This thesis describes phytochemical studies on ten of the genes Hypericum and the evaluation of their antibacterial activity. Bio-assay guided fractionation using various chromatography techniques was used in this study. Eighteen compounds were isolated and their structures were characterised by extensive 1- and 2- dimensional NMR. Antibacterial includes Mycobacterium. bovis BCG (M bovis BCG), Mycobacterium. tuberculosis H37Rv (M tuberculosis)and panel of Staphylococcus aureus strains. Minimum inhibitory concentration (MIC) assay was used to evaluate antibacterial activity of the plant extract and isolated compounds. Enzymatic inhibition of ATP-dependent MurE ligase from M tuberculosis was assayed using a colorimetric phosphate detection method, mammalian microphage cell toxicity was also evaluated of interested isolated compounds. The investigation of H acmosepalum let isolation of eight compounds including β-sitosterol, hypercalin B and lupeol from hexane extract. Its chloroform extract gave hyperenone A, 1,7- dihydroxyxanthone and 2-Hydroxyxanthone. Catechin and epicatechin were isolated from methanol extract. Hyperenone A and hypercalin B exhibited antibacterial activity against multidrug-resistant strains of Staphylococcus aureus, with minimum inhibition concentration values ranging from 2-128 μg/ml, to 0.5-128 μg/ml., respectively. Hyperenone A showed growth inhibition against M tuberculosis H37Rv and M bovis BCG at 75 ug/ml. and 100 ug/ml., Neither hyperenone A nor hypercalin B inhibited the growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells. Both compounds were tested for their ability to inhibit the A TP-dependent MurE ligase of M tuberculosis, a crucial enzyme in the cytoplasmic steps of peptidoglycan biosynthesis. Hyperenone A inhibited MurE selectively whereas hypercalin B did not have any effect on enzyme activity. Stigmasterol and tritrpenes were isolated from hexane extract of H patulum. Fractionation of methanol extract of this species afforded a series of flavanoid derivates including quercetin, rutin and flavanoid glycosides. All these isolated compounds were inactive against S. aureus at 256 μg/ml., Fractionation of methanol extract of H frondosum yielded mixture of catechin and epicatechin The hexane extract of the root of H olympicum led the isolation of two new tri-prylenated xanthones derivatives. These metabolites were active against S. aureus and MIC values ranged from 16-32 μg/ml. to 32-64 μg/ml. respectively. Chloroform extract led isolation of pentenoic acid.

In this study, an in-house built atmospheric. pressure non-thermal plasma jet has been investigated for its potential utilisation as a new alternative antimicrobial tool for a variety of medical applications. Anti - biofilm activity of this plasma jet has been evaluated against biofilms of a selected panel of bacterial species, grown on different abiotic surfaces, where complete eradication of all tested bacterial biofilms was achieved after relatively short plasma exposures of up to 10 minutes. Multiple approaches of cell viability evaluation were adopted to show the nature, extent and distribution of the remarkable anti-biofilm activity of the plasma jet including colony counting, XTT metabolic assay, scanning electron microscopy examination and differential Live/Dead fluorescent staining followed by confocal laser scanning microscopy examination. Antibacterial efficacy of the plasma jet has also been evaluated against similar bacterial species in their planktonic mode of growth where plasma exposures even shorter than those required for biofilm eradication were sufficient to cause complete inactivation of these planktonic bacteria. Such excellent bactericidal activity resulted from the ability of plasma exposure to mediate an oxidative damage to multiple cellular targets including cellular membrane, DNA and proteins of bacterial cells. However, damage of cellular membrane and the resultant disruption of its integrity and permeability were shown to be the primary rate-determining step in the plasma mediated bacterial cell death. Furthermore, in depth investigation of the plasma- mediated bacterial destruction mechanism has been carried out to identify the plasma-produced reactive species that were responsible for mediating its bactericidal activity. Based on the findings of this study, a hypothesis was formulated to describe the mechanism of bacterial cell destruction after plasma exposure. This hypothesis assumed a two-part mechanism; one part was a rapid H20 2-dependent mechanism associated with Fenton's or Fenton's-like reaction that was catalysed by metal ions released from the bacterial cells initially damaged by another proposed H20 2 - independent mechanism.

