The increasing bacterial resistance to antibiotics is prompting research into new approaches to design bacterial repellent surfaces. This work investigated the hot embossing of sub-micron direct laser interference patterning (DLIP) and laser-induced periodic surface structures (LIPSS) textures from stainless steel onto polypropylene samples to achieve bacterial repellent properties. Staphylococcus aureus bacteria were adhered to the textured surfaces, and adhered bacterial counts were compared to untextured polypropylene using colony-forming unit and scanning electron microscopy analysis. Both DLIP and LIPSS textures, with a periodicity of around 700 nm, significantly reduced bacterial colonization compared to untextured samples. These findings highlight the potential of DLIP and LIPSS textures as effective strategies for developing antimicrobial polymer materials.

Control of adhesion is a striking feature of living matter that is of particular interest regarding technological translation(1-3). We discovered that entropic repulsion caused by interfacial orientational fluctuations of cholesterol layers restricts protein adsorption and bacterial adhesion. Moreover, we found that intrinsically adhesive wax ester layers become similarly antibioadhesive when containing small quantities (under 10 wt%) of cholesterol. Wetting, adsorption and adhesion experiments, as well as atomistic simulations, showed that repulsive characteristics depend on the specific molecular structure of cholesterol that encodes a finely balanced fluctuating reorientation at the interface of unconstrained supramolecular assemblies: layers of cholesterol analogues differing only in minute molecular variations showed markedly different interfacial mobility and no antiadhesive effects. Also, orientationally fixed cholesterol layers did not resist bioadhesion. Our insights provide a conceptually new physicochemical perspective on biointerfaces and may guide future material design in regulation of adhesion.

The discovery of novel structural classes of antibiotics is urgently needed to address the ongoing antibiotic resistance crisis1-9. Deep learning approaches have aided in exploring chemical spaces1,10-15; these typically use black box models and do not provide chemical insights. Here we reasoned that the chemical substructures associated with antibiotic activity learned by neural network models can be identified and used to predict structural classes of antibiotics. We tested this hypothesis by developing an explainable, substructure-based approach for the efficient, deep learning-guided exploration of chemical spaces. We determined the antibiotic activities and human cell cytotoxicity profiles of 39,312 compounds and applied ensembles of graph neural networks to predict antibiotic activity and cytotoxicity for 12,076,365 compounds. Using explainable graph algorithms, we identified substructure-based rationales for compounds with high predicted antibiotic activity and low predicted cytotoxicity. We empirically tested 283 compounds and found that compounds exhibiting antibiotic activity against Staphylococcus aureus were enriched in putative structural classes arising from rationales. Of these structural classes of compounds, one is selective against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci, evades substantial resistance, and reduces bacterial titres in mouse models of MRSA skin and systemic thigh infection. Our approach enables the deep learning-guided discovery of structural classes of antibiotics and demonstrates that machine learning models in drug discovery can be explainable, providing insights into the chemical substructures that underlie selective antibiotic activity.

Bacterial colonization occurs on all biological and artificial surfaces in the oral cavity. The formation of multicellular biofilms or settlement of misallocated species can cause caries (diet‐depending acidification), periodontal diseases, peri‐implantitis, or denture‐associated stomatitis. Inhibition or delay of initial adhesion should strongly reduce dental interventions. However, no strategy is found to mildly manage bacterial colonization in this complex physiological environment, that is, without toxic, antibacterial, or antiseptic approaches. It is shown in a previous study that micro‐ and submicrometer surface topographies, which can effectively control initial bacterial adhesion in other application areas, do not have any significant impact within the oral cavity. Herein, a simple approach is presented to reduce initial microbial surface colonization by plain biphasic textures with defined combinations of hydrophobic and hydrophilic phases (SiO 2 , NH 2 , CH 2 , CH 3 , F 3 ). A significant reduction of microbial adhesion on textures in the nano‐ and microscale (150 nm and 2.4 μm) compared to respective monophasic substrates is observed. This might be a new design principle for dental materials, to inhibit microbial colonization in critical scenarios, at least for shorter time scales (<24 h).

