Doctor of Philosophy (Ph.D.), Bowling Green State University, 2024, Biological Sciences

Due to the drastic climatic changes, the fresh market production of vegetables like spinach, lettuce, and arugula is shifting to hydroponic greenhouse operations. In hydroponic systems, the Pythium species is a problematic plant pathogen and can be introduced on airborne dust particles from neighboring farm fields. It causes root rot that results in stunting or yellowing of leaves. Hydroponically farmed vegetables are mostly eaten raw, so chemical control is not an option for controlling Pythium infections. In this study, I sought to investigate microbial biocontrol as a safer way to manage this challenge. To identify bacterial antagonists of Pythium, I surveyed a collection of 192 pseudomonads from a Lake Erie diatom bloom, also known to contain oomycetes, and 96 bacterial strains from soils around Wood County. Using a high throughput competitive plate assay, I have identified and sequenced nine strains of Pseudomonas fluorescens that exhibit contact-dependent killing of Pythium dissotocum (A1, A2, SP3, SP2), P. oopapillum, P. ultimum, P. heterothalicum, Saprolegnia parasitica, and several other yet to be identified Pythium isolates from commercial greenhouses around the US [TBL (isolate from butter lettuce), TLC (isolate from leaf lettuce), TA (isolate from arugula), E1 and CAL] at a lowest concentration of 4000 CFUs/ml. To address the utility of LE6_D7 as a biopesticide in a complex hydroponic mixture, 50 ml of bacterial (LE6_D7) culture (OD600 2.5) was added to a 5 L tub of contaminated nutrient solution from our experimental hydroponic system and incubated for 24 hours. Aliquots of the experimental nutrient solution treated with LE6_D7 post 24 hours were filtered and the filters were grown overnight on antibiotic V8 agar plates. LE6_D7 kills over 90% of the Pythium propagules in a complex hydroponic solution containing algae, other bacteria, and Pythium. Bioinformatic analysis of the P. fluorescens (LE6_D7) sequence that was used in mutation experiments, indica (open full item for complete abstract)

Committee: Paul Morris Ph.D. (Committee Chair); Julia Halo Ph.D. (Committee Member); Vipaporn Phuntumart Ph.D. (Committee Member); Christopher Ward Ph.D. (Committee Member); Deborah Wooldridge Ph.D. (Other) Subjects: Bioinformatics; Biology; Microbiology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Biology and Microbiology

Antimicrobial resistance is one of the greatest threats to human health. The β-Lactams (including penicillins, cephalosporins, carbapenems, and monobactams) are safe, effective, widely-used antibiotics. Mimicking an essential peptide bond and inhibiting enzymes involved in peptidoglycan biosynthesis, β-lactams disrupt the cell wall, killing susceptible organisms. Bacteria have developed resistance mechanisms to β-lactams, of which β-lactamases are the most prevalent and problematic. β-Lactamases hydrolyze the namesake, four-membered, cyclic amide β-lactam ring essential to the activity of β-lactam antibiotics, rendering them ineffective. Unfortunately, β-lactamases are constantly evolving in clinics and the environment and substrate expansion means fewer antibiotics are available to treat a given infection. Pseudomonas aeruginosa is a commonly multi-drug resistant (MDR) pathogen and Pseudomonas-derived cephalosporinase (PDC) is a chromosomal β-lactamase and major resistance determinant in the species. Herein, we take a comprehensive and multidisciplinary approach to understanding and overcoming PDC-mediated antibiotic resistance. We begin by proposing a standardized numbering scheme for class C β-lactamases, seeking eliminate the confusion that commonly arises when different research groups refer to the same amino acids using different designations. Next, we microbiologically and biochemically characterize and elucidate the mechanism of two PDC variants associated with oxyimino-cephalosporin resistance: a tyrosine to histidine substitution at amino acid 221 (associated with ceftolozane-tazobactam and ceftazidime-avibactam resistance) and the deletion of threonine 289 and proline 290 (associated with cefepime resistance). These variants modify the Ω-loop and R2-loop (regions bounding opposite sides of the active site and playing a crucial role in substrate specificity), respectively. Both variants lead to substrate expansion through a kcat or k3 driven mechanism ass (open full item for complete abstract)

Committee: Robert Bonomo (Advisor); Focco van den Akker (Committee Member); Krisztina Papp-Wallace (Committee Member); W. Henry Boom (Committee Chair) Subjects: Biochemistry; Bioinformatics; Microbiology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2021, Biological Sciences

