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The first observations of biofilms were made by the “father of microbiology” Antonie van Leeuwenhoek in the 17th century. The number of publications on biofilms has grown exponentially in the last 20 years, highlighting the medical relevance of the field. The complexity of the bacterial biofilm as well as its variability across species provides a continual channel for discovery. While all biofilms differ, there are some components that remain standard such as proteins, polysaccharides and DNA allowing for linkages between seemingly distinct biofilms. Biofilm-associated infections account for more than 65% of all infections implicating the need for understanding the stages of biofilm formation and development. Our lab focuses on the amyloid component of the biofilm and has identified that curli and extracellular DNA (eDNA) complex irreversibly within the biofilms of Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we investigate the formation and pathogenicity of cytotoxic curli intermediates previously unidentified in the in vitro biofilm. The identification of multiple curli conformations within the biofilm biogenesis aides in the understanding of amyloid kinetics in the enteric biofilm. Together, these studies provide a link between biofilm-associated infections and autoimmune responses in the host. In these studies, we planned to isolate curli from different stages in biofilm development to observe their differences both structurally and through their interactions. We identified turbulence has a significant impact on the formation of mature biofilm. We were able to isolate an intermediate form of curli through increased turbulence during biofilm growth. There has never been an intermediate form of curli isolated before our studies due to the high efficiency of the nucleation-precipitation process of curli fiber formation. From this isolation, we characterized these intermediates in comparison to the mature curli complexes. We observed that higher turbulence leads to lesser biofilm formation by sedimentation assay and crystal violet staining. Additionally, we investigated the expression of the curli forming genes csgBA via flow cytometry analysis which indicated that csgBA was preferentially expressed under low turbulence conditions. When investigating the curli conformations isolated from the biofilm, we found that intermediate complexes incorporated less thioflavin T (ThT) indicating lesser amyloid content. We also differentiated the mature and intermediate curli aggregate populations using multiple microscopy techniques. Under confocal microscopy, intermediate fibers seldom measured larger than 100 µm, while mature curli did. Electron dense regions were observed under transmission electron microscopy in the mature curli indicating high fibrillization and compact structure of these aggregates, not seen in the intermediates. Due to known interactions of curli with eDNA in the biofilm, next we investigated the DNA content of the complexes. We hypothesized that the mature structured complexes would have greater DNA content supporting the maturation of the fibrils and the structural compaction. Indeed, we found more DNA could be extracted from the mature curli fibers. Interestingly, we increased the fibrillization of intermediates upon addition of exogenous genomic DNA suggesting DNA incorporation was necessary for the formation of the mature fibrillar aggregates. Intermediates of amyloid β are found to be more cytotoxic than the mature form of the amyloid. For this reason, we hypothesized that curli intermediates could also be cytotoxic. After treating bone marrow-derived macrophages with mature and intermediate curli complexes, we observed that the intermediate aggregates were significantly more cytotoxic to immune cells than mature aggregates. Together, this data implicates a role for the cytotoxic intermediate form of curli in the pathogenesis of Salmonella as well as other enteric bacteria. Curli complexes have been previously described as a novel pathogen associated molecular pattern (PAMP) by their ability to activate numerous receptors in immune cells. The host immune response to curli complexes has been elucidated in our lab. First, binding to Toll-like receptor-2 (TLR2) begins with recognition of the conserved cross-β sheet secondary structure. Mutations disrupting this structure are shown to abrogate immune cell recognition and signaling. For this reason, we next investigated the pathogenicity of the intermediates discovered and characterized above. As cytotoxic oligomers exist for human amyloids, we aimed isolate an earlier form of the intermediate curli and investigate the ability of these conformations to activate host immune responses. As mature curli has been reported to induce anti-dsDNA antibodies in murine models of systemic lupus erythematosus (SLE), we anticipated differences in