Vibrio cholerae, the etiological agent of the severe diarrheal disease cholera, is an enteric pathogen that can be found in aquatic ecosystems when not colonizing the human gastrointestinal tract. Under adverse environmental conditions, V. cholerae is capable of entering dormant states that increase its survival during these ecological fluctuations. In these states, V. cholerae slows its metabolic activity and exhibits drastically altered gene expression and morphology. Stressors that lead to entry into these states vary from nutrient limitation, suboptimal pH, or antimicrobials. Cells in these dormant states are highly resistant to antimicrobials and cannot be detected using standard microbiological techniques which poses major public health challenges such as food or water contamination. In V. cholerae, proteolysis of virulence regulator ToxR has been identified to be required for entry into a dormant state called viable but nonculturable (VBNC) under nutrient limitation and alkaline pH mediated by the sigma-E stress response. However, the mechanisms that lead to the initiation of this cascade remain unknown. The stringent response is another mechanism involved in mediating bacterial survival during late stationary phase. The stringent response involves the alarmone (p)ppGpp, which acts at the level of transcription to inhibit cellular processes that consume significant resources and activate genes responsible for biosynthetic processes. RelA is one enzyme responsible for the synthesis of (p)ppGpp, which in turn activates transcription of RpoE, suggesting a potential connection with ToxR proteolysis. Therefore, the aim of this study is to define the role of RelA in dormancy and ToxR proteolysis in V. cholerae. Our results show that RelA alone is not sufficient to control dormancy and ToxR proteolysis in V. cholerae. Nonetheless, another regulator (SpoT) is also associated with (p)ppGpp synthesis, indicating that other stringent response-associated mechanisms might be involved in ToxR proteolysis.

The emergence of human pathogens represents a major current global health concern. Characterization of the adaptations required for a given microorganism to emerge as a human pathogen is important for understanding epidemics, as we are typically aware of a pathogen's existence only after it has emerged, manifesting as an outbreak. Cholera is a severe diarrheal disease caused by the aquatic bacterium Vibrio cholerae O1 and is one paradigmatic example of disease emergence. Only a subset of V. cholerae strains can cause the disease while the majority of the strains cannot cause cholera symptoms. We found that toxigenic strains of V. cholerae encode allelic variations of core genes, termed Virulence Adaptive Polymorphisms (VAPs), that confer preadaptations towards the emergence of pathogenic traits. Interestingly, VAPs appear to naturally circulate in environmental populations of V. cholerae. One gene potentially encoding VAPs codes for the outer membrane protein U, OmpU. This major porin plays numerous roles in V. cholerae pathogenesis such as bile tolerance, antimicrobial peptide resistance or facilitates intestinal colonization. Interestingly, we found that these phenotypes appear to be allelic dependent and might provide a clue towards the emergence of toxigenic V. cholerae. To date, the distribution and prevalence of VAPs in environmental populations and the specific molecular mechanisms leading to their virulence preadaptations remain unknown. Here we examined the diversity of ompU alleles in natural V. cholerae populations in order to identify VAPs unique to the toxigenic allele of ompU to discern these preadaptations. We developed a comparative framework to address this by examining allelic variations of OmpU from an endemic population of V. cholerae that we identified for this study in Eastern Florida. We generated 14 isogenic mutant strains each encoding a unique ompU allele that largely covered the landscape of protein variability and examined their resistance profile to host antimicrobials. We determined the genotype to phenotype associations between these mutants and identified and experimentally confirmed four conserved domains that are unique to alleles of ompU that confer resistance to bile and other host antimicrobials. Interestingly, a mutant strain in which we exchanged the four domains of the clinical allele for those of a strain that was sensitive, exhibits a resistance profile closer to an OmpU deletion mutant. Our findings highlight the critical importance of allelic variations in the emergence of virulence adaptive traits and the suitability of our approach towards dissecting its emergence. This tractable approach can be naturally applied to other bacterial pathogens.

Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi, the causative agent of genital chancroid. CDT is a heterotrimeric toxin consisting of a cell-binding domain (CdtA + CdtC) and a catalytic domain (CdtB) that has DNase activity. After binding to the host plasma membrane, CDT undergoes endocytosis and travels through the endosomes en route to the endoplasmic reticulum (ER). Only CdtB and CdtC arrive in the Golgi before moving to the ER. Only then does CdtB move into the nucleus, causing DNA damage that induces cell-cycle arrest and apoptosis. The previous CDT trafficking model suggested that CdtA remains on the plasma membrane while the CdtB/CdtC heterodimer is transported inside the cell. This model is based on experiments that were unable to detect CdtA inside the host cell. Here, we reexamine this model and demonstrate that CDT is internalized as an intact holotoxin. Furthermore, the acidification of the endosomes induces CdtA release from the CdtB/CdtC heterodimer. Using a cell-based ELISA, we report that CdtA facilitates CDT binding to the plasma membrane and demonstrate that nearly the entire pool of surface-bound toxin is internalized from the plasma membrane within 20 minutes. As determined by Western blot, all of internalized CdtA and most of internalized CdtB and CdtC are rapidly degraded in the lysosomes. CdtA colocalized with EEA-1, an early endosomal marker, before lysosomal degradation and was destabilized by the acidic conditions found in the early endosomes (pH 6.0-6.3). This led to its release from the CDT holotoxin as determined by circular dichroism and surface plasmon resonance. The results in this dissertation demonstrate that CDT is internalized as an intact holotoxin, with the acidic environment of endosomes triggering the separation of CdtA from the CdtB/CdtC heterodimer – the first stage of CDT's novel two-stage disassembly.

The emergence of multidrug-resistant (MDR) strains of bacteria and the lack of a novel class of antibiotics has become a global health concern. Pseudomonas aeruginosa is one common MDR bacteria responsible for nosocomial infections and related mortality worldwide. It has developed resistance against commonly available antibiotics and is in the WHO's priority list of bacteria for which new antibiotics are desperately needed. Currently there is a growing interest in developing metal and non-metal-based nanoparticles to target multidrug-resistant bacteria. The objective of this study is to evaluate the efficacy of a novel nanocomposite of two non-traditional antimicrobials: a metal (Ga-III) and a non-metal (chitosan nanoparticle) against P. aeruginosa. It was hypothesized that Gallium (III) nitrate in combination with hydrothermally-treated chitosan biopolymer, which has been widely studied for wound-healing applications, will exhibit synergistic antibacterial activity due to increased modes of action . The Ga(III) nitrate is an FDA approved drug that is used to lower blood levels of calcium in some cancer patients. The drug has been under clinical trials as an antimicrobial agent due to its Iron(III) mimicking property. The chitosan-gallium nanocomposite was synthesized using hydrothermal treatment in acidic conditions. Particle size, surface charge, optical properties, and chemical interactions between Ga (III) and chitosan were studied using Dynamic Light Scattering (DLS), FT-IR, UV-VIS and Fluorescence techniques. Microplate Alamar Blue Assay, Colony Forming Unit assay and Crystal Violet biofilm inhibition assay were conducted to study the antibacterial and antibiofilm properties of the nanocomposite in aqueous suspension (pH 5.7). UV-Visible and fluorescence spectra suggested the formation of optically-active chitosan-gallium nanocomposite, exhibiting broad absorption band (~290-325 nm) and emission at 422 nm. FTIR study confirmed the depolymerization of chitosan and gallium complexation through primary amine groups of chitosan. DLS analysis showed that primary particles have hydrodynamic diameter of 141 nm and average zeta potential of +46 mV at pH 5.7. Microplate alamar blue assay revealed the MIC of the composite to be 32 µg/ml while CFU assay determined the MBC to be 128 µg/ml against P.aeruginosa. Compared to the controls chitosan and gallium nitrate, the chitosan-gallium nanocomposite showed enhanced antibacterial efficacy. Furthermore, there was 21.5% inhibition of biofilm formation at 8 µg/ml of the composite. These preliminary findings suggest the potential of chitosan-gallium nanocomposite as an effective antibacterial agent against P.aeruginosa infections.

