Materials and technological solutions preventing biofilms and antimicrobial resistance

Resume : State-of-the-art sensing platforms ideally benefit from miniaturized and integrated optical technologies providing direct access to molecule-specific information. With in-situ sensing strategies, e.g., in harsh environments or for point-of-care diagnostics in medicine becoming more prevalent, detection schemes that do not require reagents or labeled constituents facilitate localized on-site analysis close to real-time. Mid-infrared (MIR; 3-20 µm) photonics are therefore an increasingly adopted sensing concept in biodiagnostics due to the inherent molecular specificity enabling the discrimination of molecular constituents at exceedingly low concentration levels. The complexity of biofilm formation - and subsequent prevention strategies - requires a more fundamental understanding on the involved molecular mechanisms and demands for long-term monitoring strategies during biofilm formation. This renders infrared spectroscopy - and in particular attenuated total reflection (IR-ATR) spectroscopy - a unique and versatile analytical technique for monitoring biofilm formation in situ, non-destructively, and close to real time. Selected examples and recent progress in IR photonic technologies will be highlighted illustrating its utility for providing advanced molecular insight into biofilm formation.

Resume : Immobilization of novel recombinant antimicrobial proteins of broad spectrum on thermoplastic polyurethane using SAMs towards prevention of catheter-related infections. Nosocomial infections are a public healthcare threat and its prevention is a priority for the health-care systems worldwide. The increasing emergence of multidrug-resistant bacteria poses a global health emergency, which is especially challenging within the context of biofilms. The activity of antimicrobial peptides (AMPs) from the innate immunity of a variety of species is gaining interest as a possible alternative to antibiotics, also for biofilm-forming bacteria. Recently, we have described a new generation of antimicrobial multidomain proteins that combines several AMPs and complementary functional domains in a single polypeptide recombinantly produced [1]. Here, we present a new strategy to functionalize the surface of medical devices to prevent bacterial adhesion and biofilm formation. This strategy is based on the use of biofunctional self-assembled monolayers (SAMs) with antimicrobial function. In addition, we present the anti-biofilm activity of the multidomain proteins, which has not been explored so far. Specifically, our strategy relies on the immobilization of AMPs through bonds on surfaces of materials, which are relevant for medical devices, like metallic surfaces and medical grade thermoplastic polyurethane (TPU) used for catheters. To anchor AMPs on surfaces we use self-assembled monolayers (SAMs) [2], which are based on well-organized molecules on surfaces that can be functionalized and allow a fine control at the molecular level. We show that immobilized AMPs preserve their antimicrobial activity and allow a higher availability of AMPs on the surface and a more uniform distribution as compared with the incorporation of AMPs through other adsorption methods that give place to non-homogeneous peptide distributions. We have anchored two antimicrobial proteins (JAMF1 and HD5) using a mixed SAM strategy on a metallic (Au) and TPU surfaces, respectively. In particular, we have used the interaction of the JAMF1 protein terminal His-tag with the Ni-NTA complex found at the surface of the Au SAM, and the terminal cysteine of the HD5 with maleimide terminated SAMs on TPU. Finally, a biofilm assay has been conducted to demonstrate the actual antimicrobial effect of the surfaces modified with the novel antimicrobial protein. The antimicrobial activity was successfully tested against Escherichia coli, Methicillin-resistant Staphylococcus aureus, Pseudomonas aeurginosa and Klebsiella pneumoniae bacteria, being this last one an example of antibiotic-resistant strain. In summary, our strategy has been shown to be well-suited to anchor AMPs for a controlled design of antibiofilm surfaces to coat medical devices. [1] Roca‑Pinilla et al. Microb Cell Fact. 2020, 19:122 [2] a) W. I. Tatkiewicz, ACS Biomater. Sci. Eng. 2019, 5, 5470−5480; b) W. I. Tatkiewicz, ACS Appl. Mater. Interfaces 2018, 10, 30, 25779–25786; c) M. Martinez, J. Mater. Chem. B, 2020, 8, 5080

