Microbiology Commons^™

A Multi-Faceted Study Of Antibiotic-Resistant, Opportunistic Pathogens In Wastewater And Environmental Water, Jeanette Calarco

USF Tampa Graduate Theses and Dissertations

More than 2.8 million infections due to antibiotic-resistant bacteria occur annually in the United States, and more than 35,000 people die as a result. Domestic wastewater (sewage), which contains antibiotic-resistant bacteria and antibiotic resistance genes, is collected at treatment plants for removal of contaminants, e.g. pathogenic microorganisms and nutrients. While wastewater treatment is designed to remove antibiotic-resistant bacteria and other microorganisms through treatment and disinfection, some bacteria may escape treatment. Treated wastewater effluent is generally discharged into surface waters; however, some fraction of the effluent may be used as recycled water. Compromised wastewater collection infrastructure and sewage spills can disseminate …

Dissolved Inorganic Carbon Metabolism In Bacteria, Jana Wieschollek

USF Tampa Graduate Theses and Dissertations

Dissolved inorganic carbon (DIC: CO2, HCO3⁻, CO32⁻) plays a central role in the metabolism of all living organisms. DIC is necessary for steps in essential biosynthetic processes, such as the synthesis of amino acids and nucleobases; in autotrophic bacteria, organic molecules are synthesized entirely from DIC. To ensure a steady supply of CO2 and HCO3 - needed for biosynthesis and/or autotrophy, organisms use bicarbonate transporters to increase the cytoplasmic concentration of this anion, and carbonic anhydrase (CA) to speed the interconversion of CO2 and HCO3 -, both in the cytoplasm and at the cell surface. This dissertation investigates DIC metabolism …

Identification Of Host Nuclear Factors Exploited By Kshv Lana To Maintain Latency, Anandita Ghosh

USF Tampa Graduate Theses and Dissertations

Kaposi’s Sarcoma Associated Herpes Virus (KSHV or HHV-8) is a γ-herpesvirus implicated in multiple lymphoproliferative human cancers and immune dysregulatory diseases. Like other herpesviruses, KSHV has a biphasic mode of infection primarily orchestrated by two viral proteins: the Latency-Associated Nuclear Antigen (LANA), which promotes viral persistence through latency, and the Replication and Transcription Activator (RTA), which triggers lytic reactivation. During latency, the KSHV genome exists as a highly heterochromatinized, extrachromosomal episome in the nucleus, which can be reverted to euchromatin in response to certain stimuli, like stress. KSHV has evolved to exploit numerous host proteins to orchestrate its lifecycle, especially …

Group B Streptococcus Intestinal Barrier Disruption And The Protective Role Of Butyrate, Kristen Dominguez

USF Tampa Graduate Theses and Dissertations

Group B Streptococcus (Streptococcus agalactiae; GBS) is a leading cause of neonatal sepsis worldwide. Intrapartum antibiotics in GBS-colonized mothers significantly reduced the incidence of early-onset sepsis occurring in the first week of life; however, there are currently no prophylactic interventions for late-onset sepsis, occurring within the first 3 months of life. GBS is a pathobiont that primarily colonizes the gastrointestinal tract and can cross the intestinal barrier via both the paracellular and transcellular routes, causing invasive disease. Neonatal susceptibility to GBS intestinal translocation is mediated by inadequate epithelial barrier function and microbiome immaturity; however, the mechanisms by which GBS exploits …

Comparing The Genomes Of Multidrug And Vancomycin-Resistant Enterococcus Faecium From A Florida Wastewater Treatment Plant, Eleanor Brodrick

USF Tampa Graduate Theses and Dissertations

Vancomycin-resistant Enterococcus (VRE) are a serious health threat, causing 50,000 infections and 5,000 deaths each year in the United States (CDC, 2019). Multidrug-resistant VRE are becoming more prevalent, and treatment options for infections caused by these organisms are limited (Arias et al., 2010; CDC, 2019). Multidrug resistant VRE have been previously isolated from wastewater in Brazil, Canada, and Portugal (Araújo et al., 2010; de_Farias et al., 2022; Sanderson et al., 2019). Thirteen vancomycin-resistant Enterococcus faecium isolated from three stages of a Florida wastewater treatment plant were fully resistant to the following antibiotics: vancomycin, ampicillin, ciprofloxacin, erythromycin, tetracycline, and nitrofurantoin, and …

