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Novel Antimicrobial Agents Tuesday 15th June 2010 School of Pharmacy & Life Sciences, and Research Institute for Health and Welfare, Robert Gordon University, Aberdeen Conference Abstracts Welcome We welcome you to the Robert Gordon University today for what promises to be a very interesting conference. Having assembled the programme over the last few months, it is pleasing to have gathered such a prestigious grouping of people who will be discussing their work on novel antimicrobials. We would like to take the opportunity to thank all of those who accepted our invitation to speak today. It is only very occasionally that we can enjoy such an event in North-east Scotland and thanks must also be offered to the Society for General Microbiology and the Society for Applied Microbiology for their generous financial support. We would like to thank all our colleagues within Robert Gordon University who have contributed to the organisation of todays event. Finally, we hope you enjoy the conference. Dr Andrew Lamb Dr Derek Chapman 15th June 2010 Programme 9.45 – 10.25 Registration and Poster Assembly/Viewing Session 1 Novel Antimicrobials 10.25 -10.30 Dr Andrew Lamb, Robert Gordon University Welcome and Introduction Chair – Dr Andrew Lamb 10.30 – 11.10 Professor Peter Taylor, University of London ‘Some alternatives to conventional antibiotics: what's on offer?’ 11.10 – 11.40 Dr Derry Mercer, NovaBiotics Ltd ‘Advances in antifungal drug discovery’ 11.40 – 12.00 Dr Alan Bowman, University of Aberdeen ‘A novel GPI-linked antibacterial protein in the sheep tick, Ixodes ricinus’ 12.00 – 12.20 Ms Noelle O’Driscoll, Robert Gordon University ‘Cationic antimicrobial peptides: novel alternatives to existing antibiotics’ 12.20 – 12.55 Lunch 12.55 – 1.10 Poster Viewing Session Session 2 Synthesis and Evaluation of Antibacterials Chair – Professor Don Cairns 1.10 – 1.50 Professor Colin Suckling, University of Strathclyde ‘Antibacterial minor groove binders – the Strathclyde Solution!’ Programme 1.50 – 2.20 Dr Geoff Coxon, University of Strathclyde ‘2-Aminothiazole-4-carboxylates (ATCs) as a potential new class of anti-tuberculosis drug’ 2.20 – 2.40 Dr Cedric Charrier, NovaBiotics Ltd ‘1 + 1 ≠ 2 : Novel synergistic antibacterials’ 2.40 – 2.55 Tea/Coffee Session 3 Antiparasitics and Drug Resistance 2.55 – 3.35 Dundee Professor 3.35 – 4.05 Dr Paul Hunt, University of Edinburgh ‘Genetic and genomic identification of drug resistance pathways in malaria’ 4.05 Close Chair – Dr Derek Chapman Alan Fairlamb, University of ‘Spinach leaves, butterfly wings, nutmeg oil and antiparasitic drugs’ Speaker Abstracts Some alternatives to conventional antibiotics: what's on offer? Professor Peter W. Taylor School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX UK Over the last two decades, there has been a steady decline in the number of new antibacterial agents launched into the market; this period has coincided with a substantial rise in the frequency of isolation of multi-drug-resistant pathogens from nosocomial and, increasingly, community-acquired infections. Currently useful antibiotics impose enormous selective pressure on bacterial populations and the emergence of resistance to these drugs is all but inevitable; we need, therefore, to constantly replenish our antibacterial arsenal. The hope that the exploitation of genomics and target-based high throughput screening would yield a new generation of novel drugs addressing new molecular targets has not been realised in spite of massive investment; furthermore, a recent molecular analysis of protein expression in experimental infection raises the possibility that we may have already exploited most of the broad-spectrum targets essential for bacterial survival in vivo. As academia and government agencies are being urged to become more involved in the process of antibiotic development, the time may be right to begin to exploit more unconventional approaches to the eradication of infection. This contribution will focus on alternatives to conventional antibiotic chemotherapy for the resolution of troublesome infections - particularly those due to multi-drug-resistant pathogens such as MRSA, XDR-TB and the like. Thus, bacteriophage therapy, phage components such as lysins, constructs