Resume : Antimicrobial surfaces have broad use in multiple settings including touch surfaces in hospitals, implanted devices, or consumer products. Their aim is to support existing hygiene procedures, and to help combat the increasing threat of antimicrobial resistance. However, concerns have been raised over the potential selection pressure exerted by such surfaces, which might drive the evolution and spread of antimicrobial resistance. In my presentation, I will highlight the risks and knowledge gaps associated with resistance on antimicrobial surfaces by different processes including evolution by de novo mutations and horizontal gene transfer, and species sorting of inherently resistant bacteria dispersed onto antimicrobial surfaces. The latter process has the potential to select for antibiotic resistance via cross-resistance between traits that confer resistance to both the antimicrobial surface coating and antibiotics. Conditions in which antibiotics and antimicrobial coatings are present simultaneously (e.g. implants) will lead to more complex interactions that can either result in the selection for or against antibiotic resistance. We mapped these interactions between several antimicrobials and antibiotics on growth and selection of Pseudomonas aeruginosa. We find prevalent physiological (i.e. synergy and antagonism) and evolutionary (i.e. cross-resistance and collateral sensitivity) combination effects. Understanding these interactions opens the door to tailor therapeutic interventions to select against resistance. In additions, we need new methods and translational studies that investigate resistance development to antimicrobial surfaces under realistic conditions. Therefore, I will present recent developments in our lab on the development of such a method based on existing efficacy standards.

Resume : Designing bioactive materials, with controlled metal ion release, exerting significant biological action and associated to low toxicity for humans, is nowadays one of the most important challenges for our community. Here, active PHBV was obtained by modifying its surface with laser-ablated AgNPs. Laser ablation synthesis in solution (LASiS) has been used to produce bioactive Ag-based nanocolloids, in isopropyl alcohol, which can be used as water-insoluble nano-reservoirs of active metal ions. The nanomaterial was characterized by UV-Vis and TEM, in order to study its optical and morphological properties; afterwards, we developed a simple protocol to modify PHBV without altering its physicochemical properties. In fact, ATR-IR spectra did not show any modification in the main IR bands relevant to the polymeric matrix. The silver release analysis was used to theorize the antimicrobial action of the novel nanomaterial. AgNPs-PHBV films were also tested on inoculated apple juice samples, where they showed to be active against microbial proliferation. An increased antimicrobial effectiveness, in particular in terms of maximum growth rate, Lag time and time to achieve the maximum cell load, was recorded by increasing the surface to volume ratio. Due to the recorded results the proposed laser ablation synthesis is a promising technique to develop biodegradable active polymers intended for food packaging applications.

Resume : Most real-world microbiological problems are ubiquitous and costly and are rooted in biofilm formation. These include clinical infections, corrosion, or loss of process efficiencies. Some examples of these problems are found in heat transfer, dental plaque, food contamination, cleaning household surfaces, water processes, fabrics, and laundry. One strategy to avoid bacterial attachment is to repel the dirt (or nutrients) from the surfaces in the first place. The repellence mechanisms for dirt can also be extrapolated to molecules in the extracellular polymeric matrix of a biofilm such as proteins, polysaccharides, and nucleic acids. Polyethylenimine (PEI) is a potential component of hard-surface cleaning formulations since it forms uniform coatings that are known to have anti-biofilm properties. However, being a polycation, its efficacy in repelling the negatively charged components of the biofilm extracellular polymeric substance (EPS) cannot be taken for granted. In this study, we determined, experimentally, the affinity of a model negatively charged, protein (bovine serum albumin, BSA) towards glass surfaces both pristine and coated by PEI. Preliminarily, we compare the BSA-silica interactions occurring on two types of surface geometries: flat and highly curved colloidal nanoparticles. This comparison is possible through a combination of techniques, each dedicated to a specific geometry. Surface plasmon resonance (SPR) for flat and dynamic light scattering (DLS) for colloidal surface geometries, correcting the obtained data by the water contribution and geometrical factors. The results confirm the capability of colloidal particles to be a model surface for adsorption on hard surfaces [1]. The subsequent experiments were performed using colloidal silica (15 nm of radius). The adsorption isotherm of PEI on Ludox was determined and the optimal conditions for a stable nanoparticle-coated solution were found. Next, the adsorption isotherms of BSA on coated and pristine glass nanoparticles were determined via differential centrifugation. Mild centrifugation removes the aggregates formed by PEI (or BSA) bridging flocculation and the resulting supernatant was investigated using DLS and zeta potential measurements combined with SAXS and TEM experiments. The amount of polymer and protein adsorbed on the nanoparticles was obtained by thermal gravimetric analysis (TGA) of the pellet obtained under drastic centrifugation conditions inducing the precipitation of all the nanoparticles (but not of free PEI or BSA). The obtained results indicate that the affinity of BSA for PEI-coated glass is more than two orders of magnitude lower than for pristine glass. [1] H. Mateos, A. Valentini, F. Lopez, G. Palazzo. Biomimetics 2020, 5, 31.

