Microbiology Commons^™

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 …

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 …

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 …

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. …

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 …

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 …

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 …

To Mid-Cell And Beyond: Characterizing The Roles Of Gpsb And Ypsa In Cell Division Regulation In Gram-Positive Bacteria, Robert S. Brzozowski

USF Tampa Graduate Theses and Dissertations

The bacterial cell division protein FtsZ is a tubulin homolog that forms a ring-like structure at the site of cell division in most bacterial species. There it acts as a scaffold, aiding in the recruitment of other divisome proteins to the site of cell division. Furthermore, studies focusing on the role of FtsZ treadmilling and septal peptidoglycan synthesis implicates that FtsZ plays a direct role in the ultimate closure of the division septum. Thus, many studies in the field of bacterial cell division have focused on FtsZ in terms of its spatial and temporal regulation as well as its ability …

Targeting The Eskape Pathogens By Botanical And Microbial Approaches, Emily Dilandro

USF Tampa Graduate Theses and Dissertations

There is a vital need to find new clinical treatment options to combat ESKAPE pathogen infections. Nature has thus far been the most fruitful at providing antimicrobial compounds, which have been derived from a plethora of sources. Ranging from plants to microbial communities, these organisms create chemical compounds that are used as defense mechanisms against invasive or encroaching organisms and confer the producers with competitive advantages. In this study, cinnamaldehyde was investigated as a botanical approach to finding active antimicrobial compounds that inhibit the ESKAPE pathogens. Here, we show that all the ESKAPE pathogens are inhibited by cinnamaldehyde concentrations between …

The Ecology And Conservation Of An Urban Karst Subterranean Estuary, Robert J. Scharping

USF Tampa Graduate Theses and Dissertations

Sulphur Springs is an artesian spring and part of the limestone karst topography of metropolitan Tampa, Florida, USA. Underlying this spring is an extensive cave system that hosts saltwater vents and conspicuous microbial biofilm communities. For decades, water from Sulphur Springs has been extracted and used to supplement public water supply and dry season flows in the Hillsborough River Estuary. This dissertation describes research conducted at Sulphur Springs to determine the impacts of urban land use on the underlying cave and downstream estuary ecosystems, and presents the use of this system as a model to fill certain ecological knowledge gaps. …

The Role Of Secreted Proteases In Regulating Disease Progression In Staphylococcus Aureus, Brittney D. Gimza

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus is a highly successful pathogen capable of producing a wealth of virulence factors in the human host. Of note, ten extracellular proteases are produced alongside these virulence factors and play a multifaceted role during infection. They not only cleave host proteins to promote bacterial invasion, immune evasion and survival, but also control disease progression by modulating the stability of self-derived pathogenic determinants. The importance of the secreted proteases modulating virulence factor stability is evidenced by our groups previous finding that a protease-null strain has a substantially increased infectious capacity in a murine model of sepsis; resulting from the …

The Adaptations Of The Sulfur-Oxidizing, Gammaproteobacterium "Candidatus Endoriftia Persephone", Endosymbiont Of The Giant Tubeworm Riftia Pachyptila, To Hydrothermal Vent Habitat Heterogeneity, Juliana M. Leonard

USF Tampa Graduate Theses and Dissertations

The siboglinid tubeworm Riftia pachyptila is a dominant member of the deep-sea megafauna where seawater and hydrothermal vent (HTV) effluent interface and mix. It is one of the fastest growing invertebrates on land or in the sea. It does not have a digestive tract (e.g. mouth, gut, or anus), and is completely dependent on its sulfur-oxidizing endosymbiont, the Gammaproteobacterium “Candidatus Endoriftia persephone” for its nutritional requirements. This association was the first and is the most well studied among chemolithoautotrophic symbioses. “Ca. E. persephone” is a chemolithoautotrophic bacterium that oxidizes sulfide as an electron donor for energy, reduces oxygen as a …

Targeting Bacterial Resistance And Cancer Metastasis: A Structure Based Approach, Kyle Galen Kroeck

USF Tampa Graduate Theses and Dissertations

Current research in pharmaceutical development commonly utilizes a profusion of methods in molecular modeling in order to probe intricate biological problems. Many original and promising compounds have been identified and developed by integrating experimental and computational methods. Structural biology utilizes many different research techniques including x-ray crystallography, NMR, and electron microscopy in order to develop molecular models of macromolecules that are of biological interest. Such techniques can be used in conjunction with molecular docking, which utilizes those molecular models in order to target macromolecules of therapeutic interest by computationally analyzing the conformations adopted by ligands upon interaction with a desired …

Design And Implementation Of Degenerate Qpcr/Qrt-Pcr Primers To Detect Microbial Nitrogen Metabolism In Wastewater And Wastewater-Related Samples, Ryan F. Keeley

USF Tampa Graduate Theses and Dissertations

Nitrogen cycling processes can be tracked using quantitative Polymerase Chain Reaction (qPCR) to determine the presence and qReverse Transcriptase-PCR (qRT-PCR) to determine expression of key genes, or ‘biological markers’, for nitrogen metabolism. Nitrification is catalyzed in part, by two enzymes: ammonia monooxygenase (AMO; NH3 NH2OH) and nitrite oxidoreductase (NXR; NO2- NO3-). For denitrification, four enzymes act sequentially: nitrate reductase (NAR/NAP; NO3- NO2-), nitrite reductase (NIR; NO2- NO), nitric oxide reductase (NOR; NO  N2O), and nitrous oxide reductase (NOS; N2O  N2). A principle of wastewater treatment (WWT) is to remove excess nitrogen by taking advantage of natural nitrogen cycling …

