@phdthesis{Stephan2023,
  author    = {Stephan, Mareike Sophia},
  title     = {A bacterial mimetic system to study bacterial inactivation and infection},
  school      = {Universit{\"a}t Potsdam},
  pages     = {150},
  year      = {2023},
  abstract  = {The emerging threat of antibiotic-resistant bacteria has become a global challenge in the last decades, leading to a rising demand for alternative treatments for bacterial infections. One approach is to target the bacterial cell envelope, making understanding its biophysical properties crucial. Specifically, bacteriophages use the bacterial envelope as an entry point to initiate infection, and they are considered important building blocks of new antibiotic strategies against drug-resistant bacteria.. Depending on the structure of the cell wall, bacteria are classified as Gram-negative and Gram-positive. Gram-negative bacteria are equipped with a complex cell envelope composed of two lipid membranes enclosing a rigid peptidoglycan layer. The synthesis machinery of the Gram-negative cell envelope is the target of antimicrobial agents, including new physical sanitizing procedures addressing the outer membrane (OM). It is therefore very important to study the biophysical properties of the Gram-negative bacterial cell envelope. The high complexity of the Gram-negative OM sets the demand for a model system in which the contribution of individual components can be evaluated separately. In this respect, giant unilamellar vesicles (GUVs) are promising membrane systems to study membrane properties while controlling parameters such as membrane composition and surrounding medium conditions. The aim of this work was to develop methods and approaches for the preparation and characterization of a GUV-based membrane model that mimics the OM of the Gram-negative cell envelope. A major component of the OM is the lipopolysaccharide (LPS) on the outside of the OM heterobilayer. The vesicle model was designed to contain LPS in the outer leaflet and lipids in the inner leaflet. Furthermore, the interaction of the prepared LPS-GUVs with bacteriophages was tested. LPS containing GUVs were prepared by adapting the inverted emulsion technique to meet the challenging properties of LPS, namely their high self-aggregation rate in aqueous solutions. Notably, an additional emulsification step together with the adaption of solution conditions was employed to asymmetrically incorporate LPS containing long polysaccharide chains into the artificial membranes. GUV membrane asymmetry was verified with a fluorescence quenching assay. Since the necessary precautions for handling the quenching agent sodium dithionite are often underestimated and poorly described, important parameters were tested and identified to obtain a stable and reproducible assay. In the context of varied LPS incorporation, a microscopy-based technique was introduced to determine the LPS content on individual GUVs and to directly compare vesicle properties and LPS coverage. Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity. Employing LPS-GUVs we could demonstrate that a Salmonella bacteriophage bound with high specificity to its LPS receptor when presented at the GUV surface, and that the number of bound bacteriophages scaled with the amount of presented LPS receptor. In addition to binding, the bacteriophages were able to eject their DNA into the vesicle lumen. LPS-GUVs thus provide a starting platform for bottom-up approaches for the generation of more complex membranes, in which the effects of individual components on the membrane properties and the interaction with antimicrobial agents such as bacteriophages could be explored.},
  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{Alirezaeizanjani2020,
  author    = {Alirezaeizanjani, Zahra},
  title     = {Movement strategies of a multi-mode bacterial swimmer},
  doi       = {10.25932/publishup-47580},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-475806},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xix, 111},
  year      = {2020},