that interact with previously unexploited bacterial targets and agents that advantageously modify the antibiotic resistance and virulence of bacterial pathogens will be evaluated. Antifungal Drug Discovery: Where next? Dr Derry Mercer NovaBiotics Ltd, Cruickshank Building, Craibstone, Aberdeen, AB21 9TR, UK Fungal infections range from the superficial, causing inconvenience rather than illness, to systemic infections with high rates of associated morbidity and mortality. The everincreasing number of immunocompromised patients has led to a dramatic increase in the number of life-threatening fungal infections in the last two decades. Systemic fungal infections with Candida spp. and Aspergillus spp. can have mortality rates of up to 40% and 90%, respectively, if not diagnosed and treated quickly. The increasing incidence of fungal infections demands improvements to the current antifungal armamentarium. Current antifungal drugs are limited to four main drug classes and therefore there is a significant requirement for novel antifungal agents with disparate modes of action. A number of interesting approaches are being used in antifungal drug development including areas such as vaccine and biologics-based approaches, as well as more traditional approaches examining novel chemical entities interacting with specific targets. Inevitably, expansion of existing drug classes continues apace. In this paper I shall present an overview of fungal infections and currently available drugs before examining potential novel drug classes and highlighting a few of the more promising therapeutic candidates. A novel GPI-linked antibacterial protein in the sheep tick, Ixodes ricinus Dr Alan Bowman M.J. Burdin1, K. Forbes2, and A.S. Bowman1 1 School of Biological Sciences (Zoology) and 2Immunology & Infection, Medical School, University of Aberdeen, Scotland, UK Here we report on a new class of antimicrobial peptide (AMP) – the novelty of which concerns a GPI-anchor to hold it on the cell surface in contrast to all other reported AMPs that are directly secreted into the circulation to counter microbial infections. RicOrph17, found in the sheep tick, contains a putative 26amino acid signal peptide and a 26-coding amino acid GPIanchoring signal. The RicOrph17 protein shares some structural features of three finger protein family, including a cysteine skeleton responsible for the formation of 5 disulfide bonds. RicOrph17 was equally expressed in all tick tissues tested. Injection of RicOrph17-dsRNA into unfed ticks silenced the target gene expression and caused the tick to be more sensitive to bacterial challenge, resulting in a significantly higher mortality rate. A recombinant protein (rRicOrph17) lacking the secretion signal peptide and GPI-anchoring signal was produced. rRicOrph17 exhibited antibacterial activity (bacteriostatic and bacteriocidal) against 10 clinically relevant isolates (Gram +ve and –ve) and against 3 yeasts. Fluorescently-tagged rRicOrph17 was observed to enter and accumulate inside bacteria rather than remain associated with the bacterial wall unlike the pore-forming magainin. Additionally, rRicOrph17 associated with DNA. Overall, we have identified a novel class of AMP, previously unknown, but widely present throughout the arthropod phyla. The GPI-linkage could serve as a rapid and localised release of the protein after a bacterial infection. Cationic antimicrobial peptides: novel alternatives to existing antibiotics Ms Noelle O’Driscoll N. O’Driscoll1, K. Matthews1, D.K. Mercer2 and A. J. Lamb1 1 School of Pharmacy and Life Sciences, Robert Gordon University, Schoolhill, Aberdeen. AB10 1FR 2NovaBiotics Ltd, Cruickshank Building, Craibstone, Aberdeen, AB21 9TR, UK. Despite the use of antimicrobial agents for nearly eight decades since the introduction of sulphonamides in the 1930s, infectious diseases continue to have an impact both on human morbidity and mortality throughout the world and place a major burden on healthcare services. The World Health Organisation estimated 11 million deaths in 2002 from acute respiratory infections and major infectious diseases including tuberculosis and AIDS/HIV. These figures increase yearly. There is an increasing urgency in the search for new antibacterials that are active against a wide range of bacteria, possess