Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Novel Antimicrobial Treatments Based on the Interaction of Gallic Acid and UV Light: Characterization, Investigation of Antimicrobial Mechanism, and Application on Fresh Produce(2018) Wang, Qingyang; Tikekar, Rohan V; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Produce safety continues to be a challenge because produce undergoes minimal processing prior to consumption and existing sanitizers are not effective in inactivating pathogens. Novel decontamination technologies for produce are required as alternatives to traditional methods. In this project, two non-thermal process techniques were developed based on the interaction of UV light and gallic acid (GA) to enhance the safety of fresh produce. The first technique is the simultaneous application of UV-A light and GA (UVA+GA). UVA+GA treatment was effective against E. coli O157:H7, and the mechanism behind the synergistic antimicrobial effect was associated with the cellular uptake of GA, generation of reactive oxidative species (ROS), inactivation of enzymes superoxide dismutase, and damage to the bacterial membrane. In the second technique, the antimicrobial activity of GA was enhanced by its prior UV-C exposure (UVC-GA) against E. coli O157:H7 and was persistent for at least 4 weeks. The antimicrobial activity was affected by solution pH and the wavelength of UV-C exposure. The generation of ROS during UV light exposure and photo-oxidized compounds of GA such as quinone contributed to the antimicrobial activity of the UVC-GA solution. Both UVA+GA and UVC-GA treatments can enhance the inactivation of inoculated E. coli O157:H7 on produce such as spinach leaves and tomatoes without affecting the color and firmness. Common environmental stresses could confer complex cross-stress response in E. coli O157:H7 towards UVA+GA and UVC-GA treatments in that both resistance and sensitization can be induced depending on the stress applied and the technology studied. Repeated exposure to moderate UVA+GA or UVC-GA treatment can also select for sub-population that demonstrates higher resistance towards these treatments as well as cross-resistance to other lethal stress such as heat and acid. ROS scavenging enzymes and alternative sigma factor RpoS are highly likely to be associated with the adaptive response process. In conclusion, both UVA+GA and UVC-GA treatments are promising novel non-thermal techniques that are potential alternative methods for fresh produce disinfection. For future work, a better understanding of the inactivation mechanisms, optimizing of processing parameters, and the development of adaptive response associated with the two treatments need to be explored.Item Fate of antimicrobials and nutrients in dairy manure management systems(2018) Schueler, Jenna E; Lansing, Stephanie; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Anaerobic digestion (AD) and composting manure management strategies were explored at the field scale to monitor antimicrobial degradation, nutrient transformations, and optimize mitigation of these pollutants in manure fertilizer to decrease their entry to waterways. Removal of antimicrobials and antimicrobial resistance genes (ARGs) were explored at the bench scale, where AD degraded >85% of antimicrobials. At the field-scale, antimicrobials were not consistently removed, persisting in concentrations up to 34,000 ng/g DW in the AD effluent. The tetM genes were reduced during bench-scale AD suggesting that AD could be an effective treatment for removing tetracycline ARGs from manure. The 100% reduction of sulfadimethoxine antimicrobials during AD did not correspond with Sul1 reduction, illustrating differences in antimicrobial versus gene reductions during manure treatment. Antimicrobials did not degrade significantly during field scale composting, likely due to a shortened composting period (33-days). The field-scale results illuminate limitations of tracking antimicrobials in complex treatment systems.Item Development of methods to test drug sensitivity of fish pathogenic Flavobacterium columnare and drug sensitivity thresholds for F. columnare to the antimicrobial florfenicol.(2015) Gieseker, Charles Michael; Woods III, Lewis C.