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Materials and technological solutions preventing biofilms and antimicrobial resistance

Biofilms and antimicrobial resistant microorganisms are the main source of infections in humans, and conventional disinfectants/antibiotics rarely succeed in destroying them. Materials science can offer non-conventional solutions, complementing the standard biomolecular and pharmaceutical approaches.

Scope:

This symposium aims at:

(i) Providing a detailed and critical overview of the most successful results on novel smart materials or surface processes fighting antimicrobial-resistant strains and/or preventing biofilm formation;

(ii) Critically presenting next generation bioactive agents, targeting the ultimate prevention and destruction of antimicrobial resistant strains and /or biofilms in industrial and biomedical fields. Bioactive agents will include, for instance: inorganic nanomaterials, hybrid/synergistic nanoantimicrobials, and any other novel antimicrobial and antibiofilm compounds (bacteriophages, endolysins, exopolysaccharide depolymerases, bacteriocins, etc.);

(iii) Showcasing the different characterization approaches for the study of solid state surface-biofilm interactions at different stages of the biofilm growth;

(iv) Gaining fundamental knowledge about the (bio)physicochemical mechanisms of biofilm formation, including all the possible aspects contributing to the understanding and monitoring biofilm biogenesis and identification of new targets for detecting biofilms;

(v) Outlining all the possible materials science solutions for the assessment and/or prevention of nanotoxicology issues related to the use of novel nanoantimicrobials and related biocide agents;

(vi) Providing an integrated vision of active materials and technological solutions for early detecting and identifying biofilm formation with high sensitivity.

Hot topics to be covered by the symposium:

  • Smart functional inorganic anti-biofilm nanomaterials and coatings of any composition
  • Synergistic, hybrid and/or multifunctional materials fighting biofilms and antibiotic-resistant species
  • Novel bioactive molecular/biogenic compounds (bacteriophages, endolysins, exopolysaccharide depolymerases, bacteriocins, etc) for biofilm inhibition and removal
  • Materials science and characterization solutions for biofilm monitoring, including: sensors, electrochemical techniques, mass spectrometry, microscopy (electron, scanning probe, confocal, etc) and spectroscopy techniques of any kind

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08:30 BLANCO, CIOFFI, KRANZ, FERNÁNDEZ: Welcome message and introduction to the Symposium    
 
SESSION I : BLANCO
08:45
Authors : Boris Mizaikoff
Affiliations : Ulm University, Institute of Analytical and Bioanalytical Chemistry

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.

J.1..1
09:15
Authors : X. Rodriguez-Rodriguez,1 A. Lopez-Cano,2 R. Roca-Pinilla,2 A. Kyvik,1 P. Bierge,3 O.Q. Pich,3 O. Gasch,3 C. García-de-la-Mària,4 J. M. Miró,4 J. Veciana,1,5 J. Guasch,1,5,6 A Arís,2 E. Garcia-Fruitós,2 I. Ratera1,5
Affiliations : 1. Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus UAB, Bellaterra, 08193, Spain 2. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Caldes de Montbui, 08140, Spain 3. Department of Infectious Diseases, Institut d’Investigació i Innovació Parc Taulí (I3PT),Corporació Sanitària Parc Taulí, Universitat Autònoma de Barcelona, Barcelona, Spain 4. Department of Infectious Diseases, Hospital Clinic, Institut d’Investigacions Biomèdiques Agust Pi i Sunyer, University of Barcelona, Barcelona, Spain 5. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Campus UAB, Bellaterra, 08193, Spain 6. Max Planck Partner Group, ICMAB-CSIC, Campus UAB, Bellaterra, 08193, Spain

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

J.1..2
09:30
Authors : Isabel Pastoriza-Santos
Affiliations : CINBIO, Universidade de Vigo, Lagoas-Marcosende

Resume : Surface-enhanced Raman scattering (SERS) spectroscopy is an ultrasensitive analytical technique that can be applied non-invasively for the detection and imaging of a wide range of molecules. SERS allows identification of the specific spectral fingerprint of a probe analyte in contact with a plasmonic nanostructure and its sensitivity can go as far as the single-molecule level. Importantly, SERS offers multiplexing capability, requires no sample preparation and provides high spatial resolution. In this communication we report the SERS Detection and imaging of bioactive metabolites in live microbial populations employing plasmonic platforms based on Au nanoparticle assemblies. We present the use of SERS as a tool to visualize the production of microbial metabolites and chemical communication in bacterial populations grown on plasmonic nanostructured materials. We will also show the successful detection by SERS of chemical interactions in mixed bacterial populations demonstrating the potential of this technique for the analysis of the chemical processes underpinning multispecies microbial communities.

J.1..3
10:00
Authors : M.C. Sportelli1, 2; A. Ancona2, 3; A. Volpe2, 3; C. Gaudiuso2, 3; V. Lavicita4; A. Conte4; M.A. Del Nobile4; V. Miceli5; N. Cioffi*1,6
Affiliations : (1) Chemistry Department, University of Bari, Via Orabona, 4-70126 Bari, Italy (2) IFN-CNR, Physics Department, University of Bari, Via Amendola, 173-70126 Bari, Italy (3) Physics Department, University of Bari, Via Orabona, 4-70126 Bari, Italy (4) Department of Agricultural Sciences, Food and Environment, University of Foggia, Via Napoli 25-71122 Foggia, Italy (5) ENEA Research Center, BIOAG division - ss Appia km 700 – 72100 Brindisi, Italy (6) CSGI (Center for Colloid and Surface Science) c/o Chemistry Department, University of Bari, Via Orabona, 4-70126 Bari, Italy * Corresponding Author

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.

J.1..4
10:15
Authors : Helena Mateos, Nicola Cioffi, Gerardo Palazzo,
Affiliations : Università degli studi di Bari "Aldo Moro", Università degli studi di Bari "Aldo Moro", Università degli studi di Bari "Aldo Moro",

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.

J.1..5
10:30 coffee break    
 
SESSION II : CIOFFI
11:00
Authors : Gregory Francius, Fabienne Quilès, Mathieu Etienne
Affiliations : Université de Lorraine, CNRS, LCPME, F-54000 Nancy, France

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.

J.2..1
11:30
Authors : Martyna Michalska1, Sophia K. Laney1, Tao Li1, Mark Portnoi1, Nicola Mordan2, Elaine Allan3, Manish K. Tiwari4,5, Ivan P. Parkin6, Ioannis Papakonstantinou1
Affiliations : 1 Photonic Innovations Lab, Department of Electronic & Electrical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; 2 Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, Royal Free Campus, University College London, Pond Street, London, NW3 2QG, UK; 3 Department of Microbial Diseases, UCL Eastman Dental Institute, Royal Free Campus, University College London, Rowland Hill Street, London, NW3 2PF, UK; 4 Nanoengineered Systems Laboratory, Department of Mechanical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; 5 Wellcome/EPSRC Centre for Interventional and Surgical Sciences (WEISS), University College London, London, W1W 7TS, UK; 6 Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK.