Tetr Family Regulator, Farr, Influences Rot-Mediated Control Of Virulence Factor Expression In Staphylococcus Aureus, Julia C. Schumacher

USF Tampa Graduate Theses and Dissertations

Point-mutation variants of the TetR family regulator, FarR, have emerged across Staphylococcus aureus sequence types as conferring antibiotic and fatty acid resistance, particularly in Hospital-Associated Methicillin Resistant S. aureus (HA-MRSA) strains. FarR also exhibits an additional largely uncharacterized relationship to virulence factor production, seemingly through the global repressor of toxins—Rot, which functions to regulate S. aureus’ ability to colonize and persist in the host via a complex cascade of signaling. While Rot protein is post-transcriptionally regulated by the highly characterized Agr system, there is little known about its transcriptional regulation. Herein, we explored these dynamics by initially characterizing virulence phenotypes …

Integrated Periphyton Biofilters In Marine Recirculating Aquaculture Systems, Adam N. Bell

USF Tampa Graduate Theses and Dissertations

Marine aquaculture is a growing industry that has the potential to reduce eutrophication, overfishing, and loss of biodiversity. Recirculating aquaculture systems (RAS) are a type of aquaculture system that decreases discharge, raises biological control, and conserves water; however, RAS are challenged due to high energy cost, low biodiversity, water quality control, and waste nutrients. Periphyton biofilters are a new technology that allows for feed production, water quality control, microbial diversity, and resource recovery in marine aquaculture systems. The overarching question that this dissertation investigates is, how can periphyton biofilters be integrated into RAS to address the challenges of modern sustainable …

Regulated Intramembrane Proteolysis By M82 Peptidases: The Role Of Prss In The Staphylococcus Aureus Stress Response, Baylie M. Schott

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus is a major threat to human health, which is only exacerbated by its resistance to methicillin and other frontline antibiotics. Consequently, annual hospitalizations resulting from methicillin-resistant Staphylococcus aureus (MRSA) have been recorded in the hundreds of thousands for decades. However, the global impact of this opportunistic pathogen cannot solely be attributed to its antibiotic resistance, as S. aureus is also an expert at adapting to host conditions and responding to external stress. Here we attempt to gain a better understanding of a S. aureus factor that aids in circumventing such external insult - PrsS - a membrane protease …

Spatial Structure Of Germination And Seed-Associated Fungi In Tropical Pioneer Seeds, Daniela Varon Garcia

USF Tampa Graduate Theses and Dissertations

Negative density-dependent regulation is the process by which host-specific natural enemies reduce the survival of seeds and seedlings of conspecifics near adult trees, favoring heterospecific recruitment, and ultimately increasing local diversity. Negative density-dependent regulation by fungal pathogens has been consistently reported to promote plant diversity in the tropics, even though they tend to be multi-host and host-specificity is rarely reported. Nevertheless, evidence suggests that host-generalist fungi can have host-specific effects that produce disproportionate negative effects on some plant species but not others, promoting regional diversity. However, such studies have not tested for both host-specificity and distance-dependent effects in fully replicated …

Acetate-Encapsulated Linolenic Acid Liposomes Reduce Sars-Cov-2 And Rsv Infection, Andrew R. Mcgill

USF Tampa Graduate Theses and Dissertations

Coronavirus disease-19 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is an ongoing pandemic-endemic. In the United States, it remains the third leading cause of death in 2022. SARS-CoV-2, an RNA virus, has a genome prone to rapid mutation, generating variant species capable of resisting therapies and vaccination efforts. However, it has been estimated that more than 40% of SARS-CoV-2 infections may be asymptomatic, which calls into question any biological differences between the asymptomatic and afflicted groups. Additionally, Respiratory Syncytial Virus (RSV) is the most significant cause of bronchiolitis and asthma in neonates, with an increasing burden on the …