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antimicrobial drugs play a key role in managing the health of fish in aquaculture. However, public health concerns about antimicrobial resistance--the ability of microorganisms to resist standard antimicrobial treatments--include the potential for antimicrobial use in aquaculture to select for resistant bacteria in and around fish farms. Recent approval of two antimicrobial drugs to treat farmed freshwater fish with infections caused by the aquatic bacteria Flavobacterium columnare and F. psychrophilum created an important need for research to monitor these bacteria for changes in antimicrobial susceptibility and to decide when the antimicrobials should be used. Therefore three studies were conducted. The initial study optimized methods for broth microdilution testing F. columnare and F. psychrophilum and conducted a multi-laboratory standardization trial that set quality control parameters for nine antimicrobials commonly used in aquaculture, thus creating the first standardized testing method. In the second study, we constructed frequency distributions using minimal inhibitory concentrations determined from testing 134 F. columnare with the standardized method. Analysis of the distributions determined the drug concentration--called an epidemiological cutoff value (ECV)--which separated the wild type isolates with no acquired or selected resistance from the non-wild type isolates. The ECV indicated 22 of 134 isolates had decreased their susceptibility to at least 1 antimicrobial. In addition, we developed a laboratory disease model with juvenile channel catfish, Ictalurus punctatus. We compared the virulence of three F. columnare isolates with wild type or three isolates with non-wild type florfenicol susceptibility using the model. We found that five isolates had similar high level virulence and were not affected by differences in florfenicol susceptibility. Finally, we studied if decreased non-wild type florfenicol susceptibility affected the ability of the approved florfenicol treatment to control F. columnare infections. The approved treatment significantly reduced catfish mortality following exposure to an isolate with typical wild type florfenicol susceptibility but mortality was not reduced following exposure to an isolate with non-wild type susceptibility. Taken together, these studies provide methods and research needed to monitor F. columnare for changes in antimicrobial susceptibility and to rationally use florfenicol to control F. columnare infections.Item Engineering Enhanced Structural Stability to the Streptococcal Bacteriophage Endolysin PlyC(2014) Heselpoth, Ryan Daniel; Nelson, Daniel C; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antibiotic misuse and overuse has prompted bacteria to rapidly develop resistance, thereby hindering the efficacy of these chemotherapeutics. Due to antibiotic resistant strains expeditiously disseminating, antimicrobial resistance has been labeled as one of the greatest threats to human health globally. An emerging alternative antimicrobial strategy involves using bacteriophage-derived enzymes, termed endolysins. Endolysins are peptidoglycan hydrolases that liberate lytic bacteriophage virions late in the infection cycle by cleaving critical covalent bonds in the bacterial cell wall. As a result, the high intracellular osmotic pressure induces cell lysis. Antimicrobial strategies have been devised involving the extrinsic application of recombinant endolysins to susceptible Gram-positive pathogens. The efficacy of these enzymes has been validated in vitro and in vivo, with no resistance observed to date. One such example is the streptococcal-specific endolysin PlyC. This endolysin is currently the most bacteriolytically-active and possesses the ability to lyse human and animal pathogens known to cause serious health complications. Unfortunately, like numerous other endolysins, PlyC is relatively unstable and accordingly has short shelf life expectancy. With a long-term goal of using endolysins for industrial applications, furthering the development of a thermolabile translational antimicrobial with a short shelf life is ambitious. The main objective of this dissertation is to develop and validate bioengineering strategies for thermostabilizing bacteriolytic enzymes. Using PlyC as the model enzyme, we first used a rationale-based computational screening methodology to identify stabilizing mutations to a thermosusceptible region of the catalytic subunit, PlyCA. One mutation, T406R, caused a 2.27°C increase in thermodynamic stability and a 16 fold improvement in kinetic stability. Next, we developed a substantiated novel directed evolution protocol that involves randomly incorporating mutations into a bacteriolytic enzyme followed by a screening process that effectively identifies mutations that are stabilizing. Finally, applying multiple rounds of directed evolution to PlyC allowed for the identification of a thermostabilizing mutation, N211H, which increased the thermodynamic stability by 4.10°C and kinetic stability 18.8 fold. Combining the N211H and T406R mutations was additive in terms of thermal stability, with thermodynamic and kinetic stability enhancements of 7.46°C and 28.72 kcal/mol activation energy (EA) of PlyCA unfolding, respectively.Item Development of Encapsulation Systems from Zein and Metal-Organic Frameworks (MOFs) for Improved Functional Properties of Essential