Extensive biofilm formation on materials used in restorative dentistry is a common reason for their failure and the development of oral diseases like peri-implantitis or secondary caries. Therefore, novel materials and strategies that result in reduced biofouling capacities are urgently sought. Previous research suggests that surface structures in the range of bacterial cell sizes seem to be a promising approach to modulate bacterial adhesion and biofilm formation. Here we investigated bioadhesion within the oral cavity on a low surface energy material (perfluorpolyether) with different texture types (line-, hole-, pillar-like), feature sizes in a range from 0.7–4.5 µm and graded distances (0.7–130.5 µm). As a model system, the materials were fixed on splints and exposed to the oral cavity. We analyzed the enzymatic activity of amylase and lysozyme, pellicle formation, and bacterial colonization after 8 h intraoral exposure. In opposite to in vitro experiments, these in situ experiments revealed no clear signs of altered bacterial surface colonization regarding structure dimensions and texture types compared to unstructured substrates or natural enamel. In part, there seemed to be a decreasing trend of adherent cells with increasing periodicities and structure sizes, but this pattern was weak and irregular. Pellicle formation took place on all substrates in an unaltered manner. However, pellicle formation was most pronounced within recessed areas thereby partially masking the three-dimensional character of the surfaces. As the natural pellicle layer is obviously the most dominant prerequisite for bacterial adhesion, colonization in the oral environment cannot be easily controlled by structural means.

Nanocrystal micro/nanoarrays with multiplexed functionalities are of broad interest in the field of nanophotonics, cellular dynamics, and biosensing due to their tunable electrical and optical properties. This work focuses on the multicolor patterning of two-dimensional nanoplatelets (NPLs) via two sequential self-assembly and direct electron-beam lithography steps. By using scanning electron microscopy, atomic force microscopy, and fluorescence microscopy, we demonstrate the successful fabrication of fluorescent nanoarrays with a thickness of only two or three monolayers (7-11 nm) and a feature line width of ∼40 nm, which is three to four NPLs wide. To this end, first, large-area thin films of red-emitting CdSe/ZnyCd1-yS and green-emitting CdSe1-xSx/ZnyCd1-yS core/shell NPLs are fabricated based on Langmuir-type self-assembly at the liquid/air interface. By varying the concentration of ligands in the subphase, we investigate the effect of interaction potential on the film's final characteristics to prepare thin superlattices suitable for the patterning step. Equipped with the ability to fabricate a uniform superlattice with a controlled thickness, we next perform nanopatterning on a thin film of NPLs utilizing a direct electron-beam lithography (EBL) technique. The effect of acceleration voltage, aperture size, and e-beam dosage on the nanopattern's resolution and fidelity is investigated for both of the presented NPLs. After successfully optimizing EBL parameters to fabricate single-color nanopatterns, we finally focus on fabricating multicolor patterns. The obtained micro/nanoarrays provide us with an innovative experimental platform to investigate biological interactions as well as Förster resonance energy transfer.

Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to β-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating β-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features—turgor pressure, mechanosensitive channels, and cell shape changes—that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to β-lactams aimed at targeting cellular turgor.

The in situ synthesis of biomolecules on glass surfaces for direct bioscreening can be a powerful tool in the fields of pharmaceutical sciences, biomaterials, and chemical biology. However, it is still challenging to 1) achieve this conventional multistep combinatorial synthesis on glass surfaces with small feature sizes and high yields and 2) develop a surface which is compatible with solid-phase syntheses, as well as the subsequent bioscreening. This work reports an amphiphilic coating of a glass surface on which small droplets of polar aprotic organic solvents can be deposited with an enhanced contact angle and inhibited motion to permit fully automated multiple rounds of the combinatorial synthesis of small-molecule compounds and peptides. This amphiphilic coating can be switched into a hydrophilic network for protein- and cell-based screening. Employing this in situ synthesis method, chemical space can be probed via array technology with unprecedented speed for various applications, such as lead discovery/optimization in medicinal chemistry and biomaterial development.

