Cryo-XPS for Surface Characterization of Nanomedicines.

Nanoparticles used for medical applications commonly possess coatings or surface functionalities intended to provide specific behavior in vivo, for example, the use of PEG to provide stealth properties. Direct, quantitative measurement of the surface chemistry and composition of such systems in a hydrated environment has thus far not been demonstrated, yet such measurements are of great importance for the development of nanomedicine systems. Here we demonstrate the first use of cryo-XPS for the measurement of two PEG-functionalized nanomedicines: a polymeric drug delivery system and a lipid nanoparticle mRNA carrier. The observed differences between cryo-XPS and standard XPS measurements indicate the potential of cryo-XPS for providing quantitative measurements of such nanoparticle systems in hydrated conditions.

Biochemical characterization and mercury methylation capacity of Geobacter sulfurreducens biofilms grown in media containing iron hydroxide or fumarate.

Geobacter species are common in iron-rich environments and can contribute to formation of methylmercury (MeHg), a neurotoxic compound with high bioaccumulation potential formed as a result of bacterial and archaeal physiological activity. Geobacter sulfurreducens can utilize various electron acceptors for growth including iron hydroxides or fumarate. However, it remains poorly understood how the growth on these compounds affects physiological properties of bacterial cells in biofilms, including the capacity to produce MeHg. The purpose of this study was to determine changes in the biochemical composition of G. sulfurreducens during biofilm cultivation in media containing iron hydroxide or fumarate, and to quantify mercury (Hg) methylation capacity of the formed biofilms. Biofilms were characterized by Fourier-transform infrared spectroscopy in the attenuated total reflection mode (ATR-FTIR), Resonance Raman spectroscopy and confocal laser scanning microscopy. MeHg formation was quantified by mass spectrometry after incubation of biofilms with 100 nM Hg. The results of ATR-FTIR experiments showed that in presence of fumarate, G. sulfurreducens biofilm formation was accompanied by variation in content of the energy-reserve polymer glycogen over time, which could be cancelled by the addition of supplementary nutrients (yeast extract). In contrast, biofilms cultivated on Fe(III) hydroxide did not accumulate glycogen. The ATR-FTIR results further suggested that Fe(III) hydroxide surfaces bind cells via phosphate and carboxylate groups of bacteria that form complexes with iron. Furthermore, biofilms grown on Fe(III) hydroxide had higher fraction of oxidized cytochromes and produced two to three times less biomass compared to conditions with fumarate. Normalized to biofilm volume, the content of MeHg was similar in assays with biofilms grown on Fe(III) hydroxide and on fumarate (with yeast extract and without). These results suggest that G. sulfurreducens biofilms produce MeHg irrespectively from glycogen content and cytochrome redox state in the cells, and warrant further investigation of the mechanisms controlling this process.

Methylmercury formation in biofilms of Geobacter sulfurreducens.

Introduction: Mercury (Hg) is a major environmental pollutant that accumulates in biota predominantly in the form of methylmercury (MeHg). Surface-associated microbial communities (biofilms) represent an important source of MeHg in natural aquatic systems. In this work, we report MeHg formation in biofilms of the iron-reducing bacterium Geobacter sulfurreducens. Methods: Biofilms were prepared in media with varied nutrient load for 3, 5, or 7 days, and their structural properties were characterized using confocal laser scanning microscopy, cryo-scanning electron microscopy and Fourier-transform infrared spectroscopy. Results: Biofilms cultivated for 3 days with vitamins in the medium had the highest surface coverage, and they also contained abundant extracellular matrix. Using 3 and 7-days-old biofilms, we demonstrate that G. sulfurreducens biofilms prepared in media with various nutrient load produce MeHg, of which a significant portion is released to the surrounding medium. The Hg methylation rate constant determined in 6-h assays in a low-nutrient assay medium with 3-days-old biofilms was 3.9 ± 2.0 ∙ 10-14 L ∙ cell-1 ∙ h-1, which is three to five times lower than the rates found in assays with planktonic cultures of G. sulfurreducens in this and previous studies. The fraction of MeHg of total Hg within the biofilms was, however, remarkably high (close to 50%), and medium/biofilm partitioning of inorganic Hg (Hg(II)) indicated low accumulation of Hg(II) in biofilms. Discussion: These findings suggest a high Hg(II) methylation capacity of G. sulfurreducens biofilms and that Hg(II) transfer to the biofilm is the rate-limiting step for MeHg formation in this systems.

