The Escherichia coli OmpF pore is governed by an internal constriction consisting of the negatively charged loop 3 folded into the lumen and the positively charged barrel wall located on the opposite side across the pore, 'anti-loop 3'. To investigate the role of anti-loop 3 in solute diffusion, four site-directed mutations, K16A, K16D, R132A and R132D, were introduced into this eyelet region. The mutant porins were expressed efficiently and inserted into the outer membrane, and the thermal stabilities of the resulting trimers were determined. Diffusion of cefepime, a recently developed cephalosporin, was analysed in vivo. In vitro studies were performed on purified porins reconstituted in planar lipid bilayers to measure conductance, selectivity and voltage closure, as well as in liposomes for patch-clamp and sugar-swelling assays. All substitutions modified the ion-channel parameters, and minor conformational changes in the OmpF eyelet region were predicted from modelling studies. Our data show that Lys-16, and to a lesser extent Arg-132, are involved in voltage-gating and pore selectivity via their side-chain charges. Substitution K16D, which causes a severe decrease in critical voltage (V(c)), may generate a channel susceptible to membrane potential, which perturbs cefepime diffusion. These results suggest that the Lys-16 residue plays an important role in the process of diffusion through the OmpF lumen.

Beta-lactams use aqueous channels of porins to penetrate Gram-negative bacteria. The L3 loop of Escherichia coli OmpF porin is a key feature that actively contributes to both channel size and electrostatic properties. Acid residues D113, E117, and D121 are responsible for the negative part of the local electrostatic field on this loop. Two substitutions, D113A and D121A, located in the negatively charged cluster of the OmpF eyelet, increase the likelihood of producing bacteria susceptible to several beta-lactams. D113A substitution results in an increase in the ampicillin, cefoxitin, and ceftazidime susceptibility. Molecular modeling suggests that the charges harbored by the beta-lactam molecules interact with the charged residues located inside the porin eyelet.

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...

This is the first report showing the role of active efflux in the parabens resistance in E. gergoviae, a mechanism that may explain its frequent isolation in parabens-containing cosmetics compared with other enterobacterial species. Paraben efflux seems to be regulated by a mar-independent process in E. gergoviae.