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.

J.2..2
11:45
Authors : Diellza Bajrami *(1), Stephan Fischer (2), Holger Barth (2), Maria. F. García (3), Nicola Cioffi (4), Boris Mizaikoff (1),(5)
Affiliations : 1) Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany; 2) Institute of Pharmacology and Toxicology, Ulm University Medical Center, Ulm, Germany; 3) Institute of Dairy Products of Austurias, IPLA-CSIC, Villaviciosa, Spain; 4) Chemistry Department, University of Bari “Aldo Moro”, Bari, Italy; 5) Hahn-Schickard, Institute for Microanalysis Systems, Ulm, Germany; *Email:diellza.bajrami@uni-ulm.de

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

J.2..3
12:00
Authors : Yuri Antonio Diaz Fernandez, Ioritz Sorzabal Bellido, Luca Barbieri, Jo Fothergill, and Rasmita Raval,
Affiliations : Yuri Antonio Diaz Fernandez; Ioritz Sorzabal Bellido; Luca Barbieri; Rasmita Raval Open Innovation Hub for Antimicrobial Surfaces Surface Science Research Centre Department of Chemistry University of Liverpool United Kingdom Luca Barbieri; Jo Fothergill Institute of Infection and Global Health, University of Liverpool, UK

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.

J.2..4
12:30
Authors : Picca, R.A.*(1)(2), Izzi, M(1)(2), Sportelli, M.C.(1), Sabbatini, L.(1), Cioffi, N.(1)(2).
Affiliations : (1) Chemistry Department, University of Bari Aldo Moro, Bari, Italy; (2) CSGI (Center for Colloid and Surface Science), Bari, Italy

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.

J.2..5
12:45
Authors : Olga Jegel, Felix Pfitzner, Gazanis Athanasios, Jennifer Oberländer, Martin Lange, Marcus von der Au, Björn Meermann, Volker Mailänder, Alexander Klasen, Ralf Heermann, Wolfgang Tremel
Affiliations : Johannes Gutenberg-Universität Mainz, Department Chemie, Duesbergweg 10-14, D-55128 Mainz, Germany ; Johannes Gutenberg-Universität Mainz, Department Chemie, Duesbergweg 10-14, D-55128 Mainz, Germany; Johannes Gutenberg-Universität Mainz, Biozentrum II, Institut für Molekulare Physiologie, Mikrobiologie und Weinforschung, Hanns-Dieter-Hüsch-Weg 17, D-55128 Mainz, Germany ; Max Planck-Institut für Polymerforschung, Ackermannweg 10, D-55128 Mainz, Germany ; Johannes Gutenberg-Universität Mainz, Department Chemie, Duesbergweg 10-14, D-55128 Mainz, Germany; Bundesanstalt für Materialforschung und -prüfung (BAM), Abteilung I: Analytische Chemie, Referenzmaterialien, Anorganische Spurenanalytik, D-12489 Berlin, Germany; Bundesanstalt für Materialforschung und -prüfung (BAM), Abteilung I: Analytische Chemie, Referenzmaterialien, Anorganische Spurenanalytik, D-12489 Berlin, Germany; Max Planck-Institut für Polymerforschung, Ackermannweg 10, D-55128 Mainz, Germany , Johannes Gutenberg-Universität, Universitätsmedizin, Dermatologische Klinik, Langenbeckstr. 1, D-55131 Mainz, Germany ; Park Systems Europe GmbH, Schildkroetstraße 15, DE-68199 Mannheim, Germany; Johannes Gutenberg-Universität Mainz, Biozentrum II, Institut für Molekulare Physiologie, Mikrobiologie und Weinforschung, Hanns-Dieter-Hüsch-Weg 17, D-55128 Mainz, Germany; Johannes Gutenberg-Universität Mainz, Department Chemie, Duesbergweg 10-14, D-55128 Mainz, Germany

Resume : Multi-drug-resistant bacteria pose an increasingly dangerous risk for global health. Hospital-acquired infections are already believed to cause 100.000 death per year in the U.S. alone. However, novel surface coatings can be designed to address this emerging health crisis. For example, water-dispersible CeO2 nanorods show high catalytic activity in halogenation reactions and thus functionally mimic haloperoxidase enzymes. These enzymes are known to prevent the formation of biofilms through the halogenation of signaling compounds that interfere with bacterial cell-cell communication (“quorum sensing”). Here, we demonstrate the bacteria repellent properties of highly transparent CeO2-coated polycarbonate surfaces with atomic force microscopy. To this end, P. aeruginosa were cultivated for 72 h on CeO2-coated and uncoated substrates and subsequently imaged. Using Park Systems True Non-contact mode and PinPoint mode, we visualized dense biofilm formation on uncoated substrates, whereas CeO2 nanoparticle coatings effectively prevented proliferation. Enzyme mimetic activity was verified with phenol red enzyme assays. Furthermore, crystal violet staining assays and fluorescence assays underlined the biofilm inhibiting properties.

J.2..6
13:00 lunch    
 
SESSION III : KRANZ
14:00
Authors : Monica A. Cotta
Affiliations : University of Campinas, "Gleb Wataghin" Institute of Physics, Applied Physics Dept., Campinas, 13083-859, Brazil

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.

J.3..1
14:30
Authors : Giada Caniglia, Sven Daboss , Christine Kranz
Affiliations : Institute of Analytical and Bioanalytical Chemistry, Ulm University, Albert-Einstein-Allee, 11 89081 Ulm, Germany

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.

J.3..2
14:45
Authors : Gozde Tezcan* (1,2), Kelsey Cremin (1,2), Gabriel N. Meloni (1), Orkun Soyer (2), Munehiro Asally (2), Patrick R. Unwin (1)
Affiliations : (1) Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom (2) School of Life Sciences, Gibbet Hill Campus, University of Warwick, Coventry, CV4 7AL, United Kingdom

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.

J.3..3
15:00
Authors : Belén Cabal (1), David Sevillano (2), Elisa Fernández-García (1), Luis Alou (2), Marta Suárez (1), Natalia González (2), José S. Moya (1), Ramón Torrecillas (1)
Affiliations : 1-Nanomaterials and Nanotechnology Research Center (CINN-CSIC) – Universidad de Oviedo (UO) – Principado de Asturias (PA), Avda de la Vega 4-6, 33940, El Entrego, Spain 3-Microbiology Unit, Medicine Department, School of Medicine, Universidad Complutense, Avda. Complutense s/n, 28040 Madrid, Spain

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.

J.3..4
15:30
Authors : Verdiana Marchianò*1,2, Ana Catarina Duarte*3, María Matos2, Ana Rodríguez3, Pilar García3, Maria del Carmen Blanco Lopez1, Gemma Gutiérez2
Affiliations : (1) Department of Physical and Analytical Chemistry, University of Oviedo, 33006 Oviedo, Spain. (2) Department of Chemical and Environmental Engineering, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain. (3) Department of Technology and Biotechnology of Dairy Products. Dairy Research Institute of Asturias, IPLA-CSIC, Asturias, Spain

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.

J.3..5
15:45
Authors : Izzi, M*(1)(2), Picca, R.A.(1)(2), Sportelli, M.C.(1), Sabbatini, L.(1) & Cioffi, N.(1)(2).
Affiliations : (1) Chemistry Department, University of Bari Aldo Moro, Bari, Italy (2) CSGI (Center for Colloid and Surface Science), Bari, Italy * lead presenter

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.

J.3..6
16:00 coffee break    
 
POSTER SESSION : CIOFFI
16:30
Authors : Shayesteh Bazsefidpar1, Gemma Gutierrez2, Esther Serrano1, Pilar García3, Ana Catarina Duarte3, María Matos*2 and María Carmen Blanco-López 1,*
Affiliations : 1 Department of Physical and Analytical Chemistry & Institute of Biotechnology of Asturias, University of Oviedo, c/Julián Clavería 8, 33006 Oviedo, Spain; 2 Department of Chemical and Environmental Engineering & Institute of Biotechnology of Asturias, University of Oviedo, Spain 3 Department of Technology and Biotechnology of Dairy Products. Dairy Research Institute of Asturias, IPLA-CSIC, Asturias, Spain * Correspondence: matosmaría@uniovi.es, cblanco@uniovi.es

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.

J.4..1
16:30
Authors : M.C. Sportelli1; M. Izzi1; R.A. Picca1; N. Cioffi*1
Affiliations : (1) Chemistry Department, University of Bari, Via Orabona, 4-70126 Bari, Italy * Corresponding Author

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.

J.4..2
16:30
Authors : T. Tite, A.C. Popa, I.M. Chirica, B.W. Stuart, A.C. Galca, L.M. Balescu, G. Popescu-Pelin, D.M. Grant, J.M.F. Ferreira, G.E. Stan
Affiliations : National Institute of Materials Physics, RO-077125 Magurele, Romania

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.

J.4..3
16:30
Authors : X. Rodriguez-Rodriguez, A. Lopez-Cano, R. Roca-Pinilla, A. Kyvik, J. Veciana, J. Guasch, A Arís, E. Garcia-Fruitós, I. Ratera
Affiliations : X. Rodriguez-Rodriguez,1 A. Lopez-Cano,2 R. Roca-Pinilla,2 A. Kyvik, J. Veciana,1,3 J. Guasch,1,3,4 A Arís,2 E. Garcia-Fruitós,2 I. Ratera1,3 1. Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus UAB, Bellaterra, 08193, Spain 2. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Caldes de Montbui, 08140, Spain 3. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Campus UAB, Bellaterra, 08193, Spain 4. Max Planck Partner Group, ICMAB-CSIC, Campus UAB, Bellaterra, 08193, Spain

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.

J.4..4
16:30
Authors : Kurtyka P., Major R., Kustosz R., Kasperkiewicz K., Byrski A.
Affiliations : INSTITUTE OF METALLURGY AND MATERIALS SCIENCE, POLISH ACADEMY OF SCIENCES, KRAKOW, POLAND FOUNDATION OF CARDIAC SURGERY DEVELOPMENT, ARTIFICIAL HEART LABORATORY, ZABRZE, POLAND; INSTITUTE OF METALLURGY AND MATERIALS SCIENCE, POLISH ACADEMY OF SCIENCES, KRAKOW, POLAND; FOUNDATION OF CARDIAC SURGERY DEVELOPMENT, ARTIFICIAL HEART LABORATORY, ZABRZE, POLAND; UNIVERSITY OF SILESIA IN KATOWICE, FACULTY OF BIOLOGY AND ENVIRONMENTAL PROTECTION, KATOWICE, POLAND; UNIVERSITY OF SILESIA IN KATOWICE, FACULTY OF BIOLOGY AND ENVIRONMENTAL PROTECTION, KATOWICE, POLAND;

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

J.4..5
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SESSION V : FERNÁNDEZ
08:45
Authors : Macpherson, J.V. (1), Tully, J.J. (1), Terrero-Rodriguez, I-M. (1) & Wood, G. W. (1)
Affiliations : (1) Department of Chemistry, University of Warwick, UK CV4 7AL

Resume : Ozone (O3) is a very powerful oxidizing agent with a half-life of ca. 20–30 minutes in distilled water. Due to its strong oxidizing potential, dissolved ozone has been used effectively to reduce bacteria loads on surfaces to below detectable limits at ppm’s concentrations. Dissolved O3 also poses no environmental risk due to its significantly shorter half-life compared to in air whilst importantly leaving behind no chemical residues. However, due to the limited half-life dissolved O3 must be generated at the source. Electrochemical methods for O3 generation from the oxidation of water are particularly promising due to the low cost of the required instrumentation and portability of the approach. Lead oxide (PbO2) has been found to be an effective anode material for O3 production, however such electrodes suffer from electrode erosion and Pb contamination issues, as is typical with other metal based electrode systems. Boron doped diamond (BDD), in contrast, is an electrode material which will not corrode under the potentials required for electrochemical O3 production in distilled water, and has been reported to generate O3 from water oxidation. In this work we examine the BDD material properties which control electrochemical O3 production in distilled water at levels capable of preventing bacterial growth on surfaces. By doing so we are able to engineer the BDD to produce optimal concentrations of dissolved O3 on demand.

J.5..1
09:15
Authors : Jooho Jung, Kai Kamada, Taro Ueda, Takeo Hyodo, Yasuhiro Shimizu
Affiliations : Graduate School of Engineering, Nagasaki University, Japan

Resume : We fabricated inorganic nanosheets (NS)-polymer composite films showing antibacterial performance. Layered alpha-zirconium phosphate (Zr(HPO4)2, alpha-ZrP) as a pristine compound of inorganic NS were exfoliated by ion-exchanging protons in the interlayer space with bulky tetraalkylammonium (TRA) ions possessing antibacterial activity. During the exfoliation process, since the tetraalkylammonium cations adsorb on exfoliated alpha-ZrP NS with a negative surface charge, the alpha-ZrP NS modified with TRA ions behave as antibacterial materials. The resultant alpha-ZrP NS were mixed with methyl methacrylate (MMA), then UV light was irradiated to the mixture to polymerize the MMA. As a result of the photo-polymerization, antimicrobial PMMA-alpha-ZrP NS composite films were formed. The PMMA-alpha-ZrP NS films were fabricated using TRA with different lengths of alky chains (propyl ~ octyl), effect of alkyl chain length on antimicrobial activity of the composite films was studied. In general, the composite film including TRA ions with longer alkyl chains (octyl, hexyl) showed more excellent antibacterial performance than those with shorter alkyl chains (butyl, propyl), because the alkyl chains existing on the surface of alpha-ZrP NS pierce a peptide glycan layer of bacteria, resulting in inhibition of proliferation of bacteria. On the other hand, as Gram-positive and Gram-negative bacteria have different plasma membrane characteristics, the antibacterial efficacy of the composite films was largely changed for both bacteria. That is, growth of Gram-positive bacteria (Staphylococcus aureus) with a thick peptide glycan layer as an outer membrane could be easily inhibited by contacting the composite film. In contrast, Gram-negative bacteria covered with a thin peptide glycan and lipopolysaccharide layer (Escherichia coli) could not be sterilized enough. In the presentation, mechanism of the antibacterial effect of the composite films prepared here will be explained and discussed in detail.

J.5..2
09:30
Authors : Grazyna Majkowska-Skrobek1, Pawel Markwitz1, Ewelina Sosnowska1, Cédric Lood 2,3, Rob Lavigne2, Zuzanna Drulis-Kawa1,*
Affiliations : 1 Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology, University of Wroclaw, 51-148 Wroclaw, Poland 2 Department of Biosystems, Laboratory of Gene Technology, KU Leuven, 3001 Heverlee, Belgium 3 Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, Laboratory of Computational Systems Biology, KU Leuven, 3000 Leuven Belgium

Resume : Bacteriophage therapy is currently being evaluated as a critical complement to traditional antibiotic treatment. However, the emergence of phage resistance is perceived as a major hurdle to the sustainable implementation of this antimicrobial strategy. By combining comprehensive genomics and microbiological assessment, we show that the receptor-modification resistance to capsule-targeting phages involves either escape mutation(s) in the capsule biosynthesis cluster or qualitative changes in exopolysaccharides, converting clones to mucoid variants. These variants introduce cross-resistance to phages specific to the same receptor yet sensitize to phages utilizing alternative ones. The loss/modification of capsule, the main Klebsiella pneumoniae virulence factor, did not dramatically impact population fitness, nor the ability to protect bacteria against the innate immune response. Nevertheless, the introduction of phage drives bacteria to expel multidrug resistance clusters, as observed by the large deletion in K. pneumoniae 77 plasmid containing blaCTX-M, ant(3”), sul2, folA, mph(E)/mph(G) genes. The emerging bacterial resistance to viral infection steers evolution towards desired population attributes and highlights the synergistic potential for combined antibiotic-phage therapy against K. pneumoniae.

J.5..3
10:00
Authors : Kukushkina, E.A.*(1,2), Sportelli, M.C. (1), Ditaranto, N. (1,2), Picca, R.A. (1,2), Cioffi, N. (1,2)
Affiliations : (1) Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Italy. (2) CSGI (Center for Colloid and Surface Science) Bari Unit, Università degli Studi di Bari Aldo Moro, Italy.

Resume : Biofilms development and rise in number of resistant bacterial strains urge a need for creation of novel materials with pronounced bactericidal action. Some natural non-toxic components like Chitosan (CS) are widely used in various forms, e.g. sponges, gels and films. The latest have found application as a packaging material in food industry, due to the intrinsic antimicrobial properties, to prevent spoilage of products caused by pathogenic microorganisms. Mechanical robustness of pure CS-based films is not good. Addition of tannic acid (TA) provides beneficial effect on mechanical properties. TA keeps innate antimicrobial properties even upon cross-linkage with CS, the resulting film has improved physical performance and enhanced antimicrobial potency. Combatting antimicrobial resistance (AMR) is a challenging task, since microorganisms develop AMR faster than the new antimicrobials being discovered. Addition of inorganic nanophases, e.g. silver nanoparticles (AgNPs), can potentially prevent fast resistance development. It provides prolonged antimicrobial effect due to the release of silver ions and generation of reactive oxygen species (ROS). In situ generated AgNPs, which are embedded in biocompatible matrix, add prolonged synergistic effect. These reinforced hybrid films were developed in a fast and scalable process, characterized by various spectroscopic (ex. FT-IR, XPS) and microscopic techniques (ex. TEM, SEM). They will be tested against common food pathogens as potential antibiofilm coating additive. Acknowledgements Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439.

J.5..4
10:15
Authors : S. I. Hossain1,2*, M. C. Sportelli1, R. A. Picca1,2, L. Gentile1,2, G. Palazzo1,2, N. Ditaranto 1,2, N. Cioffi1,2
Affiliations : 1Dipartimento di Chimica and 2CSGI (Center for Colloid and Surface Science) Bari Unit, Università degli Studi di Bari “Aldo Moro”, Bari, Italy; Email: syedimdadul.hossain@uniba.it

Resume : Alarmingly, more and more infections are becoming resistant to antibiotics, which poses a great difficulty to treat infections. Notably, Biofilms infections are thought to be responsible for over 80% of infections in humans. Unless we take action, we risk seeing a silent pandemic rising. On this note, scientific communities stand shoulder to shoulder to overcome the development of antimicrobial resistance so that still safe and available medicines can be used to treat infections. One of the possible alternative strategies to slow down antimicrobial resistance and inhibit biofilms is the development of nanoantimicrobials (NAMs) [1,2]. Particularly, silver halides (AgX) have the potential to be NAMs by providing a tailored concentration of biocidal Ag+ ions in an aqueous medium [3]. In the present study, we explore a simple titration method to synthesize green, reproducible, stable, and scalable synergistic silver chloride/Benzyl-hexadecyl-dimetyl-ammonium chloride (AgCl/BAC) colloidal NAMs. Morphology and stability of the nanocolloids were investigated as a function of different molar fractions of the reagents. Nanoparticle size distribution and hydrodynamic radius were measured by dynamic light scattering. NAMs were further characterized by transmission electron microscopy, X-ray photoelectron and Uv-Vis spectroscopies. Our experimental evidences support the morphological stability of the nanocolloids, along with their antimicrobial property. Application of the as-prepared AgCl/BAC NAMs is planned to be investigated to developing antimicrobial water-insoluble hard coating providing slow-releasing active phases in active Food Packaging and Biomedical devices, mainly aiming at bacteriostatic, long term effects. References 1. Nanomaterials 10(4), (2020), 802 2. Chem. Mater. 17, (2005) 5255–5262 3. JACS, 128, (2006), 9798-9808 Acknowledgements Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439.

J.5..5
10:30 coffee break    
 
SESSION VI : KRANZ
11:00
Authors : Samantha Douman, David Collins, Loanda Cumba, Stephen Beirne, Gordon G. Wallace, Zhilian Yue, Emmanuel I. Iwuoha, Federica Melinato, Yann Pellegrin and Robert J. Forster
Affiliations : National Centre for Sensor Research, School of Chemical Sciences, Dublin City University, Dublin 9, Ireland. FutureNeuro SFI Research Centre; Intelligent Polymer Research Institute, ARC Centre of Excellence for Electromaterials Science, University of Wollongong, NSW 2522, Australia; SensorLab (UWC Sensor Laboratories), University of Western Cape, Cape Town, South Africa; Université de Nantes, CEISAM, UMR CNRS, Nantes, France

Resume : Electrochemical and electrochemiluminescent sensors for the detection of biofilm causing bacteria offer several advantages including high sensitivity, simplicity of operation, fast response times, and lower cost for quantifying biofilms. They can also be easily miniaturised and made using mechanically flexible materials allowing wearable, and perhaps even implantable, sensors to be created. 3D electrodes can significantly enhance the detection sensitivity due to enhanced mass transport and high surface areas within a small volume. In this contribution, we present our latest results on 3D electrodes, e.g., 3D pin arrays and microcavity arrays, in conventional and wireless(bipolar) electrochemical setups. We show that compared to planar electrodes, 3D arrays can significantly enhance the detection of whole bacteria and amplify the redox and electrochemiluminescent responses associated with the detection of bacterial DNA in sandwich assays. Specifically, we report on the electrochemiluminescent properties of electrodes with spherical pores produced using nanosphere lithography or 3D-printing. Using these strategies, robust porous electrodes with tailored surface areas, volumetric porosity and flow properties have been developed. Significantly, enhanced electrochemiluminescence is observed for polymeric, monolayer and solution phase reactants. COMSOL modelling of the electric field, and direct imaging of the ECL intensity within the cavities using confocal and super resolution (STED) microscopy, reveals the distribution of the electric field within the porous electrode. In wireless electrochemistry, a second possibility is to shape the local electric field strength and distribution so as to enhance the sensitivity of electrochemical and electrochemiluminescent detection of bacteria and their associated signalling molecules. For example, we will discuss the properties of a 3D titanium array for electrochemiluminescence, ECL, generation from ruthenium tris-bpy type systems through both co-reactant and annihilation mechanisms. Significantly, the presence of an oxide layer inhibits water reduction allowing ECL generation in aqueous solutions without the need for a co-reactant through annihilation of electrogenerated ruthenium tris-bpy radicals

J.6..1
11:30
Authors : Irina M. Terrero Rodríguez, Julie V. Macpherson
Affiliations : Department of Chemistry, University of Warwick

Resume : Biofilm formation is a type of biofouling where bacteria irreversibly attach and form communities on a surface. Biofilms can form in many different types of surfaces and result in millions of dollars lost to problems such as product quality impairment and biocorrosion. An in-situ monitoring system that can offer real time information is crucial to contain biofilm formation and to optimize antimicrobial use. Electrochemical sensing methods are very desirable for this application, as they easy to implement and miniaturize. In this work we are interested in exploring the potential for using capacitance, faradaic and/or resistance measurements in low ionic strength media e.g. tap water or distilled water. As bacteria grow they will partially block the electrode surface and secrete charged metabolism products and redox-active signaling molecules, altering the electrochemical properties of the system. Boron-doped diamond (BDD) is an excellent electrode material for sensing due to its low background currents, high electrode potentials can also be applied to the electrode to clean in-situ after growth of the bacteria. However, it is typically a low biofouling material. To encourage bacterial attachment, electrode surface roughness was varied during this study. SEM imaging was used to visualize electrode surfaces at different time points.

J.6..2
11:45
Authors : Nazan Altun *(1), Marta Sampayo Iglesias (1), Natalia Prado Marrón (1), Martín Hervello Costas (1), Juan Díaz García (1), Felipe Lombó (2), Pelayo González González (1).
Affiliations : (1) ASINCAR (Asociación de Investigación de Industrias Cárnicas del Principado de Asturias), 33180, Asturias, Spain (2) Research Unit “Biotechnology in Nutraceuticals and Bioactive Compounds-BIONUC”, Department of Functional Biology, Faculty of Medicine, University of Oviedo, 33006, Asturias, Spain * lead presenter

Resume : Introduction When bacteria are present in the raw food material, they adhere to the surface of equipment and tools (the main microbial adhesion and biofilm formation sites in the food industry) at the convenient conditions, forming a biofilm matrix. Biofilms cause contamination risk to the food industry because of their biotransfer potential by planktonic cells (ability of the microorganisms to disperse from biofilm surfaces after cleaning procedures). NIRS is suggested as a useful, rapid, and noninvasive tool for biofilm detection [1-2]. Material and Methods In this study, a detection model for single species biofilms has been developed. The studied biofilm species are commonly present in food industry surfaces, such as polyethylene. Biofilms of P. aeruginosa CECT108, E. coli CECT434 and S. enterica CECT4594 were generated on coupon surfaces made of high density polyethylene (HDPE). The biofilms were analysed by near infrared spectroscopy (NIRS) coupled with spectral pretreatment methods and different chemometric classification models to detect presence/absence of biofilm and to identify the type of bacterial species. Crystal violet assay was done to cross-validate biofilms detection. Results Regression results on crystal violet data show, bacteria strain was highly influential on absorbance responses. It was possible to build successful classification models on biofilms (over 85% for detection and 70% for identification) by aid of staining and image processing techniques and use of NIRS coupled with chemometrics was found to be a promising method. Conclusions NIRS can be used for biofilm detection on HDPE surfaces and studies can further be extended to other surfaces and bacteria. Bibliography 1) Wirtanen G, Husmark U, Sandholm TM: Microbial Evaluation of the Biotransfer Potential from Surfaces with Bacillus Biofilms after Rinsing and Cleaning Procedures in Closed Food-Processing Systems. J Food Prot 1996, 59(7):727-733. 2) Alexandrakis D, Downey G, Scannell AGM: Detection and Identification of Bacteria in an Isolated System with Near-Infrared Spectroscopy and Multivariate Analysis. J. Agric. Food Chem. 2008, 56, 10: 3431–3437

