Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow

Assessment of processing technologies which may improve the nutritional composition of dairy products – Overview of progress

Among consumers there is a growing demand for food products with a natural nutritional-physiological advantage over comparable conventional products. As part of an EU funded project, ALP is examining the possible impact of processing on nutritionally valuable milk components, using the example of conjugated linoleic acids (CLA). The extent to which processing influences the CLA content of the end product was determined by literature research and own investigations of organic and conventional butter. Furthermore, new chemical, sensory-based and bio crystallization methods were evaluated by ALP and the University of Kassel to determine the oxidation stability of butter. In a further step the storage stability of CLA enriched and conventional butter was examined and the different methods will be compared. As a third objective a process for low-input CLA enrichment of milk fat (with a focus on alpine butter) has been developed. Since the process selected for the work is a physical enrichment process, it is accepted by international organic farming and food groups. Among the many benefits ascribed to CLA, it is believed to be an effective agent against cancer. The demand for foods with properties that promote human health is growing. The dairy industry has the opportunity to meet this demand by developing new dairy products with a nutritional-physiological function for the functional food market

Role of adhesion forces in mechanosensitive channel gating inStaphylococcus aureusadhering to surfaces

Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials

Two-Stage Interpretation of Changes in TEER of Intestinal Epithelial Layers Protected by Adhering Bifidobacteria During E. coli Challenges

Mechanisms of gastrointestinal protection by probiotic bacteria against infection involve amongst others, modulation of intestinal epithelial barrier function. Trans-epithelial electrical resistance (TEER) is widely used to evaluate cellular barrier functions. Here, we developed a two-stage interpretative model of the time-dependence of the TEER of epithelial layers grown in a transwell during Escherichia coli challenges in the absence or presence of adhering bifidobacteria. E. coli adhesion in absence or presence of adhering bifidobacteria was enumerated using selective plating. After 4-8 h, E. coli challenges increased TEER to a maximum due to bacterial adhesion and increased expression of a tight-junction protein [zonula occludens-1 (ZO-1)], concurrent with a less dense layer structure, that is indicative of mild epithelial layer damage. Before the occurrence of a TEER-maximum, decreases in electrical conductance (i.e., the reciprocal TEER) did not relate with para-cellular dextran-permeability, but after occurrence of a TEER-maximum, dextran-permeability and conductance increased linearly, indicative of more severe epithelial layer damage. Within 24 h after the occurrence of a TEER maximum, TEER decreased to below the level of unchallenged epithelial layers demonstrating microscopically observable holes and apoptosis. Under probiotic protection by adhering bifidobacteria, TEER-maxima were delayed or decreased in magnitude due to later transition from mild to severe damage, but similar linear relations between conductance and dextran permeability were observed as in absence of adhering bifidobacteria. Based on the time-dependence of the TEER and the relation between conductance and dextran-permeability, it is proposed that bacterial adhesion to epithelial layers first causes mild damage, followed by more severe damage after the occurrence of a TEER-maximum. The mild damage caused by E. coli prior to the occurrence of TEER maxima was reversible upon antibiotic treatment, but the severe damage after occurrence of TEER maxima could not be reverted by antibiotic treatment. Thus, single-time TEER is interpretable in two ways, depending whether increasing to or decreasing from its maximum. Adhering bifidobacteria elongate the time-window available for antibiotic treatment to repair initial pathogen damage to intestinal epithelial layers.</p

Клінічні особливості генетично детермінованих гіпобета-ліпопротеїнемії та гіпохолестеролемії

Familial hypobetalipoproteinemia (FHBL) is an autosomal dominant disorder of lipid metabolism characterized by extremely low plasma levels of apolipoprotein B, total cholesterol and low-density lipoprotein cholesterol. Heterozygotes for FHBL are often asymptomatic. Clinical features of homozygous FHBL can include acanthocytosis, deficiencies in fat-soluble vitamins secondary to malabsorption, atypical retinitis pigmentosa and neuromuscular abnormalities

The growth of different body length dimensions is not predictive for the peak growth velocity of sitting height in the individual child

The aim of this study was to determine whether the differences in timing of the peak growth velocity (PGV) between sitting height, total body height, subischial leg length, and foot length can be used to predict whether the individual patient with adolescent idiopathic scoliosis is before or past his or her PGV of sitting height. Furthermore, ratios of growth of different body parts were considered in order to determine their value in prediction of the PGV of sitting height in the individual patient. Ages of the PGV were determined for sitting height (n = 360), total body height (n = 432), subischial leg length (n = 357), and foot length (n = 263), and compared for the whole group and for the individual child in particular. Furthermore, the ages of the highest and lowest ratios between the body length dimensions were determined and compared to the age of the PGV of sitting height. The mean ages of the highest and lowest ratios were significantly different from the mean age of the PGV of sitting height in 3 out of 12 ratios in girls and 8 out of 12 ratios in boys. The variation over children was large and the ratios were too small, leading to a too large influence of measurement errors. The mean ages of the PGV all differed significantly from the mean age of the PGV of sitting height. However, the variation over individual children of the age differences in PGV between body dimensions was large, and the differences in timing of the PGV were not useful to predict whether the individual child is before or past his or her PGV of sitting height