the autoimmune response to these different curli aggregates as well. First, we isolated and characterized an early form of intermediates isolated at 24 hours which were smaller in size and incorporated less DNA within their complexes than the aforementioned intermediates. Further investigations into the structure of these early intermediates described altered secondary structure by circular dichroism. The lack of fully formed secondary structure in the early intermediates we hypothesized to decrease the ability of these complexes to interact with immune receptors as mature curli. Indeed, we saw decreased response in pro-inflammatory cytokine production as well as type I IFN production. This lack of type I IFN production, lead us to investigate the autoantibody response to the early intermediates. When treating both wild-type and autoimmune-prone mice with curli complexes, early autoantibodies responses were dependent on the DNA content of the complex. However, after continual treatment for 10 weeks, intermediate complexes produced levels of anti-dsDNA similar to that of the mature curli treatment. In addition to anti-dsDNA antibodies, for the first time, other anti-nuclear antibodies, such as anti-C1q and anti-nucleosome, were produced in response to these treatments as well. Finally, chronic exposure to curli complexes led to significant histopathological changes including synovial proliferation and periosteal resorption, in the joints of mice autoimmune-prone mice. Together, this data identified that chronic exposure to curli induced autoimmune sequelae which is thought to be transient in genetically healthy individuals, but leads to joint inflammation in individuals whom are genetically predisposed to autoimmunity. In summary, these data significantly broaden the knowledge of curli amyloid formation during biofilm biogenesis in vitro through the identification of previously unidentified cytotoxic intermediate conformations of curli. Additionally, our work forwards the fields of both autoimmunity and biofilm-associated infection research by providing evidence of the direct impact that chronic exposure to the biofilm has on the host, both transient and long-lasting in those whom are pre-disposed to autoimmunity. / Biomedical Sciences

Intracellular pathogens like <i>Salmonella</i> have developed various mechanisms to evade host defenses, and they can establish infections. Treatment and eradication are difficult due to our inability in achieving the optimum concentrations of cell-impermeable aminoglycosides like gentamicin within these cells. In this dissertation, we hypothesize that developing a novel core-shell methodology for incorporating high amounts of gentamicin into the cores with either hydrophilic or amphiphilic shell will be more effective than the free gentamicin in clearing intracellular <i>Salmonella</i> infection. Hydrophilic core-shell nanostructures (N1) were made with block co-polymers of poly (ethylene oxide-<i>b</i>-sodium acrylate) blended with sodium polyacrylate (PAA^-+Na) and complexed with the polycationic antibiotic gentamicin. N1 showed 20-25 fold higher gentamicin loading than the currently existing materials and reduced numbers of viable <i>Salmonella</i> in the liver and spleen compared to free gentamicin. To further improve the rate and route of uptake, the shell of the nanostructures were made amphiphilic by incorporating pluronics F68 (PPO)₆₈ in the block copolymer. We showed that core-shell nanostructures encapsulating gentamicin having (PPO)₆₈ in the shell (N2) enhances the rate and modulates the route of uptake into macrophages, thus promoting significant reduction in the intracellular <i>Salmonella in-vitro</i> and <i>in-vivo</i>. The main drawback of N2 was its poor stability at physiological pH of 7.4, 0.1 M NaCl. Therefore, core-shell nanostructures encapsulating gentamicin containing pluronic P85 (PPO)₈₅ in the shell (N3) with improved colloidal and ionic stability were designed. N3 achieved significant intracellular reduction of vacuolar <i>Salmonella</i> (0.53 log₁₀) and cytoplasm resident <i>Listeria</i> (3.11 log₁₀) compared to free gentamicin in-vitro. However, greater reduction of <i>Listeria</i> suggested that sub-cellular localization of bacterium influences targeting by N3. Even though oral administration of N3 was not effective compared to free gentamicin, parenteral (I.P.) administration significantly reduced the intracellular <i>Salmonella</i> from liver and spleen compared to free gentamicin and appeared to have no abnormal <i>in-vivo</i> toxicity. In summary, core-shell nanostructures encapsulating gentamicin (N) with improved encapsulation efficiency and different shell chemistry (N1, N2 and N3) were developed with enhanced efficacy against intracellular Salmonella. The novel gentamicin delivery approach developed in this study may be applicable for therapy of many intracellular infections. / Ph. D.