Anthropogenic Fecal Pollution Affects Microbial Water Quality And Multidrug Resistance In Tropical Aquatic Environments, Adriana González-Fernández

USF Tampa Graduate Theses and Dissertations

Although an often-overlooked issue, fecal pollution at recreational beaches should concerneveryone. Worldwide more than 80% of wastewater is discharged into rivers or the ocean without any treatment. Untreated and inefficiently treated wastewater can introduce diseasecausing microorganisms into the aquatic environment. Fecal-borne pathogens can also originate in animal waste, and humans can be exposed to these pathogens from poorly managed animal feces that make their way into the aquatic environment. Exposure to waterborne pathogens in recreational waters is a public health hazard, as it facilitates the transmission of waterborne illness, and surface waters serve as natural reservoirs of antibiotic resistant bacteria …

To Be Or Not To Be: A Tale Of Staphylococcal Gpsb, Lauren R. Hammond

USF Tampa Graduate Theses and Dissertations

There are two big drivers motivating studies into bacterial cell division. The first is a desire to understand life around us. One of the defining characteristics of life is the ability for a cell to grow and divide, and without in-depth knowledge of this process, we cannot truly understand the complexities that allow for life on this planet. The second motivator is to identify new drug targets in the ongoing fight against antimicrobial resistance. Many infectious organisms have become resistant to commonly used antibiotics, and the bacterial cell division machinery is a largely untapped essential process with many potential therapeutic …

Lord Of The Z-Rings: Uncovering The Role Of Mraz And Ftsl In Bacillus Subtilis Cell Division, Maria Louise White

USF Tampa Graduate Theses and Dissertations

Cell division is a complex and highly orchestrated process that requires careful coordination. The process of cell division in prokaryotes primarily occurs via binary fission, where one mother cell becomes two genetically identically and equally sized daughter cells. In most bacteria a key protein, FtsZ, forms a FtsZ ring (Z-ring) to mark the division site and recruit proteins involved in cytokinesis. Whilst much has been learned about the contents and assembly of the divisome in model organisms such as Escherichia coli and Bacillus subtilis factors governing the regulation of divisome are much more poorly understood. …

Assessing The Human-Health Risk Of Exposure To Pathogens From Beach Sands, Javier Federico Gallard-Góngora

USF Tampa Graduate Theses and Dissertations

Fecal contamination at recreational beaches impacts the health of beachgoers, through the introduction of disease-causing microorganisms, and the well-being of communities dependent on income from recreational beach activities. Beach ecosystems are also impacted by sewage through the introduction of nutrients that can cause abnormal increases in autochthonous microorganisms which can impact the population of larger organisms in the ecosystem. Fecal contamination is introduced into sand via untreated sewage, direct deposition of human feces into sand, runoff, and deposition of animal feces into sand. The introduction of fecal contamination into sand exposes individuals to pathogens (disease causing microorganisms) which can result …

Seroprevalence Of Sars-Cov-2 Antibodies, The Gut Microbiome, And Peripherally Circulating Cytokines In Pregnant Hispanic Females, Tina Susanna Mutka

USF Tampa Graduate Theses and Dissertations

Pregnant Hispanic females have been disproportionately impacted by SARS-CoV-2 andhave experienced high rates of asymptomatic or mildly symptomatic, unrecognized infection. Acute SARS-CoV-2 infection has been associated with gut microbiome changes and altered peripherally circulating cytokine levels in symptomatic, non-pregnant individuals.

