Triclosan and PCMX are two broad-spectrum antimicrobial agents of particular interest to Reckitt Benckiser. Triclosan is one of the most widely used biocides today and is incorporated into increasing numbers of household products such as toothpaste, mouth washes and underann deodorants. It is generally accepted that biocides have multiple target sites within the bacterial cell; however, recent reports have identified mutations in the E. coli fabI gene as being linked to decreased susceptibility to triclosan. The fabI gene encodes for enoyl reductase, an important enzyme in fatty acid biosynthesis. Conclusions were drawn that triclosan had a single, primary target in bacteria and the previously reported effects of triclosan on membrane structure were secondary effects arising from specific inhibition of the fatty acid biosynthetic pathway. In this study the mechanisms of action of both triclosan and PCMX were investigated against the hygiene indicators E. coli NCTC 8196 and S. aureus NCTC 10788. Initially, interactions of both biocides with the bacterial cell were investigated using adsorption isotherms. Triclosan was found to possess an unusual Z-shaped isotherm, indicative of a concentration dependant breakdown of cellular structure. Disruption of E. coli cells by sonication also increased the cells sorptive capacity for triclosan, indicating that exposure to concentrations of triclosan above 6ppm induced a structurally damaging event to the cell. PCMX was shown to have a complex S-shaped isotherm, with 2 saturation plateuxs. The termination of the primary uptake phase corresponded to the threshold concentration required for bactericidal activity and leakage of cellular constituents, the secondary uptake was thought to represent penetration to new sorptive sites within the cell itself. The bacteriostatic and bactericidal properties of both biocides were probed by calculation of MICs and MBCs and by production of time survivor curves. Both compounds were found to have bacteriostatic or bactericidal activity against E. coli NCTC 8196 and S. aureus NCTC 10788 depending on concentration. Concentration exponents were detennined and were found to be in the region of S for triclosan and 6 for PCMX, indicating that their activity is greatly reduced on dilution. Non-growing cells were used to investigate membrane damage caused by the two biocides. The leakage of intracellular potassium and changes in the permeability of the membrane to specific ions and protons were used as subtle indicators of membrane damage. PCMX was shown to cause substantial membrane damage, indicated by rapid, gross potassium leakage and by the rapid loss of absorbance of E. coli spheroplasts stabilised in salt solutions, when exposed to bactericidal concentrations of the agent. The effect of triclosan on the membrane appeared to be more subtle, with cells exhibiting a more concentration dependant loss of potassium and spheroplast stability over time. Bactericidal concentrations of triclosan were shown to induce the translocation of protons and the uncoupling of oxidative phosphorylation. Both compounds were able to manifest significant damage to the bacterial cell under conditions which limit the significance of the ENR system to bacterial survival. Whilst ENR may be a target for the growth inhibitory properties of triclosan, this study indicates that the disinfectant potential of the agent derives from additional target damage. Combinations of triclosan and PCMX were also investigated for possible signs of synergy. Whilst adsorption profiles for either compound in the presence of the other clearly indicated changes in the sorptive capacity of the cell, the enhanced bacteriostatic and bactericidal effects of combinations of the biocides were thought to have arisen from the non-linear relationship between antibacterial activity and concentration, rather than 'true' synergy itself.

Investigations of the prevalence of antibiotic resistance genes and their genetic supports are essential for our understanding of the mechanisms of resistance, and their transfer. This study investigated the prevalence of tetracycline and erythromycin resistance in the Gram positive aerobic cultivable portion, and the total oral and faecal microbiota of six European countries. Only Gram positive isolates were investigated as they represent a distinct phylogenetic group. Furthermore, this project was part of a larger European-wide study on the biology of Gram positive organisms. A collection of 123 tetracycline and/or erythromycin resistant isolates was made, and through macroarray analysis the most common tet genes were found to be tet(W), let(O) and /<.'/(W) in the aerobic oral flora, and tet(M). tet(O) and tet(Q) in the aerobic faecal flora. Three isolates did not hybridise to any probes on the array. In order to investigate the contribution of the whole metagenome to antibiotic resistance, total extracted DNA was analysed on the macroarray and 12 BAG libraries were constructed. The most common let genes in the oral microbiota were tet( ), tet(Q) and /i7(30) and were /(//(W). tet(O). tet(Q) and /tV(32) in the faecal metagenome. The BAC libraries were evaluated for efficiency of cloning microbial DNA, and to ensure they were representative of each microbiota, by end-sequence analysis. The libraries were screened on tetracycline. 32 resistant clones were found, only four of these were stable. One. NFtetCl. contained tet(O). The entire insert was sequenced to determine its support, it was shown to contain orfs with similarity to tnpY from n445L and to or/6 from n916 and cppJ from the /e/(0)-harbouring Campylobacter coli plasmid pCC31. Clone SFtetCIO harboured tet( ) PCR analysis illustrated it was flanked by sequences with homology to those flanking tet(M) in Tn9/6 however, int from Jn9J6 did not amplify with specific primers. Clones IStetCl and FRStetCll did not hybridise to any probes on the array. These harbour either novel or rare tet genes. Clone IStetCl was subcloned and found to harbour a putatitive natural chimera of two tetracycline resistance plasmids: pRSB107 and pR64. This study thus provides further evidence of the prevalence of antibiotic resistant bacteria in the human Gastrointestinal (GI) tract, and the difference in prevalence of tetrcycline resistance determinants in the aerobic cultivable flora and total microbiota. Furthermore, it illustrates how antibiotic resistance genes are contained on mobile genetic elements which are mosaic in structure having undergone evolutionary changes in which functional modules are exchanged.