Resume : We can find interest in biofilms from different perspectives. On the one hand, for example, biofilms can develop powerful electrocatalytic performances that are found valuable in microbial electrochemical technologies. Some of the applications are related to energy production or soil and water remediation. On other hand, biofilms are providing a protective environment that promotes resistance to antibacterial treatments. Our laboratory has been involved in research on biofilms for many years at the interface between microbiology and physical-chemistry. In this communication we wish to summarize some recent developments at LCPME to promote antibacterial/antibiofilm surfaces by surface functionalization and texturation, e.g. for osteo-integrative prosthesis.

Resume : Bioinspired nanopatterned materials that exhibit antimicrobial properties are being synthesized and tested at increasing rates for use in healthcare, manufacturing processes, and diagnostics. Such surfaces kill bacteria upon contact by imparting mechanical forces to the bacterial cell membrane, offering safe and sustainable solution to prevent microbial contamination, without inducing antimicrobial resistance. Yet such patterning in glass (SiO2) is scarce, despite the vast market for such a product, including bathrooms, food-processing facilities or hospitals where good hygiene is required. The likely reason is a fabrication challenge, which we solve here by developing a novel glass etching process with high etching selectivity enabling a vast tuning range of nanopattern geometries. Through this process, we present, for the first time, nanopatterned glass capable of killing a major healthcare-associated pathogen, Staphylococcus aureus. We model and experimentally investigate the antibacterial properties of our designs by viable counting and surface fluorescent imaging, where live and damaged/dead cells are visualized based on their membrane integrity. Both measurements indicate significantly reduced numbers of viable bacteria after interacting with the nanostructures comparing to the control (smooth surface). The average proportion of non-viable cells on the surface is 81%, matching our theoretical predictions and previous results obtained with silicon (83-85%)1. We further probe the surface by scanning electron microscopy to elucidate the underlying mechanism. We envision that such structuring in glass will facilitate fundamental studies on mechano-bactericidal surfaces and be useful for numerous practical applications. 1 E. P. Ivanova, D. P. Linklater, M. Werner, V. A. Baulin, X. Xu, N. Vrancken, S. Rubanov, E. Hanssen, J. Wandiyanto, V. K. Truong, A. Elbourne, S. Maclaughlin, S. Juodkazis and R. J. Crawford, Proc. Natl. Acad. Sci., 2020, 117, 12598–12605.

Resume : Antimicrobial food packaging materials are a challenge for preventing the development of biofilm-associated contaminations targeted by pathogenic bacteria. Concerns related to synthetic preservatives demand for innovative and safe natural antimicrobials. Chitosan - a deacetylated polysaccharide derived from chitin - is a biocompatible compound with pronounced inhibitory activity especially suitable for addressing sessile communities of lactic acid bacteria. In the present study, we discuss in-situ investigations on the selective antimicrobial efficiency of chitosan for controlling the growth of Lactobacillus parabuchneri biofilms. The electrostatic interaction of chitosan with the bacterial cell wall typically occurs between protonated amino groups and negatively charged phospholipids, which promotes permeabilization, cell wall disruption, and finally, cell death. These charges were additionally amplified by the structural modification of the biopolymer amino groups via a methylation process, which yielded the quaternized derivative TMC (i.e., N,N,N-trimethyl chitosan). To evaluate the antimicrobial effectiveness against L. parab. biofilms, infrared attenuated total reflection (IR-ATR) spectroscopy was used providing information in molecular detail using an integrated platform complemented by orthogonal sensing technologies. Thereby, the simultaneously determination of molecular mechanisms and metabolic oxygen consumption was observable with time. In addition, scanning electron microscopy (SEM) measurements were performed on biofilms grown on polystyrene and stainless steel surfaces visualizing morphological changes of the associated biofilms. The IR studies revealed strong electrostatic interactions between chitosan/its water-soluble derivative and the bacteria vs. a significant decrease of the IR bands characteristic for extracellular polymeric matrix (EPS) production by L. parab. biofilms. Long-term monitoring of the biofilm inhibition via low minimum inhibitory concentration MIC (varying 0.05-0.1%) confirms suitable antimicrobial activity with potential of bacterial growth prevention for foodborne contaminants in dairy industry. Keywords: chitosan antimicrobial, derivative TMC, biofilm inhibition, IR-ATR spectroscopy, molecular mechanisms, L. parabuchneri biofilms