J.6..3
12:00
Authors : Elena E. Ferapontova
Affiliations : Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark

Resume : Rapid detection of microorganisms in industry and human environment is a 1st step in human health protection, preventing huge economical losses, e.g., only in US’s oil industry microbial corrosion costs >$170 billion/year. Sensitive and specific, and yet inexpensive electrochemical sensing approaches can provide the required tools for fast and robust bacterial analysis [1-4]. Here, I discuss the recent achievements in ultrasensitive electrochemical detection of bacteria in environmental and biological samples. I will discuss our recent achievements in the development of electrochemical “label-free” sensing approaches for detection of bacterial DNA and RNA, enabling detection of 1 fM bacterial DNA/RNA, and electrochemical aptamer and immunoassays for detection of bacterial cells at their single cell level. A particular emphasis will be made on magnetic-beads–based approaches that allow detecting a single bacterial cell in 1-5 mL samples at a cost below 1-2 € that favorably compete with the existing standard microbial counting and quantitative PCR analytical schemes [5]. References [1] Ferapontova E., Basic concepts and recent advances in electrochemical analysis of nucleic acids. Curr. Opin. Electrochem. 2017, 5, 218-25 [2] Campuzano S., Yáñez-Sedeño P., Pingarrón J.M., Electrochemical biosensing for the diagnosis of viral infections and tropical diseases. ChemElectroChem 2017, 4, 753-77 [3] Binte Jamal, R.; Shipovskov, S.; Ferapontova, E.E.*, Electrochemical Immuno- and Aptamer-Based Assays for Bacteria: Pros and Cons over Traditional Detection Schemes, Sensors (IF 5.0) 20(19) 2020 5561 (p.1-27) [4] Ferapontova E., Electrochemical assays for microbial analysis: how far they are from solving microbiota and microbiome challenges, Curr. Opin. Electrochem. 2020, 19, 153-161 [5] Pankratov, D.; Bendixen, M.; Shipovskov, S.; Gosewinkel, U.; Ferapontova, E. E., Cellulase-Linked Immunomagnetic Microbial Assay on Electrodes: Specific and Sensitive Detection of a Single Bacterial Cell, Analytical Chemistry (IF 6.8) 92 2020 12451–12459

J.6..4
12:30
Authors : Mylan Lam (1), Vivien Moris(1), Romain Vayron (2), Rémi Delille (2), Véronique Migonney (1), Céline Falentin-Daudré (1)
Affiliations : (1)LBPS/CSPBAT, UMR CNRS 7244, Institut Galilée, Université Sorbonne Paris Nord, 99 avenue JB Clément, 93430- Villetaneuse, France (2)LAMIH, UMR CNRS 8201, Université Polytechnique Hauts-de-France, Valenciennes, France

Resume : Silicone breast implant (BI) issues have raised many concerns and attention these past years. Part of the top 10 most implantable biomedical devices, BIs are widely used for aesthetic and reconstructive surgeries. However, as with any devices, BIs are subjected to healthcare-associated infections as the development of capsular contractures mostly or, more rarely, the apparition of large cell anaplasic lymphoma, a cancer-related to breast prosthesis.  The grafting of bioactive polymers has actively demonstrated to improve metallic and polymer surfaces’ bio-integration by conferring them an antibacterial property and improving their biocompatibility. In the scientific literature, numerous strategies are reported to overcome bacterial biofilms and improve the surface’s biocompatibility by surface modifications. Our team has recently achieved a simple way to graft the poly(styrene sodium sulfonate) – (polyNaSS) polyNaSS directly on silicone breast implants outer shell only using UV irradiation with a grafting “from” strategy. Characterization methods as SEM-EDS, XPS, AFM, ATR-FTIR, colorimetric assay, and contact angle measurements have confirmed the effective grafting. Therefore, in the biomedical field, it is necessary to fulfill the regulation in force. ISO standards state the requirements a material should respect to guarantee the safety and quality of a product. Hence, the present study aims to evaluate the grafting parameter’s impacts, including UV irradiation and the presence of the grafted bioactive polymer on both silicone shell initial mechanical properties and the biological responses using a standard L929 fibroblast cell line to evaluate the cellular viability. First, the results have shown an effective grafting of the polyNaSS on silicone breast implants' surfaces. Second, a clear improvement of the biocompatibility of the grafted surface towards cell development has been proven without damaging the material's initial mechanical properties.

J.6..5
12:45
Authors : Roman Major1, Katarzyna Kasperkiewicz2, Adam Byrski1, Maciej Gawlikowski3, , Marcin Dyner4, Juergen M. Lackner5, Łukasz Major1, Reinhard Kaindl5
Affiliations : 1 Institute of Metallurgy and Materials Science, Polish Academy of Sciences, Reymonta St. 25, 30-059 Cracow, Poland 2 University of Silesia in Katowice, Faculty of Natural Sciences, Institute of Biology, Biotechnology and Environmental Protection, Jagiellońska St. 28, 40- 032 Katowice, Poland; ORCID: 0000-0002-9851-790X 3 Silesian University of Technology, Faculty of Biomedical Engineering, Department of Biosensors and Processing of Biomedical Signals, Roosevelt Str. 40, Zabrze, Poland. 4 Faculty of Science and Technology, Jan Dlugosz University in Czestochowa, Armii Krajowej Av, 13/15, 42-200 Czestochowa, Poland, 5 JOANNEUM RESEARCH Forschungsges.m.b.H., Institute of Surface Technologies and Photonics, Functional Surfaces, Leobner Strasse 94, 8712 Niklasdorf, Austria