BACKGROUND:All soft and solid surface structures in the oral cavity are covered by the acquired pellicle followed by bacterial colonization. This applies for natural structures as well as for restorative or prosthetic materials; the adherent bacterial biofilm is associated among others with the development of caries, periodontal diseases, peri-implantitis, or denture-associated stomatitis. Accordingly, there is a considerable demand for novel materials and coatings that limit and modulate bacterial attachment and/or propagation of microorganisms.OBJECTIVES AND FINDINGS:The present paper depicts the current knowledge on the impact of different physicochemical surface characteristics on bioadsorption in the oral cavity. Furthermore, it was carved out which strategies were developed in dental research and general surface science to inhibit bacterial colonization and to delay biofilm formation by low-fouling or "easy-to-clean" surfaces. These include the modulation of physicochemical properties such as periodic topographies, roughness, surface free energy, or hardness. In recent years, a large emphasis was laid on micro- and nanostructured surfaces and on liquid repellent superhydrophic as well as superhydrophilic interfaces. Materials incorporating mobile or bound nanoparticles promoting bacteriostatic or bacteriotoxic properties were also used. Recently, chemically textured interfaces gained increasing interest and could represent promising solutions for innovative antibioadhesion interfaces. Due to the unique conditions in the oral cavity, mainly in vivo or in situ studies were considered in the review.CONCLUSION:Despite many promising approaches for modulation of biofilm formation in the oral cavity, the ubiquitous phenomenon of bioadsorption and adhesion pellicle formation in the challenging oral milieu masks surface properties and therewith hampers low-fouling strategies.CLINICAL RELEVANCE:Improved dental materials and surface coatings with easy-to-clean properties have the potential to improve oral health, but extensive and systematic research is required in this field to develop biocompatible and effective substances.

Textured implant surfaces with micrometer and sub-micrometer features can improve contact properties like cell adhesion and bacteria repellency. A critical point of these surfaces is their mechanical stability during implantation. Therefore, strategies capable to provide both biocompatibility for an improved implant healing and resistance to wear for protecting the functional surface are required. In this work, laser-based fabrication methods have been used to produce hierarchical patterns on titanium surfaces. Using Direct Laser Writing with a nanosecond pulsed laser, crater-like structures with a separation distance of 50 µm are produced on unpolished titanium surfaces. Directly on this texture, a hole-like pattern with 5 µm spatial period is generated using Direct Laser Interference Patterning with picosecond pulses. While the smaller features should reduce the bacterial adhesion, the larger geometry was designed to protect the smaller features from wear. On the multifunctional surface, the adherence of E. Coli bacteria is reduced by 30% compared to the untreated reference. In addition, wear test performed on the multiple-scale patterns demonstrated the possibility to protect the smaller features by the larger craters. Also, the influence of the laser treatment on the growth of a titanium oxide layer was evaluated using Energy Dispersive X-Ray Spectroscopy analysis.

Considering the attractive surface functionalities of springtails (Collembola), an attempt at mimicking their cuticular topography on metals is proposed. An efficient single-step manufacturing process has been considered, involving laser-induced periodic surface structures (LIPSS) generated by near-infrared femtosecond laser pulses. By investigating the influence of number of pulses and pulse fluence, extraordinarily uniform triangular structures were fabricated on stainless steel and titanium alloy surfaces, resembling the primary comb-like surface structure of springtails. The laser-textured metallic surfaces exhibited hydrophobic properties and light scattering effects that were considered in this research as a potential in-line process monitoring solution. The possibilities to increase the processing throughput by employing high repetition rates in the MHz-range are also investigated.

Functionalized metallic nanofeatures can be selectively fabricated via ultrashort laser processing; however, the cost-effective large-area texturing, intrinsically constrained by the diffraction limit of light, remains a challenging issue. A high-intensity near-field phenomenon that takes place when irradiating microsized spheres, referred to as photonic nanojet (PN), was investigated in the transitional state between geometrical optics and dipole regime to fabricate functionalized metallic subwavelength features. Finite element simulations were performed to predict the PN focal length and beam spot size, and nanofeature formation. A systematic approach was employed to functionalize metallic surface by varying the pulse energy, focal offset, and number of pulses to fabricate controlled array of nanoholes and to study the generation of triangular and rhombic laser-induced periodic surface structures (LIPSS). Finally, large-area texturing was investigated to minimize the dry laser cleaning (DLC) effect and improve homogeneity of PN-assisted texturing. Tailored dimensions and densities of achievable surface patterns could provide hexagonal light scattering and selective optical reflectance for a specific light wavelength. Surfaces exhibited controlled wetting properties with either hydrophilicity or hydrophobicity. No correlation was found between wetting and microbacterial colonization properties of textured metallic surfaces after 4 h incubation of Escherichia coli. However, an unexpected bacterial repellency was observed.