No description available.

This thesis is concerned with studies towards the design, synthesis and biological evaluation of analogues of the oxazolomycins, a class of structurally complex natural products with potent antibiotic, antiviral and in vivo antitumour activity. Initial investigations focused towards a flexible synthetic route to the left-hand triene system, utilising a series of stereoselective Wittig olefinations. This resulted in the synthesis of truncated inthomycin C analogue (±)-209, which displayed antibacterial activity against S. aureus, E. coli and S. pneumoniae. Methodology was also developed which gave rapid access to the central c.c-dimethyl-Ii-hydroxy amide fragment via ring- opening of substituted p-Iactones, derived from the corresponding aldehydes by a tandem aldol-cyclisation reaction, with primary amines. Installation of the amide functionality at this position had posed difficulties in previous syntheses as a result of the steric hindrance imposed by the neighbouring gem-dimethyl groups. Molecular modelling of oxazolomycin B and a related structure was employed to determine their lowest energy conformations and revealed that the u.o-dimethyl-Bchydroxy amide moiety is likely to control the conformational preferences of the natural products. A library of simplified amide analogues based on this motif was synthesised and their biological activity evaluated, which showed that this subunit can display intrinsic antibacterial activity when appropriately substituted. This investigation was extended to synthesis of a series of c.o-dimethyl-Bchydroxy esters, which exhibited similar J~vels of antibacterial activity to their amide congeners. The structures and conformational preferences of the synthesised amides and esters were studied using X-ray crystallography and IH NMR spectroscopy. Coupling of the u.o-dimethyl-Bchydroxy amide fragment with analogues of the right-hand pyroglutamate core, to give simplified "hybrid" analogues of the oxazolomycins, was explored using a variety of linking strategies. The most successful of these exploited the "Click" 1,3-dipolar cycloaddition of azides and nitrile oxides with terminal alkynes, yielding "hybrid" oxazolomycin mimics featuring triazole and isoxazole linking groups respectively. This approach lead to the development of several novel conjugates exhibiting potent anti-infective bioactivity not shown by either of the constituent components alone; of particular note was derivative 330, which was found to possess an MIC value of 2 ug/rnl. against H influenzae. Such routes to structurally unusual templates based upon the oxazolomycins by lactam and amide subunit conjugation may be amenable to convenient library generation for the purpose of optimisation against different bacterial targets.

The primary photophysical properties of several antibiotic drugs of the fluoroquinolone class have been studied in order to understand better and rationalize the mechanisms leading to phototoxicity due to these drugs. The light absorption and emission properties of various fluoroquinolones have been characterized and, in most cases, they were found to be dependant on medium conditions such as pH and buffer content. Fluoroquinolones have been found capable of binding to DNA, affecting their emission from the excited state. Light-induced production of excited species by FQs and their subsequent reactivity are thus influenced by the characteristics of the medium. Finally, an unprecedented behaviour has been found for the triplet state of fluoroquinolones ciprofloxacin, sarafloxacin, enoxacin norfloxacin when it is formed in the presence of certain inorganic salts commonly used as buffers. The triplet state formed at the end of the excitation pulse reacts with anions such as phosphate and bicarbonate to form another triplet state of lower energy. This previously unreported effect has implications in the photo toxic potential of fluoroquinolones in cellular environments.

This thesis aims to examine factors influencing the solubility of oxalate from dietary sources, namely cereal brans and legumes, and strategies to reduce the bioavailability of oxalate, including the influence of enzymes and a probiotic. Intake of oxalate through diet is a significant factor in the formation of kidney stones, particularly when consumed alongside mineral rich foods, due to the low solubility of calcium oxalate and some other mineral oxalates. Consumption of whole bran cereals and legumes is increasing because of potential nutritional benefits, yet both foods contain significant levels of oxalate, as well as phytate and minerals that can be determinant factors towards the risk of kidney stone formation. Phytate has been reported to form complexes with metal cations, thereby leaving less cations, particularly calcium and magnesium, available to form insoluble oxalate. Therefore, phytate and mineral composition of whole bran cereals (wheat, barley and oat) and legumes were determined, along with soluble and insoluble oxalate concentrations, in order to investigate the effects on oxalate solubility. Phytate concentration in tested food samples ranged from 227•4393 mg/ 100 g and the concentrations of cations were in the range 54•70 mg/IOOg for calcium and 75•398 mg/100g for magnesium. It has been found that soluble oxalate concentration did not increase in proportion to total oxalate, and the phytate concentration in all foods was sufficient to contribute to an increase in soluble oxalate concentration by binding calcium, since the phytate:calcium ratio was greater than 0.24. This value indicates that the phytate concentration is sufficient to reduce the calcium available for binding to oxalate, and thereby contributes to an increase in soluble oxalate.

