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

Abstract

In Enterobacter aerogenes, ␤-lactam resistance often involves a decrease in outer membrane permeability induced by modifications of porin synthesis. In ATCC 15038 strain, we observed a different pattern of porin production associated with a variable antibiotic susceptibility. We purified Omp35, which is expressed under conditions of low osmolality and analyzed its pore-forming properties in artificial membranes. This porin was found to be an OmpF-like protein with high conductance values. It showed a noticeably higher conductance compared to Omp36 and a specific location of WNYT residues in the L3 loop. The importance of the constriction region in the porin function suggests that this organization is involved in the level of susceptibility to negative large cephalosporins such as ceftriaxone by bacteria producing the Omp35 porin subfamily.The bacterial porins are major outer membrane proteins that form water-filled channels, allowing the diffusion across the outer membrane of small polar molecules, amino acids, and nutrients (14,18,23). General nonspecific porins, such as OmpF and OmpC from Escherichia coli, form homotrimers in the outer membrane. These porin trimers are constituted by large ␤-barrel monomer subunits which are constricted about halfway through the membrane by an internal loop, loop 3 (L3) (18). Resolution of the three-dimensional structure of the E. coli OmpF and Klebsiella pneumoniae OmpK36 has led to identification of the functional domains of the enterobacterial channels (5,8,13,17).Enterobacter aerogenes is one of the most frequently described gram-negative pathogens responsible for nosocomial respiratory tract infections in France and Belgium (2, 6, 10). 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). We recently isolated a strain with an unusual porin phenotype: this EA3 strain synthesized a mutated Omp36 porin containing a G112D substitution in the L3 domain (19). Despite this prominent role of porins in drug susceptibility, only one porin, Omp36, is today functionally characterized in E. aerogenes (19).The aim of this work was to investigate the structural and functional properties of the second major pore-forming protein produced by the E. aerogenes strain. The sequence and the channel properties of the new porin Omp35 were determined and agree with the biological differences concerning the ␤-lactam susceptibility, observed between the bacteria producing either Omp35 or Omp36. MATERIALS AND METHODSBacterial strains, growth conditions, and antibiotic susceptibility tests. The E. aerogenes ATCC 15038 strain was used as the standard strain. E. aerogenes EAEP289 is a Kan s derivative of the previously described clinical strain EA27 (24). Bacteria were routinely grown in Luria-Bertani (LB) medium or nutrient broth (NB). For the determination of MI...

Role of the culture medium in porin expression and piperacillin-tazobactam susceptibility in Escherichia coli

“…When the growth was carried out with the high-osmotic medium (NBS), the lower band disappeared. By analogy to previous studies, we inferred that the upper band was OmpC while the lower band corresponded to OmpF (Bornet et al, 2004). In strains 1103140 and 1103148, a doublet exhibiting a very similar-sized product to OmpC migration was detected in all of the different media and no band exhibiting OmpF migration characteristics was observed.…”

“…As reported previously (Bornet et al, 2004), changes in medium osmolarity had no significant effect on imipenem MIC except for strain 1103146, while the susceptibility to cefepime and ceftriaxone was altered by osmolarity increase except for strain 1103140. Concerning piperacillin, sorbitol addition increased MIC from one to two dilutions.…”

“…As described previously (Liu & Ferenci, 2001;Bornet et al, 2004;Dupont et al, 2007), medium osmolarity influences the OmpF:OmpC balance due to bacterial osmotic two-component system (TCS) regulators (Masi & Pagès, 2013). Under low osmolarity, OmpF is preferred to OmpC and under high osmolarity the opposite occurs.…”

“…S/R for susceptible/ resistant to describe their resulting antibiotic susceptibility phenotype; Table 1). In parallel, nutrient broth (NB) supplemented or not with 20% sorbitol (NBS) was used as a control (Bornet et al, 2004;Dupont et al, 2007). The osmolarity of the different media was determined using an osmometer apparatus according to the manufacturer's instructions (Gonotec): LB5424(+12) mOsmol kg 21 , MH5312(+4) mOsmol kg 21 , NB546(+3) mOsmol kg 21 , NBS51290(+23) mOsmol kg 21 and GN15476(+9) mOsmol kg 21 .…”

Caracterização molecular dos mecanismos de resistência aos carbapenêmicos de isolados clínicos de Enterobacter aerogenes e Enterobacter cloacae

“…Os autores descreveram duas proteínas de membrana externa distintas OMP-C (42 kDa) e OMP-F (39 kDa) como responsáveis pela resistência desse microrganismo ao Imipenem ao Meropenem e ao Cefepima (27) . (147,148) . Em 2005, Thiolas observa a mesma diminuição da porina OMP 36 em isolados resistentes a Imipenem e colistina como também o aumento do componente da bomba de efluxo AcrA em isolados resistentes ao Imipenem (29) .…”