Hospitals worldwide are becoming increasingly plagued by antibiotic-resistant pathogens; concomitantly, the number of patients who die from antibiotic-resistant pathogens is increasing. The rise of multi-drug resistant (MDR) pathogens has rendered many antibiotics obsolete. The streptogramin and aminoglycoside antibiotics are drugs of last resort against life-threatening, MDR Gram-positive (e.g., methicillin resistant Staphylococcus aureus (MRSA)) and -negative (e.g., Pseudomonas aeruginosa) pathogens, respectively; however, as microbial drug resistance continues to emerge, the efficacy of these two important drug classes is decreasing. One of the most common mechanisms by which pathogens become resistant to streptogramin and aminoglycoside antibiotics is enzymatic inactivation: the Vat and AAC(3) acetyltransferases are employed by pathogens to inactivate streptogramin and aminoglycoside antibiotics, respectively. There currently is a dire need to not only develop new antibiotics, but to find new, creative strategies to outwit microbial resistance mechanisms. One of these strategies is to rescue the activity of antibiotics through the discovery of antibiotic adjuvants. In the current study, adjuvants which rescue the activity of streptogramin and aminoglycoside antibiotics through inhibition of the resistance acetyltransferases, VatD and AAC(3)-Ia, have been discovered—through the development of a cell-based screening method, we have found the first inhibitors of VatD, as well as of AAC(3)-Ia and its homologues, AAC(3)-Ib, AAC(3)-Ic, and AAC(3)-Id. We have demonstrated that streptogramin and aminoglycoside resistance can be reversed both in vitro and in vivo by the protein kinase inhibitors, GW5074 and rottlerin. Steady state kinetics revealed these compounds to inhibit VatD and AAC(3) enzymes mainly through noncompetitive or mixed mechanisms. This study has also demonstrated that eukaryotic kinase inhibitor libraries may be repurposed for the discovery of not only new antibiotic adjuvants, but also new antimicrobial targets. The inhibitors described herein may someday serve as effective adjuvants of streptogramin and aminoglycoside antibiotics. / Thesis / Doctor of Philosophy (PhD)

Despite recent advances, radiopharmaceuticals to detect and characterize bacterial infections have a number of limitations. Many of the clinically approved radiopharmaceutical agents are not specific to bacterial infections and accumulate at lesions of inflammation. Hence, new approaches are necessary to detect bacteria with high specificity and selectivity. A library of desferrioxamine B (DFO) derivatives were prepared to create radiolabeled siderophores to create a bacteria-specific imaging probe by exploiting the mechanism bacteria use to scavenge iron, which plays a key role in bacterial growth and biofilm formation Compounds were synthesized using two convenient carbamate forming strategies in 30% to 92% yield. The cold and radioactive gallium (67Ga) complexes were prepared and characterized and their uptake by S. aureus bacteria were assessed in vitro and in vivo. In vivo studies revealed that 67GaDFOethoxycarbamate had uptake comparable to GaDFO that was blockable, showing the compound was actively taken up via the siderophore pathway. In vivo studies in a mouse model resulted in a good infected to non-infected thigh ratio (11:1) and non-specific uptake by the GI tract. Bioorthogonal chemistry was also explored as an approach for imaging infection using trans-cyclooctene (TCO) functionalized vancomycin and a tetrazine functionalized 67GaDFO (67GaDFO-Tz) complex.2,3 In vitro results revealed that allowing vancomycin-TCO to bind S. aureus prior to the addition of 67GaDFO-Tz (pretargeting) showed higher (63%) uptake than with a conjugate formed prior to incubation with the bacteria (direct targeting, 28%). For the bioorthogonal approach, the distribution of the 67GaDFO-Tz was assessed in a S. aureus infection murine model, which showed significant uptake of 67GaDFO-Tz in the GI tract 1 h post intravenous injection. However, uptake in the infected joint was evident at 71 h post infection. The data suggests targeting bacteria using TCO-labeled antibiotics and radiolabeled tetrazines is a feasible strategy, but that further optimization of the vancomycin injection dose and injection time are necessary. / Thesis / Master of Science (MSc)

Antimicrobial resistance (AMR) is a One-Health (human, animal, environment) challenge that requires collaborative, interdisciplinary action. Comparable surveillance data are needed to effectively inform policy interventions aimed at preventing the spread of AMR. Environmental monitoring lags behind that of other One Health sectors and is in need of agreed upon targets and standardized methods. A challenge is that there are numerous microorganisms, antibiotic resistance genes (ARGs), and mobile genetic elements and corresponding methods that have been proposed. In this dissertation, a framework for AMR monitoring of aquatic environments was developed through a combination of literature review and stakeholder input, via surveys and a workshop. Through this process, three targets were selected for standardization: the sulfonamide resistance gene (sul1), the class 1 integron integrase gene (intI1), and cefotaxime-resistant Escherichia coli. Quantitative polymerase chain reaction (qPCR)- and culture-based protocols were developed and pilot tested in two independent laboratories on a set of six water matrices: wastewater, recycled water, and surface water from six different wastewater utilities engaging in water reuse located in five states across the USA. The impact of wastewater treatment and advanced water treatment processes was examined in terms of removal of these targets. Finally, qPCR and culture methods were used to examine the relationship between sul1, intI1, E. coli, and fecal indicators in private household wells across four states in the Southern USA that were identified as susceptible to storm events. The overall findings provide a useful baseline occurrence of the proposed AMR monitoring indicators across a range of water types and protocols that are accessible to water utilities. / Doctor of Philosophy / Life-saving drugs and treatments are failing at an increasing rate because of antimicrobial resistance (AMR). Antimicrobials, such as antibiotics, are a double-edged sword, because they are an effective weapon for killing disease-causing pathogens, but the more they are used the greater the likelihood that microbes that are resistant to them will survive, reproduce, and spread. National action plans for AMR have been created by a majority of countries, emphasizing the importance of antibiotic stewardship and other mitigation strategies. However, numerous data gaps need to be addressed in order to identify strategies that are most likely to be effective and to implement them. Environmental surveillance, including wastewater influent, wastewater effluent, and surface water, could prove an informative means to track AMR trends with time and relate them to human activities and corresponding mitigation efforts. The purpose of this dissertation was to develop a framework for AMR surveillance of aquatic environments and to test it across an array of sample types. We considered an array of possible culture- and DNA-based targets from available scientific literature and engaged experts and stakeholders in narrowing down the list to options that were both informative and feasible. We developed protocols for quantifying an antibiotic resistance gene (sul1), a mobile genetic element that has been implicated in the spread of multi-antibiotic resistance (intI1), and an extended spectrum beta-lactamase (ESBL) producing form of Escherichia coli. We compared the methods between two independent laboratories on untreated wastewater, treated wastewater, recycled water, and surface water collected from six locations across five states. We additionally did a survey of private household well water that was hypothesized to be vulnerable to contamination due to storms and lack of resources for maintenance. The results of this research can help to support environmental monitoring of AMR across the US and globally.

Opportunistic premise (i.e., building) plumbing pathogens (OPPPs) and antibiotic resistant bacteria are emerging microbial concerns in drinking water. OPPPs, such as Legionella pneumophila, are the leading cause of drinking water disease in many developed countries. Contributing factors include the relative success in controlling fecal pathogens, the presence of complex building plumbing systems that create habitats for OPPPs, and the relative resistance of OPPPs to disinfectants, and aging populations that are susceptible to infection. Concurrently, drinking water is increasingly being scrutinized as a potential environment that is conducive to horizontal gene transfer of antibiotic resistance genes (ARGs), selection pressure for enhanced survival of resistant bacteria, and a route of transmission of antibiotic resistant pathogens. While maintaining a disinfectant residual is an established approach to controlling OPPPs in premise plumbing, some studies have indicated that co-resistance and cross-resistance to disinfectants can increase the relative abundances of resistant bacteria and ARGs. Thus, there may be trade-offs to controlling both OPPPs and antibiotic resistance in premise plumbing that call for controlled study aimed at optimizing residual disinfection application for this purpose. A critical review of the scientific literature in Chapter 2 revealed that premise plumbing is a biologically and chemically complex environment, in which the choice of pipe material has cascading effects on water chemistry and the corresponding premise plumbing microbiome. This, in turn, has broad implications for the control of OPPPs, which need to be elucidated through controlled experiments in which worst case premise plumbing conditions are held constant (e.g., warm temperature), while other variables are manipulated. Chapter 3 introduces the convectively-mixed pipe reactors (CMPRs) as a novel low-cost, small footprint approach to replicably conduct such experiments. The CMPRs were demonstrated to effectively simulate key chemical and biological phenomena that occur in distal reaches of premise plumbing. In Chapter 4, the CMPRs were leveraged to study the interactive effects of four disinfectants (chlorine, monochloramine, chlorine dioxide, and copper-silver ionization) and three pipe materials (PVC copper, and iron). The CMPRs were inoculated with two antibiotic-resistant OPPPs: Pseudomonas aeruginosa and Acinetobacter baumannii. It was found that pipe-material (PVC or PVC combined with iron or copper) profoundly impacted the water chemistry in a manner that dictated disinfection efficacy. In Chapter 5, we applied shotgun metagenomic shotgun sequencing to evaluate effects of the combination of pipe material and disinfectant type on the wider microbial community, especially their ability to select for or reduce ARGs. In Chapter 6, we used CMPRs and metagenomic sequencing in a study comparing Dutch drinking water practices to our prior testing in an American system. Dutch drinking water is of interest because of lack of historical use of disinfectants was hypothesized to result in a microbial community that is relatively depleted of ARGs or mobile genetic elements, which can enhance spread of ARGs as disinfectants are applied. Generally, it was found that OPPPs required higher doses of disinfectants for inactivation than the general microbial community, sometimes concentrations approaching the regulatory limits in the US (e.g., 4 mg/L of total chlorine). Even successful reductions were modest, typically ~1-log, and failed to eliminate either P. aeruginosa or A. baumannii. Moreover P. aeruginosa, A. baumannii, and non-tuberculous mycobacteria varied substantially in their preference for pipe material and susceptibility to disinfectants. We found that disinfectants tended to increase the relative abundance of OPPPs, ARGs, and mobile genetic elements. Disinfectants were sometimes associated with net increases in levels of these pathogens and genes when applied at low levels (e.g., 0.1 mg/L of monochloramine), which effectively acted to reduce competition from less resistant and non-pathogenic taxa. When a low dose of monochloramine was applied to PVC CMPRs in the US, we estimated from metagenomic sequencing data that this water contained roughly 100,000 cells per milliliter of taxa known to contain pathogenic members. The Dutch drinking water exhibited more diverse microbial communities and lower relative abundances of taxa containing pathogens. ARGs were two times proportionally more abundant in CMPRs operated in the US without disinfectant than in the corresponding CMPRs operated in the Netherlands. The findings of this dissertation can help to optimize the application of in-building disinfectant addition for addressing concerns related both to OPPPs and antibiotic resistance. The studies herein highlight the necessity of developing comprehensive OPPP and antibiotic resistance control strategies that emphasize not just disinfectant dose, but other key control parameters such as contact time, hydraulics, and temperature. The functional diversity of OPPPs, antibiotic resistant bacteria, and the background premise plumbing microbiome further necessitates broad, holistic programs for monitoring and control. / Doctor of Philosophy / Efforts to provide safe drinking water face two emerging threats: the rise of pathogens that thrive in the plumbing environment that delivers water to the tap and the rise of antibiotic resistance. In the US and many other parts of the world, opportunistic pathogens are the predominant agents responsible for disease spread by tap water. Opportunistic pathogens tend to infect aged or immunocompromised individuals (hence, 'opportunistic') and grow well in in-building plumbing. Globally, antibiotic resistance is on the rise and becoming a fundamental threat to modern medicine. Pathogenic bacteria become resistant to antibiotics used to treat infections when they acquire antibiotic resistance genes (ARGs), which can happen either by mutation or from other resistant bacteria sharing ARGs. Overuse or misuse of antibiotics can impose selection pressure that stimulates horizontal gene transfer and enhance survival of bacteria that are resistant. Prior studies have suggested that under some circumstances, disinfectants used to control pathogens in drinking water can also select for antibiotic resistant bacteria. Thus, the overarching goal of this research was to optimize the type and dose of disinfectant used, depending on building-level factors such as pipe material, for effectively controlling proliferation of both opportunistic pathogens and antibiotic resistance. This dissertation largely focuses on in-building plumbing systems, which are home to potentially tens of thousands of bacterial cells per milliliter of water or per square centimeter of internal pipe surfaces. These bacteria interact not only with each other and other microbes, but also with features of the plumbing environment, such as the water chemistry or the pipe materials. Building plumbing systems are highly intricate ecosystems that can undermine the effectiveness of disinfectants provided by utilities. One major contribution of this research is the development of the convectively-mixed pipe reactors (CMPRs) as a simple and easy-to-use test system that recreates combinations of features of interest encountered in in-building plumbing. We applied the CMPRs to study two common residual disinfectants (chlorine and monochloramine) supplied by water utilities, and two other disinfectants (chlorine dioxide and copper-silver ionization) which are commonly dosed by building operators, especially in hospitals and other buildings housing individuals susceptible to infection. These four disinfectants were applied to CMPRs consisting of PVC, copper, and iron pipe. Chemical, culture, and DNA methods were used to understand how these disinfectants affected the microbes and their ecology. We then took the opportunity to set up CMPRs in the Netherlands, where there has been no historical exposure to chlorine because their water quality regulations emphasize limiting nutrients in the water and elevating the hot water line temperatures as means to control microbial growth. The CMPRs effectively produced worst-case plumbing scenarios, where opportunistic pathogens were especially difficult to control through residual disinfection. Dosed disinfectants tended to be no longer measurable in the water after five hours. The CMPRs also showed that the disinfectant most effective for one pathogen could be the least effective for another. If doses were applied near regulatory limits, the concentrations of pathogens and antibiotic resistance genes decreased. However, opportunistic pathogens tended to survive better than background populations of bacteria. Bacteria carrying ARGs also survived some disinfectant conditions better as well. Thus, if doses were applied at levels that could inactivate some microbes, but not the opportunistic pathogens, pathogen abundances sometimes increased. These results were largely confirmed in the experiment with Dutch drinking water. Here, chlorine appeared to be more problematic than monochloramine in terms of enriching pathogens and antibiotic resistance. We also noted that Dutch waters garnered more diverse microbial communities, with fewer DNA markers for pathogens and antibiotic resistance. In general, this research takes a key step towards optimizing application of residual disinfectants for control of both opportunistic pathogens and antibiotic resistance. Because disinfectants can have negative impacts on drinking water microbial communities when supplied insufficiently, it is important that the other features of in-building plumbing, such as the selection of pipe material or the hydraulics, facilitate disinfectants reaching all portions of plumbing and at the necessary concentrations. It is recommended that the selection process for disinfectant type and dose considers the plumbing materials and other conditions such that disinfection can be aimed towards controlling multiple opportunistic pathogens, which can vary in their susceptibility, and antibiotic resistance.

Medical implants constitute an essential advancement in modern medicine, often restoring or replacing functionality to failed organs. Whether a medical implant is temporary or permanent, medical implants carry the risk of implant failure due to an infection. Implant-associated infections (IAI) are challenging to treat and often result in increased medical costs, prolonged hospital stays, implant failure, and, in some instances, severe infections that can lead to amputations, sepsis, or mortality. Eradicating an IAI can be challenging since bacteria can form biofilms on the implant’s surface. The biofilms comprise an extracellular matrix protecting the bacterial cells against systemic antibiotics and the host’s immune system. Treating an IAI usually entails a broad range of antibiotic treatment and surgical procedures for tissue debridement or implant replacement. For the reasons stated above, scientists and engineers continue to develop technologies to protect the surface of medical implants against infections. Amongst the new technologies, Liquid-Infused Surfaces (LIS) are renowned for their repellent and anti-fouling properties created by tethering a stable liquid layer onto the surface. However, many challenges remain to adopt this technology for implantable devices. For instance, the high repellent properties can hinder implant-tissue interaction and discourage proper integration with the body. Furthermore, the stable liquid layer is contingent on the surface properties of the coated material. In other words, the long-term stability of these coatings may be compromised if the surface chemistry is covered by biological processes such as biofilm formation from adherent bacteria. This thesis aims to expand on the applications of LIS coatings and enhance their properties for implantable materials. This thesis reviews different types of antibiotic surface coatings and further examines LIS technologies as a viable antibacterial coating for medical implants. Then, three novel multifunctional LIS coatings are presented. The first developed coating enhanced the antibacterial properties of the coating by adding bactericidal agents within the LIS coating. The developed antibiotic liquid-infused coating not only repelled bacteria but also lysed bacteria upon contact. The second coating was designed to promote tissue integration. This multifunctional coating promoted cell deposition and proliferation while remaining repellent toward bacteria, while the conventional LIS coating displayed poor cell availability. Lastly, a collagen-bacteriophage conjugated liquid-infused coating was developed to promote tissue integration while having a two-tier layer of antibacterial protection. This coating was tested in a mouse sepsis model and prevented mortality of all mice, with other groups as high as 90% mortality. These coatings constitute essential steppingstones to bring LIS technology to medical implants. / Dissertation / Doctor of Philosophy (PhD) / Implant-associated infections (IAI) remain a significant problem in modern medicine. IAIs are challenging to treat and often result in increased medical costs, prolonged hospital stays, implant failure, and, in some instances, severe infections that can lead to sepsis or mortality. For these reasons, new technologies have been developed to protect the surface of medical implants against infections. Amongst the new technologies, Liquid-Infused Surfaces (LIS) are renowned for their repellent and anti-fouling properties created by tethering a stable liquid layer onto the surface. This thesis aims to expand on the applications of LIS coatings and enhance their properties for implantable materials. This thesis reviews different types of antibiotic surface coatings, examines LIS technologies, and presents three novel multifunctional LIS coatings. The newly developed coatings enhance the LIS coatings through the addition of antibacterial properties and biomolecules to promote tissue integration.

Infections caused by the organism Staphylococcus aureus are one of the most common causes for community-associated and healthcare-acquired infections (HAI). Isolates of this bacterium found within the healthcare setting often demonstrate a higher prevalence of antibiotic resistance making these infections difficult to treat. Historically, considerable focus has been placed on methicillin-resistant S. aureus (MRSA), which are strains resistant to β-lactam antibiotics like penicillin, oxacillin and cephalosporins; however, methicillin-sensitive (MSSA) strains may also possess resistance to several first-line antibiotics. Resistance to antibiotics can be acquired through horizontal gene transfer (HGT) by means of mobile genetic elements or by random DNA mutations as product of DNA replication. Bacteria have elucidated these mechanisms to defend themselves from antibiotics and one cause that promotes resistance is the inappropriate use or prescription of antibiotics to treat infections, i.e., using antibiotics to treat COVID-19. Through the SARS-CoV-2 pandemic, the CDC reported an increased prescription for antibiotics, similarly, other previous studies reported that antibiotics were part of treatment plant in some patients with COVID-19. The aim of this thesis is to study the differences in antibiotic resistance profiles of Staphylococcus aureus strains collected from carriage and disease samples at Nemours Children's Hospital in Orlando, FL from 2019-2022. The focus will be on comparing the susceptibility of methicillin-sensitive and methicillin-resistant strains to various antibiotics. The results will provide clinicians with valuable information that will allow for better treatments and consideration for antibiotic use when creating a treatment plan for patients.

This thesis consists of two chapters, each of which is a unique research project. Chapter 1 is focused on the synthesis and biological evaluation of novel sideromycin antibiotics. Sideromycins are bifunctional “Trojan Horse” molecules that have an iron chelator “siderophore” moiety covalently bound to an antibiotic. Such molecules exploit existing bacterial mechanisms for obtaining iron from their environment. Antibiotics that would typically not pass Gram-negative membranes are allowed access via siderophore transporter proteins. This project utilized a siderophore that has not previously been used in this capacity. The synthesis and biological evaluation of multiple sideromycin conjugates is reported. Chapter 2 describes the chemical synthesis of coumarin natural products using a synthetic process recently developed in the Magolan laboratory that enables the efficient prenylation of phenols. These natural products are molecules of biological interest in various capacities but are rare and difficult to isolate from their plant sources. They have also previously been cumbersome to make via chemical synthesis. The chemistry described herein constitutes an inexpensive and efficient process to produce these compounds that is superior to previously known methods. / Thesis / Master of Science (MSc) / This thesis is divided into two chapters. The first chapter is focused on the development of new sideromycin antibiotics. Sideromycins are “Trojan Horse”-like antibiotics that exploit the mechanisms of Gram-negative bacteria for obtaining iron, an essential nutrient, to enable antibiotic entry. This chapter details the synthesis of molecules that attach functionalities called “siderophores” to antibiotics, enabling them to be “smuggled” into Gram-negative microbes. This project uses a siderophore not previously utilized in sideromycin research. The second chapter is focused on the chemical synthesis of rare natural products that are phenols with prenyl substituents. Many such compounds are plant-derived and have potential for biomedical use. However, difficulty in isolating them makes them prohibitively expensive in the purity and quantity required for research. They are also challenging to make synthetically. This chapter details the application of a recently discovered process in the Magolan laboratory to synthesize coumarin-containing prenylated phenolic natural products.

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