minimal host toxicity and do not readily select for resistant mutants. Cationic antimicrobial peptides (CAPS) comprise a group of effective, safe and chemically diverse antibacterials. These gene coded CAPs are found in the tissues of most complex organisms, comprising part of the innate immune system. Constitutively expressed or induced, endogenous CAPs provide a fast response to bacterial invasion, are mobilized quickly after microbial infection and rapidly neutralise a broad range of microbes. The aim of this research was focussed on examining the effects of novel CAPs on Escherichia coli NCTC 4174. Results will be presented illustrating the effect of these novel CAPs on this bacterium. From these results it will be evident that the bactericidal effect of these polypeptides is not solely due to membrane disruption. Cellular aggregation, gross deformation of bacterial cells and inhibition of septation are some of the effects observed. Antibacterial minor groove binders – the Strathclyde Solution Professor Colin Suckling Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, UK. The natural products distamycin and netropsin, which are oligoamides of pyrrole amino acids that bind to the minor groove of DNA, have been known for 50 years to have antibacterial and antiviral activity. Only in the last 10 years, however, have derivatives been obtained with sufficient selectivity to be serious candidates for preclinical drug development. For bacterial infections, we have discovered a novel class of minor groove binder for DNA with potent antibacterial activity against Gram positive bacteria (MIC ~ mol) and remarkably low toxicity to mammalian cells. Some compounds are also active against Gram negative bacteria but are less potent. The key strategy in developing these compounds was to increase the hydrophobic content of the minor groove binder to promote increased hydrophobic interactions with the wall of the minor groove of double stranded DNA. This has been achieved by introducing branched alkyl substituents in place of methyl groups and alkenes in place of amides. Not all substitutions lead to active compounds. With the aid of spectroscopic and physical methods, we have identified the structural requirements for binding to DNA; these features also correlate with antibacterial activity. However available information does not allow us to conclude that binding to bacterial DNA is the only component of their mechanism of action. This presentation will introduce the structures of our compounds in the context of their biological properties and describe what we know of their mechanism of action. 2-Aminothiazole-4-carboxylates (ATCs) towards a potential new class of antituberculosis drug. Dr Geoff Coxon Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, 27 Taylor Street, Glasgow, G4 0NR, UK. Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H37Rv and, dissociatively, against the β-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H37Rv with an MIC of 0.06 µg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)t hiazole-4-carboxylate inhibited mtFabH with an IC50 of 0.95±0.05 µg/ml (2.43±0.13 µM) but was not active against the whole cell organism. These findings clearly identify the 2aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of antitubercular agents. 1+1≠2 Novel Synergistic Antibacterials Dr Cedric Charrier C. Charrier, D.K. Mercer, S. Robertson, D. O’Neil. NovaBiotics Ltd, Cruickshank Building, Craibstone, Aberdeen, AB21 9TR, UK Objectives The ever-increasing emergence and spread of multi-drug resistant bacteria and the recurrence of microbial biofilm infections is a serious concern for the treatment of all infectious diseases, such as respiratory tract and skin infections. To address this unmet medical need for safe and effective therapeutics, NovaBiotics is developing novel antibacterials that have the potential for activity against all cell types encountered in bacterial biofilms, namely planktonic, sessile and persister cells. The technology, originally based on naturally-occurring antimicrobial peptides, has been developed to display a low toxicity profile, a low risk for antimicrobial resistance development and is compatible with currently available therapies and anti-biofilm agents. Results The cationic antimicrobial peptide