Pre-clinical evaluation of novel antimicrobial coatings for blood-contacting devices commonly relies on the performance of animal studies since alternative in vitro models do not adequately represent the interactions between blood, bacteria, and material surfaces as they occur in vivo. To reduce the need of these cost-intensive and controversial animal tests, this project was dedicated to the development of a new model setup that overcomes this limitation and allows in vitro evaluation under in vivo-like conditions. This newly developed model was intended to be directly applied to evaluate recently in-house developed antimicrobial coatings, so-called anchor polymers. Therefore, the project was divided into two parts. The first part of the project focused on the evaluation of the anchor polymer coatings concerning their applicability in blood-contacting devices. For this purpose, the PEGylated styrene-maleic acid copolymers were intensively studied using established laboratory tests. These examinations showed very promising results regarding adsorption and stability on relevant polymer substrates, antimicrobial efficacy, and biological safety of the coatings, thus revealing their great potential for future applications in medical devices. Moreover, this basic characterization was meant to allow a subsequent comparison of the new in vitro model with state-of-the-art in vitro tests. The second part of the thesis focused on the development of the realistic in vitro model. Here, a single-pass flow system realized the implementation of adjustable flow conditions. Furthermore, incubation with freshly drawn human blood provided a physiological nutrient environment and included the influence of an immune response. Staphylococcus aureus were chosen as representative microorganisms, as they are responsible for a majority of device-related blood stream infections. The resulting blood flow model was validated with one anti-adhesive and one contact-killing anchor polymer coating, confirming the model’s ability to differentiate the investigated surfaces. Inflammatory and coagulant blood activation correlated slightly with bacterial coverage, which in turn was strongly dependent on the investigated material surface. Incubation with varying flow conditions demonstrated the model’s capability to reflect the well-documented dependence of bacterial colonization and occurring flow conditions. In contrast to the state-of-the-art in vitro tests, the simultaneous incubation of test surface, bacteria and whole blood allowed the analysis of mutual interactions of the three parameters. Thus, the model represents an excellent method for pre-clinical evaluation of novel antimicrobial coatings for blood-contacting devices.

Sensing of pathogens by pattern recognition receptors (PRR) is critical to initiate protective host defence reactions. However, activation of the immune system has to be carefully titrated to avoid tissue damage necessitating mechanisms to control and terminate PRR signalling. Dectin-1 is a PRR for fungal β-glucans on immune cells that is rapidly internalised after ligand-binding. Here, we demonstrate that pathogen recognition by the Dectin-1a isoform results in the formation of a stable receptor fragment devoid of the ligand binding domain. This fragment persists in phagosomal membranes and contributes to signal transduction which is terminated by the intramembrane proteases Signal Peptide Peptidase-like (SPPL) 2a and 2b. Consequently, immune cells lacking SPPL2b demonstrate increased anti-fungal ROS production, killing capacity and cytokine responses. The identified mechanism allows to uncouple the PRR signalling response from delivery of the pathogen to degradative compartments and identifies intramembrane proteases as part of a regulatory circuit to control anti-fungal immune responses.