Multidrug resistant (MDR) bacterial infections cause an estimated 700,000 deaths per year worldwide. By the year 2050, it is predicted that MDR infections will account for 10 million deaths and cost 100 trillion dollars per year, becoming the leading cause of death surpassing cancer. Additionally, no major pharmaceutical companies are pursuing research on novel antibiotics, creating a global crisis in which pathogens have evolved resistance to most drugs and very few antibiotics are being discovered. We hypothesize that environmental Pseudomonas isolates that inhibit MDR pathogens produce novel antibiotic compounds with different bacterial targets involving growth and survivability. To test this hypothesis, 384 strains were isolated from the Maumee River in Northwest Ohio and were competed against MDR Pseudomonas aeruginosa and Burkholderia spp. using the Burkholder plate assays. Of 384 environmental isolates, 232 inhibited the growth of the 28 pathogens tested. To identify genes involved in antagonistic activity, we optimized transposon mutagenesis to identify loss of inhibition (LOI) mutants. Following three large-scale mutant hunts, a total of 34 LOI mutants were identified from environmental isolates RC3H12, RC4D1, and RC2C2. Arbitrary PCR and sequencing of the LOI mutants and whole genome sequencing of the wild-type strain were used to identify the biosynthetic gene clusters (BGCs) involved in antibiotic production. Using antiSMASH and JGI/IMG, the potential products of the identified BGCs were determined which include a predicted lipopeptide and sideromycin. The potential antibiotic compounds identified in this research can be further characterized using biochemical testing and bioinformatics which will help to advance the development of novel drugs for use against MDR P. aeruginosa and Burkholderia infections.

Committee: Hans Wildschutte Ph.D. (Advisor); Tim Davis Ph.D. (Committee Member); Vipa Phuntumart Ph.D. (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

The CDC predicts that 10 million deaths will occur worldwide by 2050 due to multi-drug resistant (MDR) bacterial infections. As microbial resistance increases, the number of new antibiotics coming to market, and the number of pharmaceutical companies that discover them, continues to decrease. Furthermore, recent data from the Wildschutte Lab suggests that there are few natural remaining antibiotics to be discovered from environmental bacteria that are effective against MDR pathogens, necessitating the use of alternative treatments. Bacteriophage therapy has been successfully used to treat infections for over 100 years in countries other than the United States and represents an alternative strategy for treating MDR pathogens. While phage therapy is a promising approach, like antibiotic regimes, it comes with drawbacks. For instance, phage are generally host specific, infecting only one or a few highly similar strains, and bacteria can rapidly evolve resistance to specific phage. Here, we identify a set of phage capable of infecting a broad host range (BHR) of MDR pathogens isolated from cystic fibrosis patients, with the potential for use in phage therapy. We isolated multiple phage and characterized them using restriction digests, host ranges assays, and imaging; as a prelude to whole genome sequencing. To further support our broad host range data, we sequenced both the16S rRNA and gyrB genes of the MDR Pseudomonas aeruginosa and Burkholderia spp. hosts to verify the diversity of the pathogens susceptible to these phage. Altogether, these results support that the isolation of at least 14 unique BHR phage capable of inhibiting multiple MDR pathogens. We have also begun to investigate the synergistic relationships between phage and antibiotics that may be exploited and used in tandem to treat pathogenic infections. While this work is still in its infancy, the results support the contention that phage therapy represents a valuable tool to combat the global antibiotic (open full item for complete abstract)

Committee: Hans Wildschutte Ph.D (Advisor); George Bullerjahn Ph.D (Committee Member); Raymond Larsen Ph.D (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2019, Biological Sciences

The research described here focuses on the phylogenetic characterization of water-derived pseudomonads and their antagonistic activity against multi-drug resistance (MDR) P. aeruginosa and Burkholderia species. Phylogenetic work was based on the gyrB housekeeping gene. Genetic techniques have been optimized and employed to identify genes associated with antimicrobial production via transposon (Tn) mutagenesis using a triparental mating system approach with Pseudomonas as the model organism. This study expands on theses previous studies in the lab to identify biosynthetic gene clusters (BGC) involved in production of novel antibiotics capable of inhibiting the growth of MDR pathogens. We utilize a previously optimized workflow to identify genes from environmental isolates involved in the inhibition of MDR P. aeruginosa and species within the Burkholderia cepacia complex (Bcc). We show that both MDR Bcc and P. aeruginosa pathogens were inhibit by environmental Pseudomonas strains. Out of 7,784 interactions, 210 of these were antagonistic. Superkillers (SK), defined as strains that inhibit ≥3 of MDR pathogens, were selected for optimization of Tn mutagenesis to identify gene cluster whose products inhibit these MDR pathogens. Only six out of the 24 SK's were capable of this process. Out of these six, three were selected for large scale mutagenesis to identify loss of inhibition (LOI) mutants. Four LOI mutations were found for strain S5F11, one of which had an insertion within a BGC predicted to produce an NRPS complex. Seven LOI mutants were found for S3E7. Although none of these insertions were identified within a BGC, genes have been identified that are observed to be heavily involved in antibiotic production. This study suggests that environmental Pseudomonas strains have the capacity to inhibit the growth of CF-derived MDR pathogens. Using Tn mutagenesis, we have identified novel loci that are associated with antibiotic production.