Quinolones, and especially fluoroquinolones, are one of the largest classes of antibacterial agents used worldwide. Thus, the risk of emerging resistance and toxicity increases the need for new and improved agents and synthesis methodologies. Solid phase organic synthesis has become an important technique in drug design. This research aimed to establish the advantages of solid phase syntheses in the preparation of fluoroquinolones antibacterial agents. In the present investigation, several trials using both solution and solid phase syntheses were attempted for the preparation of fluoroquinolones using the Vilsmeier reaction. Merrifield resin and p-nitrophenyl carbonate Wang resin were used in the solid phase synthesis. A number of fluoro- and non-fluoro compounds were investigated, along with a wide range of catalysts and conditions. The Vilsmeier approach had limited applicability in quinolone synthesis. On the other hand, the Vilsmeier reaction with oxalyl chloride produced a dimer product (bis-amide). Instead of the desired target quinolone product, we identified two bis-fluoroinated derivatives, N,N'-bis-(4-fluoro- 3-nitrophenyl) oxalamide and N,N'-bis-(3-chloro-4-fluorophenyl) malonamide in solution phase. Recently, Sechi et al (2008) reported an analogous non-fluorinated dimer product as a potential HIV-1 integrated inhibitor. Elemental analysis, mass spectrometry (MS), Fourier transform infra red spectroscopy (FT-IR), 'H and 13C nuclear magnetic resonance spectroscopy (NMR) and heteronuclear multiple quantum correlation (HMQC) were used to identify the structures of all newly synthesised compounds. Biological evaluation for the selected 12 compounds synthesised in this study showed a significant biocidal effect over the standard nystain and nalidixic acid. The relationships between the bioactive effects minimum inhibitory concentration (MIC), physicochemical parameters includes, partition coefficient, thermal behaviour, melting point, solubility and FT-IR spectroscopy have been investigated. The 7-bromoquinolone analogue, 7-bromo-6-(N-benzylpiperazin-1-yl)-4-oxo-3- quinolone carboxylic acid 91 showed the high potency as antibacterial inhibitor in addition to a significant effect on vitiligo phototherapy treatment. In addition, quinolone derivative analogues were synthesised and investigated as amylolytic agents towards some Aspergillus fungi.

Antibiotic resistance is a worldwide problem and has led to the emergence of the so-called 'superbugs' which are resistant to most of the clinically used antibiotics, and thus there is an urgent need for new antibiotics with new structures and/or unexploited modes of action. Pacidamycins, secondary metabolites produced by Streptomyces coeruleorubidus, possess a unique scaffold. These compounds are active against the superbug Pseudomonas aeruginosa through inhibiting translocase I; this represents a clinically-unexploited mode of action. This thesis describes the research carried out to elucidate and harness the biosynthetic flexibility of pacidamycin for generation of new analogues and to probe the biogenesis of its unique structural features. The system's inherent flexibility at both the C and N termini of the peptide was addressed and led to the generation of halogenated pacidamycins which were assessed for their antibiotic activity. The biosynthetic precursor for the nonproteinogenic amino acid m-tyrosine was identified as phenylalanine through feeding of synthesised [I, 2, 3, 4, 5_2H]_ phenylalanine to the pacidamycin producer. Biosynthesis of the pacidamycin nucleoside motif was studied through feeding of labeled precursors, mutant generation, metabolite screening of the mutant strains and chemical complementation with proposed biosynthetic intermediates that were synthesised for this purpose. Genes involved in the generation of the nucleoside were cloned and heterologously expressed, and the purified proteins were shown to be capable of the in vitro synthesis of the nucleoside motif. The nucleoside motif was shown to be derived from uridine by the catalysis of three enzymes Pac l l , Pac5 and Pac13 .

