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Microbiomes are an extremely vast and complex network of microorganisms. Bacteriophages are a key factor in the microbial health of an ecological system and impact the evolution of pathogenic bacterial strains. Bacteria and the phages that infect them have an intricate relationship due to the dependency on the bacterial host for phage replication, the ability of the phage to lyse and kill its host, and the horizontal gene transfer between the host and phage. This thesis aimed to understand how bacteria and the bacteriophages that infect them impact an ecological system, with a focus on disease states. By analyzing all bacteriophages targeting a specific host, genomic properties, physical similarities and differences a better understanding of how a group of tailed phages have evolved numerous mechanisms and tools to infect host bacteria was understood. The microbiome study of the Western honey bee Apis mellifera, comparing the microbial communities of colonies infested with the external parasite Varroa destructor against those not infested revealed a need for more directed treatment of Varroa infestations. Through our study we discovered that the honey bee microbiome is much more complex than previously reported, consisting of hundreds of bacterial species. In addition, through comparing infested and healthy colonies, we discovered that infestation of Varroa destructor mites negatively impact the colony microbiome in part by reducing microbes key in digestion and immune health of honey bees. Results in this thesis indicate that two microbes which have not been previously established as part of the key microbes in honey bee guts, Xenorhabdus and Sodalis, may in fact be key to honey bee health as they were both effected negatively by the presence of Varroa mite infestations. These bacteria have been shown to be involved in immune health in other insects, supporting this hypothesis. The final stage of this thesis involved the development of an acaricide bacteriophage therapy designed to target key microbes in the gut of V. destructor. Our therapy was shown to safely treat honey bee colonies infested with this parasite. It can be concluded that while phages are a normal part of the microbial ecosystem of the intestinal tract of organisms, altering that balance by increasing the phage load on the microbiome to target specific beneficial microbes drastically reduces the overall fitness of the organism. Results from this study indicate that multi-target cocktail and single-target phage therapy are an effective low impact biological Varroacide. The discovery of an effective therapy is important and to better understand the results of this thesis, further investigation is required, including a study of the impact of the phage therapy on the mite microbiome, as well as the safety of the therapy to humans.

Staphylococcus aureus is a common bacterium found on the skin and mucosal membranes of about 20% of the population. S. aureus growth on the skin is harmless, but if it bypasses the skin it can causes life-threatening diseases such as pneumonia, meningitis, bacteremia, and sepsis. Antibiotic-resistant strains of S. aureus, called Methicillin Resistant S. aureus (MRSA), are resistant to most antibiotics except vancomycin. However, vancomycin resistant strains of MRSA are becoming more common. In this study, 12 phages were isolated capable of infecting human S. aureus and/or MRSA strains. Five phages were discovered through mitomycin C induction of prophages and seven phages were found through enrichment of environmental samples. Primary S. aureus strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates. S. aureus isolates were tested for susceptibility to oxacillin in order to determine methicillin-resistance. Experiments were performed to assess the host range and killing potential of newly discovered phage. The M1M4 phage had the broadest host range and lysed 12% of the S. aureus strains that were tested. The host ranges were reinforced by spectrophotometric assay data which showed a reduction in bacterial optical density of 1.3 OD600. The phages were used to decontaminate MRSA from fomites (glass and cloth) and successfully reduced colony forming units by 1-2 logs, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics.

Produce is responsible for 46% of all foodborne illnesses in the USA. Salmonella enterica causes 19,000 hospitalizations each year, and has been associated with produce. Presently, chlorine based sanitizers are most often used, however organic matter reduces its antimicrobial activity. Bacteriophage treatments are an all-natural, alternative method for pathogen inactivation. The objective of this study was to determine the efficacy of a five-strain bacteriophage treatment against a S. enterica cocktail in simulated wash waters at different temperatures. Bacteriophage and S. enterica were enumerated in simulated wash water solutions. One set of experiments studied bacteriophage and S. enterica growth in TSB+vegetable solutions. Bacteriophage behavior was not statistically different (p < 0.05) in spinach, romaine, or iceberg lettuce across different concentrations of organic matter. S. enterica reduction was approximately 2 log over 135 minutes for vegetable solutions and for the TSB control. S. enterica reduction was only 0.5 log in water solutions. The next set of experiments studied bacteriophage and S. enterica growth in vegetable solutions. Spinach wash water and tryptone soy broth solutions (TSB) at 20 °C and 37 °C. S. enterica was not reduced in spinach solution studies at 20 °C and 37 °C or at broth solutions at 20 °C. However, S. enterica was effectively reduced 4 log in broth solutions at 37 °C up to 7.5 hours, but grew to high levels after 24 hours. These results indicate that bacteriophage could not effectively control bacteria levels in produce wash water, and may need to be optimized.

Bio-interface topography strongly affects the nature and efficiency of interactions with living cells and biological molecules, making hydrogels decorated with micro and nanostructures an attractive choice for a wide range of biomedical applications. Despite the distinct advantages of protein hydrogels, literature in the field has disproportionately focused on synthetic polymers to the point that most methods are inherently incompatible with proteins and heat-sensitive molecules. We report the development of multiple biomolecule-friendly technologies to construct microstructured protein and bacteriophage (bacterial virus) hydrogels. Firstly, ordered and sphericity-controllable microbumps were obtained on the surface of protein hydrogels using polystyrene microporous templates. Addition of protein nanogels resulted in the hierarchical nano-on-micro morphology on the microbumps, exhibiting bacterial repellency 100 times stronger than a flat hydrogel surface. The developed microstructures are therefore especially suitable for antifouling applications. The microstructures created on protein hydrogels paved the way for applying honeycomb template on proteinous bacterial viruses. We developed a high-throughput method to manufacture isolated, homogenous, pure and hybrid phage microgels. The crosslinked phages in each phage-exclusive microgel self-organized and exhibited a highly-aligned nanofibrous texture. Sprays of hybrid microgels loaded with potent virulent phage effectively reduced heavy loads of multidrug resistant Escherichia coli O157:H7 on food products by 6 logs. / Thesis / Doctor of Philosophy (PhD) / Bacteriophages (bacterial viruses), also known as phages, are natural bacteria predators. These viruses act as direct missiles, each phage targeting limited groups of bacteria. In addition, phages are an endless resource for self-propagating nanoparticles that can be used as building blocks for new material. I developed a platform for manufacturing a large quantity of microscale beads made of millions of phages. These micro-beads can be sprayed on fresh produce and meat to remove bacterial contamination (with the added benefit of not affecting taste or smell). I also printed phages on substrates, like an ink. The printed phage ink evolved into a patented technology for designing phage coatings on surfaces with very high surface area, like the small structures on our fingers. This phage coating was successfully used to test the existence of bacteria in liquids.

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