Committee: Hans Wildschutte PhD (Advisor); Timothy Davis PhD (Committee Member); Robert Huber PhD (Committee Member) Subjects: Biology

Master of Science (MS), Bowling Green State University, 2017, Biological Sciences

Antibiotic resistance has become a crisis of global proportions. People all over the world are dying from multidrug resistant infections, and it is predicted that bacterial infections will once again become the leading cause of death. One human opportunistic pathogen of great concern is Pseudomonas aeruginosa. P. aeruginosa is the most abundant pathogen in cystic fibrosis (CF) patients' lungs over time and is resistant to most currently used antibiotics. Chronic infection of the CF lung is the main cause of morbidity and mortality in CF patients. With the rise of multidrug resistant bacteria and lack of novel antibiotics, treatment for CF patients will become more problematic. Escalating the problem is a lack of research from pharmaceutical companies due to low profitability, resulting in a large void in the discovery and development of antibiotics. Thus, research labs within academia have played an important role in the discovery of novel compounds. Environmental bacteria are known to naturally produce secondary metabolites, some of which outcompete surrounding bacteria for resources. We hypothesized that environmental Pseudomonas from diverse soil and water habitats produce secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa. To address this hypothesis, we used a population based study in tandem with transposon mutagenesis and bioinformatics to identify eight biosynthetic gene clusters (BGCs) from four different environmental Pseudomonas strains, S4G9, LE6C9, LE5C2 and S3E10. Of the eight BGCs identified, seven had putative products of non-ribosomal peptide synthetases and one had a putative product of a phenazine. All compounds appeared to be diverse and potentially novel, but further biochemical research must be done to verify these findings. Overall, we were able to identify genes that encode secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa as well as other human pathogens. This research has creat (open full item for complete abstract)

Committee: Hans Wildschutte (Advisor); Ray Larsen (Committee Member); Jill Zeilstra-Ryalls (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Master of Science, The Ohio State University, 2008, Plant Pathology

The search for alternatives to chemical pesticides in agriculture raises the need to study substitutes as biocontrol for plant disease management. Rhizobacteria may present a front line of defense for plants against pathogens against biotic and abiotic stresses. A SYBR-green real time quantitative PCR protocol for the detection and quantification phlD+ pseudomonads in the soil with a detection limit of log4 cells per gram of root was developed. Pseudomonas fluorescens Wood1R decreased acid-soil stress related symptoms in corn plants perhaps through alterations in foliar nutrient concentration. In a different study, Pseudomonas fluorescens genotypes A1, A2 and S2 reduced tomato bacterial spot. Finally, in vitro screening of growth inhibition against a broad spectrum of pathogens demonstrated the potential of eight Bacillus spp. isolates to reduce tomato diseases. This data suggest that more research on biocontrol can result in novel traits and isolates that can ultimately be integrated in plant disease management.

Committee: Brian B. McSpadden Gardener PhD (Advisor); Michael J. Boehm PhD (Committee Member); Anne E. Dorrance PhD (Committee Member); Laurence V. Madden PhD (Committee Member) Subjects: Agriculture; Plant Pathology

Master of Science in Engineering, University of Akron, 2009, Chemical Engineering

Microbial biofilm formation on surfaces leads to significant losses in terms of energy,equipment damage, product contamination, medical infections, etc. and also raises serious environmental concerns. To prevent formation of biofilms, a detailed investigation into the initial stage of bacterial attachment was conducted using three types of bacterial species; Pseudomonas aeruginosa PAO1, Escherichia coli and Pseudomonas putida. Several approaches were evaluated and finally a reproducible procedure was adopted to study initial bacterial attachment. The procedure primarily involved monitoring and counting the number of attached cells on the glass walls of the flow chambers, through which a bacterial suspension was circulated and, subsequently, saline was passed for washing to remove loosely attached cells. Preliminary investigations in terms of fluid flow behaviors, consistency check, and the effect of various factors including the attachment locations within the chamber, on the attachment results were conducted prior to employing the flow chamber systems to study the initial bacterial attachment. Using the flow chambers systems, the effects of chamber wall shear, medium pH, chamber, and two potential antifoulants, Zosteric acid and Rhamnolipids, on attachments were examined. The three bacterial species used showed similar behavior (peaking at different shear stress values) in response to varying shear conditions in their environment. The antifouling properties of zosteric acid were not convincing enough, however promising results were seen while studying rhamnolipids in the case of all three bacterial strains. The effect of other factors like pH, presence of zosteric acid impurities, surface conditioning were also explored. These conditions however proved to have no effect on the initial bacterial attachment but provided useful information for future studies.