uinolones are a family of drugs used to treat bacterial infections by targeting bacterial type II topoisomerases (TOP2s), DNA gyrase and topoisomerase IV. Recent studies have shown that quinolones can cause genotoxicity in mammalian cells. Genotoxicity occurs when an agent causes damage to the genetic apparatus of a cell. Due to the similarities between the mammalian and bacterial TOP2 enzymes it is thought that the quinolones are targeting mammalian TOP2 to produce their genotoxic response. The aim of this study was to investigate if quinolone genotoxicity involves mammalian TOP2. Using the micronucleus assay, the genotoxicity of two quinolones ciprofloxacin and gemifloxacin was tested in three Nalm-6 cell lines containing varying amounts of TOP2. With ciprofloxacin only the Nalm-6TOP2A+/- cells showed genotoxicity, whereas for gemifloxacin only the Nalm-6 WT cells showed genotoxicity, suggesting that for gemifloxacin the removal of TOP2A or TOP2B lowers the genotoxicity of the quinolone. A selection of quinolones were tested using the Trapped in AgaRose DNA ImmunoStaining (TARDIS) assay to determine whether they stabilise the TOP2-DNA complexes in mammalian cells as known TOP2 poisons do. The analysis showed that after three hours incubation the level of complexes increased, indicating that the quinolones are able to stabilise TOP2-DNA complexes. Taken together the micronucleus and TARDIS assay data show that the quinolones are targeting mammalian TOP2 at high concentrations.

Antibacterial agents are of relevance to a number of industrial sectors including medical, healthcare, food and environment. Organic antibacterial agents kill or inhibit the growth of bacteria by interrupting the synthesis of protein, lipid, nucleic acids or the integrity of the bacterial membrane. Heavy metals and metal oxides have a long application history in inhibiting the growth of bacteria.

The primary mechanism of resistance to the antibiotic fusidic acid (FA) in clinical strains of Staphylococcus aureus is expression of a FusB-type protein (usually FusB or FusC). These proteins bind elongation factor G (EF-G), the target of FA, and protect bacterial translation from FA-mediated inhibition. However, the interaction of these proteins with EF-G is poorly characterised, their structure has not been elucidated, and it is unknown how protection from FA is mediated. This thesis reports the first structure of a FusB-type protein, and begins to define the interaction between these proteins and EF-G. The 3D structure of FusC reveals a monomer composed of two distinct domains. The N-terminal domain comprises a four-helix bundle exhibiting similarity to existing protein structures. By contrast, the C-terminal domain forms a novel fold of helices and B-sheet with four conserved cysteine residues coordinating a central zinc ion. This region of FusB-type proteins was found to mediate a high-affinity interaction with the C-terminal domains of EF-G in vitro, yielding a complex with a 1: 1 stoichiometry. The protein-protein interaction occurs away from the FA binding site of EF-G, suggesting that FusB-type proteins do not mediate FA-resistance through direct steric hindrance of the interaction between FA and its target. Instead, FusB-type proteins interact with the portion of EF-G known to make contact with the inside of the ribosome. Owing to structural constraints, EF-G appears unable to simultaneously bind to FusB-type proteins and the ribosome. Based on these findings, a model of FusB-type resistance is proposed in which FusB-type proteins drive the release of frozen FA-EF-G-GDP complexes from the ribosome by competing for binding to EF-G.

