Jean-Michel Bolla

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.

Geraniol Restores Antibiotic Activities against Multidrug-Resistant Isolates from Gram-Negative Species

ABSTRACT The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine β-naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of β-lactams, quinolones, and chloramphenicol.

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.

Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype

The present study assessed the antimicrobial activities of various natural products belonging to the terpenoids, alkaloids and phenolics against a collection of Gram-negative multidrug-resistant (MDR) bacteria. The results demonstrated that most of the compounds were extruded by bacterial efflux pumps. In the presence of the efflux pump inhibitor phenylalanine arginine β-naphthylamide (PAβN), the activities of laurentixanthone B (xanthone), plumbagin (naphthoquinone), 4-hydroxylonchocarpin (flavonoid) and MAB3 (coumarin) increased significantly against all studied MDR bacteria. Laurentixanthone B, 4-hydroxylonchocarpin and MAB3 contained the same pharmacophoric moiety as plumbagin. This study indicates that the AcrAB-TolC (Enterobacteriaceae) and MexAB-OprM (Pseudomonas aeruginosa) efflux pumps are involved in resistance of Gram-negative bacteria to most of the natural products.

How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins

Background Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. β-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression. Methodology/Principal Findings Here influx of β-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single β-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of β-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility. Conclusions/Significance We propose the idea of a molecular “passport” that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections.

A phenylalanine-arginine β-naphthylamide sensitive multidrug efflux pump involved in intrinsic and acquired resistance of Campylobacter to macrolides

The macrolide erythromycin is the antibiotic of choice in the management of Campylobacter infections. Although mutation has been reported to account for resistance to the antibiotic, resistance may also be due to an efflux pump that extrudes the drug prior to reaching its target. Moreover, the efflux pump may be one that accommodates resistance to other related or unrelated drugs (multidrug resistance). We examined the possibility that resistance to erythromycin may involve an efflux pump whose presence may be identified by the use of the unique commercial inhibitor of the previously described efflux pumps phenylalanine-arginine beta-naphtylamide (PAbetaN). We showed that PAbetaN is able to significantly increase the susceptibility of the reference strain NCTC 11168 to erythromycin, suggesting that an efflux pump functions at a basal level in the reference wild type strain. Erythromycin-resistant isolates were tested for their response to PAbetaN treatment. Among the strains tested, resistance of three isolates to erythromycin was reduced to a level comparable to that of the susceptible strain when the strains were grown in the presence of this inhibitor. To conclude, besides mutations, erythromycin resistance in Campylobacter may also be due to an efflux mechanism sensitive to PAbetaN.

Efflux pumps are involved in the defense of Gram-negative bacteria against the natural products isobavachalcone and diospyrone.

ABSTRACT The activities of two naturally occurring compounds, isobavachalcone and diospyrone, against documented strains and multidrug-resistant (MDR) Gram-negative bacterial isolates were evaluated. The results indicated that the two compounds exhibited intrinsic antibacterial activity against several Gram-negative bacteria, and their activities were significantly improved in the presence of an efflux pump inhibitor (MIC values decreased to below 10 μg/ml). In addition, the activities of isobavachalcone and diospyrone against various strains exhibiting deletions of the major efflux pump components (AcrAB, TolC) were significantly increased. The overall results indicate that isobavachalcone and diospyrone could be candidates for the development of new drugs against MDR strains and that their use in combination with efflux pump inhibitors reinforces their activity.

Antibacterial action of essential oils from corsica

Abstract The antimicrobial activity of 28 essential oil samples isolated from local plants or plants cultivated in Corsica was evaluated against a large panel of human pathogenic bacteria, including Campylobacter jejuni which appeared as a good model for this purpose. The chemical composition of the 18 oils selected for their efficiency was determined by GC and GC/MS. Among them, the oils of Cistus ladaniferus, Crithmum maritimum, Daucus carota, Juniperus communis, Mentha aquatica and Santolina corsica showed compositions without components known as active, suggesting the presence of compounds not previously described as antibacterial agents.

Efflux pumps of gram-negative bacteria, a new target for new molecules.

Antibiotic resistance mechanisms reported in Gram-negative bacteria are a worldwide health problem. The continuous dissemination of multi-drug resistant (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria the over-expression of efflux pumps expel structurally-unrelated antibiotics decreasing their intracellular concentration. It is necessary to clearly define the molecular and genetic bases of the efflux pump in order to combat this mechanism. The characterization of efflux pumps, from genetic to structural studies, allows the definition of a new, original antiresistance bullet, the efflux pump inhibitor (EPI). This new class of antibacterial molecules may act conjointly to the usual antibiotic in order to restore its activity. Several families of EPIs have been now reported and described. The use of these EPIs promotes a significant increase of susceptibility to one or more antibiotics in strains or clinical isolates which were initially resistant. These EPIs may target different efflux targets, (i) the expression of genes that induces MDR, the transporters that pump the antibiotic out of bacterium, (ii) the assembly of membrane transporter complex involved in drug efflux, (iii) the energy involved in this active transport, (iv) the inhibition of the flux of molecules inside the efflux channel by competition or blocking (via steric hindrances). With the recent thorough characterization of the efflux pump AcrB at its structural and physiological level including the identification of drug affinity sites and kinetic parameters for some antibiotics, it is now possible to rationally develop an improved new generation of EPIs.

Environmental Regulation of Campylobacter jejuni Major Outer Membrane Protein Porin Expression in Escherichia coli Monitored by Using Green Fluorescent Protein

ABSTRACT Porins allow exchanges between bacteria and their environment. In the gram-negative food-borne pathogen Campylobacter jejuni two porins, major outer membrane protein (MOMP) and Omp50, have been identified. MOMP is synthesized at a very high level under laboratory culture conditions, suggesting that its promoter functions very efficiently under these conditions. In Campylobacter samples, we observed that MOMP porin expression increased at a high temperature (42°C) or a high pH (pH 8.5) compared to expression at a low temperature (31°C) or an acidic pH (pH 5.5). To study the regulation of MOMP expression at the transcriptional level, we constructed an momp-gfp fusion in which gfp expression was put under the control of the momp promoter. Interestingly, we observed the same pattern of regulation in Escherichia coli, as monitored by green fluorescent protein production, that was found in Campylobacter. The ranges of pH and temperature tested are physiologically relevant, because they can be found in the digestive tracts of both birds and humans, which are both colonized by Campylobacter. Our results suggest that a component of the regulatory mechanism is conserved in C. jejuni and E. coli. However, medium osmolarity and sodium salicylate did not have a significant effect on C. jejuni momp promoter activity in E. coli, suggesting that major regulatory elements of E. coli porin expression do not participate in MOMP regulation. In contrast, mechanisms involving DNA supercoiling may be involved, as shown by DNA gyrase inhibition assays. These findings are a step towards determining the role of outer membrane proteins in the adaptation of C. jejuni to its environment.