  abstract  = {Bacteria are one of the most widespread kinds of microorganisms that play essential roles in many biological and ecological processes. Bacteria live either as independent individuals or in organized communities. At the level of single cells, interactions between bacteria, their neighbors, and the surrounding physical and chemical environment are the foundations of microbial processes. Modern microscopy imaging techniques provide attractive and promising means to study the impact of these interactions on the dynamics of bacteria. The aim of this dissertation is to deepen our understanding four fundamental bacterial processes - single-cell motility, chemotaxis, bacterial interactions with environmental constraints, and their communication with neighbors - through a live cell imaging technique. By exploring these processes, we expanded our knowledge on so far unexplained mechanisms of bacterial interactions. Firstly, we studied the motility of the soil bacterium Pseudomonas putida (P. putida), which swims through flagella propulsion, and has a complex, multi-mode swimming tactic. It was recently reported that P. putida exhibits several distinct swimming modes - the flagella can push and pull the cell body or wrap around it. Using a new combined phase-contrast and fluorescence imaging set-up, the swimming mode (push, pull, or wrapped) of each run phase was automatically recorded, which provided the full swimming statistics of the multi-mode swimmer. Furthermore, the investigation of cell interactions with a solid boundary illustrated an asymmetry for the different swimming modes; in contrast to the push and pull modes, the curvature of runs in wrapped mode was not affected by the solid boundary. This finding suggested that having a multi-mode swimming strategy may provide further versatility to react to environmental constraints. Then we determined how P. putida navigates toward chemoattractants, i.e. its chemotaxis strategies. We found that individual run modes show distinct chemotactic responses in nutrition gradients. In particular, P. putida cells exhibited an asymmetry in their chemotactic responsiveness; the wrapped mode (slow swimming mode) was affected by the chemoattractant, whereas the push mode (fast swimming mode) was not. These results can be seen as a starting point to understand more complex chemotaxis strategies of multi-mode swimmers going beyond the well-known paradigm of Escherichia coli, that exhibits only one swimming mode. Finally we considered the cell dynamics in a dense population. Besides physical interactions with their neighbors, cells communicate their activities and orchestrate their population behaviors via quorum-sensing. Molecules that are secreted to the surrounding by the bacterial cells, act as signals and regulate the cell population behaviour. We studied P. putida's motility in a dense population by exposing the cells to environments with different concentrations of chemical signals. We found that higher amounts of chemical signals in the surrounding influenced the single-cell behaviourr, suggesting that cell-cell communications may also affect the flagellar dynamics. In summary, this dissertation studies the dynamics of a bacterium with a multi-mode swimming tactic and how it is affected by the surrounding environment using microscopy imaging. The detailed description of the bacterial motility in fundamental bacterial processes can provide new insights into the ecology of microorganisms.},
  language  = {en}
}
@phdthesis{Numberger2019,
  author    = {Numberger, Daniela},
  title     = {Urban wastewater and lakes as habitats for bacteria and potential vectors for pathogens},
  doi       = {10.25932/publishup-43709},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-437095},
  school      = {Universit{\"a}t Potsdam},
  pages     = {VI, 130},
  year      = {2019},
  abstract  = {Wasser ist lebensnotwendig und somit eine essentielle Ressource. Jedoch sind unsere S{\"u}ßwasser-Ressourcen begrenzt und ihre Erhaltung daher besonders wichtig. Verschmutzungen mit Chemikalien und Krankheitserregern, die mit einer wachsenden Bev{\"o}lkerung und Urbanisierung einhergehen, verschlechtern die Qualit{\"a}t unseres S{\"u}ßwassers. Außerdem kann Wasser als {\"U}bertragungsvektor f{\"u}r Krankheitserreger dienen und daher wasserb{\"u}rtige Krankheiten verursachen. Der Leibniz-Forschungsverbund INFECTIONS'21 untersuchte innerhalb der interdisziplin{\"a}ren Forschungsgruppe III - „Wasser", Gew{\"a}sser als zentralen Mittelpunkt f{\"u}r Krankheiterreger. Dabei konzentrierte man sich auf Clostridioides difficile sowie avi{\"a}re Influenza A-Viren, von denen angenommen wird, dass sie in die Gew{\"a}sser ausgeschieden werden. Ein weiteres Ziel bestand darin, die bakterielle Gemeinschaften eines Kl{\"a}rwerkes der deutschen Hauptstadt Berlin zu charakterisieren, um anschließend eine Bewertung des potentiellen Gesundheitsrisikos geben zu k{\"o}nnen. Bakterielle Gemeinschaften des Roh- und Klarwassers aus dem Kl{\"a}rwerk unterschieden sich signifikant voneinander. Der Anteil an Darm-/F{\"a}kalbakterien war relativ niedrig und potentielle Darmpathogene wurden gr{\"o}ßtenteils aus dem Rohwasser entfernt. Ein potentielles Gesundheitsrisiko konnte allerdings von potentiell pathogenen Legionellen wie L. lytica festgestellt werden, deren relative Abundanz im Klarwasser h{\"o}her war als im Rohwasser. Es wurden außerdem drei C. difficile-Isolate aus den Kl{\"a}rwerk-Rohwasser und einem st{\"a}dtischen Badesee in Berlin (Weisser See) gewonnen und sequenziert. Die beiden Isolate aus dem Kl{\"a}rwerk tragen keine Toxin-Gene, wohingegen das Isolat aus dem See Toxin-Gene besitzt. Alle drei Isolate sind sehr nah mit humanen St{\"a}mmen verwandt. Dies deutet auf ein potentielles, wenn auch sporadisches Gesundheitsrisiko hin. (Avi{\"a}re) Influenza A-Viren wurden in 38.8\% der untersuchten Sedimentproben mittels PCR detektiert, aber die Virusisolierung schlug fehl. Ein Experiment mit beimpften Wasser- und Sedimentproben zeigte, dass f{\"u}r die Isolierung aus Sedimentproben eine relativ hohe Viruskonzentration n{\"o}tig ist. In Wasserproben ist jedoch ein niedriger Titer an Influenza A-Viren ausreichend, um eine Infektion auszul{\"o}sen. Es konnte zudem auch festgestellt werden, dass sich „Madin-Darby Canine Kidney (MDCK)―-Zellkulturen im Gegensatz zu embryonierten H{\"u}hnereiern besser eignen, um Influenza A-Viren aus Sediment zu isolieren. Zusammenfassend l{\"a}sst sich sagen, dass diese Arbeit m{\"o}gliche Gesundheitsrisiken aufgedeckt hat, wie etwa durch Legionellen im untersuchten Berliner Kl{\"a}rwerk, deren relative Abundanz in gekl{\"a}rtem Abwasser h{\"o}her ist als im Rohwasser. Desweiteren wird indiziert, dass Abwasser und Gew{\"a}sser als Reservoir und Vektor f{\"u}r pathogene Organismen dienen k{\"o}nnen, selbst f{\"u}r nicht-typische Wasser-Pathogene wie C. difficile.},
  language  = {en}
}
@phdthesis{Hintsche2018,
  author    = {Hintsche, Marius},
  title     = {Locomotion of a bacterium with a polar bundle of flagella},
  doi       = {10.25932/publishup-42697},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-426972},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xi, 108},
  year      = {2018},
  abstract  = {Movement and navigation are essential for many organisms during some parts of their lives. This is also true for bacteria, which can move along surfaces and swim though liquid environments. They are able to sense their environment, and move towards environmental cues in a directed fashion. These abilities enable microbial lifecyles in biofilms, improved food uptake, host infection, and many more. In this thesis we study aspects of the swimming movement - or motility - of the soil bacterium (P. putida). Like most bacteria, P. putida swims by rotating its helical flagella, but their arrangement differs from the main model organism in bacterial motility research: (E. coli). P. putida is known for its intriguing motility strategy, where fast and slow episodes can occur after each other. Up until now, it was not known how these two speeds can be produced, and what advantages they might confer to this bacterium. Normally the flagella, the main component of thrust generation in bacteria, are not observable by ordinary light microscopy. In order to elucidate this behavior, we therefore used a fluorescent staining technique on a mutant strain of this species to specifically label the flagella, while leaving the cell body only faintly stained. This allowed us to image the flagella of the swimming bacteria with high spacial and temporal resolution with a customized high speed fluorescence microscopy setup. Our observations show that P. putida can swim in three different modes. First, It can swim with the flagella pushing the cell body, which is the main mode of swimming motility previously known from other bacteria. Second, it can swim with the flagella pulling the cell body, which was thought not to be possible in situations with multiple flagella. Lastly, it can wrap its flagellar bundle around the cell body, which results in a speed wich is slower by a factor of two. In this mode, the flagella are in a different physical conformation with a larger radius so the cell body can fit inside. These three swimming modes explain the previous observation of two speeds, as well as the non strict alternation of the different speeds. Because most bacterial swimming in nature does not occur in smoothly walled glass enclosures under a microscope, we used an artificial, microfluidic, structured system of obstacles to study the motion of our model organism in a structured environment. Bacteria were observed in microchannels with cylindrical obstacles of different sizes and with different distances with video microscopy and cell tracking. We analyzed turning angles, run times, and run length, which we compared to a minimal model for movement in structured geometries. Our findings show that hydrodynamic interactions with the walls lead to a guiding of the bacteria along obstacles. When comparing the observed behavior with the statics of a particle that is deflected with every obstacle contact, we find that cells run for longer