Effect of APTES modified TiO2 on antioxidant enzymes activity secreted by Escherichia coli and Staphylococcus epidermidis.

In this work, the impact of APTES-modified TiO2 photocatalysts on antioxidant enzymes (catalase and superoxide dismutase) activity secreted by bacteria was presented. Microbial tests has been examined using Escherichia coli (ATCC 29425) and Staphylococcus epidermidis (ATCC 49461) as model organisms. It was found that APTES-TiO2 affected the activity of antioxidant enzymes. Additionally, obtained APTES-TiO2 photocatalysts were capable of total E. coli and S. epidermidis inactivation under artificial solar light irradiation. The sample modified with the concentration of APTES equals 300 mM (TiO2-4h-120°C-300mM) showed the strongest photocatalytic activity toward both bacteria species. The two-stage photocatalytic mechanism of bacteria response to photocatalysts was proposed.

Overview of Staphylococcus epidermidis cell wall-anchored proteins: potential targets to inhibit biofilm formation.

Currently, the treatment of infections by Staphylococcus epidermidis (S. epidermidis) represents a challenge because some strains have multidrug-resistance to antimicrobial products (antibiotic and biocides) and can produce biofilms. These biofilms protect bacterial cells from both antimicrobials and the host immune response. Therefore, it is crucial to encourage research on the development of new treatments. One method is immunotherapy, targeting components of S. epidermidis, such as S. epidermidis surface (Ses) proteins. Ses is expressed constitutively in most strains, and they participate in biofilm formation. This review is an update on Ses, regarding their structure, biological function, their relationship with S. epidermidis biofilm formation, and its possible role as therapeutic targets to develop immunotherapeutic treatments to prevent infections by S. epidermidis.

Hierarchical Rose Petal Surfaces Delay the Early-Stage Bacterial Biofilm Growth.

A variety of natural surfaces exhibit antibacterial properties; as a result, significant efforts in the past decade have been dedicated toward fabrication of biomimetic surfaces that can help control biofilm growth. Examples of such surfaces include rose petals, which possess hierarchical structures like the micropapillae measuring tens of microns and nanofolds that range in the size of 700 ± 100 nm. We duplicated the natural structures on rose petal surfaces via a simple UV-curable nanocasting technique and tested the efficacy of these artificial surfaces in preventing biofilm growth using clinically relevant bacteria strains. The rose petal-structured surfaces exhibited hydrophobicity (contact angle (CA) ≈ 130.8° ± 4.3°) and high CA hysteresis (∼91.0° ± 4.9°). Water droplets on rose petal replicas evaporated following the constant contact line mode, indicating the likely coexistence of both Cassie and Wenzel states (Cassie-Baxter impregnating the wetting state). Fluorescence microscopy and image analysis revealed the significantly lower attachment of Staphylococcus epidermidis (86.1 ± 6.2% less) and Pseudomonas aeruginosa (85.9 ± 3.2% less) on the rose petal-structured surfaces, compared with flat surfaces over a period of 2 h. An extensive biofilm matrix was observed in biofilms formed by both species on flat surfaces after prolonged growth (several days), but was less apparent on rose petal-biomimetic surfaces. In addition, the biomass of S. epidermidis (63.2 ± 9.4% less) and P. aeruginosa (76.0 ± 10.0% less) biofilms were significantly reduced on the rose petal-structured surfaces, in comparison to the flat surfaces. By comparing P. aeruginosa growth on representative unitary nanopillars, we demonstrated that hierarchical structures are more effective in delaying biofilm growth. The mechanisms are two-fold: (1) the nanofolds across the hemispherical micropapillae restrict initial attachment of bacterial cells and delay the direct contact of cells via cell alignment and (2) the hemispherical micropapillae arrays isolate bacterial clusters and inhibit the formation of a fibrous network. The hierarchical features on rose petal surfaces may be useful for developing strategies to control biofilm formation in medical and industrial contexts.

Regularized reconstruction of absorbing and phase objects from a single in-line hologram, application to fluid mechanics and micro-biology.