This study investigated the seroprevalence of anti-SARS-CoV-2 IgG antibodies in a sample of pregnant Hispanic females (n = 197). A sub-sample of SARS-CoV-2 seropositive and seronegative pregnant Hispanic females were followed across pregnancy and their bacterial gut microbiome was characterized, and cytokine levels measured to investigate relationships between SARS-CoV-2 serostatus, the gut microbiome, and peripherally circulating cytokine levels across pregnancy. …

Antibiotic-Resistant Escherichia Coli And EnterococcusSpp. In Sand And Water At Tampa Bay Beaches, Jennifer K. Sabater

USF Tampa Graduate Theses and Dissertations

As antibiotic resistance in the environment continues to rise there is an increased concern that infections may become harder to treat as bacteria acquire genes for multidrug resistance. Recreational beach waters in the Tampa Bay area are routinely monitored by the Florida Department of Environmental Protection for the presence of fecal indicator bacteria (FIB) such as Escherichia coli and enterococci. Exceedances of beach action values (BAV) 235 CFU/100 mL (E. coli) and 70 CFU/100 mL (enterococci) indicate the presence of fecal contamination which is associated with an increased risk of disease for beachgoers. Antibiotic-resistant E. coli and Enterococcus spp. have …

Antimicrobial Activity Of Zinc Oxide Nanoparticles Compared To Silver Nanoparticles Administered Towards Methicillin-Resistant Staphylococcus Aureus Biofilms, Kevin Núño

USF Tampa Graduate Theses and Dissertations

Zinc oxide nanoparticles (ZnO NPs), silver nanoparticles (AgNPs), and chitosan nanoparticles have strong antibacterial activity as a broad-spectrum antibiotic against multi-drug resistant (MDR) bacteria. MDR bacteria have a mechanism that increases antibiotic resistance through biofilm formations. MDR methicillin-resistant Staphylococcus aureus BAA-1720 was chosen for an in vitro assessment to calculate the biofilm reduction rate and the median effective dose (ED50) by performing biofilm quantification. Dynamic light scattering was chosen to determine the physiochemical characteristics of ZnO NPs, silver nanoparticles (AgNPs), and chitosan NPs. Transmission electron microscopy was conducted to verify each treatment’s physiochemical characteristics. Antimicrobial susceptibility testing of broad-spectrum and …

Multifaceted Approach To Understanding Acinetobacter Baumannii Biofilm Formation And Drug Resistance, Jessie L. Allen

USF Tampa Graduate Theses and Dissertations

Acinetobacter baumannii is a multi-drug resistant nosocomial pathogen known for causing wound related- and respiratory-infections. It is currently on the WHO’s list of critical pathogens due to its broadly drug resistant nature and the constant appearance of pan-resistant isolates. A majority of the infections caused by this organism are biofilm associated, however there is limited existing knowledge regarding the mechanisms used to engage in this multicellular lifestyle. As such, we set out to explore the factors influencing this behavior using an 10,000+ isolate transposon mutant library of A. baumannii strain AB5075. Of the strains tested, 6.45% demonstrated some level of …

Unraveling The Role Of Novel G5 Peptidase Family Proteins In Virulence And Cell Envelope Biogenesis Of Staphylococcus Aureus, Stephanie M. Marroquin

USF Tampa Graduate Theses and Dissertations

Virulence factors and the bacterial cell envelope are two important components in S. aureus pathogenesis and survival. More importantly, understanding the regulation of these cellular processes is crucial to further understanding and combating this successful pathogen. To date, numerous factors have been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane G5 peptidase family protein, as a novel effector of this system. Using a USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis, and …

Microbial Dark Matter: Culturing The Uncultured In Search Of Novel Chemotaxonomy, Sarah J. Kennedy

