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.

BackgroundMulti-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 FindingsHere 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/SignificanceWe 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.

Gram-negative bacteria are protected by an outer membrane barrier, and to reach their periplasmic target, penicillins have to diffuse through outer membrane porins such as OmpF. Here we propose a structure-dynamics-based strategy for improving such antibiotic uptake. Using a variety of experiments (high-resolution single channel recording, Minimum Inhibitory Concentration (MIC), liposome swelling assay) and accelerated molecular simulations, we decipher the subtle balance of interactions governing ampicillin diffusion through the porin OmpF. This suggests mutagenesis of a hot spot residue of OmpF for which additional simulations reveal drastic changes in the molecular and energetic pathway of ampicillin's diffusion. Inverting the problem, we predict and describe how benzylpenicillin diffuses with a lower effective energy barrier by interacting differently with OmpF. The thorough comparison between the theoretical predictions and the three independent experiments, which were set up to measure the kinetics of transport and biological activity, gives insights on how to combine such different investigation techniques with the aim of providing complementary validation. Our study illustrates the importance of microscopic interactions at the constriction region of the biological channel to control the antibiotic flux through it. We conclude by providing a complete inventory of the channel and antibiotic hot spots and discuss the implications in terms of antibacterial screening and design.

Mutations or deletions in ramR, leading to the overexpression of RamA predominated in clinical MDR E. aerogenes isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in ramR, and the self-regulating region proximal to ramA, probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing E. aerogenes towards the emergence of a MDR phenotype.

Intravenous therapy with purified plasma-derived alpha-proteinase inhibitor (α-PI) concentrates is the only specific treatment for α-PI deficiency. For the therapy to be safe and efficacious, α-PI concentrates should be highly pure and contain high amounts of functional protein. This study compared the four plasma-derived α-PI products commercially available in Europe (Respreeza, Prolastin, Alfalastin, Trypsone) by biochemical methods with respect to function, purity, structure, and chemical modifications. Respreeza had the highest level of functional protein (48.8 mg/mL) and the highest specific activity (0.862 mg active α-PI per mg total protein). By size exclusion chromatography, Respreeza was 97.4% pure, followed by Alfalastin 88.1%, Prolastin 76.9%, and Trypsone 70.8%. By reversed phase chromatography, Respreeza had an α-PI purity of 97.7%, followed by Trypsone 88.0%, Prolastin 78.0%, and Alfalastin 69.5%. The main protein band by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was found for all products at approximately 50 kDa. Additional protein bands were found for Prolastin, Alfalastin, and Trypsone. The α-PI products differed in cysteine oxidation state and C-terminal lysine status. α-PI products tested differ in purity, concentration, and chemical variation. Respreeza has the highest level of purity. The impact of the non-therapeutic proteins identified has not been evaluated.

In the first part of this thesis, we introduce the concept of prospective strict no-arbitrage for discrete-time financial market models with proportional transaction. The prospective strict no-arbitrage condition, which is a variant of strict no-arbitrage, is slightly weaker than the robust no-arbitrage condition. It still implies that the set of portfolios attainable from zero initial endowment is closed in probability. Consequently, prospective strict no-arbitrage implies the existence of consistent prices, which may lie on the boundary of the bid-ask spread. A weak version of prospective strict no-arbitrage turns out to be equivalent to the existence of a consistent price system. In continuous-time financial market models with proportional transaction costs, efficient friction, i.e., nonvanishing transaction costs, is a standing assumption. Together with robust no free lunch with vanishing risk, it rules out strategies of infinite variation which usually appear in frictionless financial markets. In the second part of this thesis, we show how models with and without transaction costs can be unified. The bid and the ask price of a risky asset are given by cadlag processes which are locally bounded from below and may coincide at some points. In a first step, we show that if the bid-ask model satisfies no unbounded profit with bounded risk for simple long-only strategies, then there exists a semimartingale lying between the bid and the ask price process. In a second step, under the additional assumption that the zeros of the bid-ask spread are either starting points of an excursion away from zero or inner points from the right, we show that for every bounded predictable strategy specifying the amount of risky assets, the semimartingale can be used to construct the corresponding self-financing risk-free position in a consistent way. Finally, the set of most general strategies is introduced, which also provides a new view on the frictionless case.

A standing assumption in the literature on proportional transaction costs is efficient friction. Together with robust no free lunch with vanishing risk, it rules out strategies of infinite variation, as they usually appear in frictionless markets. In this paper, we show how the models with and without transaction costs can be unified.The bid and the ask price of a risky asset are given by càdlàg processes which are locally bounded from below and may coincide at some points. In a first step, we show that if the bid-ask model satisfies "no unbounded profit with bounded risk" for simple strategies, then there exists a semimartingale lying between the bid and the ask price process.In a second step, under the additional assumption that the zeros of the bid-ask spread are either starting points of an excursion away from zero or inner points from the right, we show that for every bounded predictable strategy specifying the amount of risky assets, the semimartingale can be used to construct the corresponding self-financing risk-free position in a consistent way.