Reconstruction of phase objects is a central problem in digital holography, whose various applications include microscopy, biomedical imaging, and fluid mechanics. Starting from a single in-line hologram, there is no direct way to recover the phase of the diffracted wave in the hologram plane. The reconstruction of absorbing and phase objects therefore requires the inversion of the non-linear hologram formation model. We propose a regularized reconstruction method that includes several physically-grounded constraints such as bounds on transmittance values, maximum/minimum phase, spatial smoothness or the absence of any object in parts of the field of view. To solve the non-convex and non-smooth optimization problem induced by our modeling, a variable splitting strategy is applied and the closed-form solution of the sub-problem (the so-called proximal operator) is derived. The resulting algorithm is efficient and is shown to lead to quantitative phase estimation on reconstructions of accurate simulations of in-line holograms based on the Mie theory. As our approach is adaptable to several in-line digital holography configurations, we present and discuss the promising results of reconstructions from experimental in-line holograms obtained in two different applications: the tracking of an evaporating droplet (size ∼ 100µm) and the microscopic imaging of bacteria (size ∼ 1µm).

A synthetic channel that efficiently inserts into mammalian cell membranes and destroys cancer cells.

Despite the fact that a large number of synthetic channels have been developed in the last three decades, few of them can function in mammalian cell membranes because of their weak membrane insertion abilities. This study describes a tubular molecule with terminal positively charged amino groups that displays a strong ability to insert into lipid bilayers composed of phosphatidylcholine and consequently forming unimolecular transmembrane channels. It has been demonstrated that the insertion of the channel into the phosphatidylcholine bilayers was driven by the electrostatic interaction between the positively charged amino groups of the channel molecules and the negatively charged phosphate groups of the lipid molecules. The high affinity of the channels for lipid bilayers led to efficient mammalian cell membrane insertion. The channels showed high effective activity against HepG2 cancer cells at concentrations above 5.1 µM.

Defining Molecular Details of the Chemistry of Biofilm Formation by Raman Microspectroscopy.

Two protocols that allow for the comparison of Raman spectra of planktonic cells and biofilm formed from these cells in their growth phase have been developed. Planktonic cells are washed and flash-frozen in <1 min to reduce the time for metabolic changes during processing, prior to freeze-drying. Biofilm is formed by standing cells in 50 µL indentations in aluminum foil in an atmosphere of saturated water vapor for 24-48 h. The results for Escherichia coli type K12 cells, which do not readily form biofilm, are compared to those for Staphylococcus epidermidis cells, which prolifically synthesize biofilm. For E. coli, the Raman spectra of the planktonic and biofilm samples are similar with the exception that the spectral signature of RNA, present in planktonic cells, could not be detected in biofilm. For S. epidermidis, major changes occur upon biofilm formation. In addition to the absence of the RNA features, new bands occur near 950 cm-1 and between 1350 and 1420 cm-1 that are associated with an increase in carbohydrate content. Unlike the case in E. coli biofilm, the intensity of G base ring modes is reduced in but A and T base ring signatures become more prominent. For S. epidermis in the biofilm's amide III region, there is evidence of an increase in the level of ß-sheet structure accompanied by a decrease in α-helical content. The presence of biofilm is confirmed by microscope-aided photography and, separately, by staining with methyl violet.

Synthetic Channel Specifically Inserts into the Lipid Bilayer of Gram-Positive Bacteria but not that of Mammalian Erythrocytes.

A series of tubular molecules with different lengths have been synthesized by attaching Trp-incorporated peptides to the pillar[5]arene backbone. The tubular molecules are able to insert into the lipid bilayer to form unimolecular transmembrane channels. One of the channels has been revealed to specifically insert into the bilayer of the Gram-positive bacteria. In contrast, this channel cannot insert into the membranes of the mammalian rat erythrocytes even at the high concentration of 100 µm. It was further demonstrated that, as a result of this high membrane selectivity, the channel exhibits efficient antimicrobial activity for the Gram-positive bacteria and very low hemolytic toxicity for mammalian erythrocytes.

Effect of texture of platelet bags on bacterial and platelet adhesion.