Springtails (Collembola) have a nanostructured cuticle. To evaluate and to understand anti-biofouling properties of springtail cuticles’ morphology under different conditions, springtails, shed cuticles and cuticle replicates were studied after incubation with protein solutions and bacterial cultures using common in vitro models. In a second step, they were exposed to human oral environment in situ in order to explore potential application in dentistry. In vitro , the cuticular structures were found to resist wetting by albumin solutions for up to 3 h and colonization by Staphylococcus epidermidis was inhibited. When exposed in the oral cavity, initial pellicle formation was of high heterogeneity: parts of the surface were coated by adsorbed proteins, others remained uncoated but exhibited locally attached, ‘bridging’, proteinaceous membranes spanning across cavities of the cuticle surface; this unique phenomenon was observed for the first time. Also the degree of bacterial colonization varied considerably. In conclusion, the springtail cuticle partially modulates bioadhesion in the oral cavity in a unique and specific manner, but it has no universal effect. Especially after longer exposure, the nanotextured surface of springtails is masked by the pellicle, resulting in subsequent bacterial colonization, and, thus, cannot effectively avoid bioadhesion in the oral cavity comprehensively. Nevertheless, the observed phenomena offer valuable information and new perspectives for the development of antifouling surfaces applicable in the oral cavity.

Event Abstract Back to Event Impact of structure-dimensions on initial and short-term bacterial adhesion Ralf Helbig1*, Jens Friedrichs1, Andrés Lasagni2* and Carsten Werner1* 1 Leibniz Institue of Polymer Research, Institute of Biofunctional Polmer Materials, Germany 2 Dresden University of Technology, Large Area Based Surface micro-/nano-Structuring, Germany Introduction: Bacterial biofilms on man-made surfaces can cause a multitude of negative consequences in industrial and medical environments. A central process in the formation of biofilms is the initial transition of planktonic bacteria to a sessile state by attaching to a given substrate. Besides environmental conditions, such as pH, temperature, competing organisms and nutrition, the initial interactions between bacteria and substrate are mainly influenced by interfacial properties such as chemistry, polarity, mechanical properties and structure. Previous studies have demonstrated fundamental effects of substrate topography on the initial adhesion phase although a comprehensive picture is still missing. Here we applied periodically structured substrates with different structure dimensions, types and wetting properties to systematically study the effect of surface topography on bacterial adhesion. Material and Methods: Periodic line, post and hole structures were made by laser interference patterning from the slightly hydrophilic photoresist SU8. For each pattern geometry the spatial period was varied between 500 nm and 5000 nm. Surface wettability of the samples was varied by functionalization with aminosilan (APTES) or amorphous fluorpolymer TAF respectively. Structured samples were incubated in bacterial solutions (Staphylococcus epidermidis) at 37°C for 2 min or 24 hours with constant shaking. After the defined time periods samples were fixed, stained with DAPI and imaged. Results and Discussion: We could show that the initial interactions (minutes to hours) of bacterial cells with structured surfaces strongly depend on the structure dimensions (Figure 1). On structured surfaces with structure periods several times bigger than the cells size (i.e. 5000nm in this study) the number of adherent bacteria decreases (in comparison to an unstructured control) since cells are detached from structure tops due to increased shear forces induced by turbulent liquid flow. However, cells within depressions of the structure are protected from shear forces. For periods close to the cell size (i.e. 1000nm in this study), cell attachment increases because of a maximized net contact area between cells and surface. Smaller periods (i.e. 500nm in this study) decrease the net contact area between cells and surface resulting in increased cell retention. The observed effects were independent on the contact time between bacteria and substrate, the wetting properties of the substrate and the structure type. Conclusion: The presented results provide, for the first time, a comprehensive overview on the impact of surface topography on bacterial retention and may represent a foundation for the development of antifouling surfaces in the future. Figure 1: Schematic representation of the dependency of the structure period on initial bacterial attachment. Keywords: Bacteria, Cell Adhesion, nanotopography, Pattering Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: General Session Oral Topic: Nanotopography of biomaterials Citation: Helbig R, Friedrichs J, Lasagni A and Werner C (2016). Impact of structure-dimensions on initial and short-term bacterial adhesion. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01503 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Ralf Helbig, Leibniz Institue of Polymer Research, Institute of Biofunctional Polmer Materials, Dresden, Germany, Email1 Dr. Andrés Lasagni, Dresden University of Technology, Large Area Based Surface micro-/nano-Structuring, Dresden, Germany, Email2 Dr. Carsten Werner, Leibniz Institue of Polymer Research, Institute of Biofunctional Polmer Materials, Dresden, Germany, Email3 Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ralf Helbig Jens Friedrichs Andrés Lasagni Carsten Werner Google Ralf Helbig Jens Friedrichs Andrés Lasagni Carsten Werner Google Scholar Ralf Helbig Jens Friedrichs Andrés Lasagni Carsten Werner PubMed Ralf Helbig Jens Friedrichs Andrés Lasagni Carsten Werner Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Substrate topography can have profound effects on initial bacterial adhesion during biofilm formation. We applied Staphylococcus epidermidis and Escherichia coli cells onto periodically structured substrates with different structure dimensions, structure types and wetting properties. We found a strong dependence of cell retention on the structure dimensions of the applied substrates. Periodicities in the range of the cell size increased, whereas smaller periodicities decreased cell retention, independent of contact time (minutes to hours) and hydrophobicity. These novel insights on the role of surface topography on bacterial retention might facilitate the development of non-fouling surfaces in the future.