Resume : Antimicrobial surfaces are a powerful tool to fight bacterial infections and prevent the emergence of antimicrobial resistance within biofilms. However, translational barriers from fundamental science to commercial scale applications are still very high, compounded by stringent regulatory frameworks. The need of universal approaches, based on industrial scalable processes and able to deliver disruptive yet safer technologies, is becoming urgent for several priority areas such food security, health care, and pharma. In this talk we will focus on recent examples of “top-down” and “bottom-up” approaches used to fabricate controlled antimicrobial surfaces using fabrication methods based on macroscopic chemical processes, while delivering controlled antimicrobial responses directly at the surface and in the planktonic state. We will exemplify how these top-down approaches are amenable to analysis at the molecular level, tracking location and release of actives at the functional interface with microscopic precision. The combination of high-resolution microscopy techniques and spatially resolved spectroscopies can track modes of action of these bio-actives surfaces at the micro- and nanoscale, informing theoretical models that, in turn, contribute to deeper insights and new design rules for the next generation of smart antimicrobial surfaces.

Resume : ZnO nanomaterials are antimicrobial agents used to combat different pathogens. Among several applications, they have been exploited for artwork preservation against biodeterioration [1]. We have proposed a green electrochemical-thermal approach for the preparation of ZnO nanostructures (NSs) based on the use of a sacrificial Zn anode in an alkaline medium [2]. We have demonstrated that the type of stabilizer and synthesis parameters allow tuning morphology and size of the proposed ZnO NSs. Differently charged species have been used as capping agents [2-4]. The as-prepared powders have been applied for preparing hybrid coatings with antimicrobial/consolidating properties for stone artwork protection [5]. Flower-like NSs have been also tested against B. subtilis [4]. In this communication, morphological (TEM) and spectroscopic (UV-Vis, FTIR, XPS) characterization of the ZnO NSs will be presented outlining the importance of the stabilizer on material properties. Results about the antimicrobial properties of the as-prepared ZnO NSs will be reported as well. 1. David, M.E., et al. Materials 13 (2020): 2064. 2. Picca, R.A., et al. Electrochim. Acta 178 (2015) 45. 3. Picca, R.A., et al. J. Sol-Gel Sci. Technol. 81 (2017) 338. 4. Sportelli, M.C., et al. Nanomaterials 10 (2020) 473. 5. Ditaranto, N., et al. New J. Chem. 39 (2015) 6836. Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439.

Resume : Understanding bacterial cellular signaling and function at the nano-bio interface can pave the way towards developing next-generation smart diagnostic tools, as well as provide new targets for preventing biofilm-related infections. In the last years, we have extensively investigated the bacterial life cycle of Xylella fastidiosa, an economically-important phytopathogen which affects cultures worldwide. The pathogenicity of X.fastidiosa is related to biofilms formed in xylem vessels, which generate hydric stress with major impacts on agricultural productivity. In this work we devise and discuss different materials platforms to further understand X.fastidiosa interaction with surfaces as well as key mechanisms involved in cellular assembly leading to biofilm formation. In particular, we will show how single-crystalline nanowire arrays can be used to probe in real time cell adhesion forces in the presence of drugs reported to control infection, such as n-acetyl-cysteine, narrowing down possible molecular mechanisms to improve efficacy of these methods. In a complementary but different approach, we have used Au micropatterns with well-defined geometries and dimensions, prepared with direct laser writing lithography on SiO2 substrates, to create spatially-selective adhesion of X. fastidiosa cells. Our Au disk arrays provide close control of both cell density and distances between cell clusters. Our results elucidate the formation of filamentous cells as induced by local bacterial density; their growth is oriented toward neighboring cell clusters in a distance-dependent manner which eventually creates a network of interconnected cell clusters that facilitate the macro-scale biofilm architecture. These results indirectly confirm quorum sensing based chemical signaling involved in the formation of filamentous cells associated with bacterial clusters of X. fastidiosa. The present approach based on Au patterned arrays is not only promising to understand complex phenomena of multicellular assembly but also offers new directions to engineering biological systems.

