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

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

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

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

PHD, Kent State University, 2023, College of Arts and Sciences / Department of Biological Sciences

This dissertation is focused on the direct and indirect effects of micronutrients (Fe, Mo, Mn, Ni, Zn) in combination with macronutrients on algal community composition, growth, toxin production and alkaline phosphatase activity in lakes and streams. To assess the effect of nutrients on phytoplankton and biofilm community functions, a series of macro- and micronutrient enrichment experiments have been used both in situ and within bottle incubations. Quantification of enzyme activity, community composition and metabolic processes alongside growth permits assessment of what processes are limited or driven by these nutrients. By determining the magnitude of effect macro- and micronutrient enrichment has on various aquatic primary producer community processes, this work helps to fill the micronutrient knowledge gap in aquatic ecology and expand our understanding of the underlying physiological mechanisms controlling community and ecosystem level responses.

Committee: David Costello (Advisor); Darren Bade (Committee Member); Timothy Gallagher (Committee Member); Jim Hood (Committee Member); Christie Bahlai (Committee Member) Subjects: Freshwater Ecology

Master of Science, University of Toledo, 2022, Civil Engineering

The occurrence of harmful algal blooms (HABs) in many freshwater systems has been a significant problem for surrounding cities for decades especially as potable water supply. Algaecide applications are known as one of the effective management tactics for rapidly controlling HABs in the source waters. However, excess applications have been shown to damage the bacteria cells and to release intracellular organic matter (IOM), which is difficult to be treated during the conventional water treatment processes. There are previous studies regarding algaecide applications in terms efficacy under different conditions, however limited studies on how algaecide treatments affect algal organic matter release (AOM). Four different USEPA-registered algaecides, two copper-based (SeClear® and Algimycin® PWF) and two peroxide-based (PAK® 27 and Oximycin® P5), were selected for this study based on preliminary results of commercial algaecide testing. The efficacy of algaecides and the release of AOM from the damaged cells were tested for 108 hours in a prepared monoculture of Microcystis aeruginosa. The efficacy was again tested at the same cell concentration of Microcystis aeruginosa in mixed culture at the selected doses and the exposure time from the single culture. Total chlorophyll-a (chl-a) and total phycocyanin concentrations were monitored to evaluate cyanobacterial health and dissolved organic carbon (DOC), dissolved toxin, and dissolved phycocyanin concentrations were monitored to analyze the cell damage. A Parallel Factor Analysis (PARAFAC) model was built using the collected Excitation-Emission Matrix (EEM) spectra of the samples under different algaecide treatment types and concentrations to characterize and quantify the aqueous AOM during the treatment. Three possible components were characterized: Soluble microbial by-products (SMPs)-like, tyrosine-like, and humic-like materials. This research advances the understanding of impacts of HABs management using algaecides. The (open full item for complete abstract)

Committee: Youngwoo Seo (Committee Chair); Defne Apul (Committee Member); Daewook Kang (Committee Member) Subjects: Engineering

Master of Science, University of Toledo, 2022, Civil Engineering

Globally, harmful algal blooms (HABs) have increased in frequency due to many factors like eutrophication, increased temperature, and ecosystem instability. Typically, algaecides are used to control HABs, some of which are formulations of hydrogen peroxide and copper sulfate. Unfortunately, traditional HAB control methods using algaecides have many downsides including requiring repeated applications and adverse environmental externalities. Cyanophage have been shown to be effective at suppressing cyanobacterial growth and have been suggested to be effective after algaecide application, however this idea has yet to be tested. Given the shortcomings of chemical-based algaecides, this experiment explored the synergistic impact of cyanophage Ma-LMM01 and the low dose algaecide, Pak-27. In the combined experiment low-dose Pak-27 was more effective at suppressing growth when combined with cyanophage Ma-LMM01 and application of the low-dose Pak-27 did not impact the cyanophage's ability to lyse the cells when applied before cyanophage inoculation. These results provide insight in the potential for a biological based treatment method, and the creation of a “cyanophage cocktail” that contains many different strains of cyanophage with different host preferences is an emerging research opportunity.