Cholera is an acute disease that is a global threat to the world and can kill people within a few hours if left untreated. In the last 200 years, seven pandemics occurred, and, in some countries, it remains endemic. The World Health Organization (WHO) declared a global initiative to prevent cholera by 2030. Cholera dynamics are contributed by several environmental factors such as salinity level of water, water temperature, presence of plankton especially zooplankton such as cladocerans, rotifers, copepods, etc. Vibrio cholerae (V. cholerae) bacterium is the main reason behind the cholera disease and the growth of V. cholerae depends on its host in the water reservoir which is the zooplankton because they share a symbiotic relationship. Investigating plankton bloom could be one of the key indicators for predicting cholera outbreaks. Though there are lots of models for cholera transmission dynamics, there are few existing models focused on the environmental impacts of plankton reservoirs. In this work, we have formulated a model of cholera transmission dynamics with the environmental impacts of plankton reservoirs. We have derived the basic reproduction number and discussed various alternative threshold parameters using the next generation matrix approach. Next, we have considered the existence and stability of the disease-free and positive equilibria. Our model analysis could be helpful for scientists to better understand the impact of environmental factors on cholera outbreaks and eventually for a possible prediction of the timing and location of the next cholera outbreak.

The purpose of this thesis was to investigate new possible compounds that can be used to treat orthopedic implant infections caused by bacterial pathogens. Current treatment includes the use of antibiotics and the DAIR procedure, which stands for debridement, antibiotic therapy, irrigation, and retention. However, antibiotics are becoming less effective as a treatment due to bacteria gaining antibiotic resistance. Two bacterial species involved in orthopedic implant infections are P. aeruginosa and S. aureus. This thesis investigated cerium oxide nanoparticles and L. fermentum, a beneficial bacterium, as possible treatments to stop bacterial growth and the formation of biofilm. This was done by using the Kirby-Bauer disk diffusion method with P. aeruginosa and S. aureus. An XTT assay, a viability assay, was also performed on RAW macrophages to determine how these compounds affect human immune cells. Dextran-coated, 50/50, and 70/30 Ce4+/Ce3+ CNP (cerium oxide nanoparticles) at 1, 10, 20, 100, 500, and 800 µg/mL were investigated. These kinds of CNP were investigated to determine which type of CNP and at what concentration was most effective. The results show significant reductions (p-value ≤ 0.05) in infection totals for various treatments, such as 10 µg/mL 50/50 CNP (cerium oxide nanoparticles). This study adds to the field of research in investigating new treatments for orthopedic implant infections.

Globally, tuberculosis, a disease caused by the species of Mycobacterium tuberculosis (Mtb) complex, stands as a leading cause of death from a single infectious agent. Even though antituberculous drugs are available, treatment is challenging due to antibiotic resistance associated with point mutations in the bacterial genome. Resistance to the first-line antibiotics – rifampin and isoniazid – results in multidrug-resistant tuberculosis (MDR) requiring a more complicated treatment regimen. Timely and accurate identification of drug-resistant TB cases can help prescribe the most effective treatment and prevent the spread of infection. This research aims to develop an assay to discern multi-drug resistant forms of tuberculosis using a molecular assay based on split deoxyribozyme hybridization probes. For the probe design, a catalytic core of an RNA-cleaving deoxyribozyme is split into two parts, with each part elongated with a target-recognizing fragment ("arm"). In the presence of a fully complementary nucleic acid target, but not the one containing point mutations, the catalytic core of the deoxyribozyme can be re-formed due to the assembling of the target-probe complex, which recognizes and allows cleavage of a fluorophore- and quencher-labeled signal reporter, thereby ensuring increase in fluorescence in a target-dependent manner. The target-binding arms of the probes were optimized in terms of the signal-to-background ratio and selectivity of target recognition using synthetic targets corresponding to the fragments of the katG and rpoB genes with point-mutation sites implicated in the resistance to isoniazid and rifampin, respectively. The optimized probe sequences were used to interrogate the targets obtained by amplifying the correspondent fragments of the Mtb genes using Linear- After-The-Exponential (LATE) PCR, which allows efficient synthesis of a single-stranded amplicon. The signal triggered by cognate targets can be read using a portable fluorometer, which eliminates the need to use a sophisticated real-time PCR instrument for the assay. The success of the split deoxyribozyme assay can establish an affordable and user-friendly molecular diagnostic assay where a sample can be amplified and analyzed in a single tube.

Healthcare and ancillary workers in hospital settings are at an increased risk of Tuberculosis (TB) due to the nature of their job and exposure to persons with TB. Knowledge of healthcare workers (HCWs), (physicians, residents, nurses, midlevels etc.) who provide direct care and ancillary staff (technicians, aides, administrators, etc.) who provide indirect care in medical institutions play an important role in the diagnosis, treatment, control, and prevention of TB. Research has shown a variation in knowledge based on TB prevalence, facility type, available resources, provider training and clinical experience, education level of staff, etc. The purpose of this study is to assess the knowledge of TB among HCWs and ancillary staff in an underserved medical institution who provide care to low-income populations in the United States. This is a cross sectional observational study. A validated questionnaire that assesses TB knowledge will be used. Participants from a historically black college in Tennessee will be recruited in the study. Participants will include HCWs such as attending physicians and residents from all residency programs as well as nurses, midlevels, etc. within the institution. We will also recruit ancillary staff in the same departments. The survey will be distributed between Jan. 2018-Feb. 2018, and will contain 10 questions. Information on demographics, work history (age, sex, education, job category, duration of employment, training level) and TB knowledge (general information on TB) will be captured. The survey will be distributed via email through RedCap, a secure web application for creating and managing online surveys. Emails of participants will be obtained through the institution’s employee and student directory. A minimum of 200 participants will be surveyed, to obtain a power of 80% and CI of 95%. Survey will be entered into the REDCap electronic research database and entered data from participants will be checked for completeness and accuracy. Knowledge will be assessed as: poor (75%-85% correct); and outstanding (>85% correct). We will compare differences in TB knowledge of HCWs and ancillary staff and between practicing physicians and physicians in training. Data analysis will be performed using R software. The expected results are that healthcare workers have higher knowledge level than ancillary workers and that practicing physicians have higher knowledge level than physicians in training due to years of clinical experience and education. As TB continues to be one of the leading causes of death worldwide, it is important that HCWs exhibit a good level of knowledge. The findings from this study will generate data to guide TB education efforts for providers and healthcare facility ancillary staff. Results will help to clarify misconceptions about TB transmission and enhance the quality of care for patients with TB and reduce the risk of nosocomial transmission of TB.

Since the work of John Snow, scientists and medical professionals have understood that individuals develop cholera by means of consuming contaminated water. Despite the knowledge of cholera's route of infection, many countries have experienced and still experience endemic cholera. Cholera is caused by the Vibrio cholerae (V. cholerae) bacterium and presents with acute diarrhea and vomiting. If untreated, infected individuals may die due to dehydration. Cholera is a disease that most commonly affects countries with poor infrastructure and water sanitation. Despite efforts to control cholera in such countries, the disease persists. One such example is Haiti which has been experiencing a cholera outbreak since 2010. While there has been much research in the field of microbiology to understand V. cholerae, there has been comparably less research in the field of mathematical biology to understand the dynamics of V. cholerae in the environment. A mathematical model of V. cholerae incorporating a phage population is coupled with a SIRS disease model to examine the impact of vibrio and phage interaction. It is shown that there might exist two endemic equilibria, besides the disease free equilibrium: one in which phage persist in the environment and one in which the phage fail to persist. Existence and stability of these equilibria are established. Disease control strategies based on vibrio and phage interactions are discussed.

Vibrio vulnificus, an emergent human pathogen, causes septicemia with a mortality rate over 50%. Additionally, symptom onset occurs rapidly, with the incubation time for ingestion cases being around 26 hours. This, combined with the severity of symptoms has led to V. vulnificus being considered the deadliest seafood-associated pathogen, claiming responsibility for 95% of seafood-related deaths. Currently, the molecular mechanisms through which some strains of this bacteria emerge to become pathogens are unknown. The main focus of this study is to expand upon the base of knowledge surrounding this question through comparing virulence phenotypes in environmentally collected strains of V. vulnificus. Specifically, this study will evaluate the pathogenic potential of environmental isolates collected from water and oyster sources in Lee County, Florida, in lieu of the outbreak that occurred in October 2022. To test this, a variety of assays were performed. First, a phylogenetic tree was built to establish the relationships between strains. Next, to study in vitro responses, serum resistance assays and sialic acid growth curves were performed. Then, to further classify the pathogenic potential of these environmental strains, they were tested against THP-1 monocytes differentiated into macrophages for their ability to resist phagocytosis and induce apoptosis. This study found differential responses amongst the environmental isolates, with some exhibiting significant pathogenic potential and others being sensitive to all tested assays. Understanding which strains emerge as pathogens will help determine the prevalence of key virulence factors within natural populations of bacteria and provide critical data on the phenotypic outcomes of differing genotypes.