Resume : Biofilms are well-organized microbial communities that can stick to both biotic and abiotic surfaces and which exhibit an increased antimicrobial resistance in comparison with the planktonic cell cultures. The formation of biofilms is a complex multi-step process [1], which begins with the adhesion of the bacteria at a surface and the subsequent irreversible attachment, followed by cell division and proliferation. In the last decades, novel strategies to prevent the formation of biofilms have been developed and, among others, antimicrobial polymers (AMP) such as chitosan, poly-lysine, and polydopamine have been intensively studied, due to their ability to inhibit or eradicate biofilms. The AMPs are known to exhibit antimicrobial activities thanks to their inherent chemical structure (nitrogenic groups, poly-lysine, halamines) or can be used as a support matrix improving the effect of other antimicrobials [2]. Polydopamine is a unique nature-inspired polymer, derived from mussel foot proteins, which exhibit among other properties such as a plethora of functional groups (high content of catechol, amine, and imine groups), also exceptional adhesion. The mechanical and chemical properties of PDA are strongly dependent on the deposition method and the experimental condition in which they are studied; for instance, it has been shown that the adhesion properties of PDA and the antimicrobial properties [3] are strictly influenced by the pH and the oxidation state of the polymer [4, 5]. In this contribution, atomic force microscopy (AFM) and electrochemical force spectroscopy measurements will be used to study the adhesion properties of PDA in different conditions, and the early stages of attachment at several model samples of different bacterial strains were investigated. References 1. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ (1987) Bacterial Biofilms in Nature and Disease. Annu Rev Microbiol 41:435–464 2. Jain A, Duvvuri LS, Farah S, Beyth N, Domb AJ, Khan W (2014) Antimicrobial Polymers. Adv Healthc Mater 3:1969–1985 3. Liu H, Qu X, Tan H, Song J, Lei M, Kim E, Payne GF, Liu C (2019) Role of polydopamine’s redox-activity on its pro-oxidant, radical-scavenging, and antimicrobial activities. Acta Biomater 88:181–196 4. Daboss S, Lin J, Godejohann M, Kranz C (2020) Redox Switchable Polydopamine-Modified AFM-SECM Probes: A Probe for Electrochemical Force Spectroscopy. Anal Chem 92:8404–8413 5. Ball V (2014) Physicochemical perspective on “polydopamine” and “poly(catecholamine)” films for their applications in biomaterial coatings (Review). Biointerphases 9:030801 Acknowledgments Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439.

Resume : The cell membrane is a biological barrier against the majority of the molecules due to its highly hydrophobic nature.1,2 In this study, we aim to develop a new tool to identify the interaction between pore forming molecules and cell membranes to understand fast and dynamic biophysical mechanisms underlying cell membrane permeation at the single-cell level by using scanning ion conductance microscopy (SICM) combined with fluorescence imaging. Delivery was achieved by repeated approach-deliver-retract of an SICM nanopipette, with potential control over the delivery of dimethyl sulfoxide (DMSO) molecules to the cell of interest. Propidium iodide dye was used as an optical readout to quantify cell membrane permeation by fluorescence intensity change over time. Finite element method modelling was used to predict the flux of DMSO delivered and to quantify DMSO concentration in the cell. The results demonstrate a significant fluorescence intensity increase in 20 seconds that was localised at the cell nucleus, accompanied with morphological changes such as bleb formation at the HeLa cell indicating permeation of the cell membrane when the delivery potential was applied to SICM nanopipette. These changes were only observed on the cell to which DMSO was delivered, and surrounding cells were not permeabilised and fluorescence intensity did not show an increase. This study brings a novel application to enhance drug uptake or delivery studies to identify new drug targets for various type of cells by monitoring the cell membrane permeation of antimicrobial agents into biofilms and cancer drugs permeation into HeLa cells while maintaining the cell viability with non-contact SICM nanopipette providing in-situ and controlled single-cell membrane permeation. References 1. N.J Yang and M.J Hinner, Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Methods Mol Biol.,2015,1266,29-53. 2. S. Kundu, S. Malik, M. Ghosh, S. Nandi, A. Pyne, A. Debnath and N. Sarkar, A comparative study on DMSO-induced modulation of the structural and dynamical properties of model bilayer membranes, Langmuir, 2021, 37(6),2065-2078.

Resume : Modern healthcare employs many types of invasive devices and procedures to treat patients and to help them recover. Infections can be associated with the devices used in medical procedures, such as catheters or ventilators. Biofilms play a pivotal role in healthcare-associated infections (HAIs). Typical antibiotic therapies are ineffective against biofilm formation and promote fast development of resistances. New and novel approaches to prevent and treat biofilm infections are urgently required. Antimicrobial polymers can help to prevent the formation of biofilm-associated infections and to solve the problems associated with the use of conventional antimicrobial agents, such as residual toxicity, short-term antimicrobial activity and development of resistant microorganisms. This study investigates a novel approach to controlling biofilms of the most frequent pathogens implicated in the etiology of biomaterials-associated infections. New bactericidal filler based on a non-toxic glass, belonging to B2O3-SiO2-Al2O3-Na2O-ZnO system, was used to formulate composites of the most widely used polymers in biomedical applications [i.e. thermoplastic polyurethane (TPU) and polydimethyl siloxane (PDMS)], with varying percentage by weight of the bactericidal glass (5, 15, 25, 35, 50 %). Glass-filled polymer composites show dramatically restricted bacterial colonisation and biofilm formation. They exhibit time- and dose-dependent killing, with maximal action at 5 days. The highest activity was found against S.epidermidis biofilm (99% of reduction), one of the most common cause of nosocomial infections. In addition, all the materials revealed an excellent biocompatibility.

Resume : Antimicrobial resistance (AMR) has become a global health problem that is exarcebated by the ability of some bacteria to form biofilms. These are complex aggregations of microorganisms enclosed in a protective and adhesive matrix. Currently, different approaches are being considered such as drug delivery therapy to improve the action of traditional antibiotics, or phage therapy as alternative to antibiotics. One option for antimicrobials encapsulation are vesicles, niosomes are non-ionic vesicles that are able to reduce side effects of drugs, increasing their pharmacodynamics. In this work, positive charged niosomes were synthetized to enter easily in the biofilm structure. The antimicrobial used was the chimeric protein CHAPSH3b. Thin film hydration method was used, with some variations in parameters to overcome the problem of denaturation of the protein. Size and charge were measured using Dynamic light scattering (DLS) and morphology was studied through Transmission Electron Microscopy (TEM). After niosomes purification by ultracentrifugation, the encapsulation efficiency was calculated with High-performance liquid chromatography (HPLC). The results showed that after protein encapsulation, niosomes loaded with CHAPSH3b had a smaller size than those vesicles without protein encapsulated. Moreover, the values of zeta potential were 40 - 50 mV, demonstrating that the vesicles were stable and consequently, have more electrostatic affinity with biofilms of gram-positive bacteria.

Resume : Biodeterioration and consolidation are two crucial points in the preservation of historic monuments. The growth of microorganisms on limestone surfaces is responsible for the formation of a biofilm, which causes long term damage of the sample. In addition, the loss of cohesion of the stone matrices results in manufact erosion. To fight these problems, synergistic (nano)materials combining antimicrobial activity of ZnO and consolidating properties of calcium hydroxide are proposed here. In recent years, we have demonstrated the feasibility of zinc oxide nanoparticles (ZnO NPs) as bioactive materials for stone artworks protection [1]. On the other side, colloidal dispersions of calcium hydroxide nanoparticles (Ca(OH)2 NPs) are highly compatible with different stones [2]. In this work, we propose a one-step synthesis of hybrid ZnO-Ca(OH)2 nanostructures. This approach is based on the combination of a classical synthesis of Ca(OH)2 NPs [3] with an electrosynthesis of ZnO NPs based on sacrificial anode electrolysis method [4]. Characterization of the novel material by morphological and spectroscopic analyses is presented, along with preliminary results on the application as consolidation/antimicrobial treatments. [1] N. Ditaranto et al., New J. Chem. 39 (2015) 6836. [2] P. Baglioni et al., Langmuir. 29 (2013) 5110. [3] M. Ambrosi et al., Langmuir. 17 (2001) 4251. [4] M.C. Sportelli et al., Nanomaterials. 10 (2020) 473.

Resume : Escherichia coli (E. coli) is a gram-negative biofilm forming bacteria, which has the ability to colonize and persist in human, animal hosts and environment. E. coli O157:H7 is responsible for severe food intoxication, and it is considered a public health threat. Additionally, it shows antimicrobial resistance against broad spectrum of antibiotics. Therefore, there is a need for rapid, portable and simple methods for E. coli O157:H7 detection. The objective of this work was to design of a rapid and sensitive lateral flow immunoassays strip for E. coli O157:H7 detection using iron oxide nanoparticles (IONs) encapsulated by lipid-polymer hybrid nanoparticles as labels (LPHNPs). LPHNPs enhance strongly the encapsulation efficiency. First, monodisperse IONs were synthesized with average diameters between 3 and 7 nm by the W/O microemulsion method and further encapsulated in lipid?polymer hybrid system (IONs @ PLGA?PVA/PC) as labels for LFIA. E. coli O157:H7 was cultured in Tryptic Soy Broth (TSB) to reach a concentration of 2×1010 (cfu/ml) and serial dilutions in PB buffer were prepared. The LPHNPs allowed carrying out immunomagnetic separation and preconcentration for point-of-use sensitive detection. Funding: This work is part of a project that has received funding from the European Union?s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439 (Break Biofilms). The work was also supported by the project MAT2017-84959-C2-1-R.

Resume : Biofilms are considered a major cause of serious health issues in human medicine and food industry, due to their resistance against antibiotics. Given the antibiotic resistance of biofilms, it is of increasing importance to develop innovative methodologies for the treatment of biofilm-related infections; the utility of antimicrobial nanoparticles (NPs) has been widely shown to this aim. These antimicrobial materials were successfully applied as a key-component of several industrial goods for automotive industry to prevent microbial proliferation on common touching surfaces. However, the widespread use of nanomaterials in commercial goods poses some nano-toxicological concerns. Hence, a detailed characterization of NP-treated materials is unavoidable. Here we present the most representative results of the analytical characterization of these composite materials in terms of morphology, surface chemical composition, ionic release in contact media. The potential release of entire nanoparticles from treated materials was studied by Transmission Electron Microscopy (TEM) on contact solutions, aimed at ruling out or quantifying the extent of whole particle release under real-life usage conditions. Conclusions will be drawn about the safety and antimicrobial efficiency of the mentioned composites. Acknowledgements: Financial support acknowledged from the E.U, PON Research and Innovation 2014-2020 – Project: “E-Design” ARS01_01158.

Resume : Nowadays, there is a frenetic quest for innovative biomaterials for the functionalization of osseous implants, able to respond to astringent healthcare requirements (e.g., controlled release of therapeutic ions, match of biomaterial degradation ? bone growth rates, antimicrobial efficiency). As third-generation biomaterials, phosphate bio-glasses (PBGs) are capable to stimulate specific biological responses at molecular level, successfully accomplishing the coupling of the bioactive and resorbable material designs. These outstanding properties could open numerous possibilities for using PBGs standalone or in bio-degradable composites for advanced biomedical applications. The coating of metallic implants with biofunctional (osteoinductive, hemocompatible, or antimicrobial) layers represents a highly promising solution for orthopaedics and dentistry. Surprisingly, little attention has been payed to date to the application of PBGs as implant-coatings. No information on the biological in vitro response of PBG sputtered films has been reported yet. Of paramount importance, is the need to discover new avenues able to offer the possibility to tune without difficulty the structure of PBG coatings for obtaining flexible biodegradability and specific biological response. In this work, we report the synthesis of PBG films by radio-frequency magnetron sputtering (RF-MS). We found the physical-chemical properties of PBG films (e.g. porosity, density, composition, network connectivity) as well as their biodegradability can be adjusted by simply changing, as important RF-MS variable, the working argon pressure from 0.2 to 1.0 Pa. The properties of the films were multi-parametrically ascertained, qualitatively and quantitatively by various analytical techniques. Furthermore, the cytocompatibility of PBG coatings, as another crucial biofunctional properties, was investigated to unveil the prospects of their fine tuning. All the deposited PBG films were cytocompatible. Thereby, a path towards the engineering of PBG sputtered films structure is suggested, which, in turn, can allow to fine tune the in situ degradation and release speed of the therapeutic ions. The finding of uniform sputtered PBG films over an appreciable substrate area, in terms of thickness, composition and structure, unveil their applicability as dental implants and will positively influence the chances for a successful transfer of technology to industrial level. The results presented could serve has a useful benchmark for developing PBG bioceramics with controlled properties and tunable biodegradability.

