Modification of physico-chemical surface properties and growth of Staphylococcus aureus under hyperglycemia and ketoacidosis conditions.

Diabetes is a widely spread disease affecting the quality of life of millions of people around the world and is associated to a higher risk of developing infections in different parts of the body. The reasons why diabetes enhances infection episodes are not entirely clear; in this study our aim was to explore the changes that one of the most frequently pathogenic bacteria undergoes when exposed to hyperglycemia and ketoacidosis conditions. Physical surface properties such as hydrophobicity and surface electrical charge are related to bacterial growth behavior and the ability of Staphylococcus aureus to form biofilms. The addition of glucose made bacteria more negatively charged and with moderate-intermediate hydrophobicity. Ketone bodies increased hydrophobicity to approximately 75% and pathological concentrations hindered some of the bacterial surface charge by decreasing the negative zeta potential of cells. When both components were present, the bacterial physical surface changes were more similar to those observed in ketone bodies, suggesting a preferential adsorption of ketone bodies over glucose because of the more favorable solubility of glucose in water. Glucose diabetic concentrations gave the highest number of bacteria in the stationary phase of growth and provoked an increase in the biofilm slime index of around 400% in relation to the control state. Also, this situation is related with an increase of bacterial coverage. The combination of a high concentration of glucose and ketone bodies, which corresponds to a poorly controlled diabetic situation, appears associated with an early infection phase; increased hydrophobic attractive force and reduced electrostatic repulsion between cells results in better packing of cells within the biofilm and more efficient retention to the host surface. Knowledge of bacterial response in high amount of glucose and ketoacidosis environments can serve as a basis for designing strategies to prevent bacterial adhesion, biofilm formation and, consequently, the development of infections.

Characterization of Magnesium-Polylactic Acid Films Casted on Different Substrates and Doped with Diverse Amounts of CTAB.

Polylactic acid (PLA) is a good candidate for the manufacture of polymeric biodegradable biomaterials. The inclusion of metallic particles and surfactants solves its mechanical limitations and improves its wettability, respectively. In this work, cetyltrimethylammonium bromide (CTAB) and magnesium particles have been incorporated into PLA films to evaluate the changes produced in the polymeric matrix cast on glass and silicone substrates. For this purpose, the surface of the films has been characterized by means of contact angle measurements and ToF-SIMS. Depth profiles and SEM images of the cross sections of the films have also been obtained to study their morphology. The results show that the CTAB in the polymer matrix with and without magnesium improves the wettability of the films, making them more suitable for cell adhesion. The higher the hydrophilicity, the higher the surfactant concentration. The depth profiles show, for the first time, that, depending on the surfactant concentration and the presence of Mg, there is a layer-like distribution near the surface where, in addition to the CTAB + PLA mixture, a surfactant exclusion zone can be seen. This new structure could be relevant in in vitro/in vivo situations when the degradation processes remove the film components in a sequential form.

3D-PLA-experimental set up to display the electrical background of the so-called geometric factor of electrokinetic cells.

The so-called geometric factor defined in electrokinetic cells, L/S (L being the length and S the cross-section of the channel), is relevant for providing the surface interaction electrical potential (zeta potential, ζ) of large surfaces, such as those used in the design of biomedical devices or water purification systems. Conversely, recent studies demonstrate that this factor is also employed to determine geometrical parameters, such as porosity in membrane-like systems. This factor, which has been attributed exclusively a geometrical character, can also be obtained from the electrical conductivity and resistance of the electrokinetic channel. In this work, we assess whether these two ways of obtaining the L/S factor are equivalent and how possible deviations can affect the value of the zeta potential. For this purpose, we work with channels of different geometries obtained by 3D printing using PLA (polylactic acid) as a polymer employed in biomedical applications. The discrepancies between the L/S factor obtained by electrical and purely geometrical measurements increase as the geometrical L/S factor becomes larger, reaching differences close to 80%. The results show that the so-called geometrical L/S factor also has an important electrical contribution and would be better denoted as electrogeometric factor. The differences found between the L/S factors are also propagated to the calculation of ζ but an optimum conductivity zone (from about 10 to 40 mS m-1) can be defined to obtain the zeta potential by selecting any of the L/S factors obtained from electrokinetic measurements. The results of this work should be taken into account in those investigations that use the L/S factor to obtain the geometry-porosity of permeable materials.