Resume : Introduction Finger amputation is surgical treatment for ~69.000 patients in EU after traumatic injury, in which replantation microsurgery fails due to severity of tissue damage. However, absence of even a single finger results in major impairment in hand function (precise grasping, grip power) and affects the completely social and professional life of the frequently young victims. Surgical reconstruction is currently only possible by autograft transplantation, e.g. toe-to-hand transfer, leading to impairment at the foot site. Some motion functional restoration is possible by bone-anchored silicone prosthesis, but without sensations available. Reasonable, our current research focuses on alternatives for surgical reconstruction by novel patient-specific, durable, biomimetic, bioactive and antibacterial implants for reconstruction of lost bone and joints. The implant design – and the beyond the project improved micro(neuro)surgery – will include soft-tissue related mobility, implantation of state-of-the-art nerve conduits and aesthetic appearance for fast, successful rehabilitation. Key issues for long-term functionality of biomaterial-based reconstruction of hard tissue are based on surgical demands: (1) Perfect integration of bone substituting metal to the surrounding bone tissue (a) without loosening due to stress shielding at the interface and (b) with protection against bacterial inflammation (antimicrobial properties and formation of vascularized bone tissue (ossification)) even months to years after the injury. (2) biomimetic finger joints based on nonwearing materials without ossification tendency to prevent loss of motion function. Materials &Methods Direct cytotoxicity tests were conducted using Normal Human Dermal Fibroblasts (NHDF) C-12302 cells. Live and necrotic cells were evaluated with attached confocal microscopes using propidium iodide (marking dead cells red) and MitoTracker Green (staining live cells green). The more propidium odidide excitation, the greater the likelihood that cells located on the surface of the biomaterial are dead. Results of all 4 tested materials are presented as co-localization chart. Ti64-stress relief anodize show the most cytotoxicity towards tested fibroblast cells, whereas Ti64-as built-anodize has the least cytotoxic impact on fibroblast cells. Lactate dehydrogenase (LDH) is an enzyme found in the cytoplasm of all cells in the human body. When cells are damaged, lactate dehydrogenase is released from within them, and this reaction causes its concentration and activity in the blood to increase. (higher activity = more likely more cells are damaged). The antimicrobial activity contact test was based on ISO 22196:2007(E). Escherichia coli strain ATCC 8739 (Gram-negative) and Staphylococcus aureus strain 6538P (gram-positive) were used, as recommended in the norm. Obtained results are visualized as antibacterial activity index (R), which represents the difference between the number of viable bacteria recovered from both untreated and treated specimens. Material yields antibacterial properties if the calculated R value is greater than 2 (orders of magnitude). The higher the R index is, the better the antibacterial properties are. Acknowlegement This research was financially supported by the Polish National Centre of Research and Development (Grant no. fingerIMPLANT M-ERA.NET2/2019/7/2020, “Patient-specific, anti-microbial bioactive finger implants for durable functional reconstruction after amputation”).

J.6..6
13:00 lunch    
 
SESSION VII : BLANCO
14:00
Authors : Frank Schreiber
Affiliations : Federal Institute for Materials Research and Testing (BAM)

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.

J.7..1
14:30
Authors : C. Vitelaru, A.C Parau, A.E. Kiss, I. Pana, M. Dinu, L.R. Constantin, A. Vladescu,S. Costinas, C.S. Adochite, M. Moga, L. Floroian, M. Badea, M. Idomir, L.E. Tonofrei
Affiliations : National Institute of Research and Development for Optoelectronics - INOE 2000;National Institute of Research and Development for Optoelectronics - INOE 2000;National Institute of Research and Development for Optoelectronics - INOE 2000;National Institute of Research and Development for Optoelectronics - INOE 2000;National Institute of Research and Development for Optoelectronics - INOE 2000;National Institute of Research and Development for Optoelectronics - INOE 2000;National Institute of Research and Development for Optoelectronics - INOE 2000;Transilvania University of Brasov, Faculty of Medicine;Transilvania University of Brasov, Faculty of Medicine;Transilvania University of Brasov, Faculty of Medicine;Transilvania University of Brasov, Faculty of Medicine;Transilvania University of Brasov, Faculty of Medicine;Transilvania University of Brasov, Faculty of Medicine;ATS Novus SRL

Resume : Silver nanoparticles are widely used as potent antimicrobial agents in various applications. By embedding them into a transparent dielectric matrix it is possible to obtain coatings that are both transparent and have antimicrobial properties. Such thin films can be deposited onto self-adhesive polymer foils, adding additional functionalities to the mechanical protection provided by such foils. In order to keep the transparency of the coatings one needs to limit the amount of silver on the surface and to avoid coalescence by confining the metal only in isolated nanosized particles. Nevertheless, a sufficient amount of silver has to be available to act as antimicrobial agent on the surface. The transparent films, with total thicknesses of few tens of nm, were deposited on flexible self-adhesive polymers by magnetron discharge. RF sputtering of oxide targets was used to obtain the amorphous oxide matrix, consisting of SiO2, TiO2 or a combination of both. For the silver target the HiPIMS regime operated at low repetition rates was used, in order to finely tune the amount of silver into the growing film. Both co-sputtering and sequential sputtering from different targets were used to investigate different growth paths and to finely tune the quantity of Ag. The thin film surface was investigated by AFM, providing information on the topography of the coatings and their preferential growth on the textured polymer foil. SEM-EDX was used to assess the presence of silver into the thin films, and evaluate its relative variation as a function of process parameters. XRD revealed the presence of specific Ag peaks into an amorphous oxide matrix. UV-Vis-NIR spectroscopy of the transparent structures such as polymer foils covered by thin films, revealed that preferential absorption occurs in the 400 to 500 nm spectral range. This is typical for silver surface plasmon resonance, the position of the peak being tuned both by incorporating the silver in a different matrix, either SiO2 or TiO2, or by adjusting the amount of silver in the coating. The transparency of the thin films over visible range was optimized by controlling the amount of oxygen in the process, maintaining the transmittance above 90% making the thin films suitable as transparent coatings with antimicrobial properties. The antimicrobial properties were assessed by using antimicrobial test with Escherichia coli strain. Our studies indicated different actions of these transparent films in presence of E. coli. The highest efficiency was observed for the Ag/SiO2 combination, in the concentration range of 103- 105 CFU/mL. These conclusions could be useful for future applications designs of these materials, in order to use their discovered antimicrobial actions for possible protection properties. Acknowledgment: „This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI – UEFISCDI, project number 489PED ⁄ 2020, within PNCDI III, and Core Program, Project no. 18N/2019 .

