotype (8,29,32). Moreover, various clinical isolates show alteration of nonspecific porins associated with the presence of active drug efflux; both processes maintain a very low intracellular concentration of drugs and contribute to a high resistance level for structurally unrelated molecules including ␤-lactam antibiotics, quinolones, tetracyclines, and chloramphenicol (5,6,21,24). An important medicinal challenge is to find new compounds capable of circumventing the efflux machinery (7,19,20,22,30). The aim of this study was to analyze 4-alkoxysubstituted quinolines, termed efflux pump inhibitors (EPI), with respect to their ability to interfere with the efflux pump.The strains used in this work were E. aerogenes EA3, EA27, and EA117 and K. pneumoniae KP55 clinical isolates exhibiting active efflux of norfloxacin or chloramphenicol (6,15,21) and TolC Ϫ and AcrA Ϫ E. aerogenes EA27 derivatives previously constructed (29). MICs, chloramphenicol uptake, potassium efflux, and ␤-lactamase activities were determined as previously described (13,21).Biological effect of alkoxyquinolines on a resistant E. aerogenes strain. Documented clinical isolate EA27, overexpressing the AcrAB complex owing to a frameshift mutation in acrR (21, 29), was used to determine the activity of nine alkoxyquinolines. The alkoxyquinoline compounds and phenylalanine-arginine-␤-naphthylamide (PA␤N), a previously characterized EPI (20, 22), showed poor intrinsic antimicrobial activities with high MICs (Table 1). These low intrinsic activities allowed us to analyze the restoring effect of the molecules on the antibiotic susceptibility of several MDR strains. The various compounds were assayed for the ability to induce a decrease...

Among the three series of quinazoline derivatives synthesised and studied in this work, some molecules increase the antibiotic susceptibility of Gram-negative bacteria presenting multidrug-resistant phenotypes. N-alkyl compounds induced an increase in the activity of chloramphenicol, nalidixic acid and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps in clinical isolates.These molecules are able to increase the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. Their activity depends on the antibiotic structure, suggesting that different sites may be involved for the recognition of substrates by a given efflux pump. Quinazoline molecules exhibiting a nitro functional group are more active, and structure-activity relationship studies may be undertaken to identify the pharmacophoric group involved in the AcrB and MexB affinity sites.

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