distances than that model. Navigation in chemical gradients is one of the main applications of motility in bacteria. We studied the swimming response of P. putida cells to chemical stimuli (chemotaxis) of the common food preservative sodium benzoate. Using a microfluidic gradient generation device, we created gradients of varying strength, and observed the motion of cells with a video microscope and subsequent cell tracking. Analysis of different motility parameters like run lengths and times, shows that P. putida employs the classical chemotaxis strategy of E. coli: runs up the gradient are biased to be longer than those down the gradient. Using the two different run speeds we observed due to the different swimming modes, we classify runs into `fast' and `slow' modes with a Gaussian mixture model (GMM). We find no evidence that P. putida's uses its swimming modes to perform chemotaxis. In most studies of bacterial motility, cell tracking is used to gather trajectories of individual swimming cells. These trajectories then have to be decomposed into run sections and tumble sections. Several algorithms have been developed to this end, but most require manual tuning of a number of parameters, or extensive measurements with chemotaxis mutant strains. Together with our collaborators, we developed a novel motility analysis scheme, based on generalized Kramers-Moyal-coefficients. From the underlying stochastic model, many parameters like run length etc., can be inferred by an optimization procedure without the need for explicit run and tumble classification. The method can, however, be extended to a fully fledged tumble classifier. Using this method, we analyze E. coli chemotaxis measurements in an aspartate analog, and find evidence for a chemotactic bias in the tumble angles.},
  language  = {en}
}
@phdthesis{Codutti2018,
  author    = {Codutti, Agnese},
  title     = {Behavior of magnetic microswimmers},
  doi       = {10.25932/publishup-42297},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-422976},
  school      = {Universit{\"a}t Potsdam},
  pages     = {iv, 142},
  year      = {2018},
  abstract  = {Microswimmers, i.e. swimmers of micron size experiencing low Reynolds numbers, have received a great deal of attention in the last years, since many applications are envisioned in medicine and bioremediation. A promising field is the one of magnetic swimmers, since magnetism is biocom-patible and could be used to direct or actuate the swimmers. This thesis studies two examples of magnetic microswimmers from a physics point of view. The first system to be studied are magnetic cells, which can be magnetic biohybrids (a swimming cell coupled with a magnetic synthetic component) or magnetotactic bacteria (naturally occurring bacteria that produce an intracellular chain of magnetic crystals). A magnetic cell can passively interact with external magnetic fields, which can be used for direction. The aim of the thesis is to understand how magnetic cells couple this magnetic interaction to their swimming strategies, mainly how they combine it with chemotaxis (the ability to sense external gradient of chemical species and to bias their walk on these gradients). In particular, one open question addresses the advantage given by these magnetic interactions for the magnetotactic bacteria in a natural environment, such as porous sediments. In the thesis, a modified Active Brownian Particle model is used to perform simulations and to reproduce experimental data for different systems such as bacteria swimming in the bulk, in a capillary or in confined geometries. I will show that magnetic fields speed up chemotaxis under special conditions, depending on parameters such as their swimming strategy (run-and-tumble or run-and-reverse), aerotactic strategy (axial or polar), and magnetic fields (intensities and orientations), but it can also hinder bacterial chemotaxis depending on the system. The second example of magnetic microswimmer are rigid magnetic propellers such as helices or random-shaped propellers. These propellers are actuated and directed by an external rotating magnetic field. One open question is how shape and magnetic properties influence the propeller behavior; the goal of this research field is to design the best propeller for a given situation. The aim of the thesis is to propose a simulation method to reproduce the behavior of experimentally-realized propellers and to determine their magnetic properties. The hydrodynamic simulations are based on the use of the mobility matrix. As main result, I propose a method to match the experimental data, while showing that not only shape but also the magnetic properties influence the propellers swimming characteristics.},
  language  = {en}
}