A highly immunogenic vaccine platform against encapsulated pathogens using chimeric probiotic Escherichia coli membrane vesicles.

Vaccines against infectious diseases should elicit potent and long-lasting immunity, ideally even in those with age-related decline in immune response. Here we report a rational polysaccharide vaccine platform using probiotic Escherichia coli-derived membrane vesicles (MVs). First, we constructed a probiotic E. coli clone harboring the genetic locus responsible for biogenesis of serotype 14 pneumococcal capsular polysaccharides (CPS14) as a model antigen. CPS14 was found to be polymerized and mainly localized on the outer membrane of the E. coli cells. The glycine-induced MVs displayed the exogenous CPS14 at high density on the outermost surface, on which the CPS14 moiety was covalently tethered to a lipid A-core oligosaccharide anchor. In in vivo immunization experiments, CPS14+MVs, but not a mixture of free CPS14 and empty MVs, strongly elicited IgG class-switch recombination with a Th1/Th2-balanced IgG subclass distribution without any adjuvant. In addition, CPS14+MVs were structurally stable with heat treatment and immunization with the heat-treated MVs-elicited CPS14-specific antibody responses in mouse serum to levels comparable to those of non-treated CPS14+MVs. Notably, the immunogenicity of CPS14+MVs was significantly stronger than those of two currently licensed vaccines against pneumococci. The CPS14+MV-elicited humoral immune responses persisted for 1 year in both blood and lung. Furthermore, the CPS14+MV vaccine was widely efficacious in mice of different ages. Even in aged mice, vaccination resulted in robust production of CPS14-specific IgG that bound to the pneumococcal cell surface. Taken together, the present probiotic E. coli MVs-based vaccine platform offers a promising, generalizable solution against encapsulated pathogens.

Assuring the Biofunctionalization of Silicone Covalently Bonded to Rhamnolipids: Antibiofilm Activity and Biocompatibility.

Silicone-based medical devices composed of polydimethylsiloxane (PDMS) are widely used all over the human body (e.g., urinary stents and catheters, central venous catheters stents) with extreme clinical success. Nevertheless, their abiotic surfaces, being prone to microorganism colonization, are often involved in infection occurrence. Improving PDMS antimicrobial properties by surface functionalization with biosurfactants to prevent related infections has been the goal of different works, but studies that mimic the clinical use of these novel surfaces are missing. This work aims at the biofunctional assessment of PDMS functionalized with rhamnolipids (RLs), using translational tests that more closely mimic the clinical microenvironment. Rhamnolipids were covalently bonded to PDMS, and the obtained surfaces were characterized by contact angle modification assessment, ATR-FTIR analysis and atomic force microscopy imaging. Moreover, a parallel flow chamber was used to assess the Staphylococcus aureus antibiofilm activity of the obtained surfaces under dynamic conditions, and an in vitro characterization with human dermal fibroblast cells in both direct and indirect characterization assays, along with an in vivo subcutaneous implantation assay in the translational rabbit model, was performed. A 1.2 log reduction in S. aureus biofilm was observed after 24 h under flow dynamic conditions. Additionally, functionalized PDMS lessened cell adhesion upon direct contact, while supporting a cytocompatible profile, within an indirect assay. The adequacy of the biological response was further validated upon in vivo subcutaneous tissue implantation. An important step was taken towards biofunctional assessment of RLs-functionalized PDMS, reinforcing their suitability for medical device usage and infection prevention.

Enhanced Antibiotic Tolerance of an In Vitro Multispecies Uropathogen Biofilm Model, Useful for Studies of Catheter-Associated Urinary Tract Infections.

Catheter-associated urinary tract infections (CAUTI) are a common clinical concern as they can lead to severe, persistent infections or bacteremia in long-term catheterized patients. This type of CAUTI is difficult to eradicate, as they are caused by multispecies biofilms that may have reduced susceptibility to antibiotics. Many new strategies to tackle CAUTI have been proposed in the past decade, including antibiotic combination treatments, surface modification and probiotic usage. However, those strategies were mainly assessed on mono- or dual-species biofilms that hardly represent the long-term CAUTI cases where, normally, 2-4 or even more species can be involved. We developed a four-species in vitro biofilm model on catheters involving clinical strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca and Proteus mirabilis isolated from indwelling catheters. Interspecies interactions and responses to antibiotics were quantitatively assessed. Collaborative as well as competitive interactions were found among members in our model biofilm and those interactions affected the individual species' abundances upon exposure to antibiotics as mono-, dual- or multispecies biofilms. Our study shows complex interactions between species during the assessment of CAUTI control strategies for biofilms and highlights the necessity of evaluating treatment and control regimes in a multispecies setting.