J.7..2
14:45
Authors : L. Duta1*, M.C. Chifiriuc2,3, V. Grumezescu1, G.E Stan4, O. Gherasim1,5, G. Popescu-Pelin1, V. Craciun1, F.N. Oktar6,7
Affiliations : 1National Institute for Lasers, Plasma and Radiation Physics, Magurele, Romania 2Department of Microbiology, Faculty of Biology, University of Bucharest, Bucharest, Romania 3Research Institute of the University of Bucharest (ICUB), Earth, Environmental and Life Sciences Division, Bucharest, Romania 4National Institute of Materials Physics, Magurele, Romania 5Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, Bucharest, Romania 6Department of Bioengineering, Faculty of Engineering, University of Marmara, Istanbul, Turkey 7Advanced Nanomaterials Research Laboratory (ANRL), University of Marmara, Istanbul, Turkey

Resume : We report on simple and doped hydroxyapatite (HA) coatings of biological-origin (BioHA) synthesized by Pulsed Laser Deposition onto titanium implants. The role of different doping agents on the physical-chemical, mechanical and biological properties of structures was assessed. The morphological investigations revealed the fabrication of surfaces with rough and irregular morphologies, which were demonstrated to facilitate a good adhesion of cells and anchorage of implants in situ. Structural analyses demonstrated that the synthesized BioHA coatings consisted of a hexagonal HA phase. Compositional examination indicated the presence of trace-elements generally found in the composition of the bone mineral phase, along with a quasi-stoichiometric target-to-substrate transfer. This is consistent with the biological nature of pristine materials. The IR spectra highlighted the main bands specific to the phosphate groups, which corresponded to a HA-type structure. Moreover, after only three days of immersion in simulated body fluid, IR spectra showed a remarkable growth of a biomimetic apatitic layer, which is indicative for a high biomineralization capacity of the synthesized coatings. The inferred bonding strength adherence values were superior to the threshold imposed by international standard. All synthesized layers exhibited low cytotoxicity on human osteosarcoma and skin cells, corroborated with a long-lasting anti-staphylococcal and –fungal biofilm activity. Along with their low-cost fabrication from sustainable resources, the combined characteristics of these bioinspired HA materials could offer guidance towards their suitability of being used as viable alternatives to synthetic HA ones for implantological applications. Acknowledgements: Project no. PN-III-P1-1.1-TE2019-1449 (TE 189/2021) and Core Programme 16N/2019.

J.7..3
15:00
Authors : Dipankar Koley
Affiliations : Associate Professor Department of Chemistry Oregon State University

Resume : Dental composites may be formulated with metallic ions that, upon release, may control surface biofilm formation. To better understand this phenomenon, it is important to probe the immediate chemical microenvironment at the composite surface upon which a biofilm is growing and metabolizing. For this study, we developed carbon-based solid-state u-Ion-Selective Electrode (u-ISE) sensors. This unique low PVC content sensor can perform amperometric measurements and then be switched to potentiometric measurements. The 35-40 m diameter Ca2+-ISE showed fast response time (~1 sec or less), low limit of detection (~1 M), and broad linear range (5 M to 200 mM). In addition, the Ca2+-u-ISE was proven to demonstrate excellent selectivity towards major interfering ions (such as Na+, K+, and Mg2+, with logKCa2+,A = -5.5 to -6.0). Similarly, carbon-based u-pH sensors have also been developed that showed near Nernstian slope and been used in the chemical mapping of microbial biofilms. New findings of probing the biofilm-material chemical interface in real-time using these SECM probes along with flexible microsensors would be presented at the meeting.

J.7..4
15:30
Authors : S. Auditto, S. Carrara, F. Rouvier, F. Brunel, C. Janneau, M. Camplo, M. Sergent, I. About, J.-M. Bolla, J.-M. Raimundo
Affiliations : Aix-Marseille Univ; CNRS; CINAM; INSERM; SSA; IRBA; MCT; ISM; Inst Movement Sci; Avignon Université; IRD; IMBE

Resume : Bacterial infections are one of the major threats to public health, food safety and de-velopment today which makes it urgent to combat by developing materials or strategies limiting or preventing these bacterial prolifer-ations and biofilm infections.[1] Although, medical implants have led to dramatic im-provement in patient's health and well-being, there are often drawbacks that include surgi-cal risks during placement or removal, im-plant failure and more specifically microbial infections. These implant-associated infec-tions are mainly caused by the bacterial bio-film formation in which bacteria are more recalcitrant towards treatments. Indeed, im-plant surfaces are non-vascularized abiotic materials rendering the common strategies inappropriate and ineffective.[2] In this context we have designed and devel-oped innovative and smart interfaces based on phosphonium SAMs that can be electri-cally activated on-demand for eradicating bacterial infections on solid surfaces. Hence, upon electroactivation, using a low potential of 0.2V for 1 hour, a successful stamping out of Gram-positive and Gram-negative bacteria strains has been clearly highlighted on SAM-modified titanium surfaces. Subsequently, using these conditions, Staphylococcus aure-us and Klebsiella pneumoniae were killed up to 95% and 90% respectively and full eradi-cation if time is prolongated (Fig. 1). More importantly, no harmful activity has been observed towards eukaryotic cells which clearly demonstrates the biocompatible char-acter of these novel surfaces for further im-plementation. 1. Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S.; Mendelson, M.; Monnet, D. L.; Pulcini, C.; Kahlmeter, G.; Kluyt-mans, J.; Carmeli, Y.; Ouellette, M.; Out-terson, K.; Patel, J.; Cavaleri, M.; Cox, E. M.; Houchens, C. R.; Grayson, M. L.; Hansen, P.; Singh, N.; Theuretzbacher, U.; Magrini, N. (2018) Lancet Infect. Dis.18, 318-327. 2. a) Brunel, F.; Lautard, C.; Giorgio, S.; Garzino, F.; Raimundo, J. M.; Bolla, J. M.; Camplo, M. (2018) Bioorg. Med. Chem. Lett. 28, 926-929; b) Brunel, F.; Lautard, C.; Garzino, F.; Raimundo, J. M.; Bolla, J. M.; Camplo, M. (2020) Bioorg. Med. Chem. Lett. 30, 127389; c) Raimundo, J.-M.; Camplo, M.; Bolla, J.-M.; Brunel, F.; Lautard, C.; PCT Int. Appl. (2020) WO 2020008000 A1 20200109; d) Carrara, S.; Rouvier, F.; Auditto, S.; Brunel, F.; Janneau, C.; Camplo, M.; Sergent, M.; About, I.; Bol-la, J.-M.; Raimundo, J.-M. (2021) ACS Appl. Mater. Interfaces submitted.

J.7..5
15:45 BLANCO, CIOFFI, KRANZ, FERNÁNDEZ: announcement of the grant winner & concluding remarks    

No abstract for this day

No abstract for this day


Symposium organizers
Christine KRANZUlm University

Institute of Analytical and Bioanalytical Chemistry, Albert-Einstein-Allee 11, 89081 Ulm

Christine.kranz@uni-ulm.de
Maria Carmen BLANCO LÓPEZUniversity of Oviedo

Department of Physical and Analytical Chemistry, C/ Julián Clavería 8, 33006 Oviedo, Spain

cblanco@uniovi.es
María FERNÁNDEZIPLA-CSIC

Paseo Rio Linares s/n 33300. Villaviciosa, Asturias, Spain

mfernandez@ipla.csic.es
Nicola CIOFFIUniversity of Bari

Department of Chemistry, Campus Universitario, 4 via Orabona, I-70126, Bari, Italy

nicola.cioffi@uniba.it