2D foam film coating of antimicrobial lysozyme amyloid fibrils onto cellulose nanopapers.

Amyloid fibrils made from inexpensive hen egg white lysozyme (HEWL) are bio-based, bio-degradable and bio-compatible colloids with broad-spectrum antimicrobial activity, making them an attractive alternative to existing small-molecule antibiotics. Their surface activity leads to the formation of 2D foam films within a loop, similar to soap films when blowing bubbles. The stability of the foam was optimized by screening concentration and pH, which also revealed that the HEWL amyloid foams were actually stabilized by unconverted peptides unable to undergo amyloid self-assembly rather than the fibrils themselves. The 2D foam film was successfully deposited on different substrates to produce a homogenous coating layer with a thickness of roughly 30 nm. This was thick enough to shield the negative charge of dry cellulose nanopaper substrates, leading to a positively charged HEWL amyloid coating. The coating exhibited a broad-spectrum antimicrobial effect based on the interactions with the negatively charged cell walls and membranes of clinically relevant pathogens (Staphylococcus aureus, Escherichia coli and Candida albicans). The coating method presented here offers an alternative to existing techniques, such as dip and spray coating, in particular when optimized for continuous production. Based on the facile preparation and broad spectrum antimicrobial performance, we anticipate that these biohybrid materials could potentially be used in the biomedical sector as wound dressings.

Topical application of Lactobacilli successfully eradicates Pseudomonas aeruginosa biofilms and promotes wound healing in chronic wounds.

Chronic wounds are difficult to treat due to the presence of biofilm which prevents wound healing. Pseudomonas aeruginosa is one of the most common pathogens found in chronic wounds and conventional treatment strategies have been ineffective in the eradication of its biofilm, without harming the surrounding healthy tissue at the same time. Here, we introduced an innovative approach applying the probiotic product Bio-K+ (containing three lactobacilli) topically as an antimicrobial and antibiofilm agent. We identified lactic acid as the main active component. While antibiotics and antiseptics such as silver-ions only demonstrated limited efficacy, Bio-K+ was able to completely eradicate mature P. aeruginosa biofilms established in an in-vitro and ex-vivo human skin model. Furthermore, it demonstrated biocompatibility in the co-culture with human dermal fibroblasts and accelerated the migration of fibroblasts in a cell migration assay promoting wound healing. To enhance clinical practicability, we introduced Bio-K+ into the hydrocolloid dressing Aquacel, achieving sustained release of lactic acid and biofilm eradication. This new treatment approach applying probiotics could represent a major improvement in the management of chronic wounds and can be extended in treating other biofilm-associated infections.

In Situ Investigation of Pseudomonas aeruginosa Biofilm Development: Interplay between Flow, Growth Medium, and Mechanical Properties of Substrate.

To better understand the impact of biomaterial mechanical properties and growth medium on bacterial adhesion and biofilm formation under flow, we investigated the biofilm formation ability of Pseudomonas aeruginosa in different media on polydimethylsiloxane (PDMS) of different stiffness in real time using a microfluidic platform. P. aeruginosa colonization was recorded with optical microscopy and automated image analysis. The bacterial intracellular level of cyclic diguanylate (c-di-GMP), which regulates biofilm formation, was monitored using the transcription of the putative adhesin gene (cdrA) as a proxy. Contrary to the previous supposition, we revealed that PDMS material stiffness within the tested range has negligible impact on biofilm development and biofilm structures, whereas culture media not only influence the kinetics of biofilm development but also affect the biofilm morphology and structure dramatically. Interestingly, magnesium rather than previously reported calcium was identified here to play a decisive role in the formation of dense P. aeruginosa aggregates and high levels of c-di-GMP. These results demonstrate that although short-term adhesion assays bring valuable insight into bacterial and material interactions, long-term evaluations are essential to better predict overall biofilm outcome. The microfluidic system developed here presents a valuable application potential for studying biofilm development in situ. .

Advanced antifouling and antibacterial hydrogels enabled by controlled thermo-responses of a biocompatible polymer composite.

To optimally apply antibiotics and antimicrobials, smart wound dressing conferring controlled drug release and preventing adhesions of biological objects is advantageous. Poly(N-isopropylacrylamide) (PNIPAAm), a conventional thermo-responsive polymer, and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), a typical antifouling polymer, have therefore potential to be fabricated as copolymers to achieve dual functions of thermo-responsiveness and antifouling. Herein, a hydrogel made of PNIPAM-co-PMPC was designed and loaded with octenidine, a widely applied antimicrobial agent for wound treatment, to achieve both antifouling and triggered drug release. The thermo-switch of the fabricated hydrogel allowed 25-fold more octenidine release at 37 °C (infected wound temperature) than at 30 °C (normal skin temperature) after 120 minutes, which led to at least a 3 lg reduction of the viable bacteria at 37 °C on artificially infected wounds. Furthermore, we pioneeringly assessed the antifouling property of the material in PBS buffer using single molecule/cell/bacterial force spectroscopy, and revealed that the fabricated hydrogel displayed distinctive antifouling properties against proteins, mammalian cells, and bacteria. This work demonstrated a promising design of a hydrogel applicable for preventing and treating wound infections. The concept of dual-functional materials can be envisaged for other clinical applications related to the prevention of biofilm-associated infections, such as urinary catheters, stents, and dental implants.

Robust Antibacterial Activity of Xanthan-Gum-Stabilized and Patterned CeO2-x-TiO2 Antifog Films.

Increased occurrence of antimicrobial resistance leads to a huge burden on patients, the healthcare system, and society worldwide. Developing antimicrobial materials through doping rare-earth elements is a new strategy to overcome this challenge. To this end, we design antibacterial films containing CeO2-x-TiO2, xanthan gum, poly(acrylic acid), and hyaluronic acid. CeO2-x-TiO2 inks are additionally integrated into a hexagonal grid for prominent transparency. Such design yields not only an antibacterial efficacy of ∼100% toward Staphylococcus aureus and Escherichia coli but also excellent antifog performance for 72 h in a 100% humidity atmosphere. Moreover, FluidFM is employed to understand the interaction in-depth between bacteria and materials. We further reveal that reactive oxygen species (ROS) are crucial for the bactericidal activity of E. coli through fluorescent spectroscopic analysis and SEM imaging. We meanwhile confirm that Ce3+ ions are involved in the stripping phosphate groups, damaging the cell membrane of S. aureus. Therefore, the hexagonal mesh and xanthan-gum cross-linking chains act as a reservoir for ROS and Ce3+ ions, realizing a long-lasting antibacterial function. We hence develop an antibacterial and antifog dual-functional material that has the potential for a broad application in display devices, medical devices, food packaging, and wearable electronics.

pH-responsive silica nanoparticles for the treatment of skin wound infections.

Chronic wounds are not only a burden for patients but also challenging for clinic treatment due to biofilm formation. Here, we utilized the phenomenon that chronic wounds possess an elevated local pH of 8.9 and developed pH-sensitive silica nanoparticles (SiNPs) to achieve a targeted drug release on alkaline wounds and optimized drug utility. Chlorhexidine (CHX), a disinfectant and antiseptic, was loaded into SiNPs as the model drug. The loaded CHX displayed a release 4 - 5 fold higher at pH 8.0 and 8.5 than at pH 6.5, 7.0 and 7.4. CHX-SiNPs furthermore exhibited a distinctive antibacterial activity at pH 8.0 and 8.5 against both Gram-negative and -positive bacterial pathogens, while no cytotoxicity was found according to cell viability analysis. The CHX-SiNPs were further formulated into alginate hydrogels to allow ease of use. The antibacterial efficacy of CHX-SiNPs was then studied with artificial wounds on ex vivo human skin. Treatment with CHX-SiNPs enabled nearly a 4-lg reduction of the viable bacterial cells, and the alginate formulated CHX-SiNPs led to almost a 3-lg reduction compared to the negative controls. The obtained results demonstrated that CHX-SiNPs are capable of efficient pH-triggered drug release, leading to high antibacterial efficacy. Moreover, CHX-SiNPs enlighten clinic potential towards the treatment of chronic wound infections. STATEMENT OF SIGNIFICANCE: A platform for controlled drug release at a relatively high pH value i.e., over 8, was established by tuning the physical structures of silica nanoparticles (SiNPs). Incorporation of chlorhexidine, an antimicrobial agent, into the fabricated SiNPs allowed a distinctive inhibition of bacterial growth at alkaline pHs, but not at acidic pHs. The efficacy of the SiNPs loaded with chlorhexidine in treating wound infections was further validated by utilizing ex vivo human skin samples. The presented work demonstrates clinic potential of employing alkaline pH as a non-invasive stimulus to achieve on-demand delivery of antimicrobials through SiNPs, showcasing a valuable approach to treating bacterial infections on chronic wounds.

Self-Assembly Pathways and Antimicrobial Properties of Lysozyme in Different Aggregation States.

Antimicrobial resistance in microorganisms will cause millions of deaths and pose a vast burden on health systems; therefore, alternatives to existing small-molecule antibiotics have to be developed. Lysozyme is an antimicrobial enzyme and has broad-spectrum antimicrobial activity in different aggregated forms. Here, we propose a reductive pathway to obtain colloidally stable amyloid-like worm-shaped lysozyme nanoparticles (worms) from hen egg white lysozyme (HEWL) and compare them to amyloid fibrils made in an acid hydrolysis pathway. The aggregation of HEWL into worms follows strongly pH-dependent kinetics and induces a structural transition from α-helices to ß-sheets. Both HEWL worms and amyloid fibrils show broad-spectrum antimicrobial activity against the bacteria Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative), and the fungus Candida albicans. The colloidal stability of the worms allows the determination of minimum inhibitory concentrations, which are lower than that for native HEWL in the case of S. aureus. Overall, amyloid fibrils have the strongest antimicrobial effect, likely due to the increased positive charge compared to native HEWL. The structural and functional characterizations of HEWL worms and amyloids investigated herein are critical for understanding the detailed mechanisms of antimicrobial activity and opens up new avenues for the design of broad-spectrum antimicrobial materials for use in various applications.

Photo-activated titanium surface confers time dependent bactericidal activity towards Gram positive and negative bacteria.

Titanium (Ti)-based implants are broadly applied in the medical field, but their related infections can lead to implant failure. Photo-irradiation of metal materials to generate antimicrobial agents, an alternative to antibiotics, is a promising method to reduce bacterial infection and antibiotic usage. It is therefore important to understand how bacterial pathogens respond to Ti surfaces. Here, Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus, the most prevalent pathogens linked to healthcare-associated infections, were used as model strains. Two different kinds of Ti surfaces respectively stored in dry condition and 0.9 % NaCl solution were applied. Upon UV irradiation and in the absence of bacteria, both tested surfaces exhibited similar bactericidal activity, even though the surfaces stored in 0.9 % NaCl solution generated a slightly higher level of reactive oxygen species (ROS). Interestingly, P. aeruginosa and S. aureus responded to the irradiated Ti surfaces differently regarding interaction time: the number of viable P. aeruginosa was reduced up to 90 % after 30 min interaction with the treated surfaces compared to the untreated ones, but this reduction is lessened to 69 %-81 % after 240 min. By contrast, UV treatment of surfaces did not impact the viability of S. aureus after 30 min interaction, however, led to more than 99 % reduction after 240 min incubation. These results provide first experimental evidence that Gram negative and positive bacterial species respond to ROS with different inactivation kinetics. This work also demonstrated that treatment with photo-irradiation in the absence of bacteria conferred Ti surfaces with efficient bactericidal activity.

Bioresponsive Hybrid Nanofibers Enable Controlled Drug Delivery through Glass Transition Switching at Physiological Temperature.

To avoid excessive usage of antibiotics and antimicrobial agents, smart wound dressings permitting controlled drug release for treatment of bacterial infections are highly desired. In search of a sensitive stimulus to activate drug release under physiological conditions, we found that the glass transition temperature (Tg) of a polymer or polymer blend can be an ideal parameter because a thermal stimulus can regulate drug release at the physiological temperature of 37 °C. A well-tuned Tg for a controlled drug release from fibers at 37 °C was achieved by varying the blending ratio of Eudragit® RS 100 and poly(methyl methacrylate). Octenidine, an antimicrobial agent often used in wound treatment, was encapsulated into the polymer blend during the electrospinning process and evaluated for its controlled release based on modulation of temperature. The thermal switch of the nanofibrous membranes can be turned "on" at physiological temperature (37 °C) and "off" at room temperature (25 °C), conferring a controlled release of octenidine. It was found that octenidine can be released in an amount at least 8.5 times higher (25 mg·L-1) during the "on" stage compared to the "off" stage after 24 h, which was regulated by the wet Tg (34.8-36.5 °C). The "on"/"off" switch for controlled drug release can moreover be repeated at least 5 times. Furthermore, the fabricated nanofibrous membranes displayed a distinctive antibacterial activity, causing a log3 reduction of the viable cells for both Gram negative and positive pathogens at 37 °C, when the thermal switch was "on". This study forms the groundwork for a treatment concept where no external stimulus is needed for the release of antimicrobials at physiological conditions, and will help reduce the overuse of antibiotics by allowing controlled drug release.

Antibacterial, Cytocompatible, Sustainably Sourced: Cellulose Membranes with Bifunctional Peptides for Advanced Wound Dressings.

Progressive antibiotic resistance is a serious condition adding to the challenges associated with skin wound treatment, and antibacterial wound dressings with alternatives to antibiotics are urgently needed. Cellulose-based membranes are increasingly considered as wound dressings, necessitating further functionalization steps. A bifunctional peptide, combining an antimicrobial peptide (AMP) and a cellulose binding peptide (CBP), is designed. AMPs affect bacteria via multiple modes of action, thereby reducing the evolutionary pressure selecting for antibiotic resistance. The bifunctional peptide is successfully immobilized on cellulose membranes of bacterial origin or electrospun fibers of plant-derived cellulose, with tight control over peptide concentrations (0.2 ± 0.1 to 4.6 ± 1.6 µg mm-2 ). With this approach, new materials with antibacterial activity against Staphylococcus aureus (log4 reduction) and Pseudomonas aeruginosa (log1 reduction) are developed. Furthermore, membranes are cytocompatible in cultures of human fibroblasts. Additionally, a cell adhesive CBP-RGD peptide is designed and immobilized on membranes, inducing a 2.2-fold increased cell spreading compared to pristine cellulose. The versatile concept provides a toolbox for the functionalization of cellulose membranes of different origins and architectures with a broad choice in peptides. Functionalization in tris-buffered saline avoids further purification steps, allowing for translational research and multiple applications outside the field of wound dressings.

Role of the Surface Nanoscale Roughness of Stainless Steel on Bacterial Adhesion and Microcolony Formation.

Hospital-acquired infections can cause serious complications and are a severe problem because of the increased emergence of antibiotic-resistant bacteria. Biophysical modification of the material surfaces to prevent or reduce bacteria adhesion is an attractive alternative to antibiotic treatment. Since stainless steel is a widely used material for implants and in hospital settings, in this work, we used stainless steel to investigate the effect of the material surface topographies on bacterial adhesion and early biofilm formation. Stainless steel samples with different surface roughnesses Rq in a range of 217.9-56.6 nm (Ra in a range of 172.5-45.2 nm) were fabricated via electropolishing and compared for adhesion of bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. It was found that the number of viable cells on the untreated rough surface was at least 10-fold lower than those on the electropolished surfaces after 4 h of incubation time for P. aeruginosa and 15-fold lower for S. aureus. Fluorescence images and scanning electron microscopy images revealed that the bacterial cells tend to adhere individually as single cells on untreated rough surfaces. In contrast, clusters of the bacterial cells (microcolonies) were observed on electropolished smooth surfaces. Our study demonstrates that nanoscale surface roughness can play an important role in restraining bacterial adhesion and formation of microcolonies.