BACKGROUND: Missed detection of Staphylococcus epidermidis contamination in platelet (PLT) storage bags by the standard 24-hour-postcollection BacT/ALERT screening test has been documented. A slow growth rate and the strong tendency of this bacterium to adhere to surfaces can contribute to missed detection of the pathogen. STUDY DESIGN AND METHODS: Topography of two different PLT storage bag surfaces, textured (rough) and smooth surfaces of Terumo 80440 bags (designated A15), was studied. Adhesion of biofilm-positive and -negative S. epidermidis strains on these surfaces was evaluated under static conditions. Quality of stored PLTs in A15 bags under blood bank conditions was compared for two different bag orientations (rough vs. smooth surface down) on Days 2, 5, and 7 of storage. PLT adhesion on the surfaces was evaluated after 7 days of storage. RESULTS: Bacterial adhesion and biofilm formation were significantly higher on the rough surfaces of A15 bags compared to the smooth surfaces. After 7 days of storage in A15 bags, PLTs showed similar metabolite levels, pH, and response capacity in the bags with different orientation and more PLT adhesion and aggregation was observed on rough surfaces. CONCLUSION: Higher bacterial adhesion on rough surfaces can contribute to missed detection of bacterial strains that tend to adhere on surfaces. PLT adhesion and aggregation on rough surfaces can affect the quality and safety of PLTs by promoting more bacterial adhesion and biofilm formation on surfaces.

Automated analysis of food-borne pathogens using a novel microbial cell culture, sensing and classification system.

We hereby report the design and implementation of an Autonomous Microbial Cell Culture and Classification (AMC(3)) system for rapid detection of food pathogens. Traditional food testing methods require multistep procedures and long incubation period, and are thus prone to human error. AMC(3) introduces a "one click approach" to the detection and classification of pathogenic bacteria. Once the cultured materials are prepared, all operations are automatic. AMC(3) is an integrated sensor array platform in a microbial fuel cell system composed of a multi-potentiostat, an automated data collection system (Python program, Yocto Maxi-coupler electromechanical relay module) and a powerful classification program. The classification scheme consists of Probabilistic Neural Network (PNN), Support Vector Machines (SVM) and General Regression Neural Network (GRNN) oracle-based system. Differential Pulse Voltammetry (DPV) is performed on standard samples or unknown samples. Then, using preset feature extractions and quality control, accepted data are analyzed by the intelligent classification system. In a typical use, thirty-two extracted features were analyzed to correctly classify the following pathogens: Escherichia coli ATCC#25922, Escherichia coli ATCC#11775, and Staphylococcus epidermidis ATCC#12228. 85.4% accuracy range was recorded for unknown samples, and within a shorter time period than the industry standard of 24 hours.

Influence of Oxynitrided Surface in the Production of a Less Susceptible Titanium Surface to Skin-Borne Bacterial Adhesion.

There is a growing quest for an ideal biomaterial that shows appropriate cellular response and is not susceptible to microbial adhesion. In this study, commercial grade II titanium was submitted to RF/DC plasma surface modification at 2.2 mbar, using gas mixtures of argon, nitrogen, and oxygen at proportions 4:1:2 and 4:1:3. The surfaces were physically and chemically characterized. In order to evaluate bacterial response, the surfaces were exposed to Staphylococcus epidermidis. Oxynitrided samples, although having a higher roughness as compared with untreated samples, exhibited lower bacterial growth. This observation is probably due to the formation of different crystalline phases of nitrides and oxides caused by plasma treatment. The surface with highest contact angle and highest surface tension showed lower bacterial adhesion. These results were confirmed by scanning electron microscopy. The role of nitrogen in reducing bacterial adhesion is clear when this material is compared with untreated titanium, on which only an oxide film is present.

Effects of temperature on the morphological, polymeric, and mechanical properties of Staphylococcus epidermidis bacterial biofilms.

