A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin

Abstract

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 porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…The Omp36 G112D mutation severely impairs β-lactam diffusion through the channel, and the kinetics of cefepime uptake are greatly reduced in E. aerogenes isolates that carry this mutation 52,53 . Electrophysiological changes in the G112D porin channel, compared with wild-type E. aerogenes Omp36, are similar to those observed with the E. coli OmpF G119D mutation 49,50 .…”

“…Molecular and functional analyses characterized this G112D substitution, which is located inside internal loop 3 (rEF. 49). This substitution is similar to the G119D mutation, previously obtained by mutagenesis of E. coli ompF, which creates a major protrusion into the channel lumen, causing strong steric hindrance in the channel and a drastic reduction in β-lactam susceptibility 50,51 .…”

Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

“…Outer membranes were extracted using the spheroplast procedure (9), and the final membrane fractions were obtained by centrifugation (180,000 ϫ g) at 4°C for 1 h. The pellet was resuspended in a solution containing 20 mM NaPi (pH 7.6), 10 mM NaCl, and 1% octylpolyoxyethylene (octyl-POE; Bachem, Bubendorf, Switzerland), and the suspension was homogenized and stirred for 30 min at 4°C. The suspension was centrifuged (180,000 ϫ g) for 1 h at 4°C, and the pellet was resuspended in 20 mM NaPi (pH 7.6), 10 mM NaCl and 3% octyl-POE.…”

“…This bacterial pathogen harbors a variety of antibiotic resistance mechanisms (7,9,19,24,30). Among them, the modification of outer membrane permeability, a porin deficiency associated with the expression of cephalosporinase activity, is frequently detected in clinical resistant isolates (7,19).…”

Porines bactériennes et sensibilité aux antibiotiques

“…Chez Pseudomonas aeruginosa, la disparition d'une porine a été rapportée dans le cas de résistance à l'imipénème 3 [25]. (Figures 2 et 3), induisant le rétrécissement du canal et l'altération du champ électrostatique [29]. Des observations similaires ont été faites chez Neisseria gonorrhoeae [30] avec des mutations localisées dans l'équivalent de la boucle 3 des porines d'E.…”