Ethanol Inactivation of Enveloped Viruses: Structural and Surface Chemistry Insights into Phi6.

Lipid-enveloped viruses, such as Ebola, influenza, or coronaviruses, are a major threat to human health. Ethanol is an efficient disinfectant that is widely used to inactivate these viruses and prevent their transmission. However, the interactions between ethanol and enveloped viruses leading to their inactivation are not yet fully understood. This study demonstrates the link between ethanol-induced viral inactivation and the nanostructural and chemical transformations of the model virus Phi6, an 85 nm diameter lipid-enveloped bacterial virus that is commonly used as surrogate for human pathogenic viruses. The virus morphology was investigated using small-angle X-ray scattering and dynamic light scattering and was related to its infectivity. The Phi6's surface chemistry was characterized by cryogenic X-ray photoelectron spectroscopy, and the modifications in protein structure were assessed by circular dichroism and fluorescence spectroscopy. Ethanol-triggered structural modifications were found in the lipid envelope, detaching from the protein capsid and forming coexisting nanostructures.

Applying Cryo-X-ray Photoelectron Spectroscopy to Study the Surface Chemical Composition of Fungi and Viruses.

Interaction between microorganisms and their surroundings are generally mediated via the cell wall or cell envelope. An understanding of the overall chemical composition of these surface layers may give clues on how these interactions occur and suggest mechanisms to manipulate them. This knowledge is key, for instance, in research aiming to reduce colonization of medical devices and device-related infections from different types of microorganisms. In this context, X-ray photoelectron spectroscopy (XPS) is a powerful technique as its analysis depth below 10 nm enables studies of the outermost surface structures of microorganism. Of specific interest for the study of biological systems is cryogenic XPS (cryo-XPS). This technique allows studies of intact fast-frozen hydrated samples without the need for pre-treatment procedures that may cause the cell structure to collapse or change due to the loss of water. Previously, cryo-XPS has been applied to study bacterial and algal surfaces with respect to their composition of lipids, polysaccharides and peptide (protein and/or peptidoglycan). This contribution focuses onto two other groups of microorganisms with widely different architecture and modes of life, namely fungi and viruses. It evaluates to what extent existing models for data treatment of XPS spectra can be applied to understand the chemical composition of their very different surface layers. XPS data from model organisms as well as reference substances representing specific building blocks of their surface were collected and are presented. These results aims to guide future analysis of the surface chemical composition of biological systems.

Comparative Study of NAP-XPS and Cryo-XPS for the Investigation of Surface Chemistry of the Bacterial Cell-Envelope.

Bacteria generally interact with the environment via processes involving their cell-envelope. Thus, techniques that may shed light on their surface chemistry are attractive tools for providing an understanding of bacterial interactions. One of these tools is Al Kα-excited photoelectron spectroscopy (XPS) with its estimated information depth of <10 nm. XPS-analyses of bacteria have been performed for several decades on freeze-dried specimens in order to be compatible with the vacuum in the analysis chamber of the spectrometer. A limitation of these studies has been that the freeze-drying method may collapse cell structure as well as introduce surface contaminants. However, recent developments in XPS allow for analysis of biological samples at near ambient pressure (NAP-XPS) or as frozen hydrated specimens (cryo-XPS) in vacuum. In this work, we have analyzed bacterial samples from a reference strain of the Gram-negative bacterium Pseudomonas fluorescens using both techniques. We compare the results obtained and, in general, observe good agreement between the two techniques. Furthermore, we discuss advantages and disadvantages with the two analysis approaches and the output data they provide. XPS reference data from the bacterial strain are provided, and we propose that planktonic cells of this strain (DSM 50090) are used as a reference material for surface chemical analysis of bacterial systems.

Antimicrobial peptide induced colloidal transformations in bacteria-mimetic vesicles: Combining in silico tools and experimental methods.

