Theses and Dissertations at Montana State University (MSU)
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Item Investigation of Staphylococcus epidermidis biofilm resistance to rifampin(Montana State University - Bozeman, College of Engineering, 2001) Zheng, ZhilanItem Nuclear magnetic resonance microscopy of Staphylococcus epidermidis biofilms(Montana State University - Bozeman, College of Engineering, 2004) Gjersing, Erica Lee; Chairperson, Graduate Committee: Philip S. Stewart; Joseph Seymour (co-chair)Nuclear Magnetic Resonance (NMR) microscopy is well-suited for investigating living systems since it is innocuous and non-invasive. In addition to imaging internal structures of systems, NMR microscopy techniques can be used to obtain information about transport phenomena, such as fluid velocities and diffusion. The goal of this research was to determine the applicability of NMR imaging techniques to studying transport properties in biofilms. Staphylococcus epidermidis biofilms were investigated because of their importance in medical implant infections. NMR experiments were used to image biofilm structure and the fluid flow patterns in one millimeter square glass capillaries. Results showing the heterogeneous structure of biofilms are congruent with confocal laser microscopy images. The advantage of using the NMR techniques to image biofilm structures is that there are no light or laser penetration barriers and the innermost regions of the structures can be easily revealed. In addition to imaging the biofilms, velocity distributions have been mapped for the one millimeter capillary system. Laminar flows in clean, square capillaries display axial velocities that are both uniform and symmetrical while non-axial components of velocity are not present. In contrast, a biofilm fouled capillary displays irregular flow patterns in the axial direction along with distinct non-axial secondary flow perturbations. These results demonstrate that biofilms impact bulk flow in ways that cannot be ignored when modeling their impact on transport in bioreactors and medial devices. This work establishes NMR microscopy as an important biofilm research tool which can spatially resolve structural characteristics and transport processes in biofilm fouled systems.Item Spatial and temporal patterns of antimicrobial action against Staphylococcus Epidermidis biofilms(Montana State University - Bozeman, College of Engineering, 2008) Davison, William Marshall; Chairperson, Graduate Committee: Philip S. Stewart; Joseph D. Seymour (co-chair)This study investigated the spatio-temporal patterns of antimicrobial action against Staphylococcus epidermidis planktonic and biofilm bacteria. Bacteria were stained with a fluorogenic esterase substrate, Calcein-AM, which allowed for the visualization of cells that possessed intact cell membranes. Four different antimicrobial agents were tested for their effect upon cell viability as associated with membrane integrity. The four biocides were Barquat®, glutaraldehyde, chlorine, and nisin. Planktonic bacteria were analyzed with flow cytometry, observing fluorescence loss during 1 h antimicrobial treatment. Treatment with Barquat resulted in initial fluorescence loss, which increased during the treatment period to levels which were present prior to the introduction of biocide, along with a decrease in cell density. Treatments with glutaraldehyde and chlorine resulted in increased average fluorescence intensity for the cell population, accompanied by decreased cell density for chlorine and increased cell density for glutaraldehyde. Nisin treatment resulted in a decrease in CAM fluorescence with an increase in cell density. Viable cell plate counts showed average log reductions in CFU/mL of 3.61, 3.83, 4.12, 4.26, and 4.67 for Barquat, glutaraldehyde, high and low concentrations of chlorine, and nisin treatments, respectively. There was no apparent correlation between plate counts and flow cytometry data. Biofilm bacteria were analyzed with time-lapse confocal scanning laser microscopy, observing fluorescence loss during biocide treatment. Biofilms treated with Barquat lost an average of 91.5% of their initial fluorescence, and clusters decreased in areal coverage by 9%. Fluorescence loss during Barquat treatment suggested the presence of a tolerant subpopulation of bacteria in the interior regions of the biofilm. Glutaraldehyde treatment reduced the average fluorescence by 16%, and cluster area did not change. There was CTC staining after glutaraldehyde treatment only. The high and low concentrations of chlorine treatment showed averages of 100% and 79% reductions in CAM staining, with liquefaction of biomass causing erosion events which reduced areal coverage by 90% and 43%, respectively. Nisin treatment reduced CAM staining by an average of 100%, while shrinking the cluster area by 8%. Corner biofilms showed qualitative differences during treatment than isolated clusters. Mathematically-predicted biocide diffusion times were much faster than experimentally observed fluorescence loss in