Changes in temperature were found to affect the morphology, cell viability, and mechanical properties of Staphylococcus epidermidis bacterial biofilms. S. epidermidis biofilms are commonly associated with hospital-acquired medical device infections. We observed the effect of heat treatment on three physical properties of the biofilms: the bacterial cell morphology and viability, the polymeric properties of the extracellular polymeric substance (EPS), and the rheological properties of the bulk biofilm. After application of a 1 h heat treatment at 45 °C, cell reproduction had ceased, and at 60 °C, cell viability was significantly reduced. Size exclusion chromatography was used to fractionate the extracellular polymeric substance (EPS) based on size. Chemical analysis of each fraction showed that the relative concentrations of the polysaccharide, protein, and DNA components of the EPS were unchanged by the heat treatment at 45 and 60 °C. The results suggest that the EPS molecular constituents are not significantly degraded by the temperature treatment. However, some aggregation on the scale of 100 nm was found by dynamic light scattering at 60 °C. Finally, relative to control biofilms maintained at 37 °C, we observed an order of magnitude reduction in the biofilm yield stress after 60 °C temperature treatment. No such difference was found for treatment at 45 °C. From these results, we conclude that the yield stress of bacterial biofilms is temperature-sensitive and that this sensitivity is correlated with cell viability. The observed significant decrease in yield stress with temperature suggests a means to weaken the mechanical integrity of S. epidermidis biofilms with applications in areas such as the treatment of biofilm-infected medical devices.

Staphylococcus epidermidis Affinity for Fibrinogen-Coated Surfaces Correlates with the Abundance of the SdrG Adhesin on the Cell Surface.

Staphylococcus epidermidis is a world-leading pathogen in healthcare facilities, mainly causing medical device-associated infections. These nosocomial diseases often result in complications such as bacteremia, fibrosis, or peritonitis. The virulence of S. epidermidis relies on its ability to colonize surfaces and develop thereupon in the form of biofilms. Bacterial adherence on biomaterials, usually covered with plasma proteins after implantation, is a critical step leading to biofilm infections. The cell surface protein SdrG mediates adhesion of S. epidermidis to fibrinogen (Fg) through a specific "dock, lock, and latch" mechanism, which results in greatly stabilized protein-ligand complexes. Here, we combine single-molecule, single-cell, and whole population assays to investigate the extent to which the surface density of SdrG determines the ability of S. epidermidis clinical strains HB, ATCC 35984, and ATCC 12228 to bind to Fg-coated surfaces. Strains that showed enhanced adhesion on Fg-coated polydimethylsiloxane (PDMS) were characterized by increased amounts of SdrG proteins on the cell surface, as observed by single-molecule analysis. Consistent with previous reports showing increased expression of SdrG following in vivo exposure, this work provides direct evidence that abundance of SdrG on the cell surface of S. epidermidis strains dramatically improves their ability to bind to Fg-coated implanted medical devices.

SERS detection of bacteria in water by in situ coating with Ag nanoparticles.

The bio-sensing for the convenient detection of bacteria has been widely explored with the use of various sensing materials and techniques. It is still a challenge to achieve an ultrasensitive and selective, but simple, rapid, and inexpensive detection method for bacteria. We report on surface-enhanced Raman scattering (SERS) for the detection of living bacteria in drinking water by employing a synthesis of silver nanoparticles coating the cell wall of bacteria. We found that the Raman signals intensity of bacteria after AgNP synthesis mainly depends on the zeta potential of the cell wall. The enhancement of the Raman signal of bacteria using this strategy is about 30-fold higher than that in the case of a simply mixed colloid-bacterial suspension. The total assay time required is only 10 min and the total reactants' volume needed to analyze bacteria in a real environment is as low as 1 mL. Particularly, only one droplet of 3 µL sample is necessary for each SERS measurement. Furthermore, we can use this novel strategy to discriminate three strains of Escherichia coli and one strain of Staphylococcus epidermidis by hierarchy cluster analysis. Finally, we can detect bacteria down to 2.5 × 10(2) cells/mL on a hydrophobic glass slide by SERS mapping. Thus, our detection method offers prominent advantages, such as reduced assay time, simple handling, low reactant volumes, small amount of sample, and higher sensitivity and selectivity compared to previously reported label free methods. This novel strategy may be extended to open an avenue for developing various SERS-based biosensors.

Viscous nature of the bond between adhering bacteria and substratum surfaces probed by atomic force microscopy.