Committee: Bi-min Zhang Newby PhD (Advisor) Subjects: Chemical Engineering; Environmental Science

Doctor of Philosophy, University of Akron, 2005, Chemical Engineering

Pseudomonas aeruginosa is ubiquitous in the nature and is one of the most commonly found microorganisms in petroleum-contaminated environments. With versatile metabolic activities, it can be used to produce various industrial and pharmaceutical products. P. aeruginosa is also a clinically important, opportunistic pathogen that causes a variety of infections, particularly in patients with severe burns, cancer, AIDS and cystic fibrosis. An important metabolic trait that supports the efficient adaptation of P. aeruginosa to this wide range of environments is its ability of active denitrification. The bacterium's properties and respiratory behaviors under different growth rates and dissolved oxygen concentrations (DO) were therefore systematically studied in this research. Continuous cultures of P. aeruginosa (ATCC 9027) were maintained at different DO (0-4.8 mg/L) and dilution rates (D, 0.01, 0.026, 0.06, and 0.13 h-1). Aerobic denitrification was found to function as an electron-accepting mechanism supplementary, instead of competitive, to aerobic respiration. The experimental results suggested that the zero-DO conditions were more favorable for survival of the bacterium. A closer examination revealed that increasing DO enhanced O2 respiration only at extremely low DO (< 0.05 mg/L), beyond which the increasing DO only slightly increased its weak inhibition on denitrification. While O2 was the preferred electron acceptor, the fraction of electrons accepted (and the ATP generated) via denitrification increased with increasing D. Unlike glucose, when hexadecane was used as the sole carbon source, there was a critical DO (0.4 mg/L in this study), below which the system could not reach the steady state. Phosphate concentration appeared to be also very important to the behaviors of culture growing on the hexadecane-based media. Furthermore, intriguing metabolic fluctuations were observed during the transition from non-aerated batch culture to aerated continuously-fed cultur (open full item for complete abstract)

Committee: Lu-Kwang Ju (Advisor); Steven Chuang (Other); Teresa Cutright (Other); Amy Milsted (Other); Ping Wang (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

My research focuses on the diverse roles of programmed cell death (PCD) in host innate immune responses. My dissertation explores how PCD assists host antimicrobial defense, as well as contributes to the progression of sepsis-induced immunosuppression. The dissertation could be divided into two main sections and one derivative section. Firstly, I have identified a novel interaction between Pseudomonas aeruginosa and host necroptosis during in vitro and in vivo infection. Secondly, I have characterized the impact of a novel PCD regulator, NINJ1, in improving sepsis-induced immunosuppression by partially restoring the host defense to secondary infections. Quorum sensing (QS), a communication system evolved by Pseudomonas aeruginosa to monitor its density, is well-acknowledged to be involved in multiple activities during bacterial infection. Recent studies have revealed clues about link between Pseudomonas aeruginosa QS and host programed cell death. However, it remains limited understanding whether QS plays a role in host PCD process during the infection. In this study, I used rhl mutants of Pseudomonas aeruginosa to in vitro challenge multiple genetic knockout macrophages to explore the connection between QS and programmed cell death. According to the data from cell death assays and immunoblotting, I discovered these rhl mutants significantly promoted necroptosis which was unknown in this field. Additionally, I found that the increased necroptosis activation was caused by the upregulation of another QS subsystem, pqs, because the deletion of pqs in rhl-deficient Pseudomonas aeruginosa abolished macrophage necroptosis in vitro and in vivo. Therefore, this study revealed a novel rhl-pqs-necroptosis pathway. Sepsis is characterized by two dynamic stages occur during the initiation and progression, which are system inflammatory response syndrome (SIRS) in the acute phase and compensatory anti-inflammatory response syndrome (CARS) in the later phase. Recent study revea (open full item for complete abstract)

Committee: Haitao Wen (Advisor); Patrick Collins (Committee Member); Amal Amer (Committee Member); Daniel Wozniak (Committee Member) Subjects: Immunology

Master of Science (MS), Bowling Green State University, 2024, Biological Sciences