The aims of the PhD were to synthesize deuterium labelled epicatechin in a microreactor, discover if theaflavin and epicatechin possess antibacterial action against important hospital pathogens and to determine if synergy exists between polyphenols and between polyphenol and current antibiotics. Other aims were to determine the minimum inhibitory concentrations of theaflavin and a theaflavin:epicatechin combination against the bacteria Stenotrophomonas maltophilia, to synthesize theaflavin and deuterium labelled theaflavin in a microreactor and to link the continuous flow process to antimicrobial testing for future mode of action analysis. A 2-step microreactor synthesis using in a T-shaped microreactor successfully produced deuterium labelled epicatechin in position 8. Mass spectrometry and H-NMR confirmed deuterium labelled epicatechin had been produced. The 2- step synthesis produced a yield in excess of 90%. A 4-step micro reactor synthesis of deuterium labelled epicatechin, was found to be unsuccessful after step 2 of the synthesis. A 1-step method for the microreactor production of planar epicatechin was also shown to be unsuccessful in a microreactor. Antimicrobial testing of theaflavin, epicatechin and a 2:1 combination of theaflavin and epicatechin was performed against 4 clinical isolates of MRSA, 6 clinical isolates of Acinetobacter baumannii and 6 clinical isolates of Stenotrophomonas maltophilia. Results from the disc diffusion assay confirmed that epicatechin produced no antibacterial action and theaflavin produced strong antibacterial action. The combination of theaflavin and epicatechin produced higher antibacterial activity than theaflavin alone indicating synergy between the two polyphenols. Minimum inhibitory concentrations (MICs) of theaflavin and the theaflavin:epicatechin combination (2:1) were determined against 9 clinical isolates and one control isolate of Stenotrophomonas maltophilia using a microtiter assay. Results from the microtiter assay used indicated that the MIC for theaflavin was between 400 and 800 μg/mL. The MIC for the theaflavin:epicatechin combination (2:1) was between 200 and 400 μg/mL. A 1-step microreactor synthesis of theaflavin from epicatechin and epigallocatechin using extracted polyphenol oxidase was shown to be successful, producing high yields. Using the same methodology, the synthesis of deuterium labelled theaflavin was undertaken using epigallocatechin and the deuterium labelled epicatechin. However, this reaction was shown to be unsuccessful. The antibacterial action of microreactor synthesized theaflavin against Acinetobacter baumannii in a continuous flow process was investigated. Bacterial viability was tested using the resazurin indicator method. No viable cells were observed from bacterial samples exposed to greater than or equal to 4 hours of the continuous flow of theaflavin products. This indicated that the theaflavin produced antibacterial action after this time of exposure. At exposure times less than 4 hours, viable cells were detected.

Bacterial infection and colonisation of polymeric biomaterials represents a major problem that is on the rise within the health care industry. Bacterial attachment and biofilm formation on medical polymers is often the cause of addition discomfort, pain and in the worse case scenario sepsis and even mortality. This, in combination to the overuse of antibiotics and evolution of resistant bacterial stains, means there is a need for a more intelligent approach in the prevention of biofilm formation and bacterial infection. In this work, the antimicrobial properties of metals (in particular silver and zinc) was utilized and incorporated into polymeric biomaterial to render them antimicrobial. Several methods of functionalising polymers with antimicrobial metals were assessed. The materials developed throughout this work were designed to respond to changes in environmental as a result of infection. These changes include differences in pH and temperature all of which are altered in response to infection. This smart design allows for the reduction of unnecessary release of antimicrobial, and will reduce the likelihood of toxicity and resistance. Polymer modifications in this research include modifications made during polymer synthesis, i.e. reaction with additional antimicrobial monomer. In this case, pH responsive zinc containing crosslinker molecule was designed to crosslink into any polymeric material. Post synthesis modifications were also investigated, and include the ‘grafting to’ and ‘grafting from’ of polymers which could then be functionalised with antimicrobial metals. This work demonstrated methods to modify non-woven polypropylene. A system, for the ‘grafting from’ approach to give a pH responsive release of antimicrobial metals from a polymer brush was investigated. Secondly a ‘grafting to’ approach to give a temperature responsive release of metals was investigated. Finally, several zinc compounds were synthesised and assessed for there ability to graft via plasma assisted grafting. The results presented in this work demonstrate novel ways of incorporating antimicrobial metal functionality into polymeric biomaterials and their antimicrobial efficacy.