Here we report on the viscous nature of the bond between adhering bacteria and a substratum surface. A tailor-made script was written for an atomic force microscope, that enabled a constant loading force of 1 or 5 nN to act for 30 s upon a bacterium compressed between a cantilever and a glass surface, while measuring its deformation. Time-dependent deformation was fitted to a one element Kelvin-Voigt analogue of the bond to yield a characteristic relaxation time and viscosity of the bond. Viscosities of streptococcal bonds were smaller (<20 kPa s) than those of staphylococcal bonds (>31 kPa s). Since staphylococci are relatively rich in extracellular polymeric substances, it can be inferred that the presence of extracellular polymeric substances yields the major contribution to the viscous response. The viscous nature of the bond between adhering bacteria and substratum surfaces provides the bacteria with more time to respond and protect themselves against external stresses.

Developing antifouling biointerfaces based on bioinspired zwitterionic dopamine through pH-modulated assembly.

The use of synthetic biomaterials as implantable devices typically is accompanied by considerable nonspecific adsorption of proteins, cells, and bacteria. These may eventually induce adverse pathogenic problems in clinical practice, such as thrombosis and biomaterial-associated infection. Thus, an effective surface coating for medical devices has been pursued to repel nonspecific adsorption from surfaces. In this study, we employ an adhesive dopamine molecule conjugated with zwitterionic sulfobetaine moiety (SB-DA), developed based on natural mussels, as a surface ligand for the modification of TiO2. The electrochemical study shows that the SB-DA exhibits fully reversible reduction-oxidation behavior at pH 3, but it is irreversible at pH 8. A contact angle goniometer and X-ray photoelectron spectroscopy were utilized to explore the surface hydration, chemical states, and bonding mechanism of SB-DA. The results indicate that the binding between hydroxyl groups of SB-DA and TiO2 converts from hydrogen bonds to bidentate binding upon the pH transition from pH 3 to 8. In order to examine the antifouling properties of SB-DA thin films, the modified substrates were brought into contact with bovine serum albumin and bacteria solutions. The fouling levels were monitored using a quartz crystal microbalance with dissipation sensor and fluorescence optical microscope. Tests showed that the sample prepared via the pH transition approach provides the best resistance to nonspecific adsorption due to the high coverage and stability of the SB-DA films. These findings support the mechanism of the pH-modulated assembly of SB-DA molecules, and for the first time we demonstrate the antifouling properties of the SB-DA to be comparable with traditional thiol-based zwitterionic self-assemblies. The success of modification with SB-DA opens an avenue for developing a biologically inspired surface chemistry and can have applications over a wide spectrum of bioapplications. The strategy of the pH transition can also be applied to other functional dopamine derivatives.

Forces driving the attachment of Staphylococcus epidermidis to fibrinogen-coated surfaces.

Cell surface proteins of bacteria play essential roles in mediating the attachment of pathogens to host tissues and, therefore, represent key targets for anti-adhesion therapy. In the opportunistic pathogen Staphylococcus epidermidis , the adhesion protein SdrG mediates attachment of bacteria to the blood plasma protein fibrinogen (Fg) through a binding mechanism that is not yet fully understood. We report the direct measurement of the forces driving the adhesion of S. epidermidis to Fg-coated substrates using single-cell force spectroscopy. We found that the S. epidermidis -Fg adhesion force is of ~150 pN magnitude and that the adhesion strength and adhesion probability strongly increase with the interaction time, suggesting that the adhesion process involves time-dependent conformational changes. Control experiments with mutant bacteria lacking SdrG and substrates coated with the Fg ß(6-20) peptide, instead of the full Fg protein, demonstrate that these force signatures originate from the rupture of specific bonds between SdrG and its peptide ligand. Collectively, our results are consistent with a dynamic, multi-step ligand-binding mechanism called "dock, lock, and latch".

Surface-grafted polysarcosine as a peptoid antifouling polymer brush.