Resume : Hospital-acquired or nosocomial infections (NI) are an important public health problem, due to the associated morbidity and mortality, and the economic burden that they represent for the health-care system. According to the most recent data, approximately 8% of patients admitted in a hospital suffer a NI (EPINE 2019). In the last decades, the consequences of NI have become more severe because of the continuous emergence of antibiotic-resistant microorganisms, which often make their treatment an authentic challenge for physicians. Far from being a phenomenon that will decrease in the coming years, it is estimated that the emergence of new resistances and the number of pan-resistant microorganisms will continue to grow reaching an increase of 67% in 2030. Modern medical and surgical practices have generalized the use of implantable medical devices that has been associated with a dramatic improvement of clinical outcomes, but microbial contamination of the implanted devices is a widespread problem. Among them, catheter-related urinary tract infections are the commonest, as urinary microorganisms colonize urethral catheters, then arriving into the bladder and providing a surface for bacterial adhesion. In order to prevent device-related infections, some coatings applied to reduce bacterial adhesion by altering the physicochemical properties of the substrate. Here, we have coated thermoplastic polyurethane (TPU) surfaces, that is the catheters’ material, with mixed self-assembled monolayers (SAMs) of polyethylene glycol and maleimide terminated polyethylene glycol molecules. These surfaces have been characterized by a multi-technique approach (AFM, XPS, SEM, FTIR, WCA …). Maleimide groups will serve as anchor points for the cysteine groups of the antimicrobial protein used, Human defensin 5 (HD5), giving place to anti-biofilm surfaces. The biofilm assay against methicillin-resistant staphylococcus aureus (MRSA) and pseudomonas aeruginosa (P.aeruginosa) showed that the immobilized antimicrobial protein is able to significantly reduce the biofilm formation. In summary, the strategy used has been shown to be excellent to anchor AMPs for a controlled design of antibiofilm surfaces to coat medical catheters.

Resume : Introduction Ventricular Assist Devices (VAD) are nowadays widely used as an effective treatment for patients with advanced heart failure and are the only alternative to heart transplantation. Currently, the most commonly used devices in clinics are fully implantable, therefore it is very important to ensure high biocompatibility of such devices. Over the years many new constructions were introduced to the clinic. However, clinical experience has shown that, despite unquestionable effectiveness of VADs, many components could still be improved. Pumps from currently used generation are mostly made of titanium alloys. These devices are being optimized not only in terms of their function, but also in terms of patient comfort and reduced mortality. According to the information provided by INTERMACS, patient survival drops to 50% after four years of support. On the other hand, the VADs, are designed to be wear-resistant and in some cases are used as a destination therapy. It is therefore important to improve the biocompatibility even further in order to reduce the occurrence of complications. The possible complications may originate from the pump thrombosis and inflow obstruction, caused by the ingrowth of tissue into the lumen of inflow cannula. It seems that the solution to this problem may be the use of modification in the form of textured surface. The unknown, however, is the effect of the textured surface on the adhesion and growth of bacterial colonies, which may result in a serious systemic infection. Materials and Methods The paper presents surface modification intended for use in an VAD inflow cannula and its vulnerability to create bacterial colony. Surface was modified using vacuum sintering method. Samples were prepared from titanium alloy Ti6Al7Nb in form of cylinders Ø14mm x H 3mm. The base material was verified for compliance with the standard including the microstructure study, the chemical composition analysis and the study of mechanical properties. The samples were subjected to tumbling before performing modification to provide repeatable initial conditions. The roughness was measured with the use of contact profilometry. The base material was characterised by Ra=1,5µm and Rz=12,5µm. During the sintering process, a powder with two different grain morphologies - spherical and irregular - were used. The grain size remained the same and was in the rage of 70-15µm. The obtained surfaces were analysed by scanning electron microscope [SEM]. The surface was characterized by high roughness with the potential for cell anchoring, enabling the formation of scar tissue. Then the antimicrobial activity contact test was performed. In the study Escherichia coli (Gram-negative bacteria) strain ATCC 8739 and Staphylococcus aureus (Gram-positive bacteria) strain 6538P were used. Results and Discussion The results have shown that surface after powder sintering is characterized by high porosity and complex 3D morphology. Generally, E. coli was more sensitive to both tested materials than S. aureus. Since the obtained R values were lower than 2.0, we infer that tested materials yield bacteriostatic activity against tested Staphylococcus aureus and Escherichia coli strains. Conclusions The results indicate that the surfaces do prevent bacteria from growing further. Nevertheless, surface modification yield no bactericidal activity. The research shows the potential for application of the proposed surface modification, but it is necessary to extend the research group to surface modifications that differ in the degree of porosity and the size of pores. Acknowledgments Project supported by: NCBiR: RH-ROT/266798/STRATEGMED-II National Science Centre, Poland: 2018/31/N/ST8/01085