Bacteriophages cohabit with their bacterial hosts and shape microbial communities. To initiate infection, phages use bacterial components as receptors to recognize and attach to hosts. Flagellotropic phages utilize bacterial flagella as receptors. Studies focused on uncovering mechanistic details of flagellotropic phage infection are lacking. As the number of phage-based applications grows, it is important to understand these details to predict the potential outcomes of phage therapy. To this end, we studied two flagellotropic phages: Agrobacterium phage 7-7-1 and bacteriophage χ. Phage 7-7-1 infects Agrobacterium spp., while bacteriophage χ infects Salmonella and Escherichia coli. Chapter 1 consists of a literature review. Chapter 2 addresses factors underlying phage-bacteria coexistence. We document the emergence of a sector-shaped lysis pattern following co-inoculation of phage χ and one of its Salmonella hosts on swim plates. We propose that this pattern serves as a reporter for balanced phage-bacteria coexistence. Using a combined experimental and mathematical modelling approach, we discovered that variations to intrinsic factors (i.e., bacterial motility, phage adsorption) skews the pattern towards either bacterial or phage predominance. Thus, this computational model may be used to predict phage therapy application outcomes. Chapter 3 details the identification of cell surface receptors essential for phage 7-7-1 infection using a transposon mutagenesis approach. We identified three Agrobacterium sp. H13-3 genes involved in phage 7-7-1 infection. Using mass spectrometry and other analyses, we determined that the LPS profiles of strains lacking these genes varied compared to the wild type. Thus, LPS is a secondary cell surface receptor for phage 7-7-1. Chapter 4 focuses on the discovery of phage encoded receptor binding proteins (RBPs) in Agrobacterium phage 7-7-1. Using an RBP screen, we discovered three candidate RBPs. We learned that our top candidate, Gp4, inhibits the growth of Agrobacterium sp. H13-3 cells in a motility and glycan dependent manner. Because of its bacteriostatic activities, this protein is a promising candidate for therapeutic use. Overall, the described works contribute to a deepened understanding of flagellotropic phage infection and the factors influencing their coexistence with motile bacteria. These works will contribute towards the development of phage therapies using whole phage or their components. / Doctor of Philosophy / Bacteriophages, or phages for short, are the natural killers of bacteria. Like antibiotics, they can also be used as medicines to treat bacterial infections. Their attack on bacteria begins by recognizing specific parts of the bacterial cell and attaching to them. These parts are called receptors. To use phages as medicines it is important to understand how they recognize and kill bacteria. This information is helpful when deciding which phage should be given to treat a bacterial infection and to predict the outcomes of these treatments. In this work, we focused on two phages to answer different questions. Both phages use long helical thread-like structures, called flagella, as receptors. Flagella help the bacteria to move through the environment and reach new areas with more nutrients. One of these flagella-dependent phages, called phage 7-7-1, infects plant pathogens that cause tumor-like growth in plants. We found that this phage uses two very different host cell components during infection and identified one of the phage proteins that interacts with these receptors. This protein prevents the growth of the plant pathogen, which makes it a promising candidate for therapeutic use. We also investigated how another bacterial virus, bacteriophage χ, is spread throughout the environment and co-exists with its motile bacterial host. We built a computational model that can predict how altering different variables affects phage-bacteria coexistence. With additional research, this model will be a useful tool for predicting the outcomes following phage treatment.

Contaminated tomatoes have become a commonly implicated vehicle of Salmonella outbreaks. Exposure of tomatoes to pathogen could occur in the field. Blossom inoculation with Salmonella can result in contaminated fruit but natural routes of blossom contamination are not well known. Salmonellae have been known to survive in agricultural soil. Since dispersal of soil particulate by wind is a common phenomenon, the potential of airborne soil particulate as a vehicle of Salmonella contamination in tomato blossoms was examined. It was determined that Salmonella enterica serotype Anatum, Baildon, Braenderup, Montevideo, Newport, Javiana had similar survival patterns in both soil and water. At the end of 40 days, populations of Salmonella in soil dropped by 2.59 log CFU/g and 5.11 log CFU/g when enumerated on Tryptic Soy Agar Yeast Extract (TSAYE) and xylose lysine Tergitol 4 (XLT4) agar respectively. Salmonella populations in water reduced by 2.55 log CFU/ml (TSAYE, enumeration) and 2.94 log CFU/ml (XLT4, enumeration). Blossom to fruit formation takes 20-30 days in tomatoes hence the introduction or presence of the pathogen in agricultural soil and water could increase risk of blossom contamination. Also, it was determined that all Salmonella serotypes tested were capable of biofilm production on glass coverslips and quartz particles. Biofilm based attachment of Salmonella to sand might aid in its dispersal. To visualize transfer of pathogen from soil particulate to blossom in real-time, bioluminescent S. Baildon, S. Braenderup, S. Newport, S. Javiana and S. Anatum were created.Heat shock procedure was developed to improve electrotransformation efficiency in Salmonella. Transformed strains were compared for bioluminescence production and plasmid stability. S. Newport had the best bioluminescence properties but no difference was observed between strains for plasmid stability. Imaging of soil particulate - S. Newport mixture inoculated blossoms, indicated that the event led to pathogen transfer to blossom. It was also determined that S. Newport â soil particulate contaminated blossoms developed into fruits that were positive for S. Newport. S. Newport presence in blossom, fruit surface and internal tissue indicates that contaminated soil particulate could serve as a vehicle of tomato contamination. / Ph. D.