Nanostructured surface topographies have an effect on bactericidal activity.

BACKGROUND: Due to the increased emergence of antimicrobial resistance, alternatives to minimize the usage of antibiotics become attractive solutions. Biophysical manipulation of material surface topography to prevent bacterial adhesion is one promising approach. To this end, it is essential to understand the relationship between surface topographical features and bactericidal properties in order to develop antibacterial surfaces. RESULTS: In this work a systematic study of topographical effects on bactericidal activity of nanostructured surfaces is presented. Nanostructured Ormostamp polymer surfaces are fabricated by nano-replication technology using nanoporous templates resulting in 80-nm diameter nanopillars. Six Ormostamp surfaces with nanopillar arrays of various nanopillar densities and heights are obtained by modifying the nanoporous template. The surface roughness ranges from 3.1 to 39.1 nm for the different pillar area parameters. A Gram-positive bacterium, Staphylococcus aureus, is used as the model bacterial strain. An average pillar density at ~ 40 pillars µm-2 with surface roughness of 39.1 nm possesses the highest bactericidal efficiency being close to 100% compared with 20% of the flat control samples. High density structures at ~ 70 pillars µm-2 and low density structures at < 20 pillars µm-2 with surface roughness smaller than 20 nm reduce the bactericidal efficiency to almost the level of the control samples. CONCLUSION: The results obtained here suggests that the topographical effects including pillar density and pillar height inhomogeneity may have significant impacts on adhering pattern and stretching degree of bacterial cell membrane. A biophysical model is prepared to interpret the morphological changes of bacteria on these nanostructures.

Core-Shell Silver Nanoparticles in Endodontic Disinfection Solutions Enable Long-Term Antimicrobial Effect on Oral Biofilms.

To achieve effective long-term disinfection of the root canals, we synthesized core-shell silver nanoparticles (AgNPs@SiO2) and used them to develop two irrigation solutions containing sodium phytate (SP) and ethylene glycol-bis(ß-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), respectively. Ex vivo studies with instrumented root canals revealed that the developed irrigation solutions can effectively remove the smear layer from the dentinal surfaces. Further in vitro experiments with single- and multispecies biofilms demonstrated for the first time that AgNPs@SiO2-based irrigation solutions possess excellent antimicrobial activities for at least 7 days, whereas the bare AgNPs lose the activity almost immediately and do not show any antibacterial activity after 2 days. The long-term antimicrobial activity exhibited by AgNPs@SiO2 solutions can be attributed to the sustainable availability of soluble silver, even after 7 days. Both solutions showed lower cytotoxicity toward human gingival fibroblasts compared to the conventionally used solution (3% NaOCl and 17% EDTA). Irrigation solutions containing AgNP@SiO2 may therefore be highly promising for applications needing a long-term antimicrobial effect.

Antibacterial Au nanostructured surfaces.

We present here a technological platform for engineering Au nanotopographies by templated electrodeposition on antibacterial surfaces. Three different types of nanostructures were fabricated: nanopillars, nanorings and nanonuggets. The nanopillars are the basic structures and are 50 nm in diameter and 100 nm in height. Particular arrangement of the nanopillars in various geometries formed nanorings and nanonuggets. Flat surfaces, rough substrate surfaces, and various nanostructured surfaces were compared for their abilities to attach and kill bacterial cells. Methicillin-resistant Staphylococcus aureus, a Gram-positive bacterial strain responsible for many infections in health care system, was used as the model bacterial strain. It was found that all the Au nanostructures, regardless their shapes, exhibited similar excellent antibacterial properties. A comparison of live cells attached to nanotopographic surfaces showed that the number of live S. aureus cells was <1% of that from flat and rough reference surfaces. Our micro/nanofabrication process is a scalable approach based on cost-efficient self-organization and provides potential for further developing functional surfaces to study the behavior of microbes on nanoscale topographies.