With the growing challenges of bacteria becoming resistant to conventional antibiotics, antimicrobial peptides (AMPs) may offer a potential alternative. One of the most studied AMPs, the human cathelicidin derived AMP LL-37 is notable for its antimicrobial activity even though its mechanism of action is not fully understood yet. This work investigates the interaction of LL-37 with 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) vesicles, which were employed as a bacterial membrane model given the common presence of this phospholipid in the bacterial membrane. Experimental techniques including small angle X-ray scattering, transmission electron microscopy and dynamic light scattering were used to characterize the interactions among LL-37 and POPG. Molecular dynamics simulations complement the experimental studies with molecular-level insights into the process. LL-37 was discovered to actively and critically interact with the POPG vesicles, modifying the membrane curvature that eventually leads to structural transformations from vesicles to mixed micelles. The results shed light on the mechanisms underlying the interactions among LL-37 and bacteria mimetic vesicles and can guide the further development of AMP based antimicrobial materials and therapies.

Do environmental pharmaceuticals affect the composition of bacterial communities in a freshwater stream? A case study of the Knivsta river in the south of Sweden.

Pharmaceutical substances present at low concentrations in the environment may cause effects on biological systems such as microbial consortia living on solid riverbed substrates. These consortia are an important part of the river ecosystem as they form part of the food chain. This case study aims to contribute to an increased understanding of how low levels of pharmaceuticals in freshwater streams may influence sessile bacterial consortia. An important point source for pharmaceutical release into the environment is treated household sewage water. In order to investigate what types of effects may occur, we collected water samples as well as riverbed substrates from a small stream in the south of Sweden, Knivstaån, upstream and downstream from a sewage treatment plant (STP). Data from these samples formed the base of this case study where we investigated both the presence of pharmaceuticals in the water and bacterial composition on riverbed substrates. In the water downstream from the STP, 19 different pharmaceuticals were detected at levels below 800 ng/dm3. The microbial composition was obtained from sequencing 16S rRNA genes directly from substrates as well as from cultivated isolates. The cultivated strains showed reduced species variability compared with the data obtained directly from the substrates. No systematic differences were observed following the sampling season. However, differences could be seen between samples upstream and downstream from the STP effluent. We further observed large similarities in bacterial composition on natural stones compared to sterile stones introduced into the river approximately two months prior to sampling, giving indications for future sampling methodology of biofilms.

Activity of airway antimicrobial peptides against cystic fibrosis pathogens.

Antimicrobial peptides are important players of the innate host defence against invading microorganisms. The aim of this study was to evaluate the activity of airway antimicrobial peptides against the common cystic fibrosis (CF) pathogen Pseudomonas aeruginosa, and to compare it to the emerging multi-drug resistant CF pathogens Achromobacter xylosoxidans and Stenotrophomonas maltophilia. Clinical bacterial isolates from CF patients were used, and the antimicrobial activity of human beta-defensin 2 and 3, LL37 and lysozyme was evaluated using radial diffusion assay and viable counts. The cell surface zeta potential was analysed to estimate the net charge at the bacterial surface. Of the bacterial species included in the study, A. xylosoxidans was the most resistant to antimicrobial peptides, whereas P. aeruginosa was the most susceptible. The net charge of the bacterial surface was significantly more negative for P. aeruginosa compared to A. xylosoxidans, which may in part explain the differences in susceptibility.

Evaluating Efficacy of Antimicrobial and Antifouling Materials for Urinary Tract Medical Devices: Challenges and Recommendations.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

Conformational Dynamics and Responsiveness of Weak and Strong Polyelectrolyte Brushes: Atomistic Simulations of Poly(dimethyl aminoethyl methacrylate) and Poly(2-(methacryloyloxy)ethyl trimethylammonium chloride).

The complex solution behavior of polymer brushes is key to control their properties, including for biomedical applications and catalysis. The swelling behavior of poly(dimethyl aminoethyl methacrylate) (PDMAEMA) and poly(2-(methacryloyloxy)ethyl trimethylammonium chloride) (PMETAC) in response to changes in pH, solvent, and salt types has been investigated using atomistic molecular dynamics simulations. PDMAEMA and PMETAC have been selected as canonical models for weak and strong polyelectrolytes whose complex conformational behavior is particularly challenging for the development and validation of atomistic models. The GROMOS-derived atomic parameters reproduce the experimental swelling coefficients obtained from ellipsometry measurements for brushes of 5-15 nm thickness. The present atomistic models capture the protonated morphology of PDMAEMA, the swollen and collapsed conformations of PDMAEMA and PMETAC in good and bad solvents, and the salt-selective response of PMETAC. The modular nature of the molecular models allows for the simple extension of atomic parameters to a variety of polymers or copolymers.

