Master of Science, The Ohio State University, 2007, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2021, Food Science and Technology

Salmonella and Campylobacter are leading bacterial agents both nationally and globally, making them high public health concerns. Both are significant important zoonotic pathogens commonly found in livestock. Poultry and poultry products are the most common sources of human infections. Infected poultry show little to no clinical signs, and risk the chance of entering the food system, potentially contaminating consumers. Direct links between the high load of bacteria in the chicken intestinal tract and the high contamination of poultry carcasses support the need for pre-harvest control. Previously, antibiotics were used to control bacterial infections and growth; but increasing occurrence of antibiotic resistant bacteria has caused laws and practices to shift. Targeting the control of foodborne pathogens in the pre-harvest stage can improve animal welfare and public health. Alternative treatment methods are needed to combat Salmonella and Campylobacter in production animals, improve antibiotic stewardship, and subsequently strengthen the economy. We discovered the antimicrobial efficacy of Lactobacillus acidophilus (LA), Lactobacillus rhamnosus GG (LGG), and Bifidobacterium animalis subsp. lactis (Bb12) in vitro. More importantly, we showed LGG significantly reduced Salmonella in the chicken cecum by 1.9 logs (P<0.001) at 10 days post infection. LGG was also able to inhibit the growth/ presence of other food safety significant Salmonella serovars in an agar well diffusion assay. Characterization of antibacterial activity of LGG revealed that although organic acids are present, the most crucial part of inhibition are the heat and protease stable peptides that were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). After which, we tested the efficacy of Lactobacillus rhamnosus GG (LGG) derived small peptides (P1-NPSRQERR, P2- PDENK, and P4-MLNERVK) against Salmonella Typhimurium (ST) in chickens and examined their antibacterial effects a (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor); Anastasia Vlasova (Committee Member); Melvin Pascall (Committee Member); Scott Kenney (Committee Member) Subjects: Food Science; Microbiology

Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program

Salmonella Typhimurium, similarly to other enteric pathogens, produce a group IV O-antigen (O-ag) capsule exhibiting structural resemblance to the lipopolysaccharide (LPS). Polysaccharide capsules are known virulence factors of many bacterial pathogens, facilitating evasion of immune recognition and systemic dissemination during acute infection. Capsular polysaccharides can also aid establishment of chronic infection and environmental spread of disease by increasing bacterial surface adherence, self-aggregation and resistance to stress, all of which are critical steps in the continued cycle of infection. In this work, we sought to further characterize the O-ag capsule, a recently described surface polysaccharide of Salmonella Typhimurium. We have established that functional surface assembly of the O-ag capsule shares several common enzymatic pathways with lipopolysaccharide (LPS) biosynthesis and that disruption of the O-ag capsule does not interfere with production of LPS. Whole cell imaging has revealed heterogeneous capsular expression within sessile and biofilm bacterial populations and indicates that capsular expression may shield recognition of the LPS O-ag by specific antibodies. Additionally, we have observed that absence of the O-ag capsule results in dysregulated surface expression of highly immunostimulatory phase I flagellin (FliC) and increases bacterial susceptibility to killing by human serum. Due to their high level of attenuation, strains lacking the alternative RNA polymerase sigma factor RpoS have been extensively investigated for possible future inclusion in a live vaccine strain and the currently licensed live-attenuated vaccine strain for Salmonella Typhi TY21A is RpoS-deficient. Efforts to produce attenuated vaccines with RpoS-deficient phenotypes have focused on achieving a balance of attenuation and immunogenicity, reporting that a lack of systemic replication of rpoS mutants necessitates multiple doses of greater than 10^10CFU to achi (open full item for complete abstract)

Committee: John Gunn PhD (Advisor); Robert Munson PhD (Committee Member); Jesse Kwiek PhD (Committee Member); Daniel Wozniak PhD (Committee Member) Subjects: Biomedical Research; Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2013, Microbiology

Salmonella enterica is the leading cause of death from foodborne illness in the United States. The mechanisms of its virulence have been well studied but key details have remained elusive. Salmonella expresses a type 1 fimbriae when transiting through the distal small intestine which enables it to bind to mannosylated glycoproteins present on the surface of intestinal epithelial cells. This allows the bacterium to dock to the cell utilizing the Salmonella pathogenicity island 1 encoded type three secretion system (SPI1-T3SS). Upon docking, Salmonella injects effector proteins into the host cell interrupting host cell function. This leads to an actin rearrangement at the site where the bacterium is docked which causes the host cell to engulf the bacterium in a process termed macropinocytosis. These processes are regulated tightly to ensure optimal expression at the time of invasion. The BarA/SirA two-component system in coordination with the Csr system are responsible for the regulation of these virulence traits. BarA is a sensor kinase that monitors the environment for the appropriate signal to start the virulence cascade. Though the signal remains elusive it is hypothesized to be the short chain fatty acids formate and acetate which are found in the distal ileum. Upon sensing these signals BarA phosphorylates SirA, which in turn can activate iii its target genes. To date only two direct targets have been identified for SirA, the small RNA’s csrB and csrC. These small non-coding RNAs are the antagonists of the RNA binding protein CsrA. Typically CsrA binds its target transcripts and prevents translation by blocking the ribosome. Through our studies we have attempted to unravel the pathways by which SirA and CsrA regulate the SPI1-T3SS and type 1 fimbriae to lead to successful invasion of the host cell. We show here that SirA has no direct binding targets in either SPI1 or the fim operon. SirA regulates SPI1 in an indirect manner through the activation of (open full item for complete abstract)

Committee: Brian Ahmer Ph.D. (Advisor); Larry Schlesinger MD/Ph.D. (Committee Member); John Gunn Ph.D. (Committee Member); Robert Munson Ph.D. (Committee Member) Subjects: Genetics; Microbiology

Doctor of Philosophy, The Ohio State University, 2004, Food Science and Nutrition

Presence of Salmonella Enteritidis in shell eggs has serious public health implications. Current procedures to control Salmonella in shell eggs are partially effective, time-consuming, or negatively impact egg quality. This study focuses on the development of methods to inactivate Salmonella in eggs by using gaseous ozone, ultraviolet radiation (UV), and heat. Externally contaminated eggs (6-log CFU/g eggshell) were treated with ozone at 0-15 psig for 0-20 min, or with UV at 100-2500 µW/cm2 for 0-5 min. Other contaminated eggs were treated with UV (1500-2500 µW/cm2) for 1 min, and then with ozone at 5 psig for 1 min. Treating eggs with ozone at 15 psig for 10 min, or with UV (1500-2500 µW/cm2) for 5 min reduced Salmonella on eggshells by ≥ 5.9 or 4.3 log, respectively. Treating contaminated eggs with UV, followed by ozone, caused synergistic inactivation by ≥ 4.6 log in 2 min. A thermal pasteurization procedure was tested to inactivate Salmonella , and its impact on egg quality was evaluated. Eggs internally inoculated with Salmonella (6-log CFU/g egg), using simulated natural contamination, were heated in water at 57, 58, or 59°C for ≤ 40 min. Sigmoid survivor's curves, with shoulders and tails, were observed during treatments at 57, 58, and 59°C with microbial inactivation by 4.8, 5.1, and 5.4 log, respectively, during 30-40 min heating. Treatments that reduced ≥ 5 log Salmonella caused albumen turbidity or protein denaturation. Salmonella was inactivated within shell eggs using heat, vacuum, and ozone. Internally contaminated eggs (7-log CFU/g egg) were heated at 57-59°C for ≤ 40 min, placed at -7 to -10 psig, and then treated with ozone (12-14% wt/wt; ≤ 20 psig) or with mixtures of ozone and carbon dioxide for ≤ 40 min. Treatments were optimized in a response surface model developed from inactivation data. Optimized treatment with heat and ozone reduced ≥ 6.3 log Salmonella in 65 min total treatment time, without drastically affecting egg quality. In conclusion, (open full item for complete abstract)

Committee: Ahmed Yousef (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2024, Microbiology

Bacteria can communicate with each other through the production, release, and detection of small molecules called N-acyl homoserine lactones (AHLs). In a subset of the family Enterobacteriaceae, including the well-known genera Salmonella and Escherichia, AHLs are not produced but these bacteria retain the ability to detect them through the LuxR-type protein SdiA. This strategy is referred to as eavesdropping: where one species may listen in on the communication of another. The role of SdiA-mediated eavesdropping in the lifecycle of these bacteria is unknown. To determine the function of eavesdropping, we first reviewed the available literature on SdiA. Since the initial discovery of SdiA, many studies have attempted to gain insight into its role by looking for mutant defects in various host systems, elucidating the SdiA regulon, or finding in vitro phenotypes. The literature on each topic is complex and interpretation must be measured and considerate of the methodology used. We next examined the role of Salmonella SdiA in several host systems, including house flies, mice, and plants. We also determined the SdiA regulons of Salmonella, E. coli, and Enterobacter cloacae. The house fly is a known mechanical vector of Salmonella with some evidence of a more dynamic interaction between host and bacteria. Based on the abundance of AHL synthase homologs in insect metagenomes, we hypothesized that SdiA played a role in the survival of Salmonella within house flies. After a series of experimental infections, the evidence suggests that sdiA mutants are highly advantaged over their wild-type competitor and that SdiA may have a negative effect on survival within house flies. Using a randomly barcoded transposon library (Barseq), we examined Salmonella fitness in mice that were co-infected with the AHL producing pathogen Yersinia enterocolitica. Consistent with previous reporting, sdiA and its regulon suffered no fitness defects during gastroenteritis. Finally, an experiment (open full item for complete abstract)

Committee: Brian Ahmer (Advisor); Chad Rappleye (Committee Member); Sarah Short (Committee Member); John Gunn (Committee Member) Subjects: Microbiology

Master of Science, The Ohio State University, 1961, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1970, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1969, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 2006, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1968, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 2007, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1961, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 2006, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1969, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1971, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2024, Animal Sciences

Salmonella, a foodborne zoonotic pathogen, is the leading cause of death associated with foodborne illness in the USA. Salmonellosis is primarily transmitted through the consumption of contaminated poultry meat and eggs. The irrational use of antibiotics to control Salmonella infection in poultry has led to the emergence of multidrug-resistant (MDR) strains and its horizontal transmission to humans. Additionally, vaccines used to prevent Salmonella infection do not provide cross-protection to heterologous serotypes of Salmonella. Therefore, there is an urgency of the development of novel antimicrobials to control Salmonella infection. Studies have shown that small specialty crop farms (SSCFs), which practices mixed farming, are reservoirs of MDR Salmonella. Regular monitoring is needed to reduce the risk of MDR Salmonella transmission from SSCFs to humans. To this end, we isolated Salmonella from 29 of 1174 environmental samples such as dairy and poultry manure, soil, and water of SSCFs. From 29 positive samples, 80 isolates were subjected to whole genome sequencing (WGS) to determine the serotypes, antimicrobial resistance genes, virulence genes, plasmids to understand the potential risk of its transmission. Our results detected the presence of ACSSuT cassette in six Salmonella isolates, which confers resistance to ampicillin, chloramphenicol, sulfonamide, streptomycin, and tetracycline, demonstrating the urgency to develop the novel antimicrobials to combat MDR Salmonella. Therefore, we assessed the activity of novel antibiotics alternatives such as probiotics and anti-microbial peptides (AMPs) against Salmonella. The anti-Salmonella activity of probiotics and AMPs characterized in our study could lay foundation as novel antimicrobials against Salmonella. In silico serotyping of Salmonella isolated from SSCFs identified 15 serotypes of Salmonella. Eight out of 15 serotypes reported in our study are among the top 20 serotypes frequently reported in human salmone (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor) Subjects: Biology

Master of Science, The Ohio State University, 2024, Animal Sciences

Salmonellosis continues to be one of the major public health concerns worldwide causing a gastrointestinal disease. Poultry meat and eggs are recognised as one of the major sources of Salmonella food poisoning in humans. Our study evaluated the protective efficacy of mannose-conjugated chitosan-nanoparticle (mChitosan-NP) based oral subunit vaccine consisting of outer membrane proteins and flagella of Salmonella Enteritidis against Salmonella colonization in the intestines of broilers by incorporating two known mucosal adjuvants, c-di-GMP (stimulator of interferon gene agonist) and whole cell lysate (WCL) of Mycobacterium smegmatis. We try to identify the optimal dose of c-di-GMP and WCL adjuvants by using three different amounts (2.5µg, 10µg and 50µg/dose) in vivo to potentiate the efficacy of Salmonella subunit vaccine formulation. In vitro analysis revealed that mChitosan-NP Salmonella vaccine and mChitosan-NP adjuvant formulations were carrying high positive charge (Zeta potential +20-25mV), size 235-260nm, and polydispersity index 0.35-0.52, conducive for in vivo studies. Subsequently, the vaccine-adjuvant formulations were evaluated for efficacy in vivo in broiler chickens by challenging with Salmonella Enteritidis. Our data showed that mChitosan (OMP+FLA)/FLA-NP WCL 10µg/dose formulation reduced over 0.5 log10 reduction of challenge bacterial load comparable to a commercial live vaccine at both day post challenge 4 and 10. The systemic and mucosal antibody responses were found superior in adjuvanted mChitosan-NP Salmonella vaccine groups. Additionally, most of the vaccine groups had an increased frequency of B cells compared to mock group at day post-challenge 4, associated with upregulation of TGF-β mRNA at day post-challenge 10. Overall, mChitosan (OMP+FLA)/FLA-NP WCL 10 µg/dose and mChitosan (OMP+FLA)/FLA-NP GMP 50µg/dose performed well in inducing immune responses and efficacy.

Committee: Renukaradhya Gourapura (Advisor); Liesa Bielke (Committee Member); Gireesh Rajashekara (Committee Member) Subjects: Animal Sciences

Master of Science, The Ohio State University, 2023, Human Ecology: Human Nutrition

Safe and nutritious fresh produce is crucial for public health, and hydroponic farming systems play a vital role in sustainable crop production. However, these farming systems also present a favorable environment for the growth of human pathogens, leading to foodborne infections. The objective of this study was to assess the impact of chemical sanitizers on quality (yield, chlorophyll, and carotenoid) of lettuce and basil grown in hydroponic Nutrient Film Technique (NFT) systems. Additionally, the effectiveness of pHmanipulation of nutrient solution and SaniDate 12.0 (200 ppm) in eliminating human pathogen Salmonella Typhimurium and Listeria monocytogenes and their impact on lettuce quality and nutritional attributes (lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin and chlorophyll a) were evaluated. Using a randomized complete block design, various sanitizer treatments were applied to nutrient solution of hydroponically grown lettuce and basil. The crops were monitored for five weeks, and their yield and nutritional composition were assessed. Results showed a detrimental effect of all sanitizer treatments on both lettuce and basil, compared to the nontreated control (p<0.01). However, lettuce and basil treated with 200ppm SaniDate 12.0 showed higher yields than those treated with chlorine sanitizers (p<0.01). An adverse effect was observed for lettuce and basil exposed to 200ppm Na-hypochlorite (lettuce; p=0.041and basil; p=0.027). In terms of human pathogen elimination, none of the treatments (including pH modification and 200ppm SaniDate 12.0) successfully eliminated Salmonella Typhimurium and Listeria monocytogenes from the NFT system. The highest reduction in Salmonella Typhimurium was achieved by SaniDate 12.0 (p<0.001), followed by pH=4(p=0.002), and finally pH=5 (p=1.00) as compared to non-treated control and for Listeria monocytogenes, the highest reduction in pathogen was SaniDate 12.0 (p<0.001), followed by pH=5 (p=0.04), as compared to non-treat (open full item for complete abstract)

Committee: Sanja Ilic (Advisor); Melanie L.L Ivey (Committee Member); Rachel Kopec (Committee Member) Subjects: Agriculture; Education; Food Science; Microbiology

Doctor of Philosophy, The Ohio State University, 2023, Chemistry

Actin is a 42 kDa ATPase that polymerizes into filaments, which are brought together into larger structures by actin-binding proteins. Actin and its partners comprise the actin cytoskeleton, which is ubiquitous in nature and is required for cell processes such as migration, division, and endocytosis. Bacteria have evolved their own set of actin-binding proteins, that they use to reorganize a eukaryotic host's cytoskeleton for their own ends. In this work, we explore the mechanisms of actin interaction by the bacterial protein Salmonella invasion protein A (SipA), and the human plastin proteins PLS2 and PLS3. SipA is a toxin expressed by Salmonella enterica serovar Typhimurium (Salmonella), which is delivered into host intestinal cells during infection. SipA is essential for Salmonella to invade host cells in the epithelium, where it becomes engulfed into a specialized compartment called the Salmonella-containing vacuole (SCV). The C-terminal half of SipA (SipA-C) has potent actin-binding ability and was previously shown to protect actin filaments from depolymerization by host proteins. We solved a high-resolution cryo-EM structure of SipA-C bound to F-actin and characterized their interaction. We found that SipA-C binds actin with a Kd of 26 pM, protects filaments from severing and depolymerization by human actin depolymerizing factor (ADF) at low mole ratios to actin, increases filament stiffness, and increases its thermal stability. Each of these behaviors are caused by the unique binding mechanism of SipA-C's Arm2 domain which penetrates the interprotofilament space of F-actin, a binding mode previously only observed with small peptide toxins. The cell regulates pH during migration. The actin-binding proteins ADF and talin are known to be sensitive to pH. These proteins enhance the turnover of focal adhesions during cell migration at alkaline pH, generated by the membrane-bound Na+/H+ exchanger NHE1. We found that human plastin 2 (PLS2) is also sensitive to (open full item for complete abstract)

Committee: Dmitri Kudryashov (Advisor); Samir Ghadiali (Committee Member); Tom Magliery (Committee Member); Venkat Gopalan (Committee Member) Subjects: Biochemistry