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.

Infectious diseases represent one of the leading causes of death globally. Prompt diagnosis is essential for the onset of clinical treatment but certain cases of underlying bacterial infection deep in the body can remain undiagnosed for weeks. Hidden bacterial infection is the leading cause of fever of unknown origin (FUO), which is observed in 2 % of all hospital admissions around the world. Molecular imaging of bacterial infections is the ideal non-invasive diagnostic tool, but all available probes also detect inflammation. Two targets were selected for development of bacteria-specific molecular imaging probes, namely iron-uptake pathways and peptidoglycans involved in the synthesis of the cell wall. Both, Gram-positive and Gram-negative bacteria use iron-binding molecules called siderophores to scavenge iron from their surroundings. The structural similarities between Fe3+ and Ga3+ allow siderophores to be radiolabelled with 67/68Ga and visualized by nuclear medicine techniques. The clinically proven siderophore Deferoxamine (Dfo) has a plasma half-life of only 5.5 min that does not favor its direct use as a probe. Dfo derivatives with improved pharmacokinetics properties were designed and tested on Staphylococcus aureus cultures. The ciprofloxacin and the ethyloxycarbonyl derivatives of DFO at the primary amino position were among the most successful conjugates targeting the siderophore active-transport mechanism and reaching high relative uptake rates. Furthermore, the peptidoglycan pathway of Gram-positive bacteria was in vitro targeted with vancomycin conjugated to 67Ga-Dfo which showed even higher labelling capacity than 67Ga-Dfo within a few minutes of exposure. In vitro siderophore studies remain challenging due to the lack of methods for the preparation of rigorously iron-depleted media. We developed an iron chelating method with the goal of creating iron-free growth media. / Thesis / Master of Science (MSc)

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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.

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