Die Ausbreitung antimikrobieller Resistenzen stellt ein zunehmendes gesundheitliches und gesellschaftliches Risiko dar. Alternative antimikrobielle Verfahren mit einem geringen Resistenzpotenzial, breiten Wirkspektrum und geringen Umweltrisiko gewinnen an Bedeutung. Ein solches Verfahren stellt die Photodynamische Inaktivierung (PDI) dar. Ihr Wirkmechanismus beruht auf der photosensibilisierten Anregung von Singulettsauerstoff (1O2), der durch oxidativen Stress zum Zelltod führt. Der für die katalytische Aktivierung des Sauerstoffs verantwortliche Photosensibilisator (PS), muss nicht in die Mikroorganismen eindringen und wird durch sichtbares Licht angeregt. Die Übertragung einer stationär vermittelten PDI auf Oberflächen erscheint daher besonders sinnvoll. In der vorliegenden Arbeit werden erstmalig zwei Ansätze untersucht, die sowohl kommerzielle Substrate als auch industrielle Standardverfahren zur Oberflächenveredelung verwenden: Eine Elektronenstrahl-Funktionalisierung von Mikrofiltrationsmembranen und eine textiltechnologische Funktionalisierung von Polyestergeweben, insbesondere Reinraumtextilien. Für die Charakterisierung der Polymeroberflächen werden neben Versuchen zur Zellviabilität, optisch-spektroskopische Methoden und erstmalig orts- sowie zeit-aufgelöste Messungen der 1O2-Lumineszenz herangezogen. Im Resultat erweisen sich beide Funktionalisierungsansätze als geeignet für eine stationär vermittelte PDI. Dabei sind die textiltechnologisch funktionalisierten Polyestergewebe besonders Wirkungsvoll und erzielen bereits nach kurzer Weißlichtbestrahlung von unter 30 Minuten eine antimikrobielle Wirkung. Die Messungen der 1O2-Lumineszenzkinetik erweisen sich als eine vielversprechende Methode eine mögliche PDI-Aktivität vorab zu bewerten und bei der Entwicklung wichtige Impulse für die Oberflächenfunktionalisierung zu setzen. Schließlich ist eine systematisierte Methodologie zur Bewertung PDI-aktiver Oberflächen ein wesentliches Resultat dieser Arbeit. / The spread of antimicrobial resistance is an increasing health and social risk. Alternative antimicrobial methods with a low resistance potential, broad spectrum of activity and low environmental risk are gaining importance. Photodynamic inactivation (PDI) is one such method. Its mechanism of action is based on the photosensitised excitation of singlet oxygen (1O2), which leads to cell death through oxidative stress. The photosensitizer (PS), which is responsible for the catalytic activation of the oxygen, does not have to penetrate the microorganisms and is excited by visible light. The transfer of a stationary-mediated PDI to surfaces therefore seems particularly useful. In the present work, two approaches are investigated for the first time that use both commercial substrates and standard industrial processes for surface modification: An electron beam functionalisation of microfiltration membranes and a textile-technological functionalisation of polyester fabrics, especially cleanroom textiles. In addition to experiments on cell viability, optical spectroscopic methods and, for the first time, spatially and temporally resolved measurements of 1O2 luminescence are used to characterise the polymer surfaces. As a result, both functionalisation approaches prove to be suitable for a stationary-mediated PDI. The textile-technologically functionalised polyester fabrics are particularly effective and achieve an antimicrobial effect after only a short white light irradiation of less than 30 minutes. Measurements of 1O2 luminescence kinetics are proving to be a promising method of evaluating possible PDI activity in advance and providing important impetus for surface functionalisation during development. Finally, a systematised methodology for the evaluation of PDI-active surfaces is an essential result of this work.

No description available.

In der humanen Haut spielen antimikrobielle Peptide (AP) bei Entzündungsgeschehen bakteriellen und nicht-bakteriellen Ursprungs eine bedeutende Rolle. Neben einer konstitutiven Expression AP können Zytokine deren vermehrte oder abgeschwächte Expression bewirken. In dieser Arbeit wurden die AP humanes β-Defensin (HBD) -2, HBD-3 und Psoriasin (PSO) in Bezug auf deren Expression in gesunder Haut und deren Modulation durch Zytokine in vivo anhand des xenogenen NOD-SCID-Maus-Transplantationsmodells untersucht. Nach erfolgreicher Transplantation von humaner Haut auf NOD-SCID Mäuse wurden die Zytokine TNF-α, IFN-γ und IL-13 in unterschiedlicher Dosierung einzeln und in Kombination intradermal appliziert. Für TNF-α konnte eine erhöhte Expression von HBD-2, HBD-3 und PSO auf RNA-Ebene mittels in-situ-Hybridisierung und Protein-Ebene mittels immunhistochemischer Nachweismethoden gezeigt werden. Eine erhöhte Expression nach Injektion von IFN-γ ließ sich für HBD-3 auf RNA-Ebene und Protein-Ebene und für HBD-2 auf RNA-Ebene erfolgreich belegen. PSO zeigte auf Protein-Ebene nach Modulation mit IFN-γ eine bei höherer Dosierung leicht abnehmende Expression. Eine Änderung der Expression durch IL-13 ließ sich nicht eindeutig belegen. In dieser Arbeit konnte die in der Literatur in vitro beschriebene Modulationsfähigkeit der untersuchten AP durch die verwendeten Zytokine in vivo belegt werden.

Solar-driven photocatalysis is of great interest in terms of a sustainable use of energy and its application in wastewater treatment. The UV light-driven photogeneration of H2O2 by solar irradiation is an advanced strategy for the treatment of bacteria and recalcitrant pollutants in wastewater, but suffers from low efficiencies. In this work, a solar-driven multifunctional nanocomposite consisting of Tm3+ upconverting nanoparticles, poly(vinyl alcohol), poly(acrylic acid) and hydroxylated sulfonated poly(ether ether ketone) was prepared. The components were crosslinked via a heating treatment at 170 °C, resulting in a non-leaching porous material. This nanocomposite exhibited excellent adsorption ability (89 % in 150 min) toward a 100 mg/L ciprofloxacin aqueous solution and proved to photodegrade it (50 %) upon 4 h artificial solar irradiation, exploiting photon upconversion processes. Moreover, an 80 % bactericidal effect against E. coli was registered upon sunlight irradiation. Altogether, these results suggest the feasibility of a solar-driven wastewater treatment based on upconverting nanoparticles.