Characterization of clinically relevant model bacterial strains of Pseudomonas aeruginosa for anti-biofilm testing of materials.

There is a great interest in developing novel anti-biofilm materials in order to decrease medical device-associated bacterial infections causing morbidity and high healthcare costs. However, the testing of novel materials is often done using bacterial lab strains that may not exhibit the same phenotype as clinically relevant strains infecting medical devices. Furthermore, no consensus of strain selection exists in the field, making results very difficult to compare between studies. In this work, 19 clinical isolates of Pseudomonas aeruginosa originating from intubated patients in an intensive care unit have been characterized and compared to the lab reference strain PAO1 and a rmlC lipopolysaccharide mutant of PAO1. The adhesion and biofilm formation was monitored, as well as cell properties such as hydrophobicity, zeta potential and motility. Two groups of isolates were observed: one with high adhesion to polymer surfaces and one with low adhesion (the latter including PAO1). Furthermore, detailed biofilm assays in a flow system were performed using five characteristic isolates from the two groups. Confocal microscopy showed that the adhesion and biofilm formation of four of these five strains could be reduced dramatically on zwitterionic surface coatings. However, one isolate with pronounced swarming colonized and formed biofilm also on the antifouling surface. We demonstrate that the biofilm properties of clinical isolates can differ greatly from that of a standard lab strain and propose two clinical model strains for testing of materials designed for prevention of biofilm formation in the respiratory tract. The methodology used could beneficially be applied for screening of other collections of pathogens to identify suitable model strains for in vitro biofilm testing. STATEMENT OF SIGNIFICANCE: Medical-device associated infections present a great challenge in health care. Therefore, much research is undertaken to prevent bacterial colonization of new types of biomaterials. The work described here characterizes, tests and presents a number of clinically relevant bacterial model strains for assessing biofilm formation by Pseudomonas aeruginosa. Such model strains are of importance as they may provide better predictability of lab testing protocols with respect to how well materials would perform in an infection situation in a patient. Furthermore, this study uses the strains to test the performance of polymer surfaces designed to repel bacterial adhesion and it is shown that the biofilm formation for four out of the five tested bacterial strains was reduced.

In vitro methods for the evaluation of antimicrobial surface designs.

Bacterial adhesion and subsequent biofilm formation on biomedical implants and devices are a major cause of their failure. As systemic antibiotic treatment is often ineffective, there is an urgent need for antimicrobial biomaterials and coatings. The term "antimicrobial" can encompass different mechanisms of action (here termed "antimicrobial surface designs"), such as antimicrobial-releasing, contact-killing or non-adhesivity. Biomaterials equipped with antimicrobial surface designs based on different mechanisms of action require different in vitro evaluation methods. Available industrial standard evaluation tests do not address the specific mechanisms of different antimicrobial surface designs and have therefore been modified over the past years, adding to the myriad of methods available in the literature to evaluate antimicrobial surface designs. The aim of this review is to categorize fourteen presently available methods including industrial standard tests for the in vitro evaluation of antimicrobial surface designs according to their suitability with respect to their antimicrobial mechanism of action. There is no single method or industrial test that allows to distinguish antimicrobial designs according to all three mechanisms identified here. However, critical consideration of each method clearly relates the different methods to a specific mechanism of antimicrobial action. It is anticipated that use of the provided table with the fourteen methods will avoid the use of wrong methods for evaluating new antimicrobial designs and therewith facilitate translation of novel antimicrobial biomaterials and coatings to clinical use. The need for more and better updated industrial standard tests is emphasized. STATEMENT OF SIGNIFICANCE: European COST-action TD1305, IPROMEDAI aims to provide better understanding of mechanisms of antimicrobial surface designs of biomaterial implants and devices. Current industrial evaluation standard tests do not sufficiently account for different, advanced antimicrobial surface designs, yet are urgently needed to obtain convincing in vitro data for approval of animal experiments and clinical trials. This review aims to provide an innovative and clear guide to choose appropriate evaluation methods for three distinctly different mechanisms of antimicrobial design: (1) antimicrobial-releasing, (2) contact-killing and (3) non-adhesivity. Use of antimicrobial evaluation methods and definition of industrial standard tests, tailored toward the antimicrobial mechanism of the design, as identified here, fulfill a missing link in the translation of novel antimicrobial surface designs to clinical use.