The role of magnesium in biomaterials related infections.

Magnesium is currently increasing interest in the field of biomaterials. An extensive bibliography on this material in the last two decades arises from its potential for the development of biodegradable implants. In addition, many researches, motivated by this progress, have analyzed the performance of magnesium in both in vitro and in vivo assays with gram-positive and gram-negative bacteria in a very broad range of conditions. This review explores the extensive literature in recent years on magnesium in biomaterials-related infections, and discusses the mechanisms of the Mg action on bacteria, as well as the competition of Mg2+ and/or synergy with other divalent cations in this subject.

Impact of PLA/Mg films degradation on surface physical properties and biofilm survival.

New biocompatible and bioabsorbable materials are currently being developed for bone regeneration. These serve as scaffolding for controlled drug release and prevent bacterial infections. Films of polylactic acid (PLA) polymers that are Mg-reinforced have demonstrated they have suitable properties and bioactive behavior for promoting the osseointegration process. However little attention has been paid to studying whether the degradation process can alter the adhesive physical properties of the biodegradable film and whether this can modify the biofilm formation capacity of pathogens. Moreover, considering that the concentration of Mg and other corrosion products may not be constant during the degradation process, the question that arises is whether these changes can have negative consequences in terms of the bacterial colonization of surfaces. Bacteria are able to react differently to the same compound, depending on its concentration in the medium and can even become stronger when threatened. In this context, physical surface parameters such as hydrophobicity, surface tension and zeta potential of PLA films reinforced with 10% Mg have been determined before and after degradation, as well as the biofilm formation capacity of Staphylococcus epidermidis. The addition of Mg to the films makes them less hydrophobic and the degradation also reduces the hydrophobicity and increases the negative charge of the surface, especially over long periods of time. Early biofilm formation at 8 h is consistent with the physical properties of the films, where we can observe a reduction in the bacterial biofilm formation. However, after 24 h of incubation, the biofilm formation increases significantly on the PLA/Mg films with respect to PLA control. The explosive release of Mg ions and other corrosion products within the first hours were not enough to prevent a greater biofilm formation after this initial time. Consequently, the Mg addition to the polymer matrix had a bacteriostatic effect but not a bactericidal one. Future works should aim to optimize the design and biofunctionality of these promising bioabsorbable composites for a degradation period suitable for the intended application.

Relevance of Topographic Parameters on the Adhesion and Proliferation of Human Gingival Fibroblasts and Oral Bacterial Strains.

Dental implantology allows replacement of failing teeth providing the patient with a general improvement of health. Unfortunately not all reconstructions succeed, as a consequence of the development of infections of bacterial origin on the implant surface. Surface topography is known to modulate a differential response to bacterial and mammalian cells but topographical measurements are often limited to vertical parameters. In this work we have extended the topographical measurements also to lateral and hybrid parameters of the five most representative implant and prosthetic component surfaces and correlated the results with bacterial and mammalian cell adhesion and proliferation outcomes. Primary human oral gingival fibroblast (gum cells) and the bacterial strains: Streptococcus mutans, Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans, implicated in infectious processes in the oral/implant environment were employed in the presence or absence of human saliva. The results confirm that even though not all the measured surface is available for bacteria to adhere, the overall race for the surface between cells and bacteria is more favourable to the smoother surfaces (nitrided, as machined or lightly acid etched) than to the rougher ones (strong acid etched or sandblasted/acid etched).

Kinetic of Adhesion of S. epidermidis with Different EPS Production on Ti6Al4V Surfaces.