Bacteria resistant to multiple antibiotics pose a number of therapeutic problems. This situation is likely to worsen in the future, as the development of new classes of antibiotics has declined sharply in recent years. Acinetobacter species are an example of antibiotic-resistant bacterial pathogens with increasing clinical significance, particularly with respect to infections in high-risk patients such as those with severe burns. The treatment options for these patients are severely limited, and they often can only be treated with highly toxic antibiotics such as colistin. It is therefore evident that there is a need to investigate alternative approaches to therapy in these cases. This thesis represents some preliminary investigations into the use of bacteriophages for the treatment of Acinetobacter infections of severe burns patients. Bacteriophages are viruses that have bacteria as their host. They cannot attack human cells and indeed they are highly specific for given species of bacteria. The aim of this thesis was to investigate the potential use of bacteriophages in these infections and their possible toxic effects on mammalian (including human skin) cells. Since bacteriophages lytic against Acinetobacter species were not available through conventional culture collections it was necessary to identify other sources. Clinical isolates of Acinetobacter species were obtained from Sussex hospitals and characterised. Several unsuccessful attempts were made to isolate corresponding bacteriophages from the environment and ultimately 10 isolates of Acinetobacter spp. and their corresponding phage were obtained from Laval University, Canada. These bacterial strains were identified and host matched to each phage using a refined screening technique and purified phage preparations were produced for those potentially therapeutic phages. The results of this study formed the basis for the selection of a small number of phage to take forward for cytotoxicity studies. Very little scientific evidence has been published on the cytotoxic potential of bacteriophage or their bacterial lysis products, although phages have been routinely used for therapeutic purposes over many years. The establishment of phages as a mainstream treatment choice for bacterial infections in the West will require stringent testing and proof that phage preparations are not harmful. Human dermal fibroblast (HDF) and keratinocytes (HDK) were isolated from human biopsies. A 3T3 mouse fibroblast cell line, HDF and HDK with 3T3 feeder layers were exposed to dilutions of purified phage. The cytotoxic impact and effect on cell proliferation was measured using a range of assays. No statistically significant difference could be detected between controls and phage with the Trypan blue method (3T3 cells). Hoechst propidium iodide stain experiments remained inconclusive (3T3 cells). Lactate dehydrogenase (LDH) and MTS tetrazolium compound reduction (MTS) results showed no evidence of statistically significant cytotoxic effect of phage on 3T3 cells, HDF or HDK. Some data suggested phage may have some beneficial effect on cell survival of HDK and proliferation of HDF and 3T3 cells compared to controls. Endotoxin levels present in phage lysates and purified phage preparations were compared to those found in bacterial suspensions subjected to lysis by other methods including autoclaving, bead beating, sonication and high concentrations of antibiotics. There was no evidence to suggest that bacteria lysed by phage liberated significantly more endotoxins than the other methods of cell disruption. A variety of endotoxin preparations and phage lysates were incubated with HDF and cytotoxicity together with cell proliferation were measured using LDH and MTS assays. Changes in interleukin levels of the same samples were monitored, measuring IL-1β, IL-6, IL-8 and TNF-α levels. IL-1β or TNF-α could not be detected in any samples. Highly concentrated phage gave rise to significantly higher IL-6 outputs than any other samples. Ten-fold dilutions of the same phage preparation were not statistically different to any other samples, including controls. This suggested components introduced during the phage purification process, rather than the phage itself may have contributed to higher IL-6 readings. Purified and crude phage lysate gave rise to significantly higher IL-8 outputs than all other samples. Colony formation assays utilising a V79 hamster cell line were used to indicate the cytotoxicity of purified phage in different diluents and provided comparison with endotoxin containing preparations used in other experiments. There was no statistically significant difference in terms of colony numbers or colony size (where measured) between phage samples and controls. This thesis has therefore demonstrated that because of the lack of cytotoxic effect of phage on mammalian cells in culture, there is potential for therapeutic applications of bacteriophage.