Protein Nanosheet Mechanics Controls Cell Adhesion and Expansion on Low-Viscosity Liquids.

Adherent cell culture typically requires cell spreading at the surface of solid substrates to sustain the formation of stable focal adhesions and assembly of a contractile cytoskeleton. However, a few reports have demonstrated that cell culture is possible on liquid substrates such as silicone and fluorinated oils, even displaying very low viscosities (0.77 cSt). Such behavior is surprising as low viscosity liquids are thought to relax much too fast (

Levofloxacin-loaded bone cement delivery system: Highly effective against intracellular bacteria and Staphylococcus aureus biofilms.

Staphylococcus aureus is a major pathogen in bone associated infections due to its ability to adhere and form biofilms on bone and/or implants. Moreover, recrudescent and chronic infections have been associated with S. aureus capacity to invade and persist within osteoblast cells. With the growing need of novel therapeutic tools, this research aimed to evaluate some important key biological properties of a novel carrier system composed of acrylic bone cement (polymethylmethacrylate - PMMA), loaded with a release modulator (lactose) and an antibiotic (levofloxacin). Levofloxacin-loaded bone cement (BC) exhibited antimicrobial effects against planktonic and biofilm forms of S. aureus (evaluated by a flow chamber system). Moreover, novel BC formulation showed high anti-bacterial intraosteoblast activity. This fact led to the conclusion that levofloxacin released from BC matrices could penetrate the cell membrane of osteoblasts and be active against S. aureus strains in the intracellular environment. Furthermore, levofloxacin-BC formulations showed no significant in vitro cytotoxicity and no allergic potential (measured by the in vivo chorioallantoic membrane assay). Our results indicate that levofloxacin-loaded BC has potential as a local antibiotic delivery system for treating S. aureus associated bone infections.

Analysis of Bacterial Cell Surface Chemical Composition Using Cryogenic X-Ray Photoelectron Spectroscopy.

This chapter describes a method for measuring the average surface chemical composition with respect to lipids, polysaccharides, and peptides (protein + peptidoglycan) for the outer part of the bacterial cell wall. Bacterial cultures grown over night are washed with a buffer or saline at controlled pH. The analysis is done on fast-frozen bacterial cell pellets obtained after centrifugation, and the analysis requires access to X-ray photoelectron spectroscopy instrumentation that can perform analyses at cryogenic temperatures (for example using liquid nitrogen). The method can be used to monitor changes in the cell wall composition following environmental stimuli or genetic mutations. The data obtained originate from the outermost part of the cell wall. Thus, it is expected that for gram-negative bacteria only the outer membrane and part of the periplasmic peptidoglycan layer is probed during analysis, and for gram-positive bacteria only the top nanometers of the peptidoglycan layer of the cell wall is monitored.

Influence of chelation strength and bacterial uptake of gallium salicylidene acylhydrazide on biofilm formation and virulence of Pseudomonas aeruginosa.

Development of antibiotic resistance in bacteria causes major challenges for our society and has prompted a great need for new and alternative treatment methods for infection. One promising approach is to target bacterial virulence using for example salicylidene acylhydrazides (hydrazones). Hydrazones coordinate metal ions such as Fe(III) and Ga(III) through a five-membered and a six-membered chelation ring. One suggested mode of action is via restricting bacterial Fe uptake. Thus, it was hypothesized that the chelating strength of these substances could be used to predict their biological activity on bacterial cells. This was investigated by comparing Ga chelation strength of two hydrazone complexes, as well as bacterial Ga uptake, biofilm formation, and virulence in the form of production and secretion of a toxin (ExoS) by Pseudomonas aeruginosa. Equilibrium constants for deprotonation and Ga(III) binding of the hydrazone N'-(5-chloro-2-hydroxy-3-methylbenzylidene)-2,4-dihydroxybenzhydrazide (ME0329), with anti-virulence effect against P. aeruginosa, were determined and compared to bacterial siderophores and the previously described Ga(III) 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (Ga-ME0163) and Ga-citrate complexes. In comparison with these two complexes, it was shown that the uptake of Ga(III) was higher from the Ga-ME0329 complex. The results further show that the Ga-ME0329 complex reduced ExoS expression and secretion to a higher extent than Ga-citrate, Ga-ME0163 or the non-coordinated hydrazone. However, the effect against biofilm formation by P. aeruginosa, by the ME0329 complex, was similar to Ga-citrate and lower than what has been reported for Ga-ME0163.