Controlling initial bacterial adhesion is essential to prevent biofilm formation and implant-related infection. The search for surface coatings that prevent initial adhesion is a powerful strategy to obtain implants that are more resistant to infection. Tracking the progression of adhesion on surfaces from the beginning of the interaction between bacteria and the surface provides a deeper understanding of the initial adhesion behavior. To this purpose, we have studied the progression over time of bacterial adhesion from a laminar flow of a bacterial suspension, using a modified Robbins device (MRD). Comparing with other laminar flow devices, such as the parallel plate flow chamber, MRD allows the use of diverse substrata under the same controlled flow conditions simultaneously. Two different surfaces of Ti6Al4V and two strains of Staphylococcus epidermidis with different exopolymer production were tested. In addition, the modified Robbins device was examined for its convenience and suitability for the purpose of this study. Results were analyzed according to a pseudofirst order kinetic. The values of the parameters obtained from this model make it possible to discriminate the adhesive behavior of surfaces and bacteria. One of the fitting parameters depends on the bacterial strain and the other only on the surface properties of the substrate.

In vivo bactericidal efficacy of the Ti6Al4V surface after ultraviolet C treatment.

BACKGROUND: Biomaterial-associated infections are one of the most important complications in orthopedic surgery. The main goal of this study was to demonstrate the in vivo bactericidal effect of ultraviolet (UV) irradiation on Ti6Al4V surfaces. MATERIALS AND METHODS: An experimental model of device-related infections was developed by direct inoculation of Staphylococcus aureus into the canal of both femurs of 34 rats. A UV-irradiated Ti6Al4V pin was press-fit into the canal by retrograde insertion in one femur and the control pin was inserted into the contralateral femur. To assess the efficacy of UV radiation, the mean colony counts after inoculation in the experimental subjects and the control group were compared at different times of sacrifice and at different inoculum doses. RESULTS: At 72 h, the mean colony counts after inoculation in experimental femurs were significantly lower than those of the control group, with a reduction percentage of 76 % (p = 0.041). A similar difference between control and experimental pins was observed at 24 h using an inoculum dose <104 colony-forming units (CFU), for which the reduction percentage was 70.48 % (p = 0.017). CONCLUSION: The irradiated surface of Ti6Al4V is able to reduce early bacterial colonization of Ti6AlV pins located in the medullar channel and in the surrounding femur. The reductions depend on the initial inoculums used to cause infection in the animals and the greatest effects are detected for inoculums <104 CFU. LEVEL OF EVIDENCE: Not applicable.

Dendronized Anionic Gold Nanoparticles: Synthesis, Characterization, and Antiviral Activity.

Anionic carbosilane dendrons decorated with sulfonate functions and one thiol moiety at the focal point have been used to synthesize water-soluble gold nanoparticles (AuNPs) through the direct reaction of dendrons, gold precursor, and reducing agent in water, and also through a place-exchange reaction. These nanoparticles have been characterized by NMR spectroscopy, TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy (XPS), UV/Vis spectroscopy, elemental analysis, and zeta-potential measurements. The interacting ability of the anionic sulfonate functions was investigated by EPR spectroscopy with copper(II) as a probe. Different structures and conformations of the AuNPs modulate the availability of sulfonate and thiol groups for complexation by copper(II). Toxicity assays of AuNPs showed that those produced through direct reaction were less toxic than those obtained by ligand exchange. Inhibition of HIV-1 infection was higher in the case of dendronized AuNPs than in dendrons.

Electrochemical analysis of the UV treated bactericidal Ti6Al4V surfaces.