NP108 and the mucolytic agent NM001 demonstrated synergistic activity against a number of bacterial species responsible for causing skin and respiratory tract infections. These synergistic compounds demonstrated antibacterial activity against planktonic, sessile as well as persister cells, which are the main cause of infection recurrence. Additionally, this novel antimicrobial combination demonstrated low cytotoxicity and haemolytic activity along with a low risk of resistance development. Conclusion The synergistic activity of NovaBiotics novel antimicrobials against the various cell types encountered in microbial biofilms represents a novel therapeutic option to address the unmet need for effective and safe treatments for the eradication of microbial biofilms. Spinach leaves, butterfly wings, nutmeg oil and antiparasitic drugs Professor Alan H. Fairlamb Division of Biological Chemistry & Drug Discovery, College of Life Sciences, University of Dundee, DD1 5EH. New drugs are urgently required for the treatment of many killer parasitic diseases, which, for economic reasons, are “neglected” by the pharmaceutical industry. In 2006, the Drug Discovery Unit was established in Dundee to fill the translational gap between discovery and validation of novel drug targets through to development of clinical drug candidates for African sleeping sickness, visceral leishmaniasis and Chagas’ disease. The specific goal is to deliver at least one preclinical candidate for human African trypanosomiasis by March 2011. Some successes and failures, challenges and lessons learned will be illustrated with enzyme targets related to metabolites originally identified in spinach leaves, butterfly wings and nutmeg oil. Genetic and genomic identification of drug resistance pathways in malaria Dr Paul Hunt Centre for Immunity, Infection and Evolution, University of Edinburgh, Ashworth Laboratory, Kings Buildings, Edinburgh, EH9 3JT Drug resistance continues to exert a major impact on the treatment and control of malaria. A knowledge of the genetic changes conferring drug resistance is essential for optimising drug delivery and disease control. A rapid proactive identification of relevant genetic markers might aid efforts to minimise the evolution of resistance. One approach combines experimental evolution to generate drug-resistant mutant parasites with whole genome resequencing to identify the mutations. We have used the rodent malaria Plasmodium chabaudi to generate a lineage of mutant parasites with in vivo resistance to a number of drugs and their combinations. A comprehensive inventory of the mutations arising in this lineage has been defined, using the Solexa platform. Importantly, the functional significance of these mutations can be evaluated using a whole-genome genetics approach, called Linkage Group Selection. This has the features of a quantitative genome-wide scan of selection, multiple natural transfection and Quantitative Trait Loci analysis. In these ways, the genetic architecture of resistance to drugs can be elucidated. I will describe how this model system has been used to determine the precise mutations conferring resistance to artemisinin, chloroquine, mefloquine and anti-folates, and indicate possible novel pathways of parasite adaptation to drugs. Poster Presentations The X-ray crystal structures of PfIspF and PaPabC: Targets for the discovery of antimicrobial agents. 1 P.E.F. O’Rourke1, T.C. Eadsforth1, P.K. Fyfe1, J. Kalinoskwa-Tlusckic1 and W.N. Hunter1 Division of Biological Chemistry and Drug Discovery, University of Dundee, Wellcome Trust Building, Dow Street, Dundee, Scotland, DD1 5EH The X-ray crystal structures of two essential microbial enzymes were solved to assist early-stage drug discovery. The enzymes selected were IspF (2C-methyl-D-erythritol cyclodiphosphate synthase) from Plasmodium falciparum and PabC (4-amino-4deoxychorismate lyase) from Pseudomonas aeruginosa. IspF is involved in isoprenoid biosynthesis and PabC is part of the folate biosynthetic pathway. The proteins were recombinantly expressed (E. coli) and suitable crystallisation conditions were identified using an automated liquid-handling system. Optimisation of these initial conditions produced crystals that diffracted x-rays. Screening chemical fragment libraries against these proteins is in progress. Any fragments that are identified will be used in co-crystallisation trials. Antimicrobial activity of Bog myrtle (Myrica Gale) essential oil against Staphylococcus epidermidis and Staphylococcus aureus. 