Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Characterization of host-pathogen interactions during early Staphylococcus aureus biofilm formation on surfaces(Montana State University - Bozeman, College of Agriculture, 2022) Pettygrove, Brian Alexander; Chairperson, Graduate Committee: Philip S. Stewart; This is a manuscript style paper that includes co-authored chapters.Implanted biomaterials such as orthopedic screws, prosthetic joints, pacemakers, and catheters are essential components of modern medicine. Unfortunately, implanted foreign bodies are susceptible to biofilm infection, leading to a persistent and difficult to treat disease state. Biofilm infections readily tolerate clearance from the immune system, however much of our understanding of the mechanisms governing persistence are formulated around the biofilm state during advanced infection. By comparison we have a poor understanding of the early stages of infection. Specifically, how contaminating organisms initially evade host immune defenses and establish a robust infection remains ill-defined. In this work, we interrogated interactions between Staphylococcus aureus (S. aureus), a frequent culprit in biomaterial infections, and early contributors to host immunity. Using in vitro time-lapse microscopy, we observed that human neutrophils readily phagocytose and kill single cells or small clusters of S. aureus cells that are attached to a surface. S. aureus cells that go undiscovered during the initial stages of neutrophil surveillance form biofilm aggregates that rapidly gain tolerance to neutrophil killing. In vivo models of implant infection demonstrated that surface adherent bacteria can evade discovery due to delayed or heterogeneous neutrophil recruitment to the surface. Biofilm aggregate formation was impaired in a strain deficient in the two-component gene regulatory system SaeR/S and the resulting cells were highly susceptible to neutrophil killing. Inhibition of aggregation was dependent on serum complement proteins C3 and factor B, suggesting that SaeR/S regulated factors actively inhibit host complement to facilitate aggregation. Taken together, these data suggest that the formation of immune-tolerant biofilm aggregates may contribute to chronic device related infections by protecting bacteria from phagocyte killing. These studies provide vital insight into the host pathogen interactions on contaminated biomaterial surfaces and highlight early events that may determine infection outcome.Item Improving osseointegration of PEEK through surface textures(Montana State University - Bozeman, College of Engineering, 2019) Scott, Renn Patrick; Chairperson, Graduate Committee: Cecily RyanPEEK (Polyetheretherketone) is one of the most promising alternatives to titanium in cortical bone implants due to being biologically inert and having an elastic modulus similar to that of bone. It also has favorable reactions conducive to common medical imaging methods such as X-ray and magnetic resonance imaging (MRI) as compared to commonly used metals such as titanium and stainless steel. However PEEK is not inherently osseoconductive, leading to longer healing times and a greater chance of infection. Many different methods exist for improving osteoblast growth, such as the addition of bio-active materials like hydroxyapatite. Manipulating the surface texture of PEEK could provide better environments for cells to attach and can be used as another layer with other techniques, making the tissue interface more robust. The main objective of this project is to observe cell adhesion to a textured surface to identify cell preference for surface geometry as a first step to improve full integration of non-resorbable implants into bone tissue. The methods explored were also chosen for their repeatability, reliability and lack of chemical modification compared to other successful surface modulation techniques. The surface textures were embossed into PEEK using micro-etched aluminum molds. Textures vary in their shape, spacing, size, depth and surface convexity/concavity. The cell adhesion was recorded through fluorescent confocal microscopy and the cell-substrate interaction was observed under electron microscopy. The results were that 25 micron and 10 micron features discouraged cell adhesion while 325 micron and 120 micron features encouraged cell adhesion with pillars performing better than holes. The best feature was the 325x40 micron square pillars. With a cell volume to surface area ratio of 5.13, a live cell count of 276.5, a dead cell count of 9.00, and a non-dimensional distance to feature of 0.67.Item Role of transport limitation in the resistance of Pseudomonas aerunginosa biofilms to ciprofloxacin and levofloxacin(Montana State University - Bozeman, College of Engineering, 1996) Vrany, Julia DawnBacterial biofilm infections are often far less susceptible to antibiotic therapy than their planktonic, or freely suspended counterparts. Transport limitation of the antibiotic within the biofilm has been proposed as one explanation for this resistance. To explore the possibility that transport limitation contributes to a decreased antibiotic efficacy, a Comparison was made between the efficacies of two fluoroquinolone antibiotics, ciprofloxacin and levofloxacin, against freely suspended and biofilm bacteria. Transport of the antibiotics to the biofilm-substratum interface was monitored using ATR-FTIR spectroscopy techniques. Both biofilms and planktonic organisms were treated with antibiotic concentrations of 100, 250, and 500 μg ml-1 for 30 min and rinsed for 1 h with fresh media. Antibiotic efficacy was determined by the ratio of culturable bacteria to total cell counts. The experimental ATR-FTIR transport data was then simulated with a mathematical computer model containing the processes of molecular diffusion, adsorption, and desorption to provide possible explanations for differences in antibiotic delivery. Levofloxacin was found to be more efficacious against planktonic organisms than biofilm cells. However, no difference in efficacy was seen when planktonic and biofilm bacteria were treated with ciprofloxacin. ATR-FTIR results showed that the biofilm provided very little transport limitation for each antibiotic, thus suggesting that the reduced susceptibility of a biofilm to antimicrobial treatment may be due to factors other than transport.