This research investigates in detail the bactericidal effect exhibited by the surface of the biomaterial Ti6Al4V after being subjected to UV-C light. It has been recently hypothesized that small surface currents, occurring as a consequence of the electron-hole pair recombination taking place after the excitation process, are behind the bactericidal properties displayed by this UV-treated material. To corroborate this hypothesis we have used different electrochemical techniques, such as electrochemical impedance spectroscopy (EIS), potentiodynamic polarization plots and Mott-Schottky plots. EIS and Mott-Schottky plots have shown that UV-C treatment causes an initial increase on the surface electrical conduction of this material. In addition, EIS and polarization plots demonstrated that higher corrosion currents occur at the UV treated than at the non-irradiated samples. Despite this increase in the corrosion currents, EIS has also shown that such currents are not likely to affect the good stability of this material oxide film since the irradiated samples completely recovered the control values after being stored in dark conditions for a period not longer than 24h. These results agree with the already-published in vitro transitory behavior of the bactericidal effect, which was shown to be present at initial times after the biomaterial implantation, a crucial moment to avoid a large number of biomaterial associated infections.

Surface-dependent mechanical stability of adsorbed human plasma fibronectin on Ti6Al4V: domain unfolding and stepwise unraveling of single compact molecules.

In this study, the structure and mechanical stability of human plasma fibronectin (HFN), a major protein component of blood plasma, have been evaluated in detail upon adsorption on the nonirradiated and irradiated Ti6Al4V material through the use of atomic force microscopy. The results indicated that the material surface changes occurring after the irradiation process reduce the disulfide bonds that typically preclude the mechanical denaturation of individual HFN domains and interfere significantly with the intraionic interactions stabilizing the compact conformation of the adsorbed HFN molecules. In particular, upon adsorption on this material, the molecules adopt a more flexible conformation and become mechanically more compliant. Unexpected observations also indicated that, regardless the material surface, a single HFN molecule can be pulled into an extended conformation without the unfolding of its domains through a series of three unraveling steps. The forces involved in the unraveling process were found to be generally lower than the forces required to unfold the individual protein domains. This report is the first one to present the force displacement details associated to the straightening of a single compact protein at the molecular level.

Adsorption behavior of human plasma fibronectin on hydrophobic and hydrophilic Ti6Al4V substrata and its influence on bacterial adhesion and detachment.

Biomaterial implant-associated infections, a common cause of medical devices' failure, are initiated by bacterial adhesion to an adsorbed protein layer on the implant material surface. In this study, the influence of protein surface orientation on bacterial adhesion has been examined using three clinically relevant bacterial strains known to express specific binding sites for human plasma fibronectin (HFN). HFN was allowed to adsorb on hydrophobic Ti6Al4V and physically modified hydrophilic Ti6Al4V substrata. Ellipsometric data reveal that the characteristics of the adsorbed protein layers primary depend on solid surface tension and the initial protein concentration in solution. In particular, HFN molecules adopt a more extended conformation on hydrophobic than hydrophilic surfaces, an effect that is more pronounced at low than at high initial protein concentrations. Moreover, the extended conformation of the protein molecules on these surfaces likely facilitates the exposure of specific sites for adhesion, resulting in the higher bacterial-cell attachment observed regardless of the strain considered. Contact angle measurements and the analysis of the number of remaining adhering cells after being subjected to external forces further suggest that both specific and nonspecific (hydrophobic) interactions play an important role on bacterial attachment. This study is the first one to evaluate the influence of surface hydrophobicity on protein adsorption and its subsequent effect on bacterial adhesion using a material whose hydrophobicity was not modified using chemical treatments that potentially led to surface properties changes other than hydrophobicity.

The zeta potential of extended dielectrics and conductors in terms of streaming potential and streaming current measurements.

