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.

In Enterobacteriaceae, membrane permeability is a key in the level of susceptibility to antibiotics. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems has been reported. These phenomena are frequently associated with other resistance mechanisms such as alteration of antibiotics or modification of the drug targets, in various clinical isolates showing a Multi Drug Resistant phenotype (MDR). In Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of MDR isolates. These bacterial isolates exhibit a noticeable reduction of functional porins per cell due to a decrease, a complete shutdown of synthesis, or the expression of an altered porin and a high expression of efflux systems (e.g. overexpression of the pump). The combined action of these mechanisms during an infection confers a significant decrease in bacterial sensitivity to antibiotherapy ensuring dissemination and colonization of the patient and favours the acquisition of additional mechanisms of resistance. MarA and ramA are involved in a complex regulation cascade controlling membrane permeability and actively participate in the triggering of the MDR phenotype. Mutations in regulator genes have been shown to induce the overproduction of efflux and the down-regulation of porin synthesis. In addition, various compounds such as salicylate, imipenem or chloramphenicol are able to activate the MDR response. This phenomenon has been observed both in vitro during culture of bacteria in the presence of drugs and in vivo during antibiotic treatment of infected patients. These effectors activate the expression of specific global regulators, marA, ramA, or target other genes located downstream in the regulation cascade.

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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Two clinical strains of Enterobacter aerogenes that exhibited phenotypes of multiresistance to ␤-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two known outer membrane proteins, OmpX and LamB. In addition, the full-length Opolysaccharide phenotype was replaced by a semirough Ra phenotype. Moreover, in one isolate the intracellular accumulation of chloramphenicol was increased in the presence of the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone, suggesting an energy-dependent efflux of chloramphenicol in this strain. The resistance strategies used by these isolates appear to be similar to that induced by stress in Escherichia coli cells.Bacteria have developed various regulatory systems which coordinate their adaptive responses with the different environmental stresses to which they are exposed. Recently, the wholegenome transcriptional profiles, or "transcriptomes," were determined in vitro for an Escherichia coli strain exposed to an inducer of the soxRS or marRAB system and an E. coli strain constitutively expressing MarA (7,39). Under these conditions, the expression of several genes appeared to be significantly activated or down-regulated. These modulations of gene expression alter the sensitivities of the bacteria to a broad range of antibiotics (1, 2).Enterobacter aerogenes is one of the more frequently described gram-negative bacteria responsible for nosocomial respiratory tract infections (5, 17). In the last 5 years, it has been shown that clinical isolates of this species, which are naturally resistant to aminopenicillins through their production of a chromosomal cephalosporinase, often express an extendedspectrum ␤-lactamase, TEM-24, which gives rise to resistance to ␤-lactam antibiotics (5,17,19,31). Moreover, E. aerogenes exhibits acquired resistance to other families of antimicrobial agents. Previous studies have reported that clinical strains exhibiting an efflux process are resistant to ␤-lactam antibiotics, quinolones, tetracycline, and chloramphenicol (12, 29). Drug efflux can be coincident with a drastic reduction in drug uptake due to the loss of porin content (16,22,23,29).Two E. aerogenes clinical isolates, isolates 117 and 119, were selected from up to 100 strains isolated in our laboratory (12). These two strains exhibited a phenotype of multiresistance to ␤-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin comparable to that of E. aerogenes strains lacking nonspecific porins or expressing mutated porins (12, 29).The aim of this work was to examine some factors that may contribute to the resistance to antimicrobial agents in these two E. aerogenes clinical strains. MATERIALS AND METHODSBacterial strains, growth conditions, and antibiotic suscepti...

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The prevalence of active drug efflux pump and porin alterations was investigated in Turkish nosocomial strains of Klebsiella pneumoniae exhibiting a multidrug-resistant phenotype. MICs of various antibiotics, including quinolones, chloramphenicol, tetracycline, and ␤-lactams, for those strains were determined either with or without the efflux pump inhibitor phenylalanine arginine ␤-naphthylamide (PA␤N). Thirty-nine percent of the strains exhibited a PA␤N-modulated resistance for quinolones, chloramphenicol, and tetracycline. In these strains, a significant increase of chloramphenicol accumulation was gained in the presence of the efflux pump inhibitor PA␤N or with the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone. Moreover, high-level expression of the membrane fusion protein AcrA, which was immunodetected in most of those isolates, suggests that the AcrAB/TolC efflux machinery contributed to their antibiotic resistance. Studies of K. pneumoniae porins indicated that the majority of the strains, including extended-spectrum ␤-lactamase producers and efflux-positive ones, presented an alteration in their sorbitol-sensitive porin (OmpK35) expression. This is the first report showing the prominent role of active drug efflux in the antibiotic resistance of nosocomial K. pneumoniae strains from Turkey.