Antimicrobial loading of nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization

Titanium is frequently used for dental implants, percutaneous pins and screws or orthopedic joint prostheses. Implant surfaces can become peri-operatively contaminated by surgically introduced bacteria during implantation causing lack of surface coverage by mammalian cells and subsequent implant failure. Especially implants that have to function in a bacteria-laden environment such as dental implants or percutaneous pins, cannot be surgically implanted while being kept sterile. Accordingly, contaminating bacteria adhering to implant surfaces hamper successful surface coverage by mammalian cells required for long-term functioning. Here, nanotubular titanium surfaces were prepared and loaded with Ag nanoparticles or gentamicin with the aim of killing contaminating bacteria in order to favor surface coverage by mammalian cells. In mono-cultures, unloaded nanotubules did not cause bacterial killing, but loading of Ag nanoparticles or gentamicin reduced the number of adhering Staphylococcus aureus or Pseudomonas aeruginosa CFUs. A gentamicin-resistant Staphylococcus epidermidis was only killed upon loading with Ag nanoparticles. However, unlike low-level gentamicin loading, loading with Ag nanoparticles also caused tissue-cell death. In bi-cultures, low-level gentamicin-loading of nanotubular titanium surfaces effectively eradicated contaminating bacteria favoring surface coverage by mammalian cells. Thus, care must be taken in loading nanotubular titanium surfaces with Ag nanoparticles, while low-level gentamicin-loaded nanotubular titanium surfaces can be used as a local antibiotic delivery system to negate failure of titanium implants due to peri-operatively introduced, contaminating bacteria without hampering surface coverage by mammalian cells

On-demand pulling-off of magnetic nanoparticles from biomaterial surfaces through implant-associated infectious biofilms for enhanced antibiotic efficacy

Biomaterial-associated infections can occur any time after surgical implantation of biomaterial implants and limit their success rates. On-demand, antimicrobial release coatings have been designed, but in vivo release triggers uniquely relating with infection do not exist, while inadvertent leakage of antimicrobials can cause exhaustion of a coating prior to need. Here, we attach magnetic-nanoparticles to a biomaterial surface, that can be pulled-off in a magnetic field through an adhering, infectious biofilm. Magnetic-nanoparticles remained stably attached to a surface upon exposure to PBS for at least 50 days, did not promote bacterial adhesion or negatively affect interaction with adhering tissue cells. Nanoparticles could be magnetically pulled-off from a surface through an adhering biofilm, creating artificial water channels in the biofilm. At a magnetic-nanoparticle coating concentration of 0.64 mg cm-2, these by-pass channels increased the penetrability of Staphylococcus aureus and Pseudomonas aeruginosa biofilms towards different antibiotics, yielding 10-fold more antibiotic killing of biofilm inhabitants than in absence of artificial channels. This innovative use of magnetic-nanoparticles for the eradication of biomaterial-associated infections requires no precise targeting of magnetic-nanoparticles and allows more effective use of existing antibiotics by breaking the penetration barrier of an infectious biofilm adhering to a biomaterial implant surface on-demand

Homogeneous Distribution of Magnetic, Antimicrobial-Carrying Nanoparticles through an Infectious Biofilm Enhances Biofilm-Killing Efficacy

Magnetic, antimicrobial-carrying nanoparticles provide a promising, new and direly needed antimicrobial strategy against infectious bacterial biofilms. Penetration and accumulation of antimicrobials over the thickness of a biofilm is a conditio sine qua non for effective killing of biofilm inhabitants. Simplified schematics on magnetic-targeting always picture homogeneous distribution of magnetic, antimicrobial-carrying nanoparticles over the thickness of biofilms, but this is not easy to achieve. Here, gentamicin-carrying magnetic nanoparticles (MNPs-G) were synthesized through gentamicin conjugation with iron-oxide nanoparticles and used to demonstrate the importance of their homogeneous distribution over the thickness of a biofilm. Diameters of MNPs-G were around 60 nm, well below the limit for reticuloendothelial rejection. MNPs-G killed most ESKAPE-panel pathogens, including Escherichia coli, equally as well as gentamicin in solution. MNPs-G distribution in a Staphylococcus aureus biofilm was dependent on magnetic-field exposure time and most homogeneous after 5 min magnetic-field exposure. Exposure of biofilms to MNPs-G with 5 min magnetic-field exposure yielded not only homogeneous distribution of MNPs-G, but concurrently better staphylococcal killing at all depths than that of MNPs, gentamicin in solution, and MNPs-G, or after other magnet-field exposure times. In summary, homogeneous distribution of gentamicin-carrying magnetic nanoparticles over the thickness of a staphylococcal biofilm was essential for killing biofilm inhabitants and required optimizing of the magnetic-field exposure time. This conclusion is important for further successful development of magnetic, antimicrobial carrying nanoparticles toward clinical application