Committee: Youngwoo Seo (Committee Chair); Dae-Wook Kang (Committee Member); Katelyn McKindles (Committee Member) Subjects: Engineering

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

Doctor of Philosophy, The Ohio State University, 0, Biochemistry Program, Ohio State

Metabolomics provides a global analysis of metabolites from many biochemical pathways, giving an unbiased view of cellular activity. The metabolome is acted upon by endogenous factors such as genome-encoded enzymes and influenced by the environment, thus it is reflective of the phenotypic state (Chapter 1). Nuclear magnetic resonance (NMR) spectroscopy is a key tool for metabolomics measurements due its high reproducibility, quantitative nature, and ability to detect all abundant known and unknown metabolites in a single set of measurements. One of the major challenges in NMR-based metabolomics is accurate and efficient metabolite identification and quantification (Chapter 2). We have developed the COLMARq web server which facilitates semi-automated metabolite identification, quantification, and statistical analysis of cohorts of samples. COLMARq is the first openly accessible web server targeted toward quantitative metabolomics analysis of cohorts of 2D NMR spectra (Chapter 3). P. aeruginosa is a Gram-negative, opportunistic pathogen that exhibits resistance to many antibiotics leading to acute and chronic infections in immunocompromised individuals. P. aeruginosa readily forms biofilms in diverse environments, which are a self-produced gel-like matrix of extracellular polymeric substances encasing the cells. Biofilms are difficult to detect and eradicate, greatly contributing to the persistence of infection. Therefore, there is a critical need for new approaches to accurately identify, regulate, and prevent biofilm formation (Chapter 4). We used an untargeted 2D NMR- based metabolomics approach to identify statistically significant differences in 52 metabolites between P. aeruginosa grown in the planktonic and biofilm states. Among them, the metabolites of the cadaverine branch of the lysine degradation pathway were systematically decreased in biofilm. Exogenous supplementation of cadaverine caused significantly increased planktonic growth, decreased biofilm a (open full item for complete abstract)

Committee: Rafael Brüschweiler (Advisor); Christopher Jaroniec (Committee Member); Mark Foster (Committee Member); Amal Amer (Committee Member) Subjects: Biochemistry; Microbiology

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

The overarching menace of drug resistance of Cystic Fibrosis (CF) pathogens, especially by members of Burkholderia cepacia complex and P. aeruginosa strains is a matter of grave concern. However, there is a glimmer of hope in the discovery of novel therapeutics; exploitation of naturally existing competition for survival through antagonism among the microbes could provide an effective therapeutic alternative. Stress response-mediated antagonistic activity could be triggered by variety of selection pressures. In this study, stress was induced in CF pathogens in vitro by exposure to either ultraviolet (UV) irradiation or the DNA cross-linker mitomycin C. Induction by UV irradiation was far more effective in triggering the induction of genes encoding antagonistic elements. Additional experiments optimized the recovery of functional lytic elements. Production requirements, as part of custom-optimization of the method, and identification of nature of the substances have been assessed systematically by different qualitative and semi-quantitative metrics such as timed early production, clear zone of inhibition (ZOI) formation tracked by serial-fold dilution or MIC. Moreover, the lytic substances have been partially identified as non-bacteriophages but more likely as heat labile proteins having a single-hit mechanism of lytic properties similar to that of bacteriocins and tailocins. Target host range susceptibility and panel-wide screening for recovery of more putative toxins with cross-sensitivity tests was also performed. In total, two strains from Burkholderia cepacia complex panel and eight from P. aeruginosa panel have been identified as producers of such putative toxins.

Committee: Raymond Larsen (Advisor); Christopher Ward (Committee Member); Hans Wildschutte (Committee Member) Subjects: Biology; Health; Microbiology; Molecular Biology

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

Pharmaceutical companies have slowed the discovery and development of antibiotics due to low-profit margins. Therefore, antibiotic discovery is at an all-time low, and pathogens have evolved resistance to all currently available drugs. As a result, multi-drug resistant (MDR) bacterial infections are becoming more difficult to treat, especially in individuals at a high risk for infection such as cystic fibrosis (CF) patients. CF is a genetically inherited disease that inhibits or decreases chloride ion transport across epithelial cell membranes, resulting increased mucus viscosity, impairing normal clearance in the lungs. This environment is ideal for bacterial colonization and leads to a chronic lung infection. A major pathogen that colonizes the CF lung over time is Pseudomonas aeruginosa. A promising alternative treatment against MDR P. aeruginosa is bacteriophage therapy which has several advantages compared to antibiotics. First, phage therapy exhibits minimal side effects because phage are highly host-specific and do not inhibit other bacteria that are part of the human microbiome. Second, phage replicate itself exponentially when killing its host; and third, phage can be applied directly to the site of infection. However, like antibiotics, bacteria can evolve resistance to phage. To circumvent the problem of phage and drug resistance, trade-off effects may promote opportunities against both entities that may be exploited to treat MDR infections. I hypothesize that the effectiveness of antibiotics can be restored after selective pressure from bacteriophage. To test this hypothesis, MDR P. aeruginosa strains were exposed to phage in trade-off experiments, and results showed that the evolved phage resistant P. aeruginosa strain became antibiotic susceptible. In one trade-off experiment, a temperate phage recombined in the P. aeruginosa pathogen at a location downstream of a multidrug resistance efflux pump that may directly affect antibiotic susceptibility. In an (open full item for complete abstract)

Committee: Hans Wildschutte Ph.D (Advisor); George Bullerjahn Ph.D (Committee Member); Ray Larsen Ph.D (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; 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, University of Toledo, 2021, Biology (Ecology)

Chapter 2: Stratification and hypoxia in the western basin of Lake Erie (WBLE) has been shown to result in phosphorus flux from the underlying sediment, which could provide necessary nutrients for harmful algal bloom (HAB) growth. Studying the duration and frequency of hypoxic events would provide pivotal information for estimations of phosphorus flux from underlying sediments. However, due to the ephemeral nature of hypoxic events in the WBLE, planned weekly vessel-based sampling trips are inadequate for alerting researchers of the onset of hypoxia, making sampling such events difficult. Instead, water quality instruments can be deployed to collect and relay live data to researchers in a much more frequent timeline. In this study, a buoy equipped with a thermistor string and an EXO3 sonde (Yellow Springs Institute) was deployed to monitor for potential stratification and depleting lake bottom oxygen concentrations. This system measured water quality parameters and posted the data online every 20 minutes. Using these data, immediate vessel-based sampling trips to 7 sites were made according to observed hypoxia. Data captured show a hypoxic event occurred in the WBLE during early July 2020 that persisted for several days before being mixed by a storm on July 11, 2020. This hypoxic event coincided with 8 days of stratification. In addition, hypolimnion water warmed to over 23 ℃ while remaining stratified from the overlying waters, which could facilitate higher phosphorus flux from sediments. On average, phosphorus concentrations in the hypolimnion were 1.06 µ/L (~43%) higher than in the epilimnion by the end of the event, suggesting that sediments were releasing phosphorus into the overlying waters. Chapter 3: The western basin of Lake Erie (WBLE) has been experiencing Harmful Algal Blooms (HABs) for over a decade. These blooms have been detrimental to the health of Lake Erie and the safety of drinking water for surrounding communities. Nutrient inputs (namel (open full item for complete abstract)

Committee: Thomas Bridgeman Dr. (Committee Chair); Michael Weintraub Dr. (Committee Member); William Hintz Dr. (Committee Member) Subjects: Ecology; Environmental Science; Limnology

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

Cyanobacterial harmful algal blooms (cHABs) have occurred regularly in Lake Erie for the past two decades. The blooms are commonly dominated by Microcystis aeruginosa, a cyanobacterium with the ability to produce a group of hepatotoxins known as microcystins (MC). Current forecasting of the blooms predicts the spatiotemporal biovolume based on chlorophyll-a concentrations collected via remote sensors and known drivers of cyanobacteria cell growth. Forecasting however, cannot predict MC concentration of a bloom. The focus of this research was to examine MC production and loss rates to provide information that would be useful to develop forecasting of MC concentration for Microcystis blooms in the western basin of Lake Erie. Throughout 2018 and 2019 summers, 26 microcosm experiments were conducted at a near river mouth and off-shore location to quantify rates of MC production and loss. Eleven trials of production experiments with nutrient amendments were conducted each year during the annual cHAB. Nutrient additions of phosphorous with different nitrogen sources were added to 2 L of whole lake water at the beginning of the experiment to understand the effects of nitrogen on MC production. Bottles were incubated in-situ at each site for 72 hours with daily sample collection. Four trials of MC loss experiments were conducted monthly in 2019 to understand if loss of MC was due to biotic factors. Triplicate two liters of whole (biotic treatment) and 0.2µm filtered lake water (filtered control) were spiked with 1µg/L of 15N-labeled dissolved microcystin LR to quantify loss rates of this congener under ambient incubation. Subsamples were collected every 3 hours for the first 24 hours, then at hour 36 and 48 for measurement of microcystins. Production studies with nutrient amendments indicated various nitrogen sources did not significantly impact the production of MC during 2018 and 2019. Ambient rates of MC production were higher earlier in the bloom season and decreas (open full item for complete abstract)

Committee: Timothy Davis Dr (Advisor); George Bullerjahn Dr (Committee Member); Justin Chaffin Dr (Committee Member) Subjects: Aquatic Sciences; Biology; Environmental Science; Limnology

