Jacqueline Chevalier

Porin alteration and active efflux : two in vivo drug resistance strategies used by Enterobacter aerogenes

Enterobacter aerogenes 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.

Membrane Permeability and Regulation of Drug “Influx and Efflux” in Enterobacterial Pathogens

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.

Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria

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.

Quinoline Derivatives as Promising Inhibitors of Antibiotic Efflux Pump in Multidrug Resistant Enterobacter Aerogenes Isolates

Efflux pumps protect the bacterial cell by expelling toxic compounds before they reach intracellular targets. Because this mechanism actively contributes to the resistance of a given bacterium to more than one class of antibiotics, molecules that are able to block the relevant efflux pump are of potential significance to combat drug resistance caused by efflux pumps. Different quinoline derivatives including alkoxy, alkylamino, thioalkoxy and chloroquinolines have been previously reported to make Enterobacter aerogenes resistant isolates that over express the mechanism of efflux, noticeably more susceptible to structurally unrelated antibiotics. In addition, various quinoline derivatives significantly increase the intracellular concentration of chloramphenicol as reported with other inhibitors, thereby suggesting the inhibition of the drug transport by AcrAB-TolC pump, which is fully active in the clinicaly resistant isolates investigated. Here, we discuss the respective properties of this molecular family, taking into account the recent insights into the structural data of AcrB pump.

Oxacillinase-Mediated Resistance to Cefepime and Susceptibility to Ceftazidime in Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa clinical isolate SOF-1 was resistant to cefepime and susceptible to ceftazidime. This resistance phenotype was explained by the expression of OXA-31, which shared 98% amino acid identity with a class D β-lactamase, OXA-1. Theoxa-31 gene was located on a ca. 300-kb nonconjugative plasmid and on a class 1 integron. No additional efflux mechanism for cefepime was detected in P. aeruginosa SOF-1. Resistance to cefepime and susceptibility to ceftazidime in P. aeruginosawere conferred by OXA-1 as well.

Detection and Prevalence of Active Drug Efflux Mechanism in Various Multidrug-Resistant Klebsiella pneumoniae Strains from Turkey

ABSTRACT 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 β-lactams, for those strains were determined either with or without the efflux pump inhibitor phenylalanine arginine β-naphthylamide (PAβN). Thirty-nine percent of the strains exhibited a PAβN-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 PAβN 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 β-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.

The AcrAB-TolC Pump Is Involved in Macrolide Resistance but Not in Telithromycin Efflux in Enterobacter aerogenes and Escherichia coli

ABSTRACT The role of the AcrAB-TolC pump in macrolide and ketolide susceptibility in Escherichia coli and Enterobacter aerogenes was studied. Efflux pump inhibitor restored erythromycin, clarithromycin, and telithromycin susceptibilities to multidrug-resistant isolates. No modification of telithromycin accumulation was detected in E. aerogenes acrAB or tolC derivatives compared to that in the parental strain. Two independent efflux pumps, inhibited by phenylalanine arginine β-naphthylamide, expel macrolides and telithromycin in E. aerogenes.

Antibacterial activity of Thymus maroccanus and Thymus broussonetii essential oils against nosocomial infection - bacteria and their synergistic potential with antibiotics.

The aim of this study was to evaluate the antibacterial effect of the association between conventional antibiotics and essential oils (EOs) of endemic Moroccan thyme species, Thymus maroccanus and T. broussonetii, on antibiotic-resistant bacteria involved in nosocomial infections. Synergistic interactions between antibiotics (ciprofloxacin, gentamicin, pristinamycin, and cefixime) and EOs, and between T. maroccanus and T. Broussonetii EOs were determined by the checkerboard test. Serial dilutions of two antimicrobial agents were mixed together so that each row (and column) contained a fixed amount of the first agent and increasing amounts of the second one. The results indicate that the oils had a high inhibitory activity against tested bacteria, except for Pseudomonas aeruginosa. In parallel with the increase of cellular killing, the release of 260nm-absorbing materials from bacterial cells, treated with EOs, increased in response to oil concentration. Out of 80 combinations tested between EOs and antibiotics, 71% showed total synergism, 20% had partial synergistic interaction and 9% showed no effect. Combination with carvacrol, the major constituent of T. maroccanus and T. broussonetii, showed also an interesting synergistic effect in combination with ciprofloxacin. The effect on Gram-positive bacteria was more important than on Gram-negative bacteria. These findings are very promising since the use of these combinations for nosocomial infections treatment is likely to reduce the minimum effective dose of the antibiotics, thus minimizing their possible toxic side effects and treatment cost. However, further investigations are needed to assess the potential for therapeutic application.

RamA Is an Alternate Activator of the Multidrug Resistance Cascade in Enterobacter aerogenes

ABSTRACT Multidrug resistance (MDR) in Enterobacter aerogenes can be mediated by induction of MarA, which is triggered by certain antibiotics and phenolic compounds. In this study, we identified the gene encoding RamA, a 113-amino-acid regulatory protein belonging to the AraC-XylS transcriptional activator family, in the Enterobacter aerogenes ATCC 13048 type strain and in a clinical multiresistant isolate. Overexpression of RamA induced an MDR phenotype in drug-susceptible Escherichia coli JM109 and E. aerogenes ATCC 13048, as demonstrated by 2- to 16-fold-increased resistance to β-lactams, tetracycline, chloramphenicol, and quinolones, a decrease in porin production, and increased production of AcrA, a component of the AcrAB-TolC drug efflux pump. We show that RamA enhances the transcription of the marRAB operon but is also able to induce an MDR phenotype in a mar-deleted strain. We demonstrate here that RamA is a transcriptional activator of the Mar regulon and is also a self-governing activator of the MDR cascade.

Inhibitors of Antibiotic Efflux in Resistant Enterobacter aerogenes and Klebsiella pneumoniae Strains

ABSTRACT In Enterobacter aerogenes and Klebsiella pneumoniae, efflux provides efficient extrusion of antibiotics and contributes to the multidrug resistance phenotype. One of the alkoxyquinoline derivatives studied here, 2,8-dimethyl-4-(2′-pyrrolidinoethyl)-oxyquinoline, restores noticeable drug susceptibility to resistant clinical strains. Analyses of energy-dependent chloramphenicol efflux indicate that this compound inhibits the efflux pump mechanism and improves the activity of structurally unrelated antibiotics on multidrug-resistant E. aerogenes and K. pneumoniae isolates.