1 N. Picamilho-Rua1, D. Hogan2, A.J. Lamb2 and M.C.E. McFadyen2 Universite Blaise Pascal, Polytech Clermont-Ferrand, 63173 Aubiere, France. 2School of Pharmacy and Life Sciences, Robert Gordon University, Schoolhill, Aberdeen, AB10 1FR. Bog myrtle (Myrica gale), an aromatic shrub widely distributed in Northern wetlands is known to possess antimicrobial properties. The essential oil component is a complex mixture of mono and sesquiterpenes, with considerable variation in chemical composition between different populations. The aim of this study was to assess the antibacterial activity of European and North American essential oil of Myrica gale against the Gram positive bacteria Staphylococcus epidermidis NCTC 8558 and Staphylococcus aureus NCTC 6571 (MSSA). Broth macrodilution and microdilution assays were performed to establish bacterial susceptibility to the antimicrobial agents. Potential synergy between the antimicrobials was evaluated via the chequerboard method and broth macrodilution antimicrobial combination studies. Antimicrobial activity of Myrica gale oil vapour against MSSA was investigated via a vapour activity assay. Myrica gale inhibited S. epidermidis in the broth macrodilution assay, with minimum inhibitory concentration (MICs) of 0.25 % v/v and 0.5 %v/v for the European and North American oils respectively. Both oils had MICs of 1% v/v in the broth microdilution assay. Combining chlorhexidine with Myrica gale produced neither synergy nor antagonism against S. epidermidis. Myrica gale, inhibited MSSA in the broth microdilution assay with MICs of 0.5% v/v and 0.125% v/v for the European and North American oils respectively. In addition, clear inhibition of growth was observed with the vapour phase of Myrica gale against MSSA. These finding highlight the potential of Myrica gale essential as putative novel topical antimicrobial against S. epidermidis and MSSA. Antimicrobial effect of Patchouli essential oil (Pogostemon cablin), alone and in combination with chlorhexidine. 1 D. Hogan1, A.J. Lamb1 and M.C.E. McFadyen1 School of Pharmacy and Life Sciences, Robert Gordon University, Schoolhill, Aberdeen, AB10 1FR. Essential oils are naturally occurring organic compounds used since the middle ages for their antibacterial, antiviral and antifungal properties. The aim of this current study was to investigate the antimicrobial activity of one such oil patchouli (Pogostemon cablin), alone and in combination with a conventional antimicrobial; chlorhexidine, against S.epidermidis 8558. The antimicrobial activity of this combination therapy against methicillin-resistant S. aureus 11940 (MRSA) was also established. Broth macrodilution and microdilution assays were performed to establish bacterial susceptibility to the antimicrobial agents. Potential synergy between the antimicrobials was evaluated via the chequerboard method and broth macrodilution antimicrobial combination studies. Chlorhexidine demonstrated antimicrobial activity against S.epidermidis and MRSA in broth macrodilution and microdilution assays, yielding MIC (minimum inhibitory concentration) values of 0.5 mg/ml against S. epidermidis and 2mg/ml against MRSA. Patchouli exhibited MICs of 0.031% v/v and 0.125% v/v against S. epidermidis, in the macrodilution and microdilution assays, respectively, and an MIC of 0.5% v/v against MRSA in the microdilution assay. MICs of chlorhexidine and patchouli oil were reduced when the two were combined against S. epidermidis (MIC of 0.5mg/ml reduced to 0.25mg/ml and MIC of 0.031% v/v reduced to 7.8 x 10-3 % v/v, for chlorhexidine and patchouli oil, respectively). These findings highlight the potential use of patchouli alone and in combination with chlorhexidine as a novel topical antimicrobial agent against S. epidermidis and MRSA. In vitro antibacterial evaluation of synthetic flavone analogues against quinolone- and methicillin-resistant Staphylococcus aureus. 