The electrical characterization of surfaces in terms of the zeta potential (ζ), i.e., the electric potential contributing to the interaction potential energy, is of major importance in a wide variety of industrial, environmental and biomedical applications in which the integration of any material with the surrounding media is initially mediated by the physico-chemical properties of its outer surface layer. Among the different existing electrokinetic techniques for obtaining ζ, streaming potential (V(str)) and streaming current (I(str)) are important when dealing with flat-extended samples. Mostly dielectric materials have been subjected to this type of analysis and only a few papers can be found in the literature regarding the electrokinetic characterization of conducting materials. Nevertheless, a standardized procedure is typically followed to calculate ζ from the measured data and, importantly, it is shown in this paper that such a procedure leads to incorrect zeta potential values when conductors are investigated. In any case, assessment of a reliable numerical value of ζ requires careful consideration of the origin of the input data and the characteristics of the experimental setup. In particular, it is shown that the cell resistance (R) typically obtained through a.c. signals (R(a.c.)), and needed for the calculations of ζ, always underestimates the zeta potential values obtained from streaming potential measurements. The consideration of R(EK), derived from the V(str)/I(str) ratio, leads to reliable values of ζ when dielectrics are investigated. For metals, the contribution of conductivity of the sample to the cell resistance provokes an underestimation of R(EK), which leads to unrealistic values of ζ. For the electrical characterization of conducting samples I(str) measurements constitute a better choice. In general, the findings gathered in this manuscript establish a measurement protocol for obtaining reliable zeta potentials of dielectrics and conductors based on the intrinsic electrokinetic behavior of both types of samples.

Insights into bacterial contact angles: difficulties in defining hydrophobicity and surface Gibbs energy.

One of the principal techniques for evaluating the surface hydrophobicity of biological samples is contact angle. This method, applied readily to flat-smooth-inert surfaces, gives rise to an important debate when implemented with microbial lawns. After its initial description, in 1984, several authors have carried out modifications of the technique but the results obtained have not been previously judged. This work focuses on the particularities of contact angle measurements on bacterial lawns and enhances the idea of the impossibility of using water contact angle as a universal measurement of bacterial hydrophobicity. Contact angles can only be used as relative indicators of hydrophobicity, in a similar way to tests based on microbial adhesion to solvents. The strong dependence of contact angles on dried bacterial lawns with measuring time and environmental conditions (e.g. pH of the media) preclude the estimation of their absolute values, and so, of the cells surface Gibbs energy. Yet, for a given measuring time, it is found that the hydrophobicity and the apparent bacterial surface Gibbs energy components are qualitatively related to the bacterial surface electrical potential. In particular, a hydrophobic increase is always accompanied by an increase of the cells Lifshitz-Van der Waals component and a decrease of their acid-base component and absolute zeta potential. Therefore, the present study shows that the physico-chemical surface properties that characterize bacteria are not independent, and one of them can be qualitatively described in terms of the others when measuring contact angles at a fixed time after the drying of the microbial beds.

In vitro biocompatibility and bacterial adhesion of physico-chemically modified Ti6Al4V surface by means of UV irradiation.

UV irradiation leads to a "spontaneous" wettability increase of the Ti6Al4V surface while preserving bulk properties of the alloy that are crucial for its performance as an orthopedic and dental implant. We hypothesized that UV treatment of Ti6Al4V may impair bacterial adhesion without compromising the good response of human bone-forming cells to this alloy. The in vitro biocompatibility of the Ti6Al4V surface, before and after UV irradiation, was analyzed by using human cells related to the osteoblastic phenotype. The adhesion processes of bacterial strains related to clinical orthopedic infections, i.e., Staphylococcus aureus and Staphylococcus epidermidis, were studied theoretically and in vitro, under dynamic and static conditions as well as in the presence or absence of shear forces. While human cell adhesion was not altered by UV irradiation of Ti6Al4V alloy, this treatment reduced not only initial bacterial adhesion rates but also the number of bacteria retained on the surface after the passage of two air-liquid interfaces on the previously adhered bacteria. This study proposes the use of UV treatment prior to implantation protocols as an easy, economic and effective way of reducing bacterial adhesion on the Ti6Al4V surface without compromising its excellent biocompatibility.

Atomic force microscopy of mechanically trapped bacterial cells.

