The AcrAB-TolC Efflux Pump Contributes to Multidrug Resistance in the Nosocomial Pathogen
            <i>Enterobacter aerogenes</i>

Pradel, Elizabeth; Pagès, Jean‐Marie

doi:10.1128/aac.46.8.2640-2643.2002

Cited by 139 publications

(135 citation statements)

References 21 publications

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“…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).…”

Section: Methodsmentioning

confidence: 99%

“…Omp35 purification and functional analyses. To express Omp35, we transformed EAEP289 strain, a porin deficient strain with pCB35 and the resulting cells were assayed for drug susceptibilities (19,24). The synthesis of Omp35, verified by immunoanalysis (data not shown), restored a noticeable ␤-lactam susceptibility in bacterial producing cells (Table 2).…”

Section: ␤-Lactammentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Bornet

Saint

Fetnaci

et al. 2004

Antimicrob Agents Chemother

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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...

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Section: Methodsmentioning

confidence: 99%

Section: ␤-Lactammentioning

confidence: 99%

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

Bornet

Saint

Fetnaci

et al. 2004

Antimicrob Agents Chemother

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“…Immunoblots were stained by colorimetric detection with alkaline phosphatase-conjugated secondary antibodies. Three reference strains, EAEP289 (a Kan s derivative of EA27, which is a clinical MDR strain), EAEP294 (EAEP289 acrA::Kan r ), and EAEP298 (EAEP289 tolC::Kan r ), were used as internal controls (18). Concerning K. pneumoniae, similar AcrA and TolC profiles were obtained with the ATCC reference strain and isolates.…”

mentioning

confidence: 77%

“…It was reported previously that the overproduction of AcrA and TolC pump elements is involved in the resistance levels of clinical isolates (4,8,9). The membrane proteins of selected isolates were extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection with polyclonal antibodies directed against AcrA and TolC components (9,18). Immunoblots were stained by colorimetric detection with alkaline phosphatase-conjugated secondary antibodies.…”

mentioning

confidence: 99%

Occurrence of Efflux Mechanism and Cephalosporinase Variant in a Population of Enterobacter aerogenes and Klebsiella pneumoniae Isolates Producing Extended-Spectrum β-Lactamases

Tran

Dupont

Lavigne

et al. 2009

Antimicrob Agents Chemother

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We investigated the occurrence of multidrug resistance in 44 Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Efflux was involved in resistance in E. aerogenes isolates more frequently than in K. pneumoniae isolates (100 versus 38% of isolates) and was associated with the expression of phenylalanine arginine ␤-naphthylamide-susceptible active efflux. AcrA-TolC overproduction in E. aerogenes isolates was noted. An analysis of four E. aerogenes isolates for which cefepime MICs were high revealed no modification in porin expression but a new specific mutation in the AmpC ␤-lactamase.Enterobacter aerogenes and Klebsiella pneumoniae, two of the most prevalent nosocomial enterobacterial species, can frequently express a multidrug resistance (MDR) phenotype by the acquisition or high-level production of ␤-lactamases in combination with the overproduction of efflux pumps, associated or not with porin alterations in the outer membrane (1,3,7,9,13). Such modifications of envelope permeability, including efflux and influx (porin), in 28 Enterobacter aerogenes and 16 Klebsiella pneumoniae clinical isolates from Nîmes University Hospital, previously studied for extended-spectrum ␤-lactamase (ESBL) profiling and clonal identification, were investigated (11). Moreover, we ascertained the mechanism involved in cefepime resistance in four E. aerogenes isolates showing high-level resistance (MIC ϭ 128 mg/liter).Characterization of the MDR phenotype was carried out by determining the MICs of various structurally unrelated antibiotics selected for their capacities to be expelled and/or to enter cells via porins (Table 1). We used the twofold broth dilution method with Mueller-Hinton broth according to the CLSI guidelines: the tests were carried out with or without the broad-spectrum efflux pump inhibitor (EPI) phenylalanine arginine ␤-naphthylamide (PA␤N). This diamine compound, used at a low concentration (26.3 mg/liter), can increase the activities of chloramphenicol, tetracycline, norfloxacin, and sparfloxacin against various enterobacterial isolates which overproduce efflux pumps such AcrAB-TolC (4, 9, 16). The test results confirmed the existence of a PA␤N-susceptible efflux mechanism in all E. aerogenes and many K. pneumoniae strains, with marked decreases in the MICs of fluoroquinolones and chloramphenicol in the presence of PA␤N (Table 1). Efflux pump activity was identified when the PA␤N addition induced a threefold decrease in the MIC of an antibiotic molecule (4, 9). For the two fluoroquinolones, the PA␤N addition had better efficiency in decreasing the MICs of sparfloxacin than those of norfloxacin. The observed difference in PA␤N efflux inhibition between the two fluoroquinolones may reflect the respective locations of ligands inside the AcrB cavity and the flux competition (20). Yu et al. demonstrated, during previous cocrystalization analyses, the presence of different affinity sites located inside the AcrB pump and suggested that the benefit generated by the conjoint use of an EPI and an antibioti...

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Bacterial Efflux Pumps and Their Inhibitors

Zgurskaya

Rybenkov

Walker

et al. 2021

Burger's Medicinal Chemistry and Drug Discovery

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Bacterial multidrug efflux transporters have distinct molecular features and mechanisms that enable their impressive substrate polyspecificity. These transporters are involved in all physiological processes where nonspecific but active transport across membranes is critical for survival and proliferation. Importantly, efflux pumps contribute to bacterial virulence and pathogenesis, as well as the spread of antibiotic resistance in clinics. Transporters belonging to several protein superfamilies have been selected for polyspecificity by evolution and augmented with accessory proteins that extend and couple transport reactions across the inner and outer membranes of the bacterium. As a result, bacteria are equipped with diverse efflux machines that protect cells from a broad variety of toxic compounds including antibiotics and other small molecule therapeutics. The inhibition of such polyspecific biomolecular machines is a challenging task, which still awaits an efficient solution. Here, we review the molecular mechanisms of efflux pumps from Gram‐negative bacteria and recent advances and challenges associated with the discovery of effective inhibitors of these pumps.

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The AcrAB-TolC Efflux Pump Contributes to Multidrug Resistance in the Nosocomial Pathogen Enterobacter aerogenes

Cited by 139 publications

References 21 publications

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

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

Occurrence of Efflux Mechanism and Cephalosporinase Variant in a Population of Enterobacter aerogenes and Klebsiella pneumoniae Isolates Producing Extended-Spectrum β-Lactamases

Bacterial Efflux Pumps and Their Inhibitors

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