USF Tampa Graduate Theses and Dissertations

Although <1% of environmental isolates can be cultured, insights into the culturable fraction refine future techniques to unlock the cryptic biodiversity. This cryptic biodiversity is omnipresent within culture-independent surveys of environmental bacterial populations. Subsequently, these elusive organisms are termed “microbial dark matter” due to the intrigue surrounding their potential taxonomic and biosynthetic impacts. To this end of investigating bacterial-specific dark matter, we employed high-throughput culturing techniques to examine Gulf of Mexico sediments and sponges for chemotaxonomically important bacteria. Through stepwise implementation of permissive and selective recovery techniques, we succeeded in recovering over 150 unique bacterial isolates. We observed several isolates that pose as chemotaxonomically interesting, including several strains that belong to critical bioremediation taxa. In addition to these biotechnological-amenable isolates, we have also succeeded in culturing taxa historically attributed with antibacterial discoveries. One such isolate was a previously sponge-associated organism belonging to the rarely cultured Verrucosispora genus. Through extensive comparative genomics, we identified it as a novel species and subsequently named it Verrucosispora sioxanthis sp. nov. with the published genome serving as a resource for continued microbial dark matter investigations. Following continued passaging of the organism, we observed improved growth, biomass, and liquid growth dispersal within each increasingly sub-passaged generation of the isolate. As such, the laboratory attenuation, or domestication, of the organism warranted in-depth whole genomic analyses to generate insight into the increased laboratory sustainability. Through phenotypically-guided bioinformatics, we determined that the domestication was mediated through genome-wide selective pseudo- or non-pseudogenization of genes. As the previously sponge-associated organism evolved with the selection pressures of the static laboratory, we demonstrate that isolate effectively underwent genomic atrophy to resolve into a largely stable, sustainable laboratory-domesticated strain. Collectively, our chemotaxonomic discovery pipeline expands on the culturable bacterial biodiversity and ultimately provides a previously uncultured model organism for initial forays into resolving the microbial dark matter knowledge gap.

Investigation Of An Alternative Protocol For The Production Of Sars-Cov-2 Antigenic Proteins, Nichole Ninaltowski

USF Tampa Graduate Theses and Dissertations

With the COVID-19 pandemic showing no signs of slowing down, large-scale antigenic protein production is still needed for surveillance using serologic assays. From screening to vaccines to biotherapeutics, being able to produce the proteins for these assays is essential; however, the current gold standard method for producing SARS-CoV-2 spike proteins is prohibitively expensive for most research groups.

Alternative methods of transfecting mammalian cells to produce recombinant proteins that are relatively inexpensive have been used for years. Unlike the expensive, commercially available lipid-based methods, other established methods such as polyethyleneimine (PEI), are considerably easier, and cheaper to meet the needs of …

A Functional Characterization Of The Omega (Ω) Subunit Of Rna Polymerase In Staphylococcus Aureus, Shrushti B. Patil

USF Tampa Graduate Theses and Dissertations

In bacteria, RNA polymerase (RNAP) is a well-characterized and highly conserved multi-subunit enzyme complex responsible for transcription of DNA into RNA. The ω subunit (rpoZ/RpoZ), one of the smaller accessory subunits of RNAP, is often overlooked and under-studied, however, particularly in Gram-positive species. We have previously shown that deleting ω impacts the integrity of RNA polymerase in S. aureus, especially the β' subunit, and alters preference of the core enzyme for sigma factors, skewing heavily towards σB instead of the housekeeping σ factor. Consequently, this causes deregulation of myriad transcriptional processes, strongly rewiring gene expression circuits, and ultimately impairing the …

Transcriptomic And Functional Investigation Of Bacterial Biofilm Formation, Brooke R. Nemec

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus and Acinetobacter baumannii are two highly successful human pathogens, which have adopted very different, but effective survival strategies. The success of S. aureus is attributed to the tight regulation of an arsenal of virulence factors. Conversely, A. baumannii lacks what would be considered traditional virulence factors and, instead, has developed a high tolerance for environmental stress, which allows it to persist in unforgiving environments, including nosocomial settings and the human body. One common characteristic of these two organisms is their proclivity for biofilm formation. Herein, we discuss the diverse mechanisms governing biofilm formation for A. baumannii and S. …

Exploring Prokaryotic And Viral Communities Within Florida’S Freshwater Springs, Kema Saydoon Malki

USF Tampa Graduate Theses and Dissertations

Florida’s freshwater springs serve as a crucial ecosystem for the state, having both ecological and economic importance. Springs occur where there is an opening where aquifers discharge groundwater. Florida’s springs are fed by the Floridan aquifer system (FAS), which underlies the entire state and extends into several neighboring states. The FAS provides potable water to over 10 million people. The constant flow of groundwater through the springs feeds major river systems and creates unique freshwater systems that support numerous plants and animals. The combination of natural beauty and charismatic fauna attract tourism and generate millions of dollars in revenue for …

Development And Validation Of An Isothermal Amplification Assay For Eastern Equine Encephalitis Virus, Mikayla D. Maddison