This article presents a study on the influence of the protocol used for immobilization of bacterial cells onto surfaces by mechanically trapping them into a filter. In this sense, the surface and structure of trapped cells are analyzed. Bacteria can be present solely or with extracellular polymeric substances (EPS). To test the behavior of the EPS layer duing the filtering process, different strains of a well-known EPS-producer bacteria (Staphylococcus epidermidis), which produce an extracellular matrix clearly visible in AFM images, have been used. Results show that this immobilization method can cause severe structural and mechanical deformation to the cell membrane. This altered mechanical state may possibly influence the parameters derived from AFM force curves (which are micro/nano-mechanical tests). Also, our results suggest that the EPS layer might move during the filtering process and could accumulate at the upper part of the cell, thus favoring distorted data of adhesion/pull-off forces as measured by an AFM tip, especially in the case of submicron-sized microbial cells such as bacteria.

Nano-mechanical exploration of the surface and sub-surface of hydrated cells of Staphylococcus epidermidis.

The surface of hydrated cells of Staphylococcus epidermidis has been probed using an atomic force microscope. While local force measurements over the surface of bacteria reveal a heterogeneous chemical surface, with heterogeneous mechanical properties, different kinds of force curves appear with high frequency, and are thought to provide information on features contributing strongly to the overall mechanical and surface behaviour of the cell. Force curves often present two different mechanical regimes, being the first one (outer) of about 48 nm thick, and presenting a local relative elasticity of about 0.08 N/m, which is about a third of the relative elasticity of the inner part of the cell wall, harder, with a relative elasticity of about 0.24 N/m, in water. Both regimes appears as straight lines in the force versus distance curves (the 'corresponding' stress-strain curves in contact mechanics), but hysteresis is observed between the approach and the retraction line in the inner regime, indicating a degree of viscoelasticity. No viscoelasticity is observed in the outer regime, however, which presents quite linear and juxtaposed approach-retraction lines. These kinds of force curves do not present measurable pull-off forces nor snap-in forces, which indicates an almost null interaction between tip and bacterial surface, which could be in agreement with the measured very high hydrophobicity of this strain. Another kind of force curve has been observed recurrently, showing peaks in the retraction curves. Adhesive pull-off forces were measured giving an average of about 2 nN. Interestingly, however, these force curves appear only when quite irregular and wavy retraction curves are present, from the very beginning of its trace (maximum indentation). This leads us to think that these pull-off forces measured by our AFM do not give information on surface forces-unbinding events at the surface of the bacteria, but could be related to events at the sub-surface of the cell surface. Oscillations seen in the retraction curve in the portion corresponding to the contact with the bacteria surface could be due to rupture phenomena within the multilayered cell wall architecture expected in Gram-positive bacteria as Staphylococcus epidermidis, which could result in local irreversible deformations of the cell surface. Imaging with a sharp tip in contact mode sometimes leads to surface damage. Force curves recorded over damaged parts of the cell surface showed a completely different behaviour, in many cases with two well-defined high-adhesion peaks, and also interestingly, with snap-in forces of about 0-2 nN, which seems to indicate a completely different electrical/hydrophobicity state only a few nanometers down from the surface. Similar indentation effects can occur in the contact of a bacterial cell with a solid surface, even when showing only atomic-molecular-scale roughness, thus interacting not only with the very surface of the cell, especially when soft layers are present in the outer. Our results highlight the importance of the cell surface mechanical properties and their interplay with purely surface properties when analyzing cell-material interaction, and show the AFM as a useful method for investigating this.

Serum as a factor influencing adhesion of Enterococcus faecalis to glass and silicone.

The purpose of this work was to analyze the effect of serum on the physicochemical surface properties and adhesion to glass and silicone of Enterococcus faecalis ATCC 29212 at 37 degrees C. As is presented using thermodynamics analysis, serum minimizes the interaction of cells with water, which correlates well with the increase in hydrophobicity and in bacterial adhesion to glass and silicone.

Thermodynamic analysis of growth temperature dependence in the adhesion of Candida parapsilosis to polystyrene.

The purpose of this work was to study the adhesion to polystyrene of two Candida parapsilosis strains, grown at 22 and 37 degrees C, in terms of hydrophobicity, surface charge, and interaction free energy. Growth temperature changed the surface properties of microorganisms, yielding a good correlation between thermodynamic predictions and adhesion behavior.