1 E. Medu1, R. Brown2 and A.J. Lamb1 School of Pharmacy and Life Sciences, Robert Gordon University, Schoolhill, Aberdeen. AB10 1FR. 2School of Chemistry, University of Southampton, Highfield, Southampton. SO17 1BJ. Objectives: This study was undertaken to screen for in vitro antibacterial activity of a series of synthetic flavones and chromones, and examine any potential synergism in combination with polymyxin. Methods: Microbroth MIC/MBC determination and modified agar dilution methods were used to screen the compounds for in vitro antibacterial activity. A chequerboard technique and timekill assays were used to examine synergism with polymyxin. Result: The compound, 2-(cyclohexylmethyl)-6-methoxy-4Hchromen-4-one (F1), gave MIC/MBC value of 32µg/ml against quinolone-resistant Staphylococcus aureus (QRSA) and MIC and MBC values of 128 and 512µg/ml against methicillin-resistant Staphylococcus aureus (MRSA). When evaluated using chequerboard technique, MIC/MBC of F1 reduced to 8 and 16µg/ml against QRSA and MRSA respectively. The chequerboard combination reduced MIC/MBC of polymyxin against both species from 128 to 32µg/ml. In time-kill viability assays, either 32µg/ml (MIC) F1 or 128µg/ml (MIC) polymyxin demonstrated bacteriostatic activity with 2 log reductions in density of exposed bacterial population within 24 hours. Increased F1 concentration up to 32 × MIC maintained bacteriostatic activity. But, combination of 32µg/ml (MIC) F1 with 128µg/ml (MIC) polymyxin was bactericidal with 7 log reductions of exposed bacterial population within 8 hours. Conclusion: The antibacterial potency of 2-(cyclohexylmethyl)6-methoxy-4H-chromen-4-one and synergism exhibited with polymyxin demonstrate this compound to have potential antibacterial activity against these resistant staphylococcal species. Antifungal effect of lactic acid bacteria on Rhizopus stolonifer 1 A.D. Bayliss1 and E. Cowie1 School of Pharmacy and Life Sciences, Robert Gordon University, St Andrews Street, Aberdeen, UK, AB25 1HG. The ability of lactic acid bacteria (LAB) to inhibit the main fungal contaminants of bread including Aspergillus, Fusarium and Penicillium, has previously been reported. Introduction of antifungal LAB isolated from these studies into bread dough allowed for a reduction of the preservative calcium propionate by 50%. An improvement in bread volume was also found. The aim of this study was to identify LAB with antifungal activity against Rhizopus stolonifer (R. stolonifer) which could potentially be used in the preservation of bakery products. A total of 19 presumptive LAB isolates (Gram-positive and catalase-negative) were obtained from 8 samples of commercially available flours. Antifungal activity was analysed by testing the bacteria against target mould using the dualoverlay assay method. Nine LAB strains from culture collections were also tested. Results indicated low inhibitory activity of 10 isolates and 5 LAB strains against conidial germination (no fungal growth on 0.1-3% of the plate area per bacterial streak). One isolate and 1 LAB strain (Lactobacillus cremoris NIZO B40) exhibited medium inhibitory activity (no fungal growth on 3-8% of the plate area per bacterial streak). Lactobacillus cremoris NIZO B40 has not previously been reported to have antifungal activity against this mould species. The strain related antifungal ability of LAB isolates was demonstrated here. LAB may have a role in maintaining microbial safety and shelf-life whilst reducing the need for chemical preservation of baked products. Future work will include further characterising LAB isolates and the antifungal agents they produce. Isolates of LAB could be incorporated into bread production and the preservation effect measured. Structural confirmation by NMR of 3-Ooctanoyl-(-)-epicatechin, a novel antibacterial 1 A. Di Salvo1, E. Medu1 and A.J.Lamb1 School of Pharmacy and Life Sciences, The Robert Gordon University, Schoolhill, Aberdeen, AB10 1FR. Recent widespread reports of high bacterial resistance to common antimicrobial agents have resulted in studies aimed at the production of more sophisticated molecular agents to address the problem. 