FranceEntembacter aemgenes is among the five most frequently isolated nosocomial pathogens in France, and this bacterium also shows increasing multidrug resistance. In this study, various E. aerogenes strains isolated from hospital units were characterized for their outer-membrane proteins, antibiotic susceptibilities (inhibition diameters and MICs) and resistance mechanisms associated with modification of envelope permeability (porin alteration and active efflux). Diminished outer-membrane permeability due to porin alterations was found in conjunction with the expression of an enzymic barrier in resistant isolates. Interestingly, changes in the functional expression of porins appeared to play a special role in susceptibility to cefepime. An active efflux to quinolones was also identified. Simultaneous changes in envelope permeability, i.e. a porin deficiency (in) and an efflux mechanism (out), were clearly evident in two clinical strains.

The Escherichia coli OmpF porin is a nonspecific channel involved in the membrane translocation of small hydrophilic molecules and especially in the passage of ␤-lactam antibiotics. In order to understand the dynamic of charged-compound uptake through bacterial porins, specific charges located in the E. coli OmpF channel were mutated. Substitutions G119D and G119E, inserting a protruding acidic side chain into the pore, decreased cephalosporin and colicin susceptibilities. Cefepime diffusion was drastically altered by these mutations. Conversely, substitutions R132A and R132D, changing a residue located in the positively charged cluster, increased the rate of cephalosporin uptake without modifying colicin sensitivity. Modelling approaches suggest that G119E generates a transverse hydrogen bond dividing the pore, while the two R132 substitutions stretch the channel size. These charge alterations located in the constriction area have differential effects on cephalosporin diffusion and substantially modify the profile of antibiotic susceptibility.The outer membrane of gram-negative bacteria shelters them from external toxic compounds. In the membrane, porins are channel-forming proteins allowing diffusion of small hydrophilic solutes through this barrier (10,18,20). With bacterial resistance to various antibiotics due to the permeability barrier impairing chemotherapy (19), it is important to define the biochemical and biophysical parameters governing target access and intracellular drug concentration. In particular, since outer membrane porins are key to ␤-lactam penetration (19,22), it is essential to understand the various possible interactions. The native Escherichia coli OmpF porin is a trimer, and the three-dimensional structure shows a monomeric ␤-barrel built of 16 antiparallel ␤-strands containing the pore (6). The longest loop, L3, is bent into the channel, forming a gate; in this constriction area, a positively charged cluster of amino acid residues protruding from the barrel wall faces the L3 negatively charged side chain residues. This generates a strong electrostatic field parallel to the membrane surface, and such an organization could facilitate the diffusion of molecules and modulate voltage gating (6,12,36). Several mutations have been selected on residues located in the channel; among them, G119D is a substitution located in L3 obtained from colicin N resistance screening after random mutagenesis (8). Structural and functional analyses of the G119D porin indicate that the mutation affects channel properties without causing large molecular alterations (11). To address the question of the effects of steric hindrance and charge movement in the flux through the pore lumen, mutant porins with site-specific mutations in positions 119 and 132 have been constructed: 119D and 119E, which are located in the negatively charged cluster, and 132A and 132D, which belong to the facing positive region. Using immunological probes directed against wild-type porin, we established the correct membrane insertion of t...

SummaryIn Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild-type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF-like protein and analysed its pore-forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild-type protein and we observed (i) that its single-channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling b-lactam diffusion via porins.

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.

We evaluated the prevalence of impermeability as a mechanism associated with resistance against beta-lactam antibiotics in members of the family Enterobacteriaceae. During a 1-year period, 80 strains were selected from 3,110 routinely isolated strains according to their noticeable cross-resistance pattern to cephalosporins. They were tested for (i) outer membrane nonspecific porins involved in the entry of small hydrophilic molecules; (ii) the MICs of cefepime, cefotaxime, imipenem, and moxalactam; and (iii) beta-lactamase production. Immunological investigations using specific probes showed that 23 of 80 strains presented an alteration of the porin content, most of them expressing an additional resistance mechanism. The prevalence of this porin-deficient phenotype is especially high in Enterobacter aerogenes and concerns 6.4% of the clinical isolates.