The overuse and misuse of antibiotics has led to a global crisis in which most bacterial pathogens have evolved resistance to the available drugs used for treatments. As a result, antibiotic resistant bacterial infections are expected to cause 10 million deaths per year by 2050, passing cancer as the leading cause of mortality. Complicating the issue is that large pharmaceutical companies have stopped antibiotic discovery and development due to marginal profits, so very few new drugs are available for life-threatening infections. From previous studies in our lab, we have shown that environmental isolates of Pseudomonas strains can inhibit cystic fibrosis (CF) derived clinical pathogens, including Pseudomonas aeruginosa and members of Achromobacter and Stenotrophomonas species. This study is part of a larger project that aims to determine if certain water habitats select for antibiotic producing bacteria. Here, 271 environmental Pseudomonas strains were isolated from Lake Superior and tested for their ability to inhibit a CF-patient derived panel of 25 multidrug-resistant pathogens, including nine Burkholderia species, nine P. aeruginosa, and seven Staphylococcus aureus; also tested were five strains of Aeromonas fish pathogens. Using an antagonistic plate assay, 8,130 individual competition assays were performed, and 515 instances of competitive inhibition were observed among the pathogens. From these events, 59 were against P. aeruginosa, 66 were against Burkholderia, 273 were against S. aureus, and 53 were against Aeromonas. To identify genes involved in antagonistic activity, transposon mutagenesis was performed with environmental antagonistic Pseudomonas strains TL1E8 and TL2A8 and loss of killing mutants were identified. TL1E8 was found to inhibit strains of Pseudomonas aeruginosa, Burkholderia, and Staphylococcus aureus. Transposon mutated genes identified included sulfite reductase flavoprotein, methyl-accepting chemotaxis protein, and diguanylate cyclase-lik (open full item for complete abstract)

Committee: Hans Wildschutte Ph.D. (Committee Chair); Raymond Larsen Ph.D. (Other); Daniel Wiegmann Ph.D. (Other) Subjects: Biology; Microbiology

MS, University of Cincinnati, 2024, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Abstract Pseudomonas aeruginosa (PA) is a gram-negative bacterium and an opportunistic nosocomial pathogen associated with significant morbidity and mortality in infected individuals. PA commonly infects those with cystic fibrosis, burn wounds, cancer, and severe infections requiring ventilation, including COVID-19. The minimal nutritional requirements and wide range of physical conditions tolerated by PA allow its successful invasion in hospital environments. Because of this, and its developing multidrug resistance, it has become increasingly important to understand the virulence factors of PA. The expression of the virulence factors KatB and AnkB are highly upregulated in response to oxidative stress, e.g. respiratory burst mediated by phagocytes during PA infection. KatB is a catalase that uses the cofactor heme to convert H2O2 into H2O and O2, and AnkB is a putative ankyrin repeat protein of unknown function. Importantly, previous studies have demonstrated that AnkB is required for the catalytic activity of KatB; however, the mechanism by which KatB becomes active via AnkB is largely unknown. Prior work in the Kovall and Hasset labs has identified a possible mechanism, in which AnkB functions as a novel heme binding protein that is required to load heme into KatB, resulting in the catalytically active KatB tetramer. To test this, we produced an AlphaFold3 model of the AnkB-heme complex and designed several structure-based AnkB point mutants that potentially disrupt the AnkB-heme binding interactions. In an AnkB null background, PA expressing wild-type or mutant AnkB constructs were subjected to a variety of assays to assess their effect on KatB activity. In addition, we purified recombinant AnkB (wild-type and mutants) and used an ELISA assay to characterize AnkB-heme complex formation, as well as screened crystallization conditions for AnkB-heme. Altogether, our results suggest that the residues Y75A, R98A, F108A, and F142A of AnkB are highly significant (open full item for complete abstract)

Committee: Rhett Kovall Ph.D. (Committee Chair); Xiaowei Hou Ph.D. (Committee Member); Paul Rosevear Ph.D. (Committee Member) Subjects: Microbiology

Master of Science, The Ohio State University, 2024, Immunology and Microbial Pathogenesis

Nontuberculous mycobacteria (NTM) are an increasingly common cause of respiratory infection in people with cystic fibrosis (PwCF). Relative to those with CF and no history of NTM infection (CF-NTMNEG), PwCF and a history of NTM infection (CF-NTMPOS) are more likely to develop severe lung disease, experience complications over the course of treatment and are precluded from lung transplant eligibility. We recently reported lung lobe-specific immune profiles of 3 cohorts (CF-NTMNEG, CF-NTMPOS and non-CF adults) and found that the CF-NTMPOS airways are distinguished by accumulations of B cells produced. Confirming the presence of antibodies, in the bronchioalveolar fluid (BAL), would prove a mechanistic connection between B cell abundance and lung tissue damage. This would be due to an overreactive antibody response or the abundance of antibodies being able to exacerbate pulmonary damage. This is caused by the release of cytokines and other inflammatory signals to recruit more immune cells to the site. We anticipate these findings will lead to the development of a clinical diagnosis of NTM infection and the creation of host-directed therapies to improve NTM disease treatment in PwCF. We hypothesize that the presence of total antibodies in BAL for these three cohorts will provide a mechanistic connection to the exacerbation of lung tissue damage in PwCF and a concurrent NTM infection.