USF Tampa Graduate Theses and Dissertations

Eastern Equine Encephalitis virus (EEEV) is a neurotrophic alphavirus for which there is no effective treatment or vaccine for humans. Periodic outbreaks in the Eastern United States represent an ongoing public health problem; Florida serves as the reservoir for EEEV for the rest of the country. Reverse transcription-polymerase chain reaction (RT-PCR) is the current gold standard for molecular diagnostic testing of the presence of EEEV in vectors. However, RT-PCR is technically complex and can be difficult for mosquito control districts to utilize. In order to provide a simple, cost-effective alternative for mosquito surveillance and control, a novel one step reverse …

Novel Co2 Transporters From Autotrophic Bacteria From Extreme Environments, Sarah E. Schmid

USF Tampa Graduate Theses and Dissertations

Autotrophic organisms are responsible for introducing carbon into food webs. These organisms take up dissolved inorganic carbon (DIC) and use it as their major carbon source, converting it to biomass using a variety of carbon fixing pathways (e.g., the Calvin-Benson-Bassham cycle). In nature, DIC concentrations can be low or temporally heterogeneous, so autotrophic organisms adapt with CO2 concentrating mechanisms (CCMs) to facilitate the acquisition and fixation of this dissolved gas. CCMs consist of membrane-spanning DIC transporters, which elevate intracellular DIC concentrations, which in turn are harvested by carboxysomes, proteinaceous microcompartments that contain the carboxylase Ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) and carbonic anhydrase. …

Environmental Influences On Expression Of Virulence- And Survival-Associated Genes And Epigenetic Modifications Of Dna In Vibrio Vulnificus, James W. Conrad

USF Tampa Graduate Theses and Dissertations

Vibrio vulnificus is an autochthonous inhabitant of warm, brackish coastal waters and is an opportunistic pathogen that causes the highest mortality of all seafood-related illnesses (1). These fatal infections are generally caused by the clinically-associated vcgC/16S rRNA type B genotypes within the biotype I group (2–5). However, the reasons for the heightened infectiousness of the clinically-associated strains over environmentally-associated ones remains elusive, as no unique clinically-associated virulence genes have been identified through genomic sequencing or other strategies. DNA methylation may contribute to regulation of virulence by affecting gene transcription, and was investigated in the highly virulent V. vulnificus strain CMCP6. …

Posttranslational Modification And Protein Disorder Regulate Protein-Protein Interactions And Dna Binding Specificity Of P53, Robin Levy

USF Tampa Graduate Theses and Dissertations

p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome …

Spatial And Temporal Determinants Associated With Eastern Equine Encephalitis Virus Activity In Florida, Kristi M. Miley

USF Tampa Graduate Theses and Dissertations

Eastern Equine Encephalitis virus (EEEV) is considered the most pathogenic mosquito-borne illness in the United States. Human mortality has been estimated to range between 35 to 75%, with horses experiencing mortality rates greater than 90%. A large number of EEEV cases occur in Florida. Though we have come a long way since the first human infection was identified in the 1930s, there is still much to learn regarding the virus’s ability to maintain transmission year-round in Florida. Phylogenic studies support that Florida may serve as the geographic reservoir for EEEV. This research investigated spatiotemporal and ecological variables associated with risk …

Microbial Community Structures In Three Bahamian Blue Holes, Meghan J. Gordon

USF Tampa Graduate Theses and Dissertations

This study used 16S rDNA metagenomics and water chemistry to conduct an examination of microbial community dynamics and biogeochemistry in three physically adjacent, sunlit blue holes with variable hydrologic regimes on San Salvador Island, Bahamas. Church and Watling’s Blue Holes are holomictic with relatively clear waters, while Inkwell Blue Hole hosts density stratification and waters stained brown with tannins. Based on water color and clarity and physicochemical profiles, I hypothesized Church and Watling’s Blue Holes would be dominated by oxigenic photoautotrophs, and that the bottom layer of Inkwell would be characterized by euxinic (anoxic and sulfidic) conditions and host primarily …