biofilms.Item Quantifying the viscoelastic properties of treated and untreated Pseudomonas aeruginosa and Staphylococcus epidermidis biofilms using a rheological creep analysis(Montana State University - Bozeman, College of Engineering, 2008) Sutton, Michael Philip; Chairperson, Graduate Committee: Warren L. JonesMicrobial biofilms are quite difficult to kill and control, and present many problems to industry and medicine. The ability to alter the mechanical properties of biofilms could aid in the control of biofilm. The goal of this research project was to develop techniques for measuring the mechanical properties of biofilms so that the effects of chemical treatments could be assessed. Constitutive material models were developed and applied to assist in this effort to quantify the effects. Biofilms are viscoelastic in nature, therefore, rheological testing techniques were utilized for this research. Creep testing was performed on a parallel plate rheometer to determine biofilm mechanical properties. The rheometer is a mechanical device that can accurately measure and apply shear stress and strain on viscoelastic samples. The Burger material model closely approximated material behavior of most chemical treatments. This model was used for determining constitutive properties. Pseudomonas aeruginosa (FRD1) and Staphylococcus epidermidis colony biofilms were used for testing. Several treatment methods were used to investigate their effect on biofilm mechanical properties. As a source of different cations, solutions of NaCl, FeCl3, AlCl3, MgCl2, CaCl2, FeCl2 were used for testing. Multivalent cation treatments stiffened the FRD1 biofilm, but weakened the S. epidermidis. Urea treatments weakened both biofilm species. Glutaraldehyde treatments weakened the FRD1 biofilm, but had little effect on the S. epidermidis. Several treatments - EDTA, Barquat, chlorine, antibiotics (rifampin, and ciprofloxacin) - weakened biofilms of both species. The effect of the same chemical treatment between the two species of biofilm sometimes had nearly opposite effects on the biofilms mechanical properties. This research illustrated that it is possible to alter the mechanical properties of biofilm through chemical addition. Further, there are significant differences between the ways that the material properties of biofilms of different species of bacteria will be affected by a chemical treatment. Finally, it was observed that the 4-parameter Burger model for constitutive mechanical properties of biofilms fit the vast majority of the collected data, so that this model proves useful in comparing properties of biofilms grown or treated under various conditions.Item Impact of Staphylococcus aureus biofilm conditioned medium on inflammation and epithelialization in human keratinocytes(Montana State University - Bozeman, College of Letters & Science, 2011) Secor, Patrick Robert; Chairperson, Graduate Committee: Thomas Hughes.Chronic wounds are characterized by prolonged inflammation and failure to epithelialize and do not respond well to conventional treatment. Bacterial biofilms are a major impediment to wound healing. The effective treatment of chronic wounds requires a better understanding of the relationship between bacterial biofilms and human skin. Human keratinocytes are the most abundant cell type in the epidermis and play essential roles in the inflammatory and epithelialization phases of wound repair. The aim of this dissertation was to determine the effect of biofilm-conditioned medium (BCM) and planktonic-conditioned medium (PCM) on inflammatory and epithelialization processes in keratinocytes. The phylogeny of chronic wounds was characterized to select a model pathogen. Staphylococcus aureus was found to be prevalent in chronic wounds. A novel in vitro model was developed to facilitate host-pathogen investigations between S. aureus biofilms and human keratinocytes. S. aureus BCM contained fermentation products and metabolites that regulate virulence. After four hours of exposure to BCM, pro-inflammatory genes were upregulated in keratinocytes relative to PCM. ELISA analysis of cytokine production in BCM-treated keratinocytes confirmed that after four hours of exposure, cytokine levels were higher relative to PCM-treated keratinocytes. However after 24 hours of exposure, BCM stalled the production of cytokines, suppressed activation of the mitogen activated protein kinases JNK and p38, and induced the release of intracellular calcium in keratinocytes. Processes relating to epithelialization such as the disruption of cytoskeletal components and induction of apoptosis were induced by BCM in keratinocytes. BCM induced a distinct inflammatory response and inhibited processes related to epithelialization. Collectively, the results provide insight into the formation and persistence of chronic wounds. The use of biofilm-based models of disease such as the in vitro model described herein will aid in the development of new biofilm based treatment strategies, not only for chronic wound infections, but all biofilm-based disease.