In the past 15 years, many efforts were made to create functionalized artificial surfaces showing special anti-bacterial and anti-biofouling properties. Thereby, the topography of medical relevant materials plays an important role. However, the targeted fabrication of promising surface structures like hole-, lamella-and pyramid-like patterns with feature sizes in the sub-micrometer range in a one-step process is still a challenge. Optical and e-beam lithography, molding and self-assembly layers show a great potential to design topographies for this purpose. At the same time, most of these techniques are based on sequential processes, require masks or molds and thus are very device relevant and time consuming. In this work, we present the Direct Laser Interference Patterning (DLIP) technology as a capable method for the fast, flexible and direct fabrication of periodic micrometer-and submicrometer structures. This method offers the possibility to equip large plain areas and curved devices with 1D, 2D and 3D patterns. Simple 1D (e.g. lines) and complex 3D (e.g. lamella, pillars) patterns with periodic distances from 0.5 mu m to 5 mu m were fabricated on polymeric materials (polyimide, polystyrene). Subsequently, we characterized the adhesion behavior of Staphylococcus epidermidis and S. aureus bacteria under in vitro and in vivo conditions. The results revealed that the topographies have a significant impact on bacteria adhesion. On the one side, one-dimensional line-like structures especially with dimensions of the bacteria enhanced microbe attachment. While on the other hand, complex three-dimensional patterns prevented biofilm formation even after implantation and contamination in living organisms.

Modification of the biomaterial surface topography is a promising strategy to prevent bacterial adhesion and biofilm formation. In this study, we use direct laser interference patterning (DLIP) to modify polystyrene surface topography at sub-micrometer scale. The results revealed that three-dimensional micrometer structures have a profound impact on bacterial adhesion. Thus, line- and pillar-like patterns enhanced S. aureus adhesion, whereas complex lamella microtopography reduced S. aureus adhesion in static and continuous flow culture conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus adhesion both when the surface is coated with human serum proteins and when the material is implanted subcutaneously in a foreign-body associated infection model.

Springtail skin morphology is translated into robust omniphobic polymer membranes by reverse imprint lithography. The combination of overhanging cross-sections and their arrangement in a self-supporting comblike pattern are crucial for mechanically stable coatings that can be even applied to curved surfaces.