Fresh produce has been implicated in several foodborne outbreaks. A primary site of microbial contamination for produce occurs on the surface during production and handling. An approach to reduce contamination is to sample the surface of produce. This study used different detergent agents at 22&#176;C and 40&#176;C to determine their efficacy for recovery of pathogenic bacteria, from surfaces of several produce types and examined survival of organisms in detergents over time. Strawberries, tomatoes and green leaf lettuce were dip inoculated in a 6-6.5 LOG CFU/ml cocktail of nalidixic acid resistant organisms. After drying, produce were rinsed with either 0.1 % sodium lauryl sulfate (SLS), 0.1% Tween 80, or water at different temperatures. Rinse solutions were plated onto Tryptic Soy agar supplemented with 50-ppm nalidixic acid (TSAN). About 4 LOG CFU/ml of Salmonella, and 3-LOG CFU/ml Shigella were recovered, with slightly lower recovery from tomatoes. Inoculated strawberries rinsed with SLS, displayed minimal recovery at ~1.5-LOG CFU/ml at 22&#176;C, and <1-LOG CFU/ml at 40&#176;C. When whole strawberries treated with SLS were analyzed, few Salmonella were recovered. Lack of recovery of Salmonella rinsed with SLS, suggests SLS may be inactivating Salmonella, especially at elevated temperatures. Detergent solutions were inoculated with 3-LOG CFU/ml cocktail and incubated for up to 32 hours at 22&#176;C, and 40&#176;C. Aliquots were plated onto TSAN at varying times. All solutions at 40&#176;C allowed Shigella to grow. SLS gave initial drops in Salmonella populations followed by slight recovery. SLS may cause an initial injury of Salmonella. While organisms were able to survive in detergents, the application of detergents to produce was no more effective in recovery of organisms from produce than water. / Master of Science

Escalating product recalls as a consequence of Salmonella-contaminated poultry products have resulted in detrimental economic impacts. One long-term alternative to Salmonella prevention, receiving increased attention, is selection to improve genetic resistance. This study evaluated the effects of an oral Salmonella Heidelberg (SH) challenge on bacterial colonization, and the ex vivo effects of SH on phagocytic and bactericidal leukocyte function in turkeys from six pedigree lines (A-F). Data were analyzed using JMP Pro (SAS) and differences were determined using Student’s t-test following ANOVA with significance reported at P ≤ 0.05. Interaction effects of treatment X gender X genetic line were significant on bacterial colonization in the ceca. Cumulatively, females exhibited higher phagocytosis potential than males. The main effect of genetic line was significant bactericidal activity of PBMCs. Microbial profiling of cecal DNA was performed to examine differences in colonization of Salmonella, E. coli, and Enterococcus species among the genetic lines. Results indicated line E having the highest Enterococcus but lowest Salmonella colonization than all other lines, while line A birds displayed the highest Salmonella colonization. These results suggest that gender and genetic line have a marked effect on susceptibility to Salmonella colonization, while genetic line X gender has a more eminent effect on Enterococcus cecal colonization. If able to determine genetic markers associated with these immune responses to Salmonella, genetic selection for increased resistance could be feasible in turkeys. / Master of Science

Bacteriophage Felix O1 was studied for applicability as a Salmonella intervention. Felix O1's potential as a Salmonella therapeutic was explored, as was its utility as a food application. Felix O1 is specific for and infects most serovars within the genus Salmonella. The entire 86.155-kb sequence of the phage's linear, double-stranded chromosome was determined. 213 open reading frames (ORFs) were found, including 23 homologues of phage genes (e<0.008). Homology searches do not indicate genes that would be expected to increase virulence of Salmonella. Thirteen T4 homologues were found, including rIIA and rIIB, rapid lysis genes of T-even phages. Site-directed mutagenesis of the rIIB region was attempted by homologous recombination with plasmids containing luxAB of Vibrio harveyi. No DrIIB luxAB+ recombinants resulted from the methods tried. Serial in vivo passage was used to select for a longer-circulating Felix O1 mutant using the modified methods of Merril et al., (1996). No difference was found in the clearance of wild-type (WT) and Felix O1 following nine serial passages. Injection of 10⁹pfus yielded 24-hour concentrations of 6.5 and 4.9 log10 pfus/ml plasma for WT and 9th passage, respectively. Both isolates were undetectable in plasma by 72 hours, but remained in spleens at 96 hours. A large-plaque Felix O1 variant (LP) isolated during in vivo serial passage was compared with WT for Salmonella growth suppression. Spectrophotometric measurement of BHI cultures indicated greater suppression of S. typhi by LP than by WT, a difference not seen with S. typhimurium DT104. Both isolates suppressed 24-hour S. typhimurium DT104 growth on experimentally-contaminated chicken frankfurters at 22°C. Untreated frankfurters yielded 6.81 log10 Salmonella cfus/g, whereas WT and LP-treated samples yielded 5.01 and 4.70 log10 cfus/g, respectively. Both phages suppressed the Salmonella typhimurium DT104 growth (p<0.0001), but the isolates did not perform differently (p=0.5088). Presence of Salmonella caused a higher yield of WT phage than from the uninoculated group (p=0.0011), but did not affect LP yield (p=0.4416). With Salmonella present, the 24-hour LP concentration was lower than WT concentration. This supports the surmised LP rapid-lysis phenotype since T4 rapid-lysis mutants typically exhibit lower burst sizes than wild-type phage. / Ph. D.

Expt. 1 evaluated effects of 0 or 10 ppm CAP in the starter phase (d 1-16) on chicks challenged with SE on d of age. Therapeutic inclusion of 10ppm CAP increased (P < 0.05) L/S and ceca positives. In Expt. 2, capsaicin oleoresin (CO) was included in finisher diets (d 30-37) at 0, 5, or 20 ppm with SE challenge on day 31. Inclusion of 5 ppm CO increased (P < 0.05) ceca SE positives and demonstrated 1.05 and 1.39-log fewer SE cfu at CO concentration of 5 or 20 ppm, respectively. A linear decrease (P < 0.05) in lamina propria thickness of SE challenged birds was observed with increased CO. Expt. 3 evaluated prophylactic CO treatment at 0, 5, or 20 ppm in starter, grower, and finisher diets for resistance to SE or ST challenge on d 14 or 29. With challenge on d 14, 5 ppm CO reduced ceca (P<0.005) SE positives and 1.1-log fewer SE cfu. Likewise, 20 ppm CO reduced (P < 0.05) SE ceca positives. Salmonella typhimurium isolation rate was reduced (P<0.05) with 5 ppm CO, and ST cfu were reduced 1.4-log with 5 ppm CO compared to 20 ppm. Lamina propria thickness increased (P < 0.05) linearly as CO concentration increased. With d 29 challenge birds fed 5 ppm CO exhibited 1.08-log fewer SE cfu, and 20 ppm CO reduced L/S positives (P < 0.025) for SE and resulted in 1.39-log fewer SE cfu. Lamina propria thickness decreased with 5 ppm CO and SE or ST challenge compared to non-challenged birds fed 5 ppm (P < 0.0005). An increase was observed in ST or SE, birds fed 20 ppm CO compared to non-challenged, birds fed 20 ppm CO (P < 0.01). No differences were observed in mast cell number in either Expt. 2 or 3. These data provide evidence that prophylactic or therapeutic dietary CAP differentially affect broiler susceptibility to Salmonella and prophylactic administration may provide non-antibiotic means to reduce Salmonella in broilers. / Master of Science