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

Multidrug resistant (MDR) pathogens are predicted to cause more than ten million annual deaths worldwide by 2050, making bacterial infections the leading cause of death. Although bacteria are evolving resistance to all known antibiotics, no large pharmaceutical companies are involved in drug discovery due to high cost and low profitability; thus, no new antibiotics are available, and the current ones are increasingly becoming ineffective at treating MDR infections. Moreover, recent studies suggest that there are few remaining new antibiotics in the environment left for discovery. Since MDR bacterial infections are predicted to be a major crisis, a new or augmented therapy to treat infections is imperative. Bacteriophage therapy is an alternative solution and has been internationally used for over 100 years. In bacteriophage therapy, phage bind to bacteria through specific protein-protein interactions that result in narrow host range infectivity. Although this specificity is beneficial because the interaction precisely targets a single pathogen, it is also problematic because a single phage usually cannot infect multiple strains of the same bacterial species. The phage discovery process to treat a specific pathogen is both time consuming and phage can fall short of the ability to antagonize multiple infections. Thus, phage with broad host range killing phenotypes are more beneficial when using phage therapy to treat infections caused by a particular pathogen. Therefore, this study set out to isolate phage with broad host range killing phenotypes such that phage that could inhibit more than one MDR pathogen could be found. In this study, 29 phage that antagonize cystic fibrosis (CF) derived MDR Pseudomonas aeruginosa were isolated from equine fecal water, purified, characterized through host range assays, and shown to kill two to eight CF derived MDR P. aeruginosa strains. Since phage have been shown to drive bacterial evolution toward increased antibiotic susce (open full item for complete abstract)

Committee: Hans Wildschutte (Advisor); George Bullerjahn (Committee Member); Raymond Larsen (Committee Member) Subjects: Biology; Biomedical Research; Health; Microbiology; Therapy

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, 2020, Biological Sciences

Sandusky Bay and Lake Erie are plagued with harmful algal blooms every summer. Sandusky Bay is a drowned river mouth that is very shallow and turbid and is dominated by Planktothrix agardhii, while Lake Erie is dominated by Microcystis aeruginosa. Both species of cyanobacterium are non-diazotrophic and produce microcystin, a hepatotoxin. A competition experiment was conducted culturing both species alone and in coculture at nitrogen (nitrate) replete, nitrate restricted, and nitrogen-free environments. Planktothrix grew better alone at nitrogen restricted medium than in co-culture with Microcystis. In coculture, Microcystis was dominant over Planktothrix however, that dominance decreased as nitrogen was reduced in each treatment. In the nitrogen replete environment, the coculture produced significantly more toxin than the monocultures and in the no nitrogen environment the Planktothrix monoculture produced more toxin than the Microcystis monoculture or the coculture. The community composition in Sandusky Bay was monitored over the winter and spring months (January-April) to see how it changed as time progressed. Nutrient amendment experiments were also conducted adding nitrate, phosphate, and a combination of nitrate and phosphate to stimulate growth and identify any possible nutrient limitations. The initial community yielded low cell densities until the temperature increased and cell abundances followed shortly thereafter. Planktothrix dominated over the winter followed by a transitional period of cryptomonad and diatom dominations before transitioning back to Planktothrix. Both nitrate and phosphate were limiting Planktothrix growth in the spring, while nitrate alone was limiting the overall community.

Committee: George Bullerjahn PhD (Advisor); Timothy Davis PhD (Committee Member); Robert McKay PhD (Committee Member) Subjects: Biology; Environmental Science; Limnology; Microbiology

Master of Science (MS), Wright State University, 2019, Chemistry

A fourth of the world's population lack access to safe water, thus the need for a more effective water treatment is imperative. Interest in silver nanoparticles (AgNPs) has grown in the last decade. Unlike chlorine, AgNPs do not form disinfection by products (DBPs), making them a prime candidate for drinking water treatment. The main aim of this study was to compare the antibacterial activity of electrochemical silver nanoparticles (eAgNPs-f) of ~5 nm in diameter against well-established pathogens: Escherichia coli (E. coli), Klebsiella variicola (K. variicola), and Pseudomonas aeruginosa (P. aeruginosa) to chlorine and Ag+ for drinking water. This was achieved by determining the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of eAgNPs, which were synthesized electrochemically and then concentrated (eAgNPs-f). The MIC values for E. coli, K. variicola, and P. aeruginosa were 4 ± 3, 3 ± 2, and 3 ± 2 mg/L, respectively. The MBC values for the same bacteria were 4 ± 3, 5 ± 2, and 5 ± 4 mg/L, respectively. When tested against chlorine, the MIC and MBC values increased over 1000-fold. CytoViva Hyperspectral Microscopy demonstrated the eAgNPs-f's affinity for the cellular membrane of E. coli after 30 minutes and physical cellular damage after 1 hour. Membrane disruption was confirmed through monitoring K+ leakage on ICP-OES. It was found that eAgNPs-f have a rapid and time consistent effect on K+ leakage, when compared to untreated control cells and Ag+. These results suggest that eAgNPs-f containing Ag+ ions are a more effective antibacterial agent than Ag+ alone, or chlorine.

Committee: Ioana E. Pavel Ph.D. (Advisor); David A. Dolson Ph.D. (Committee Member); Steven R. Higgins Ph.D. (Committee Member); Marjorie M. Markopoulos Ph.D. (Committee Member) Subjects: Chemistry; Nanotechnology

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