Committee: Richard Robinson PhD (Advisor); Emily Hemann PhD (Committee Member); Adriana Forerro PhD (Committee Member); Mark Drew PhD (Advisor) Subjects: Health Care; Immunology; Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2023, Plant Pathology

Plants exist in a constantly evolving microbial environment that significantly influences their growth, development, and overall well-being. Within this microbial milieu, certain bacteria play a pivotal role in enhancing plant health and growth. These beneficial bacteria are collectively referred to as plant growth-promoting bacteria (PGPB). They offer valuable services to plants, including improved nutrient absorption, heightened growth stimulation, and increased resilience against pathogens and the other environmental adversities. PGPB engage with plants through diverse modes of interaction, such as root colonization, endophytic association, or rhizosphere competence. An in-depth comprehension of the molecular mechanisms and ecological dynamics governing these interactions is essential for unlocking the potential of PGPB in promoting sustainable agriculture and environmental remediation. In Chapter 1, I provide an overview of current methods used to detect and diagnose Pseudomonas syringae. This encompasses traditional approaches like culture isolation and microscopy, as well as modern techniques such as PCR and ELISA. Furthermore, I explore the upcoming advancements in this domain, emphasizing the necessity for highly sensitive and specific methods to detect pathogens even at low concentrations. Additionally, I delve into approaches for diagnosing P. syringae infections when they coexist with other pathogens. Chapter 1 Figures can be found in Appendix A. In Chapter 2, I present a significant protocol for monitoring the progression of gray mold fungal infection at various developmental stages of strawberries. I detail three distinct in vivo inoculation methods for Botrytis cinerea on strawberry plants, focusing on early, middle, and late stages of strawberry growth. Chapter 2 Figures can be found in Appendix B. In Chapter 3, I introduce Bacillus proteolyticus OSUB18 as a novel inducer of ISR (Induced Systemic Resistance). This bacterium enhances plants' r (open full item for complete abstract)

Committee: Ye Xia (Advisor); Christopher Taylor (Committee Member); Yu (Gary) Gao (Committee Member); Lisa (Beck) Burris (Committee Member); Jonathan Jacobs (Committee Member) Subjects: Agriculture; Agronomy; Biochemistry; Bioinformatics; Biology; Botany; Cellular Biology; Plant Biology; Plant Pathology; Plant Sciences

Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Pythium pathogens that can cause disease on leafy greens are well-adapted to hydroponic production systems due to their ability to produce swimming zoospores. These spores that rapidly disperse in the recirculating water. Current strategies to reduce disease losses primarily involve hygienic practices and UV sterilization of water when infections become problematic. Prior work had demonstrated that the Pseudomonas fluorescens isolate LE6_D7 was capable of contact-dependent killing of Pythium isolates from hydroponic greenhouse operations from California, Indiana, Ohio, and New Jersey. Whole genome sequencing revealed that this isolate possesses two complete Type Six Secretion Systems (T6SS). In this study, the focus was on proteins associated with T6SS, due to their role in delivering proteins into the oomycete cytoplasm, providing the simplest explanation of the contact-dependent phenotype. Bioinformatic analysis identified hcp_05421 as one of four genes in this strain that produced the nanotube of T6SSs. To determine the role of this gene in virulence against Pythium isolates, a DNA fragment of a truncated version of the target gene was synthesized through PCR, including upstream and downstream flanking regions. This DNA fragment was then ligated into the vector DONR1K18ms using a Gateway™ BP clonase reaction. The vector was introduced into LE6_D7 strain either by conjugation using the E. coli helper strain RHO5, or by electroporation in 10% glycerol. Among the ten vgrg1 type genes present in this P. fluorescens strain, the largest one (vgrg_00081) was selected for characterization using a similar approach. Insertional mutants resulting from a single crossover event after the integration of the plasmid into the genome were selected on antibiotic media. Insertional mutants of both Δhcp_05421 and Δvgrg_00081 genes resulted in the loss of contact-dependent killing in two tested strains of Pythium. Double crossover mutants of Δhcp_05421 and Δvgrg_00081 gene deletions (open full item for complete abstract)

Committee: Paul Morris Ph.D. (Committee Chair); Zhaohui Xu Ph.D. (Committee Member); Vipaporn Phuntumart Ph.D. (Committee Member) Subjects: Genetics; Molecular Biology