Holomycin is a poor antibiotic (Oliva et al, 2001) that is made in reasonable quantities by S. clavuligerus - which is better known for production of clavulanic acid, a component of the successful clinical drug, Augmentin. Holomycin is a wasteful metabolite in the context of commercial clavulanic acid production, and mutants that no longer make holomycin have increased clavulanic acid productivity, Likewise, mutants that no longer make clavulanic acid will over-produce holomycin (de la Fuente et al, 2002). The biosynthesis of holomycin has not been studied.

Introduction: Antimicrobial prescribing has been reported to be sub-optimal in care homes; however, prior to this research there was no information in relation to antimicrobial use in Northern Ireland's (NI's) care homes. Furthermore, the factors having an impact on infection control practices in NI's care homes had not been identified. Methods: Two point prevalence surveys (PPS) were conducted in 30 nursing homes and 30 residential homes, to determine the prevalence of antimicrobial prescribing and any associations with resident or institutional factors. Findings from the PPS in nursing homes informed the development of a pilot intervention study for the management of urinary tract infections (UTIs). Qualitative research was conducted to investigate the experience of nursing home staff who were involved in a previous infection control education and training intervention study and to explore the feasibility of introducing widespread Meticillin-resistant Staphylococcus aureus (MRSA) decolonisation to the nursing home setting. Appropriate quantitative and qualitative analyses were performed for all studies. Results: The PPS identified a high rate of antimicrobial prescribing in nursing (April 2009, l3%; November 2009, 11%) and residential homes (November 2010, 9%; April 2011, 9%) with variability evident both within and between homes. Antimicrobials were most frequently prescribed for UTIs. In the pilot UTI intervention study, fewer (-38%) antimicrobials were prescribed and fewer (-7%) residents were treated for suspected UTIs. However, the number of urine dipstick tests and urine samples which were sent to the laboratory remained high. The qualitative study found that the factors influencing infection control and MRSA decolonisation in nursing homes were organisational (e.g. time, financial resources, environmental, management and culture); external (e.g. hospitals and general practitioners); and residents and families. Conclusion: The four studies have shown that care homes require further support to optimise antimicrobial prescribing, the management of UTIs and infection control

The research presented in this thesis explores antibiotic use and strategies to manage infection related issues within the hospital setting. In Chapter 2, the prevalence of the use of antibiotics and of HAl in hospitalised patients within Northern Ireland was explored using a point prevalence study design. This research provided details on the rate of antibiotic use and prevalence af HAI in the study sites as well as helped in the identification of targets for quality improvement of antibiotic prescribing and allowed the explanation of potential risk factors of HAl. In Chapter 3, the impact of an enhanced antibiotic stewardship programme on reducing MRSA and Clostridium difficile infection (CDI) in hospitalised patients was evaluated using segmented regression analysis within an interrupted time series. This work also involved evaluating the impact of antibiotic stewardship on the use of hi gh-risk antibiotics in the study setting. This study showed that the restriction of high-risk antibiotics contributed to both a reduction in their use and a reduction in the incidence of MRSA and CDJ in the study site hospital. Chapter 4 includes a report on the evaluation of the clinical and cost implications of blood culture contamination (Bee) within the hospital setting utilising a case-control study, comparing case samples with control samples. Length of hospital stay and total hospital resource utilisation were the main outcome measures. The results of this study indicated that BCC increased the length of hospital stay and incurred significant additional hospital costs including needless antibiotic use and extra laboratory tests. Finally, in Chapter 5, a prospective investigation was performed to evaluate the impact of an educational intervention in tackling the problem of BCC in an intensive care unit (leU). This study confinns the benefit (lower BeC rates) of continuous training and education on the proper technique for taking blood cultures in critically ill, ICU patients.

Many natural products contain a tetramic acid (pyrrolidine-2,4-dione) ring system as an integral part of their structure. They exhibit a wide range of biological activities and are structurally diverse, making them an attractive target for the synthetic chemist. The pyrroindomycin antibiotics are an example of such natural products which contain a spirotetramate core. Studies towards the total synthesis of the pyrroindomycins are described within this thesis, which involve inter- and intra- molecular Diels-Alder approaches towards the synthesis of the spirotetramate core of the natural products. In addition to these synthetic strategies, the relative stereochemistry of the aglycone (previously unknown) was elucidated using NMR spectroscopy, and the findings reported herein. Diels-Alder studies were performed on a model tetramic acid dienophile and the reactivity of this type of system compared to dehydroalanine derivatives. Several spirotetramates were prepared utilising this method, in order to determine if a similar synthetic approach could be used to synthesise the spirotetramate core of the pyrroindomycins. In addition, a spirotetramate was prepared using a nitroalkene dienophile precursor. A number of intramolecular Diels-Alder reactions involving dehydroalanine and nitroalkene dienophiles were also attempted, in order to synthesise a spirotetramate precursor which could be elaborated to the natural products.