Over the course of the last decades, the continuous exposure of bacteria to antibiotics—at least in parts due to misprescription, misuse, and misdosing—has led to the widespread development of antimicrobial resistances. This development poses a threat to the available medication in losing their effectiveness in treating bacterial infections. On the drug development side, only minor advances have been made to bring forward novel therapeutics. In addition to increasing the efforts and approaches of tapping the natural sources of new antibiotics, synthetic approaches to developing novel antimicrobials are being pursued. In this study, BDTL049 was rationally designed using knowledge based on the properties of natural antibiotics. BDTL049 is a carbosilane dendritic system with bow-tie type topology, which has antimicrobial activity at concentrations comparable to clinically established natural antibiotics. In this report, we describe its mechanism of action on the Gram-positive model organism Bacillus subtilis. Exposure to BDTL049 resulted in a complex transcriptional response, which pointed toward disturbance of the cell envelope homeostasis accompanied by disruption of other central cellular processes of bacterial metabolism as the primary targets of BDTL049 treatment. By applying a combination of whole-cell biosensors, molecular staining, and voltage sensitive dyes, we demonstrate that the mode of action of BDTL049 comprises membrane depolarization concomitant with pore formation. As a result, this new molecule kills Gram-positive bacteria within minutes. Since BDTL049 attacks bacterial cells at different targets simultaneously, this might decrease the chances for the development of bacterial resistances, thereby making it a promising candidate for a future antimicrobial agent.

Salmonella spp. sind fakultative intrazelluläre Pathogene, die gastrointestinale und systemische Erkrankungen in einem umfassenden Wirtsbereich, einschließlich Tier und Mensch, hervorrufen. Salmonella benötigt verschiedene Virulenzgene für die Infektion welche auf sogenannten Salmonella Pathogenitäts-Inseln (SPI) kodiert sind. Hinzu kommt, dass auch zahlreiche im Salmonella Genom verstreuten Gene an verschiedenen Aspekten von Virulenz und Pathogenese beteiligt sind. In der vorliegenden Studie wurde die Funktion eines zuvor nicht beschriebenen putativen transkriptionellen Regulators (STM0029) charakterisiert und definiert. Dieser scheint für die Abwehr von zellulären bakterizid wirkenden Verbindungen und das Überleben des Bakteriums innerhalb einer intrazellulären Nische von entscheidender Bedeutung zu sein. Die STM0029-deletierte Mutante wies eine gesteigerte Sensitivität gegenüber antimikrobiellen Peptiden und bakteriziden Verbindungen auf. Dazu zählten α-Defensin-1, β- Defensin-1, β-Defensin-2, LL-37 und Polymyxin B sowie Komponenten des Komplementsystems. Unerwartet war die Beobachtung, dass die Expression von STM0029 durch das PmrA/B Zwei Komponenten System reprimiert vorlag, während das PhoP/Q Zwei Komponenten System keinen Einfluss auf die Expression von STM0029 zu scheinen hat. Beide Komponent Systeme spielen bekanntlich eine entscheidende Rolle bei der Expressionsregulation von Genen die für das intrazelluläre Überleben von Salmonella wichtig sind. Bemerkenswert ist, dass ein Set von Genen welche an der Biosynthese und/oder der Modifikation für das LPS O-Antigen sowie des Peptidoglykans in der bakteriellen Zellwand beteiligt ist, im STM0029 Deletionshintergrund herab reguliert vorlag. Dieses Ergebnis deutet darauf hin, dass das STM0029 Genprodukt die Persistenz des Pathogen in Wirtszellen beeinflusst. Möglicherweise geschieht dies durch das Umgehen von wirtseigenen Abwehrmechanismen. / Salmonella spp. are facultative intracellular pathogens, which cause gastrointestinal and systemic diseases in a broad range of hosts including animals and humans. In addition to virulence genes clustered within pathogenicity islands, numerous additional genes scattered throughout the genome are also involved in various aspects of Salmoenlla virulence and pathogenesis. In this study, I identified a Salmonella putative transcriptional regulator encoded by a previously uncharacterized open reading frame designated STM0029. Deletion of STM0029 altered the expression of genes involved in both the resistance to host bactericidal challenges, and bacterial cell wall biosynthesis in S. Tyhpimurium. The ΔSTM0029 strain showed a defect in the resistance to host antimicrobial peptides, including α-defensin-1, β-defensin-1, β-defensin-2, LL-37, and polymyxin B as well as serum challenges compared to the wildtype. Unexpectedly, expression of STM0029 was found to be repressed by the PmrA/B two component system, but appeared to be independent of the PhoP/Q two component system, both of which are well-known regulatory systems involved in the regulation of expression of genes involved in Salmonella intracellular survival. Notably, the expression of a set of genes involved in bacterial LPS O-antigen and peptidoglycan biosyntheses and modifications showed decreases in the absence of STM0029. These experimental results indicate that the STM0029 gene product in S. Typhimurium contributes to resistance against host cell defense mechanisms, likely through regulation of genes involved in LPS O-antigen and peptidoglycan biosynthesis and modifications.