Poly(N-substituted glycine) "peptoids" are a class of peptidomimetic molecules receiving significant interest as engineered biomolecules. Sarcosine (i.e., poly(N-methyl glycine)) has the simplest side chain chemical structure of this family. In this Article, we demonstrate that surface-grafted polysarcosine (PSAR) brushes exhibit excellent resistance to nonspecific protein adsorption and cell attachment. Polysarcosine was coupled to a mussel adhesive protein-inspired DOPA-Lys pentapeptide, which enabled solution grafting and control of the surface chain density of the PSAR brushes. Protein adsorption was found to decrease monotonically with increasing grafted chain densities, and protein adsorption could be completely inhibited above certain critical chain densities specific to different polysarcosine chain lengths. The dependence of protein adsorption on chain length and density was also investigated by a molecular theory. PSAR brushes at high chain length and density were shown to resist fibroblast cell attachment over a 7 week period, as well as resist the attachment of some clinically relevant bacterial strains. The excellent antifouling performance of PSAR may be related to the highly hydrophilic character of polysarcosine, which was evident from high-pressure liquid chromatography measurements of polysarcosine and water contact angle measurements of the PSAR brushes. Peptoids have been shown to resist proteolytic degradation, and polysarcosine could be produced in large quantities by N-carboxy anhydride polymerization. In summary, surface-grafted polysarcosine peptoid brushes hold great promise for antifouling applications.

Mussel-inspired anchoring for patterning cells using polydopamine.

This Article introduces a simple method of cell patterning, inspired by the mussel anchoring protein. Polydopamine (PDA), artificial polymers made from self-polymerization of dopamine (a molecule that resembles mussel-adhesive proteins), has recently been studied for its ability to make modifications on surfaces in aqueous solutions. We explored the interfacial interaction between PDA and poly(ethylene glycol) (PEG) using microcontact printing (µCP). We patterned PDA on several substrates such as glass, polystyrene, and poly(dimethylsiloxane) and realized spatially defined anchoring of mammalian cells as well as bacteria. We applied our system in investigating the relationship between areas of mammalian nuclei and that of the cells. The combination of PDA and PEG enables us to make cell patterns on common laboratorial materials in a mild and convenient fashion.

Adherence of Staphylococcus epidermidis to human endothelial cells is associated with a polysaccharidic component of its extracellular mucous layer.

Bacterial adherence to eukaryotic cells is highly contributing to microbial pathogenesis. Bacterial adhesins, macromolecules, and glycosaminoglycan chains of the endothelial cell surface have been implicated in staphylococcal attachment. Our research group has isolated an antigenic polysaccharidic component of Staphylococcus epidermidis extracellular layer, known as 20-kDa PS (PS), and showed that antibodies against this polysaccharide protect from infections. Therefore, the role of PS in S. epidermidis adherence to endothelial cells was studied. For this purpose we examined the impact of PS on the ability of two S. epidermidis strains (a PS-producing and a non-PS-producing strain) to adhere to human endothelial cells in the presence or absence of specific antibodies to this polysaccharide. Hence, it is established that exogenous chondroitin sulfate (CS) decreases, in part, the S. epidermidis' attachment to endothelial cells and the antagonistic binding effect of CS and PS was also studied. The results obtained demonstrate that PS facilitates the adherence of S. epidermidis to both strains. CS abolished the PS-induced adherence in PS-producing strain and partially in the non-PS-producing one. Conclusively, the adherence of S. epidermidis to human endothelial cells is associated with its extracellular PS component and it is suggested that the bacterial binding via glycosaminoglycan chains is an important mechanism underlining the PS-induced binding to endothelial cells.

Adhesive properties of Staphylococcus epidermidis probed by atomic force microscopy.

Mapping of the surface properties of Staphylococcus epidermidis and of biofilm forming bacteria in general is a key to understand their functions, particularly their adhesive properties. To gain a comprehensive view of the structural and chemical properties of S. epidermidis, four different strains (biofilm positive and biofilm negative strains) were analyzed using in situ atomic force microscopy (AFM). Force measurements performed using bare hydrophilic silicon nitride tips disclosed similar adhesive properties for each strain. However, use of hydrophobic tips showed that hydrophobic forces are not the driving forces for adhesion of the four strains. Rather, the observation of sawtooth force-distance patterns on the surface of biofilm positive strains documents the presence of modular proteins such as Aap that may mediate cell adhesion. Treatment of two biofilm positive strains with two chemical inhibitor compounds leads to a loss of adhesion, suggesting that AFM could be a valuable tool to screen for anti-adhesion molecules.