Doctor of Philosophy (Ph.D.), University of Dayton, 2023, Biology

Antibiotic resistance has been declared a global concern by the World Health Organization and is increasing the rate of mortality of once-treatable, common infections. Antibiotic resistance is conferred by multiple mechanisms both intrinsic (horizontal gene transfer) and extrinsic (production of biofilms). The eradication of biofilms produced by bacterial colonization remains a serious threat to human infections. Bacterial biofilms produce an extracellular matrix composed of proteins, polysaccharides, and extracellular DNA (eDNA). This matrix acts as a scaffold for growth and imparts a form of protection against predators, harsh conditions, and chemicals (e.g., bacteriophage, pH, and antibiotics). The biofilm-associated cells of Pseudomonas aeruginosa (PsA) are up to 1000-fold more resistant to antibiotics than planktonic cells. Additionally, PsA has been linked to many infections that can be mortally dangerous for individuals with compromised immune systems such as Cystic Fibrosis (CF). PsA colonization in individuals with CF causes a decreased quality of life. Thus, there is a search for alternative strategies for antimicrobial management. Our lab has produced a patented zinc-containing porphyrin, Zn(II)meso-5,10,15-triyl-tris(1-methylpyridin-1-ium)-20-(pentafluorophenyl) porphine tritosylate (ZnPor), which exhibits broad antibacterial activity against planktonic and biofilm-associated cells of PsA. ZnPor presents itself as a unique possible surrogate for traditional antibiotics by its interaction with eDNA of biofilms. ZnPor intercalates between base pairs and binds to the outside of the helix, resulting in a more porous biofilm that dissembles and detaches from substrata. Furthermore, ZnPor has potent photoactivity that increases both its bactericidal and viricidal properties when exposed to light. The ability to disrupt the inherent matrix structure makes biofilm-associated cells more accessible to other treatments such as antibiotics and bacteriophage (open full item for complete abstract)

Committee: Jayne Robinson (Committee Chair); Karolyn Hansen (Committee Member); Shawn Swavey (Committee Member); Madhuri Kango-Singh (Committee Chair); Kristen Krupa (Committee Chair) Subjects: Biology; Molecular Biology; Virology

Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Bacterial pathogens are evolving resistance to all available antibiotics and are a leading cause of death worldwide. Thus, the discovery of new antibiotics that inhibit multi-drug resistant (MDR) pathogens is urgent. We hypothesize that environmental Pseudomonas strains that can inhibit the growth of MDR pathogens may be source of novel antibiotics that may have new or multiple mechanisms of action. We isolated 288 environmental Pseudomonas strains from the Portage River in northwest Ohio which represents a polluted water source. Pseudomonas strains were isolated and phylogenetically characterized using the gyrB housekeeping gene. To test our hypothesis, the environmental strains were competed against a collection of human and animal MDR pathogens including nine Burkholderia species, seven Staphylococcus aureus, and nine Pseudomonas aeruginosa strains, all isolated from lungs of cystic fibrosis patients, and five Aeromonas fish pathogens. Results from the 8,640 individual antagonistic assays showed that 115 of the environmental Pseudomonas strains demonstrated the ability to inhibit at least one pathogen. This suggests that these environmental strains may produce novel compounds which effectively inhibit these MDR pathogens. We performed transposon mutagenesis and screened for loss of inhibition mutants with strains J4D8, JA1H2, J1A5 and J3E11 to determine biosynthetic gene clusters involved in antibiotics production. Whole genome sequencing and arbitrary PCR coupled with antiSMASH were used to identify specific gene clusters involved in antagonistic activity. The compounds were predicted to include pyochelin, pyoverdine, MA026, lankacin C and cepacin A which are all derived from non-ribosomal peptides. Further analysis of these compounds is required to determine their novelty.

Committee: Hans Wildschutte Ph.D. (Committee Chair); Raymond Larsen Ph.D. (Committee Member); Christopher Ward Ph.D. (Committee Member) Subjects: Biochemistry; Bioinformatics; Biology; Biomedical Research; Microbiology; Molecular Biology

PHD, Kent State University, 2022, College of Arts and Sciences / Department of Biological Sciences

Nickel (Ni) is a widespread and persistent contaminant in riverine systems that can impair biological diversity and ecological function. The bioavailability and toxicity of Ni are strongly influenced by its complexation with solid-phase ligands in sediments. Riverine sediments are often vertically stratified with thin oxic layers overlying anoxic horizons, and the distinct physicochemical conditions in these sediment layers modify their Ni binding capacity. In anoxic sediment, reduced sulfur (AVS) is the primary metal ligand, whereas iron (Fe) and manganese (Mn) oxide minerals can be important binding fractions for Ni under oxic conditions. The mixture of competing oxidized and reduced ligands in natural sediments is largely driven by physical conditions and the redox metabolism of the sediment microbial community. However, microbes are concurrently susceptible to the toxic effects of nickel, which can ultimately modify the availability of solid-phase ligands. Current sediment quality criteria consider AVS as the major binding phase for Ni but have not yet incorporated ligands that are present in oxic sediments. The overall objective of this dissertation was to improve our understanding of the role that metal oxides play in regulating Ni bioavailability to benthic organisms in natural sediments. The first study used a field-based approach to evaluate the role of metal oxides on Ni bioavailability to the benthic invertebrate community in riverine sediments exposed to effluent from two mining operations in Thompson, Manitoba, Canada. We found that oxide minerals in natural oxic sediments bind a substantial amount of Ni. Oxic sediment chemistry more strongly represented conditions experienced by benthic invertebrates and the inclusion of oxic solid-phase ligands was critical to refine predictions of Ni bioavailability and its impact on benthic community structure. The second study further investigated the geochemical drivers of Ni sorption to natural sediments across a (open full item for complete abstract)