Oils(2013) Wu, Yunpeng; Wang, Qin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Essential oils (EOs), which are derived from plants, have antifungal, insecticidal and antimicrobial activities, but they are slightly soluble in water and impart to the water their odor and taste, which limit their applications in food area. Zein, a prolamin from corn, is able to form nanoparticles by liquid-liquid dispersion process. These nanoparticles are well dispersed in water and stable, which can be further applied to encapsulate functional materials that are insoluble in water. We have developed zein nanoparticles to encapsulate thymol and carvacrol in order to improve their solubility. The DLS (dynamic light scattering) and SEM (scanning electron microscopy) proved that zein nanoparticles encapsulated with EO were formed. The particles size was between 200~300nm before lyophilizing. 65-75% EOs have been encapsulated in the nano-sized particles. DPPH assay results proved good antioxidant property of the product. For the Ferric-ion spectrophotometric assay, hydroxyl free radicals had been cleared by 60~90% in overall. In the antimicrobial experiment, the nanoparticles encapsulating EOs reduced 0.8-1.8 log units of E. coli after 48h incubation. Furthermore, we have applied Metal-Organic Frameworks (MOFs) to encapsulate thymol. Metal-Organic Frameworks (MOFs) or porous coordination polymers (PCPs) is a new class of hybrid materials, which are formed by the self-assembly of metal-connecting points and polydentate bridging ligands. MOFs in this study was synthetized by Zinc nitrate hexahydrate and 2-aminoterephthalic acid in N, N-dimethylformamide (DMF). Thymol was then loaded inside the MOFs at the loading rate of 3.95%. The structure of porous crystal MOFs was confirmed by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Inhibition to E. coli O157:H7 was measured both in TSB medium and on TSA agar. An E. coli O157:H7 reduction of 4.4 log CFU/mL have been achieved at a thymol to broth ratio of 0.04g/100g. An inhibition area of 223.73 mm2 was observed after 12h incubation. With the two methods (zein nano-particles and MOFs), EOs can be encapsulated and well dispersed in water solution. The enhanced antioxidant activity and antimicrobial ability of the encapsulated EOs promise their further applications in food industries.Item Intervention Strategies for Escherichia coli O157:H7 and Salmonella in Organic Soil and on Fresh Produce(2012) Nguekam Yossa, Irene Nadine; Lo, Martin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recently, foodborne diseases caused by Escherichia coli O157:H7 and Salmonella have been increasingly associated with the consumption of fresh produce. Consumers' demand for safe, natural products has led to research on natural antimicrobials for effective control of foodborne pathogens on fresh produce, which can be inadvertently contaminated by soil. Therefore, there is a need to control microbial loads in soil to minimize contamination. The objectives of this study were to evaluate the antimicrobial activity of cinnamaldehyde, Ecotrol®, eugenol, Sporan® and acetic acid against E. coli O157:H7 and Salmonella in organic soil, and to evaluate the antimicrobial effects of cinnamaldehyde and Sporan® alone, or in combination with acetic acid against E. coli O157:H7, Salmonella, and the native microflora of iceberg, romaine and spinach leaves. The quality parameters of the treated fresh produce were monitored, whereas the modes of action of cinnamaldehyde and Sporan® were investigated. The results showed that cinnamaldehyde had the highest bactericidal activity against E. coli O157:H7 and Salmonella in organic soil. Increases in oil concentration resulted in further reduction of both microorganisms. Up to 5 and 6 log CFU/g of E. coli O157:H7 and Salmonella, respectively, were reduced with 2% Sporan® and acetic acid after 24 h. Sporan® in combination with acetic acid (1000SV) and 800 ppm cinnamaldehyde-Tween reduced significantly E. coli O157:H7 (~3 log CFU/g) on iceberg and spinach leaves following treatment at day 0. Likewise, 1000SV treatment reduced Salmonella ~ 2.5 log CFU/g at day 0. E. coli O157:H7 and Salmonella populations in treated iceberg, spinach and romaine leaves were reduced during storage at 4°C. The native microflora of untreated and treated spinach and lettuce leaves increased during the storage time. The texture and the color of iceberg, romaine and spinach leaves treated with essential oils were not significantly different from the control lettuce after 14 days. The scanning and transmission electron microscopy of oil-treated bacterial cells indicated possible cell structural damage and leakage of cellular content. This study shows the potential use of essential oils to effectively reduce E. coli O157:H7 and Salmonella populations in soil and on fresh produce without adversely affecting leaf color and texture.