@phdthesis{Dreymann2023,
  author    = {Dreymann, Nico},
  title     = {Identification and functional characterization of aptamers targeting human urokinase and NDM-1 for therapeutic and diagnostic applications},
  doi       = {10.25932/publishup-61291},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-612919},
  school      = {Universit{\"a}t Potsdam},
  pages     = {IX, 130},
  year      = {2023},
  abstract  = {Aptamers are single-stranded DNA (ssDNA) or RNA molecules that can bind specifically and with high affinity to target molecules due to their unique three-dimensional structure. For this reason, they are often compared to antibodies and sometimes even referred to as "chemical antibodies". They are simple and inexpensive to synthesize, easy to modify, and smaller than conventional antibodies. Enzymes, especially hydrolases, are interesting targets in this context. This class of enzymes is capable of hydrolytically cleaving various macromolecules such as proteins, as well as smaller molecules such as antibiotics. Hence, they play an important role in many biological processes including diseases and their treatment. Hydrolase detection as well as the understanding of their function is therefore of great importance for diagnostics and therapy. Due to their various desirable features compared to antibodies, aptamers are being discussed as alternative agents for analytical and diagnostic use in various applications. The use of aptamers in therapy is also frequently investigated, as the binding of aptamers can have effects on the catalytic activity, protein-protein interactions, or proteolytic cascades. Aptamers are generated by an in vitro selection process. Potential aptamer candidates are selected from a pool of enriched nucleic acid sequences with affinity to the target, and their binding affinity and specificity is investigated. This is one of the most important steps in aptamer generation to obtain specific aptamers with high affinity for use in analytical and diagnostic applications. The binding properties or binding domains and their effects on enzyme functions form the basis for therapeutic applications. In this work, the binding properties of DNA aptamers against two different hydrolases were investigated. In view of their potential utility for analytical methods, aptamers against human urokinase (uPA) and New Delhi metallo-β-lactamase-1 (NDM-1) were evaluated for their binding affinity and specificity using different methods. Using the uPA aptamers, a protocol for measuring the binding kinetics of an aptamer-protein-interaction by surface plasmon resonance spectroscopy (SPR) was developed. Based on the increased expression of uPA in different types of cancer, uPA is discussed as a prognostic and diagnostic tumor marker. As uPA aptamers showed different binding sites on the protein, microtiter plate-based aptamer sandwich assay systems for the detection of uPA were developed. Because of the function of urokinase in cancer cell proliferation and metastasis, uPA is also discussed as a therapeutic target. In this regard, the different binding sites of aptamers showed different effects on uPA function. In vitro experiments demonstrated both inhibition of uPA binding to its receptor as well as the inhibition of uPA catalytic activity for different aptamers. Thus, in addition to their specificity and affinity for their targets, the utility of the aptamers for potential diagnostic and therapeutic applications was demonstrated. First, as an alternative inhibitor of human urokinase for therapeutic purposes, and second, as valuable recognition molecules for the detection of urokinase, as a prognostic and diagnostic marker for cancer, and for NDM-1 to detect resistance to carbapenem antibiotics.},
  language  = {en}
}
@phdthesis{Muench2021,
  author    = {M{\"u}nch, Steffen},
  title     = {The relevance of the aeolian transport path for the spread of antibiotic-resistant bacteria on arable fields},
  doi       = {10.25932/publishup-53608},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-536089},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XVI, 140},
  year      = {2021},
  abstract  = {The spread of antibiotic-resistant bacteria poses a globally increasing threat to public health care. The excessive use of antibiotics in animal husbandry can develop resistances in the stables. Transmission through direct contact with animals and contamination of food has already been proven. The excrements of the animals combined with a binding material enable a further potential path of spread into the environment, if they are used as organic manure in agricultural landscapes. As most of the airborne bacteria are attached to particulate matter, the focus of the work will be the atmospheric dispersal via the dust fraction. Field measurements on arable lands in Brandenburg, Germany and wind erosion studies in a wind tunnel were conducted to investigate the risk of a potential atmospheric dust-associated spread of antibiotic-resistant bacteria from poultry manure fertilized agricultural soils. The focus was to (i) characterize the conditions for aerosolization and (ii) qualify and quantify dust emissions during agricultural operations and wind erosion. PM10 (PM, particulate matter with an aerodynamic diameter smaller than 10 µm) emission factors and bacterial fluxes for poultry manure application and incorporation have not been previously reported before. The contribution to dust emissions depends on the water content of the manure, which is affected by the manure pretreatment (fresh, composted, stored, dried), as well as by the intensity of manure spreading from the manure spreader. During poultry manure application, PM10 emission ranged between 0.05 kg ha-1 and 8.37 kg ha-1. For comparison, the subsequent land preparation contributes to 0.35 - 1.15 kg ha-1 of PM10 emissions. Manure particles were still part of dust emissions but they were accounted to be less than 1\% of total PM10 emissions due to the dilution of poultry manure in the soil after manure incorporation. Bacterial emissions of fecal origin were more relevant during manure application than during the subsequent manure incorporation, although PM10 emissions of manure incorporation were larger than PM10 emissions of manure application for the non-dried manure variants. Wind erosion leads to preferred detachment of manure particles from sandy soils, when poultry manure has been recently incorporated. Sorting effects were determined between the low-density organic particles of manure origin and the soil particles of mineral origin close above the threshold of 7 m s-1. In dependence to the wind speed, potential erosion rates between 101 and 854 kg ha-1 were identified, if 6 t ha-1 of poultry manure were applied. Microbial investigation showed that manure bacteria got detached more easily from the soil surface during wind erosion, due to their attachment on manure particles. Although antibiotic-resistant bacteria (ESBL-producing E. coli) were still found in the poultry barns, no further contamination could be detected with them in the manure, fertilized soils or in the dust generated by manure application, land preparation or wind erosion. Parallel studies of this project showed that storage of poultry manure for a few days (36 - 72 h) is sufficient to inactivate ESBL-producing E. coli. Further antibiotic-resistant bacteria, i.e. MRSA and VRE, were only found sporadically in the stables and not at all in the dust. Therefore, based on the results of this work, the risk of a potential infection by dust-associated antibiotic-resistant bacteria can be considered as low.},
  language  = {en}
}
@phdthesis{MogrovejoArias2021,
  author    = {Mogrovejo Arias, Diana Carolina},
  title     = {Assessment of the frequency and relevance of potentially pathogenic phenotypes in microbial isolates from Arctic environments},
  school      = {Universit{\"a}t Potsdam},
  pages     = {125},
  year      = {2021},
  abstract  = {The Arctic environments constitute rich and dynamic ecosystems, dominated by microorganisms extremely well adapted to survive and function under severe conditions. A range of physiological adaptations allow the microbiota in these habitats to withstand low temperatures, low water and nutrient availability, high levels of UV radiation, etc. In addition, other adaptations of clear competitive nature are directed at not only surviving but thriving in these environments, by disrupting the metabolism of neighboring cells and affecting intermicrobial communication. Since Arctic microbes are bioindicators which amplify climate alterations in the environment, the Arctic region presents the opportunity to study local microbiota and carry out research about interesting, potentially virulent phenotypes that could be dispersed into other habitats around the globe as a consequence of accelerating climate change. In this context, exploration of Arctic habitats as well as descriptions of the microbes inhabiting them are abundant but microbial competitive strategies commonly associated with virulence and pathogens are rarely reported. In this project, environmental samples from the Arctic region were collected and microorganisms (bacteria and fungi) were isolated. The clinical relevance of these microorganisms was assessed by observing the following virulence markers: ability to grow at a range of temperatures, expression of antimicrobial resistance and production of hemolysins. The aim of this project is to determine the frequency and relevance of these characteristics in an effort to understand microbial adaptations in habitats threatened by climate change. The isolates obtained and described here were able to grow at a range of temperatures, in some cases more than 30 °C higher than their original isolation temperature. A considerable number of them consistently expressed compounds capable of lysing sheep and bovine erythrocytes on blood agar at different incubation temperatures. Ethanolic extracts of these bacteria were able to cause rapid and complete lysis of erythrocyte suspensions and might even be hemolytic when assayed on human blood. In silico analyses showed a