A Small Rna And Dna Binding Protein Contribute To Biofilm Development In Bartonella Henselae, Udoka Okaro

USF Tampa Graduate Theses and Dissertations

A biofilm, which is associated with 80% of chronic infections in humans, is formed when bacteria aggregate, attach to a substrate and secrete a matrix protecting the bacteria from host cell defenses and antibiotics. Bartonella henselae (B. henselae) is the causative agent of cat scratch disease, persistent bacteremia, and one of the most frequently reported causes of blood-culture negative endocarditis (BCNE) in patients. The ability of B. henselae to adhere to the heart valve, form a biofilm and vegetation to cause endocarditis increases the morbidity and mortality rate in infected patients. The presence of a trimeric autotransporter adhesin (TAA) called …

Investigating The Proteinaceous Regulome Of The Acinetobacter Baumannii, Leila G. Casella

USF Tampa Graduate Theses and Dissertations

Acinetobacter baumannii is an opportunistic pathogen that overtime has evolved into one of the most problematic pathogens due to its ability to overcome antibiotic pressures and harshly environments in the host and hospital environments. In this context, its genomic evolution due to its capacity to acquire genes that contribute to its pathogenic and antibiotic resistance nature has been the subject of research in the last decades providing with the identification of several proteins aiding in the process of pathogenicity. Although these findings have contributed to our understanding of A. baumannii pathogenic traits, the regulatory network that control their expression are …

Evaluating Human-Associated Escherichia Coli Marker Genes For Microbial Source Tracking Applications In Florida, Jacob K. Senkbeil

USF Tampa Graduate Theses and Dissertations

Fecal pollution of surface water can lead to human health issues because waterborne transmission of pathogens is a common cause of gastroenteritis. Genetic markers developed for human-associated Escherichia coli (H8, H12, H14, and H24) are promising tools for microbial source tracking (MST) of sewage in environmental waterbodies but are untested in the US. H gene marker performance characteristics (specificity, sensitivity, and prevalence) were assessed, with the goal of developing a quantitative PCR assay for the markers with the best performance. Individual E. coli isolates from reference samples were tested to determine end-point PCR assay performance. Three hundred E. coli strains …

Microbial Associations Of Four Species Of Algal Symbiont-Bearing Foraminifera From The Florida Reef Tract, Usa, Makenna May Martin

USF Tampa Graduate Theses and Dissertations

Marine microbiome research is a rapidly expanding field of study, as scientists investigate the functions of microbial associations in eukaryotic organisms. Foraminifera are among the most abundant shelled organisms in the oceans, yet little is known of their associated microbiomes. This study investigated microbes associated with four species of Foraminifera that host three kinds of algal endosymbionts. The Order Miliolida, Family Soritidae, was represented by three species: Archaias angulatus and Cyclorbiculina compressa, which both host chlorophyte symbionts, and Sorites orbiculus, which hosts dinoflagellate symbionts. The fourth species, Amphistegina gibbosa, belongs to the Order Rotaliida and hosts diatom endosymbionts. Bacterial DNA …

Evaluation Of A Microsphere-Based Immunoassay (Mia) In Measuring Diagnostic And Prognostic Markers Of Dengue Virus Infection, Jason H. Ambrose

USF Tampa Graduate Theses and Dissertations

Infections with dengue viruses (DENV) constitute both a global problem as well as locally in Florida. DENV comprise four distinct serotypes of single-stranded RNA viruses and belong to the family Flaviviridae. DENV are among the most medically important arboviruses in the world and cases may currently exceed 400 million per annum. Additionally, dengue established its first recorded endemic transmission cycle in the state of Florida in over a half century, first within the Florida Keys during 2009-10 followed by an unrelated outbreak in Martin County in 2013. The clinical profile of DENV infections ranges from a mild febrile illness …

The Ecology Of Antibiotic Resistance: Sources And Persistence Of Vancomycin-Resistant Enterococci And Antibiotic Resistant Genes In Aquatic Environments, Suzanne M. Young

USF Tampa Graduate Theses and Dissertations

The growing crisis of antibiotic resistance is a major threat to ecosystems and human health. Infections caused by known and emerging antibiotic resistant pathogens are on the rise globally, with approximately 700,000 deaths per year caused by antibiotic resistant bacteria (1). In the United States, infections from antibiotic resistant bacteria cause more than 2 million illnesses and 23,000 deaths (2). Antibiotic resistant bacteria and antibiotic resistance genes are released into aquatic ecosystems through hospital waste, residential sewer lines and animal agricultural waste streams. Animal agriculture accounts for approximately 70% of antibiotic use in the United States (3). In agricultural ecosystems, …

Mechanism Elucidation And Inhibitor Discovery Against Serine And Metallo-Beta-Lactamases Involved In Bacterial Antibiotic Resistance, Orville A. Pemberton

USF Tampa Graduate Theses and Dissertations

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes …