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Outbreaks of infectious disease are difficult to predict. Infection control relies on both preventive and reactive measures. Reactive actions are easier to implement, but less favourable in the long-term. Treatment of infectious outbreaks is dependent on the efficacy of the pharmaceuticals available. Discovery and development of new antimicrobials is surpassed by an increasing occurrence of pathogenic drug tolerance. This phenomenon is typically referenced via antibiotic resistance; however, antifungals and antivirals are also susceptible, i.e., antimicrobial resistance (AMR). Advancing our understanding of the AMR emergence and dissemination in urban waters may be used to enhance the measures of prevention control. The aim to develop early-warning systems for infectious disease was broken down into the construction of several diagnostic tools. The purpose of the thesis is to introduce new monitoring techniques for the presence of antimicrobials in urban waterways; and the subsequent evaluation of community-wide human drug consumption, over time. The objectives included the development of analytical methods for the quantitation of antimicrobials and their human metabolites; longitudinal monitoring of an urban catchment in South Africa; and design and application of correction factors for drug excretion into sewage systems. Novel analytical methods and wastewater-based epidemiology (WBE) approaches were required for the determination of drug concentration in samples from wastewater treatment plant and river systems. Longitudinal monitoring of a case study catchment area is used to establish spatiotemporal trends of antimicrobial usage, in both formal and informal settlements. Coordination and statistical review of multiple datasets are used to add context to the results, such as system flow rates; population estimates; prescription records; and pharmacokinetic data from published literature. Analytical methods were developed for the quantitation of broad- and narrow-spectrum antibiotics; one antifungal; two antiretrovirals; and several drug metabolites, via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation studies were conducted for multiple solid and aqueous matrices associated with urban waterways; enabling comprehensive analyses throughout any urban catchment. The case study location, (Eerste River catchment, South Africa) was selected due to broad socioeconomic diversity and infrastructure. The health of both formal and informal settlements could be observed over time via the community-wide monitoring technique, WBE. Spatiotemporal patterns were identified for individual antimicrobials and per drug class, including distinctions between climatic and behavioural trends. Analyte concentrations and mass loads can be used to characterise pollution and the removal efficacy of wastewater treatment plants, respectively; but correction factors for metabolism and excretion must be applied in order to accurately calculate drug consumption in a community. A systematic literature review of pharmacokinetic metabolite excretion research was conducted to determine correction factors for seventeen antimicrobials. Selection of the most favourable metabolite (unchanged/parent or structural analogue) was conducted using a UK WBE dataset against corresponding National Health Service (NHS) prescription records. Metabolite excretion percentage, stability, recovery, and biotransformation were the major variables effecting the calculation of daily intake. The efficacy of each metabolite was correlated against the catchment prescription data. These correction factors were then able to be applied to catchments without/with only partial prescription records, with a better understanding of the specific strengths and limitations. Partial records were obtained for the Eerste River catchment. Comparisons of municipal and informal settlement waste demonstrated the difference in antimicrobial consumption between communities. Population estimates for the number of individuals contributing waste to the settlement runoff were approximated from multiple sources. However, even upper estimates suggest that the antimicrobial consumption patterns vary significantly from those observed in municipal waste. Further work should include utilisation of the high-resolution semi-targeted mass spectrometry dataset to priortise the analysis of additional metabolites and transformation products. In order to develop early- warning systems for infectious disease outbreak, continuous, regular monitoring is required. A baseline for average spatiotemporal variance has been established for one region. Additional catchments and further monitoring will contribute towards the development of these systems. Newer, collaborative pharmacokinetic research would be required to improve current metabolite excretion correction factors, as well as the application of the systematic literature review methodology for other drug metabolites. Finally, the combination of chemical and biological approaches will further our understanding of the emergence and dissemination of AMR.

Biofouling is the undesired biofilm formation on surfaces at a liquid interface that interferes with the affected substrate’s function. It is a ubiquitous problem in many engineered systems in industry. Biofouling causes contamination, essential damage to materials, and impedances to crucial industrial processes. These adverse effects lead to health hazards, gross increase in energy consumption, and significant decrease in overall productivity, all of which result in higher operational costs and environmentally destructive consequences. Interest in discovering effective alternatives to conventional antimicrobial agents has gained momentum. Current anti-biofouling strategies have significant disadvantages, such as the generation of toxic by-products, indiscriminate corrosion of surrounding materials and the environment, and promotion of resistance development. Alternative methods of controlling biofouling are in high demand because present-day solutions are far from sustainable. Plant secondary metabolites are promising candidates as novel biocides because they are (i) highly effective in killing microbes while being non-toxic to humans at antimicrobially active concentrations, and (ii) safer and non-damaging to the natural environment. Herein, antimicrobial efficacies of five plant-derived compounds were assessed against various species of planktonic bacteria as well as biofilms at various maturity stages. Allyl isothiocyanate (AIT) and cinnamaldehyde (CNAD) displayed the greatest inhibitory effects against all planktonic species tested. The minimum inhibitory concentration is defined as the lowest concentration of a substance that inhibits visible microbial growth, and the MBC is defined as the lowest concentration at which 99.9% of the population is killed. AIT yielded MICs of 156.25 mg/L and MBCs of 156.25 to 312.5 mg/L, and CNAD yielded MICs of 78.125 to 156.25 mg/L and MBCs of 78.125 to 312.5 mg/L. Furthermore, 312.5 mg/L AIT and 625 mg/L CNAD successfully reduced > 80% of biofilm adhesion as compared to negative controls. AIT and CNAD were therefore further evaluated extensively. Hindered by their volatile nature and immiscibility, plant secondary metabolites typically do not reach their maximum antimicrobial capacity due to low bioavailability. Thus, they would benefit from being protected and delivered in nano-sized carriers. In this study, mesoporous silica nanoparticles (MSNs) were evaluated as carriers for AIT and CNAD delivery. In one, employment of MSNs as carriers doubled the antibacterial efficacy of free form AIT and increased kill rate of free form CNAD by six times. Furthermore, free form AIT caused ~70% of 60 day-old biofilm to detach, whereas AIT-loaded MSNs essentially removed all of the biofilm. As for CNAD, its free form had no significant effect, whereas CNAD-loaded MSNs caused ~80% reduction in biofilm biomass. MSNs were further engineered to incorporate lactose pore caps to achieve specific, on-command delivery. These MSNs were designed to respond to external stimuli intelligently, with gatekeepers that degrade only in the vicinity of certain target bacteria that are able to metabolise lactose. Capped AIT-loaded MSNs reduced bacterial viability by ~85% as compared to the negative control, while capped CNAD-loaded versions reduced viability by ~40%. This stimuli-triggered MSN delivery technology would be more sustainable than current methods because resistance development would be lowered, and the delivery vehicles could be recycled and reused. Herein, the complete AIT- or CNAD-loaded, lactose-capped MSNs delivery complex proved to be an effective and environmentally conscientious system for killing unwanted bacteria.

This thesis describes the development of new pathways towards the synthesis of novel antimicrobial (and anticancer) agents. Two synthetic protocols based on free radical chemistry are studied, which are used to access polycyclic heterocyclic compounds of potential biological importance. Both these procedures involve the generation of radicals using Bu3SnH and AIBN initiators, and the subsequent intramolecular radical cyclisation onto the imidazole ring. Radical cyclisations onto benzimidazoles and pyrroles are also described.

Due to the increase prevalence of antimicrobial resistance (AMR) antibiotic alternatives have been of great interest. Antimicrobial peptides (AMPs) and polymers like polymethacrylates that mimic AMPs are two non-traditional antimicrobial agents that have been investigated thoroughly over the years as a potential solution to the AMR problem. This study will further the understanding of the L1 peptide by investigating the role hydrophobic amino acids have on the antimicrobial activity. Biophysical and microbiological techniques were utilized to show that the L1 hydrophobic derivative showed enhanced binding to anionic lipid bilayers while maintaining low hemolytic activity. This study also elucidates the effect of charge and hydrophobicity on polymethacrylate polymers by imploring the same techniques and methods used to investigate the Ponericin L1 peptide. These results indicate that both charge and hydrophobicity have an impact on activity and selectivity. As hydrophobicity increased, so did the hemolytic activity, suggesting a balance between hydrophobicity and charge is needed to optimize antimicrobial activity as well as selectivity. Overall, the results show that these AMPs and polymethacrylates have great potential as a platform against AMR.

Bacterial plaque is a primary etiological factor of periodontitis, and a specific group of pathogens is strongly associated with this destructive inflammatory entity. Although conventional periodontal scaling and root planing usually halts the disease process, the loss of periodontal attachment continues in some patients. Aggressive and recurrent forms of periodontitis are associated with persistent infection by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. These pathogens can invade gingival epithelium and fibroblasts, allowing them to evade the host immune system and re-colonize pockets after debridement. Although the use of adjunctive systemic antibiotics to treat invasive infection by these pathogens is recommended, an agreement on the most effective regimen has not been reached. Azithromycin is not commonly used in periodontal therapy. Its favorable properties include inhibition of a broad spectrum of periodontal pathogens, a high volume of distribution, a long half-life, and relatively low incidence of side effects. Studies have indicated that human cells take up azithromycin through active transport. In this project, we hypothesize that gingival epithelial cells, fibroblasts, and neutrophils possess active transporters that accumulate azithromycin. These transporters may enhance and sustain levels of azithromycin in cellular compartments of gingiva and in gingival crevicular fluid. The accumulated azithromycin may facilitate elimination of intracellular periodontal pathogens. The chapters of this dissertation are 1) to study the mechanism by which azithromycin is transported by cells in the gingiva, and 2) to evaluate the benefits of this transport system with regards to pharmacokinetics and antimicrobial activity against intracellular A. actinomycetemcomitans and P. gingivalis. The characterization of azithromycin transport was conducted with radiolabeled azithromycin. To study the mechanism of transport, agents with similar molecular features were added to test their effects on azithromycin transport. To study the pharmacological benefits of azithromycin transport, human subjects with clinically healthy periodontal tissues were recruited. Following systemic administration of azithromycin, serum and gingival crevicular fluid were sampled and the content of azithromycin was determined by agar diffusion bioassays. The intracellular antimicrobial activity of azithromycin was evaluated in gingival epithelial cells, fibroblasts, and neutrophils. Periodontal pathogens were first internalized in these cells, and the surviving colony-forming units after antibiotic treatment were measured by growing the cellular lysates on appropriate agar plates. Our data suggest that gingival epithelial cells, fibroblasts, and neutrophils possess saturable, concentrative active transport systems for azithromycin that are shared with organic cations. The transport systems lead to high degrees of intracellular accumulation in gingival epithelial cells, fibroblasts, and neutrophils, with cellular/extracellular concentration ratios of ~20, ~11, and ~5, respectively. Azithromycin concentration in gingival crevicular fluid is significantly higher and more sustained than that in serum over 2 weeks. Accumulation of azithromycin in these cells enhances the elimination of invasive A. actinomycetemcomitans and P. gingivalis. Compared to antibiotics that are not concentrated by host cells, azithromycin exhibits either equivalent or more potent antimicrobial activities. Competitive inhibitors of azithromycin transport significantly reduce the intracellular content of azithromycin and attenuate its antimicrobial effects. Overall, this project provides a rational basis for adjunctive use of azithromycin in periodontal therapy.

There is an urgent need for the development of new antimicrobial drugs. In previous studies, inert ruthenium(II) polypyridyl complexes (Rubbn) and complexes where each metal centre is coordinatively labile (Cl-Ru-bbn-Ru-Cl) showed good antimicrobial activity. To explore the potential of using the ruthenium(II) complexes as antibacterial agents, in this thesis, a new class of dinuclear ruthenium complexes that contained one inert metal centre and one coordinatively labile metal centre Ru-bbn-Ru-Cl (Rubbn-Cl) was synthesised, and then their biomolecule binding properties, antimicrobial activity and interaction with mammalian cells examined. The biomolecule binding study of Rubbn-Cl complexes showed that the complexes will covalently bind with isolated DNA (including mononucleotides and oligonucleotides) and RNA. Rubbn-Cl complexes also showed covalent binding with the thiol-containing amino acid methionine and strong affinity to human serum albumin. Similar to Rubbn complexes, in preliminary antimicrobial tests, Rubbn-Cl complexes were effective against both Gram positive strains and Gram negative strains. Rubb₇-Cl showed significant strain selectivity, it was the only complex that was 8-fold more active against E.coli than against the two Gram positive strains. As potential antimicrobial agents, it is important to be selectively active against bacteria but less toxic to the host mammalian cells. To investigate this possibility, time course cytotoxicity assays against liver and kidney cells were carried out. The results indicated that the ruthenium complexes were more toxic towards the liver cancer cell line HepG2 than the kidney cell lines BHK and HEK-293, but less toxic compared with bacteria. Particularly, Rubb₇-Cl showed 340-fold and 99-fold differences in toxicity between BHK and E. coli, and HEK-293 and E. coli, respectively. The major uptake mechanism of Rubb₁₂ and Rubb₇-Cl in liver and kidney cells was demonstrated to be active, with a significant contribution of carrier-assisted diffusion for ΔΔ-Rubb₁₂ but some passive diffusion for Rubb₇-Cl, both of which are different from the uptake in bacterial and leukaemia cells. The ruthenium complexes elicit reactive oxygen species (ROS), and caused mitochondrial dysfunction. The cell death was determined to be from apoptosis or from membrane damage caused by the ruthenium complexes.

A series of dinuclear ruthenium(II) complexes containing labile chlorido ligands (Cl-Rubbn) have been synthesised and their potential as anticancer agents examined. Some of the Cl-Rubbn species showed good anticancer activity against breast cancer cell lines, with the Cl-Rubb₁₂ complex being four-times more active than cisplatin. The results of this study suggest that the cytotoxicity of the dinuclear ruthenium complexes is a combination of covalent and reversible binding with DNA. A series of inert mono-, tri- and tetra-nuclear polypyridylruthenium(II) complexes have been synthesised and their potential as antimicrobial agents examined. Geometric isomers of the mononuclear complexes were separated. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against a range of Gram positive and Gram negative bacteria. The linear tetranuclear complexes were generally more active, with MIC values < 1 μM against Gram positive bacteria. Of particular note, the cellular accumulation of the oligonuclear ruthenium complexes was greater in the Gram negative strains compared to that in the Gram positive strains. The mononuclear complexes [Ru(phen)(bbn)]²⁺ (phen=1,10-phenanthroline) exhibited excellent activity against Gram positive bacteria, but only the cis-α-[Ru(phen)(bb₁₂)]²⁺ species showed good activity against Gram negative species. In particular, the cis-α-[Ru(phen)(bb₁₂)]²⁺ complex was two to four times more active than the cis-β-[Ru(phen)(bb₁₂)]²⁺ complex against the Gram negative strains. The cis-α- and cis-β-[Ru(phen)(bb₁₂)]²⁺ complexes readily accumulated in the bacteria, but significantly, showed the highest level of uptake in P. aeruginosa. The antimicrobial activities of a series of di-, tri- and tetra-nuclear ruthenium complexes against a wider range of clinically relevant pathogenic bacteria were examined. The toxicity of some of these complexes against eukaryotic cells was also investigated. Results of this study indicate that these complexes are highly active against Gram positive bacteria but they were less active against Gram negative bacteria. The dinuclear Rubb₁₂complex exhibits less toxicity to liver and kidney cells when compared to the tri- and tetra-nuclear complexes. In addition, in vivo experiments demonstrate serum level concentrations for both Rubb₁₆ and Rubb₁₂-tetra were lower than the MIC values against Gram positive and Gram negative bacteria, with both complexes accumulating mainly in the liver and kidney after dosing.

Bone regeneration or augmentation is often required prior to or concomitant with implant placement. With the limitations of many existing technologies, a biologically compatible synthetic bone grafting substitute that is osteogenic, bioerodible, and provides spacing-making functionality while acting as a drug delivery vehicle for bioactive molecules could provide an alternative to ‘gold standard’ techniques. In the first part of this work, calcium sulfate (CS) space-making synthetic bone grafts with uniformly embedded poly(β-amino ester) (PBAE) biodegradable hydrogel particles was developed to allow controlled release of bioactive agents. The embedded gel particles’ influence on the physical and chemical characteristics of CS was tested. Namely, the compressive strength and modulus, dissolution, and morphology, were studied. All CS samples dissolved via zero-order surface erosion consistent to one another. Compression testing concluded that the amount, but not size, of embedded gel particles significantly decreased (up to 75%) the overall mechanical strength of the composite. Release studies were conducted to explore this system’s ability to deliver a broad range of drug types and sizes. Lysozyme (model protein for larger growth factors like bone morphogenic protein [BMP]) was loaded into PBAE particles embedded in CS matrix. The release of simvastatin, a small molecule drug capable of up regulating BMP production, was also examined. The release of both lysozyme and simvastatin was governed by dissolution of CS. The second part of this work proposed a bilayered CS implant. The physical and chemical properties were characterized similarly to the CS composites above. Release kinetics of directly loaded simvastatin in either the shell, core, or both were investigated. A sequential release of simvastatin was witnessed giving foresight of the composite’s tunability. The sequential release of an antibacterial, metronidazole, loaded into poly(lactic-co-glycolic acid) (PLGA) particles embedded into the shell along with directly loaded simvastatin either in the shell, core, or both layers was also observed. Through controlled release of bioactive agents, as well as a tunable layered geometry, CS-based implants have the potential to be optimized in order to help streamline the steps required for the healing and regeneration of compromised bone tissue.

The aim of this thesis was to obtain antimicrobial compounds from marine eukaryote-associated bacteria. The unique environment, present on the surfaces of marine eukaryotes, creates conditions that promote and favour the production of bioactive compounds, such as antimicrobials, by giving the producers a clear advantage in the competition for nutrients. This study has demonstrated the abundance of antimicrobial producing bacteria on two marine algae, Delisea pulchra and Ulva australis. Two antimicrobial compounds, violacein and tropodithietic acid (TDA), have been successfully purified and chemically identified from two different bacterial isolates. Moreover, the production of multiple bioactive compounds was observed for both these bacteria. This study also made an attempt to understand the role of the antimicrobial compounds for the producer organisms. Consequently, the effect of violacein on biofilm formation, as well as the possible role of TDA in the defence of both the producer bacterium and the host, have been proposed. The importance of environmental conditions for the expression of bioactive compounds has also been demonstrated, for example, by showing the necessity of high iron concentrations for the production of bioactives in isolates Ul56 and 0245. Notably, despite the absence of a close phylogenetic relationship between these two isolates, they have shown similar trends in terms of production of bioactive compounds. For the first time, this work also described the construction and analysis of a large insert DNA library in E. coli using genomic DNA from a collection of cultured isolates with demonstrated antimicrobial activity. This approach proved to be successful and led to a substantial increase in the positive hit rates in the functional screening of the library, and provided invaluable information concerning the genes, potentially involved not only in the biosynthesis, but also in the processes associated with the production of bioactive compounds, such as transport and resistance.

Community-acquired urinary tract infections (CA-UTI) are common in young women. Reports of increasing resistance to the antimicrobial drugs commonly prescribed to treat CA-UTI, evidence of wide-spread dissemination of strains of multi-drug resistant Escherichia coli that can cause community outbreaks and expanding appreciation of the importance of the rational use of antibiotics are challenging the traditional management of this disease. Two population-based studies were performed to investigate the epidemiological features of CA-UTI with an emphasis on the antimicrobial resistance of causative bacteria. An eight-year retrospective cohort study was conducted in a large health maintenance organization to identify changes in uropathogen etiology and antimicrobial resistance and in empirical antimicrobial treatment practices and outcomes. A cross-sectional study was performed in a university population to investigate the relationship between changes in the prevalence of genotype-based clonal groups of uropathogen E. coli and the prevalence of antimicrobial resistance. From 1998 through 2005, less than 20% of the Escherichia coli causing uncomplicated CA-UTI (UCA-UTI) were resistant to the first line empirical treatment antimicrobial, trimethoprim/sulfamethoxazole (TMP/SMX). No trends were detected in the proportions of Escherichia coli that were resistant to TMP/SMX or to nitrofurantoin. In contrast, a small but steady increase in the proportion of Escherichia coli that were resistant to ciprofloxacin was observed. Over the same period of time, the use of ciprofloxacin as empirical treatment for UCA-UTI steadily increased while the use of TMP/SMX decreased. No sustained decreases in treatment failure or in microbiologically incompatible treatment were detected. Thus TMP/SMX remains a viable empirical treatment for women with UCA- UTI in these populations. Molecular typing of Escherichia coli causing CA-UTI revealed that the prevalence of antimicrobial resistance was influenced by a small number of Escherichia coli clonal groups. This suggests that the prevalence of antimicrobial resistant UTI in a community is not only the result of community prescribing practices and individual antimicrobial use but can be significantly impacted by the introduction and circulation of strains of uropathogens that are already drug resistant. Thus, strategies developed to maintain the usefulness of empirical treatment options for CA-UTI must include interventions that target sources of antimicrobial resistant uropathogens.

In general, a dressing is used to protect and help heal wounds. There are several types of dressings on the market such as hydrocolloid, hydrogel, and medicated dressings. One technique for making a dressing is electrospinning, which is a very simple procedure used to produce fibers. Due to much smaller fiber-diameters than produced with the conventional technique, the fibers from electrospinning have unique properties: high porosity and high surface areas, which are advantageous for wound healing. In this research, the fibers were electrospun using polyurethane, Tecophilic® or Tecoflex®, with various additives. First, multilayer transdermal electrospun dressings, four and five-layers, were developed that allowed for the controlled release of nitric oxide (NO) from a NO2-/ascorbic acid system encapsulated in polymer nanofibers. The amount of NO released from each wound dressing was investigated. Both the four and five-layer dressings were tested for morphology of fibers, water absorption, nitrite distribution, NO release profile after sterilization by gamma radiation, and stability. In the case of the four- layer transdermal dressing, the dressing was tested in diabetic, periodontal, and leishmaniasis patients. Furthermore, the color change of dressing was investigated. Tecophilic® was also spun with an antimicrobial agent, which was added to the Tecophilic® solution to electrospin an antimicrobial dressing. The morphology of fibers was tested using an optical microscope and the water absorption, uniformity, and percent extraction of dressing were also determined. In addition, the efficiency of the antimicrobial agent in the dressing was determined according to SN 195920-1994 and ASTM 2149-01. Another NO-releasing dressing was developed employing the NO donor molecules, sodium salt of linear polyethlenime NONOates (LPEINO-Na) and calcium salt of linear polyethlenime NONOates (LPEINO-Ca), which were electrospun with Tecophilic®. The NO release profiles for the LPEINO fibers were generated using a nitric oxide analyzer (NOA) and distribution of the particles in the dressing was examined. Moreover, the amount of NO released after patch storage for 1 and 2 months was determined.

lnappropriate use of antimicrobials has gradually increased and results in heath, economic and ecological problems. Antimicrobial use evaluation is pivotal in controlling the problems. lt is employed as a measurement of experimental research or surveillance of the problem trends. The current review focuses on hospital settings. lt comprises quantity of consumption and methods to evaluate quantity and quality of utilization.