variety of resistance elements, some of them novel, against natural and synthetic antimicrobial compounds. In vitro experiments against a number of antimicrobial compounds showed resistance phenotypes belonging to wild-type populations and some non-wild type which clearly denote human influence in the acquisition of antimicrobial resistance. The results of this project demonstrate the presence of virulence-associated factors expressed by microorganisms of natural, non-clinical environments. This study contains some of the first reports, to the best of our knowledge, of hemolytic microbes isolated from the Arctic region. In addition, it provides additional information about the presence and expression of intrinsic and acquired antimicrobial resistance in environmental isolates, contributing to the understanding of the evolution of relevant pathogenic species and opportunistic pathogens. Finally, this study highlights some of the potential risks associated with changes in the polar regions (habitat melting and destruction, ecosystem transition and re-colonization) as important indirect consequences of global warming and altered climatic conditions around the planet.},
  language  = {en}
}
@phdthesis{Vossenkuhl2015,
  author    = {Vossenkuhl, Birgit},
  title     = {Transmission of MRSA along the meat supply chain},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-85918},
  school      = {Universit{\"a}t Potsdam},
  pages     = {141},
  year      = {2015},
  abstract  = {Methicillin-resistente Staphylococcus aureus (MRSA) z{\"a}hlen zu den bedeutendsten antibiotikaresistenten Pathogenen, die vor allem in Krankenh{\"a}usern aber auch außerhalb von Einrichtungen des Gesundheitswesens weit verbreitet sind. Seit einigen Jahren ist eine neue Generation von MRSA auf dem Vormarsch, die vor allem Nutztierbest{\"a}nde als neue Nische besiedelt. Diese sogenannten Nutztier-assoziierten MRSA wurden wiederholt bei wirtschaftlich bedeutenden Nutztieren sowie daraus gewonnenem Fleisch nachgewiesen. Im Rahmen der vorliegenden Arbeit wurde ein methodischer Ansatz verfolgt, um die Hypothese einer m{\"o}glichen {\"U}bertragung von Nutztier-assoziierten MRSA entlang der Lebensmittelkette vom Tier auf dessen Fleisch zu best{\"a}tigen. Angepasst an die Unterschiede in den verf{\"u}gbaren Daten wurden daf{\"u}r zwei neue Konzepte erstellt. Zur Analyse der {\"U}bertragung von MRSA entlang der Schlachtkette wurde ein mathematisches Modell des Schweineschlachtprozesses entwickelt, welches dazu geeignet ist, den Verlauf der MRSA-Pr{\"a}valenz entlang der Schlachtkette zu quantifizieren sowie kritische Prozessschritte f{\"u}r eine MRSA-{\"U}bertragung zu identifizieren. Anhand von Pr{\"a}valenzdaten ist es dem Modell m{\"o}glich, die durchschnittlichen MRSA-Eliminations- und Kontaminationsraten jedes einzelnen Prozessschrittes zu sch{\"a}tzen, die anschließend in eine Monte-Carlo-Simulation einfließen. Im Ergebnis konnte gezeigt werden, dass es generell m{\"o}glich ist, die MRSA Pr{\"a}valenz im Laufe des Schlachtprozesses auf ein niedriges finales Niveau zwischen 0,15 bis 1,15\% zu reduzieren. Vor allem das Br{\"u}hen und Abfl{\"a}mmen der Schlachtk{\"o}rper wurden als kritische Prozesse im Hinblick auf eine MRSA-Dekontamination identifiziert. In Deutschland werden regelm{\"a}ßig MRSA-Pr{\"a}valenz und Typisierungsdaten auf allen Stufen der Lebensmittelkette verschiedener Nutztiere erfasst. Um die MRSA-Daten dieser Querschnittstudie hinsichtlich einer m{\"o}glichen {\"U}bertragung entlang der Kette zu analysieren, wurde ein neuer statistischer Ansatz entwickelt. Hierf{\"u}r wurde eine Chi-Quadrat-Statistik mit der Berechnung des Czekanowski-{\"A}hnlichkeitsindex kombiniert, um Unterschiede in der Verteilung stammspezifischer Eigenschaften zwischen MRSA aus dem Stall, von Karkassen nach der Schlachtung und aus Fleisch im Einzelhandel zu quantifizieren. Die Methode wurde am Beispiel der Putenfleischkette implementiert und zudem bei der Analyse der Kalbfleischkette angewendet. Die durchgehend hohen {\"A}hnlichkeitswerte zwischen den einzelnen Proben weisen auf eine m{\"o}gliche {\"U}bertragung von MRSA entlang der Lebensmittelkette hin. Die erarbeiteten Methoden sind nicht spezifisch bez{\"u}glich Prozessketten und Pathogenen. Sie bieten somit einen großen Anwendungsbereich und erweitern das Methodenspektrum zur Bewertung bakterieller {\"U}bertragungswege.},
  language  = {en}
}
@phdthesis{Patra2013,
  author    = {Patra, Pintu},
  title     = {Population dynamics of bacterial persistence},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-69253},
  school      = {Universit{\"a}t Potsdam},
  year      = {2013},
  abstract  = {The life of microorganisms is characterized by two main tasks, rapid growth under conditions permitting growth and survival under stressful conditions. The environments, in which microorganisms dwell, vary in space and time. The microorganisms innovate diverse strategies to readily adapt to the regularly fluctuating environments. Phenotypic heterogeneity is one such strategy, where an isogenic population splits into subpopulations that respond differently under identical environments. Bacterial persistence is a prime example of such phenotypic heterogeneity, whereby a population survives under an antibiotic attack, by keeping a fraction of population in a drug tolerant state, the persister state. Specifically, persister cells grow more slowly than normal cells under growth conditions, but survive longer under stress conditions such as the antibiotic administrations. Bacterial persistence is identified experimentally by examining the population survival upon an antibiotic treatment and the population resuscitation in a growth medium. The underlying population dynamics is explained with a two state model for reversible phenotype switching in a cell within the population. We study this existing model with a new theoretical approach and present analytical expressions for the time scale observed in population growth and resuscitation, that can be easily used to extract underlying model parameters of bacterial persistence. In addition, we recapitulate previously known results on the evolution of such structured population under periodically fluctuating environment using our simple approximation method. Using our analysis, we determine model parameters for Staphylococcus aureus population under several antibiotics and interpret the outcome of cross-drug treatment. Next, we consider the expansion of a population exhibiting phenotype switching in a spatially structured environment consisting of two growth permitting patches separated by an antibiotic patch. The dynamic interplay of growth, death and migration of cells in different patches leads to distinct regimes in population propagation speed as a function of migration rate. We map out the region in parameter space of phenotype switching and migration rate to observe the condition under which persistence is beneficial. Furthermore, we present an extended model that allows mutation from the two phenotypic states to a resistant state. We find that the presence of persister cells may enhance the probability of resistant mutation in a population. Using this model, we explain the experimental results showing the emergence of antibiotic resistance in a Staphylococcus aureus population upon tobramycin treatment. In summary, we identify several roles of bacterial persistence, such as help in spatial expansion, development of multidrug tolerance and emergence of antibiotic resistance. Our study provides a theoretical perspective on the dynamics of bacterial persistence in different environmental conditions. These results can be utilized to design further experiments, and to develop novel strategies to eradicate persistent infections.},
  language  = {en}
}
@phdthesis{Oey2008,
  author    = {Oey, Melanie},
  title     = {Chloroplasts as bioreactors : high-yield production of active bacteriolytic protein antibiotics},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-28950},
  school      = {Universit{\"a}t Potsdam},
  year      = {2008},
  abstract  = {Plants, more precisely their chloroplasts with their bacterial-like expression machinery inherited from their cyanobacterial ancestors, can potentially offer a cheap expression system for proteinaceous pharmaceuticals. This system would be easily scalable and provides appropriate safety due to chloroplasts maternal inheritance. In this work, it was shown that three phage lytic enzymes (Pal, Cpl-1 and PlyGBS) could be successfully expressed at very high levels and with high stability in tobacco chloroplasts. PlyGBS expression reached an amount of foreign protein accumulation (> 70\% TSP) that has never been obtained before. Although the high expression levels of PlyGBS caused a pale green phenotype with retarded growth, presumably due to exhaustion of plastid protein synthesis capacity, development and seed production were not impaired under greenhouse conditions. Since Pal and Cpl-1 showed toxic effects when expressed in E. coli, a special plastid transformation vector (pTox) was constructed to allow DNA amplification in bacteria. The construction of the pTox transformation vector allowing a recombinase-mediated deletion of an E. coli transcription block in the chloroplast, leading to an increase of foreign protein accumulation to up to 40\% of TSP for Pal and 20\% of TSP for Cpl-1. High dose-dependent bactericidal efficiency was shown for all three plant-derived lytic enzymes using their pathogenic target bacteria S. pyogenes and S. pneumoniae. Confirmation of specificity was obtained for the endotoxic proteins Pal and Cpl-1 by application to E. coli cultures. These results establish tobacco chloroplasts as a new cost-efficient and convenient production platform for phage lytic enzymes and address the greatest obstacle for clinical application. The present study is the first report of lysin production in a non-bacterial system. The properties of chloroplast-produced lysins described in this work, their stability, high accumulation rate and biological activity make them highly attractive candidates for future antibiotics.},
  language  = {en}
}