Committee: David Costello (Advisor); Mark Kershner (Committee Member); Alison Smith (Committee Member); Christopher Blackwood (Committee Member); David Singer (Committee Member) Subjects: Freshwater Ecology

Master of Science, The Ohio State University, 2022, Comparative and Veterinary Medicine

Pseudomonas syringae pv. syringae (Pss) is an emerging seed-borne pathogen that causes Pseudomonas leaf spot (PLS) disease in bell peppers. It causes severe necrotic lesions on pepper leaves that can spread to 50-80% of the field under favorable environmental conditions. PLS can cause significant economic losses to pepper production if the disease is left uncontrolled. However, not much is known about the genes that Pss carries to be able to cause disease in peppers. It is important to understand the virulence genes that Pss carries so that appropriate measures can be developed to control Pss in peppers. Therefore, part of my research aimed to use comparative genomic analysis to understand the genes in Pss that are important for virulence in pepper seedlings. The Pss strains (n=16) evaluated showed varying levels of virulence (disease severity and Pss population) at 3-, 7-, and 14-days post-infection (dpi) on the susceptible 'California Wonder' pepper variety in a controlled growth chamber environment. The Pss strains also displayed varying growth, biofilm development, and motility in vitro in M9 minimal broth at 28˚C, however, the variation in in vitro performance did not explain the variation in the virulence of the Pss strains in pepper seedlings. Whole genome sequencing was performed on these Pss strains. The genes were functionally characterized, and core genomes were separated from the variable genomes between the Pss strains. A total of 812 genes were variable among the Pss strains including known virulence genes. Additionally, a multivariate correlation analysis identified 285 genes that were significantly correlated to the virulence of Pss in pepper seedlings (r2 of  0.5 to 0.675; P<0.01). The genes that were significantly correlated with the virulence of Pss strains included known virulence genes associated with motility (n=2), biofilm (n=5), and Type III and VI secretion systems (T3SS and T6SS) (n=9). Further, the two strains (SM156-18 and SM226-1) that (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor); Sally Miller (Committee Member); James Fuchs (Committee Member) Subjects: Biology; Microbiology; Plant Pathology

Doctor of Philosophy, University of Toledo, 2022, Medicinal Chemistry

The gram-negative bacteria, Pseudomonas aeruginosa, is an opportunistic pathogen. P. aeruginosa forms biofilms by altering gene expression, through cellular signaling, to protect the cells from environmental stressors. Our goal is to investigate the role of biomolecules and genes contributing to the formation of P. aeruginosa biofilms. Insight into the pathways associated with biofilm production can illuminate targetable methods to fight future P. aeruginosa infections. Genes encode proteins that control cellular function in biological systems. Each gene contributes to specific pathways during cellular development. To understand the role of each gene, targeted gene knockout methodologies have been developed for bacterial and eukaryotic systems. We have designed a protocol for the creation of specific gene knockouts in P. aeruginosa with the use of plasmids incorporated with the lambda-red recombinase system. We have proven the effectiveness of these methodologies with the generation of a PA14 knockout mutant. Matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) has emerged as a useful for the identification of molecules associated with microorganisms.The diversity of molecules in bacterial cultures leads to difficult analysis in whole cell samples. We have tested multiple common MALDI matrices to determine ionization preference for major molecules including rhamnolipids, phospholipids, and quinolones in P. aeruginosa. We have putatively identified nominal ions by collision induced dissociation (CID) fragmentation in all matrices tested. Biofilms are complex aggregations of cells encased in an extracellular matrix. This matrix assists in cellular signaling by small molecules in the event of environmental signals to produce a response. Different cell subpopulations are localized throughout the biofilm to respond to these environmental signals. We have identified 3 different cell subpopulations in a P. aeruginosa biofilm. We have determ (open full item for complete abstract)

Committee: Erin Prestwich (Advisor); Hermann von Grafenstein (Committee Member); Jennifer Gadient (Committee Member); James Slama (Committee Member); Katherine Wall (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Microbiology; Molecular Biology