3-O-octanoyl-(-)-epicatechin is the semi synthetic derivative of a well know flavonoid with antibacterial properties. Acylation of the hydroxyl group in C6 position has been preliminarily associated with anchoring of the derivative onto the bacterial cell surface, resulting in more efficient biological activity. Therefore the need has arisen to streamline the synthetic procedure and obtain viable quantities of the compound for mechanistic antimicrobial studies. This work presents full structural elucidation of the molecular target by NMR for quality checking purposes and to extend the knowledge base of this class of compounds for further synthetic development. Investigation of the activity of Triclosan on growth, morphology and cell integrity of bacteria 1 N. O’Driscoll1, K. Matthews1 and A. J. Lamb1 School of Pharmacy and Life Sciences, Robert Gordon University, Schoolhill, Aberdeen, AB10 1FR. Triclosan (2,4,4`-trichloro-2`-hydroxydiphenyl ether) is a broad spectrum antibacterial agent known to act upon enoyl-ACPreductase as the primary target thereby inhibiting fatty acid synthesis. This investigation was undertaken to further examine the antibacterial activity of Triclosan. Bacteriostatic and bactericidal effects of Triclosan were established by measuring viability of E. coli NCTC 4174 and S. aureus NCTC 6571 using standard microbroth assays. The effect of antimicrobial exposure on bacterial viability was also examined by flow cytometry from cultures treated with propidium iodide and Syto 9. Scanning electron microscopy monitored bacterial morphology for possible changes manifest by bacteria in response to Triclosan incubation. Damage to the cytoplasmic membrane was examined by measuring amount of internal potassium lost. Triclosan inhibits onset of the log phase of bacterial growth in a concentration dependent manner. Significant changes in dynamics of bacterial growth, dependant on length of exposure to/concentration of Triclosan, were identified via flow cytometry. Incubation of E. coli or S. aureus with both sub-MIC and MIC of Triclosan revealed, via SEM, presence of cellular aggregates. Incubation of bacteria with Triclosan induced minimal potassium loss, suggesting that structural integrity of the bacterial membrane is not compromised by this agent. Whilst previous investigations have reported Triclosan to induce potassium loss, our investigation clearly demonstrates potassium leakage does not occur, even at 100xMIC. This data confirms the finding of Villalain et al (2001) that structural integrity of the membrane is not compromised by Triclosan. Modified Franz diffusion for the in vitro determination of the efficacy of antimicrobial wafers against methicillin-resistant Staphylococcus aureus (MRSA) 1 O. Labovitiadi1, K. H. Matthews1 and A.J. Lamb1 School of Pharmacy and Life Sciences, Robert Gordon University, Schoolhill, AB10 1FR, Aberdeen, UK Antimicrobial wafers offer great potential for sustained topical drug delivery directly to an infected wound bed. It is necessary to verify the efficacy of these lyophilised delivery systems, containing a range of broad spectrum antimicrobial compounds, against methicillin-resistant Staphylococcus aureus (MRSA). Lyophilised wafers were produced by casting karaya gels containing clinical concentrations of either neomycin sulphate (NS), povidone iodine (PVP-I), chlorhexidine digluconate (ChD) or silver sulfadiazine (SS) into polystyrene plates and freezedried. The effect of the released antimicrobials against MRSA was determined in vitro using a modified Franz diffusion cell. The dissolution medium of the receptor chamber was inoculated with 5×105 cfu/mL and antimicrobial wafers placed on top of a cellulose membrane (12-14 kDa) in contact with the inoculated medium. Time course samples were diluted to subinhibitory levels and plated onto nutrient agar plates. Activity was examined in relation to an established disc diffusion method. Compared to control wafer MRSA population density decreased dramatically over a 24-hour period. PVP-I and ChD displayed a four-log reduction of MRSA within 5-6 hrs and 7-8 hours respectively, whereas NS took between 8 and 24 hours. The insoluble nature of SS limited its passage through the membrane from wafer to dissolution medium. In conclusion, the release of antimicrobials from impregnated lyophilised wafers has been clearly demonstrated . Such antimicrobial release has a profound effect upon the viability of planktonic MRSA. The modified Franz diffusion cell represented an effective in vitro method, compared to disc diffusion, for evaluating efficacy of antimicrobial wafers.