No description available.

This research focuses on the antimicrobial activity of the mouse chemokine CCL28. In addition to their well characterized chemotactic activity, many chemokines have been shown to be antimicrobial in vitro, including the mucosally expressed chemokine CCL28. I have investigated the primary sequence features required for antimicrobial activity, salt sensitive nature of killing/binding mechanism, and in vivo microbial interactions of CCL28. Through the use of protein mutation and expression techniques, I have shown that the holoprotein (108 amino acids) is necessary for full antimicrobial activity of CCL28. Furthermore, the C terminal region of CCL28 is essential for microbial killing as an almost complete loss of antimicrobial activity is seen following the removal of the C terminal 24 amino acids. The positively charged amino acids of the C-terminus directly contributed to the antimicrobial activity of CCL28. These experiments are the first to investigate the role of primary structure on the killing activity of an antimicrobial chemokine. Using flow cytometry analysis, I found that the salt-sensitive nature of CCL28 killing activity corresponds to its binding ability. Additionally, I have shown direct evidence for in vivo interaction between commensal bacteria and endogenously expressed CCL28 in the mouse large intestine. This interaction may directly correlate to the in vivo antimicrobial activity of CCL28. Lastly, I have begun to generate a CCL28 knockout mouse model to directly address the in vivo antimicrobial activity of CCL28. Vector construction and ES cell targeting by the vector has been completed, chimeric mouse generation remains to be done. This work represents the first systematic study of antimicrobial chemokine function. This work extends our understanding of antimicrobial proteins and their role in innate immune protection of the host and provides guidance for making better alternative antimicrobials.

No description available.

Yes / The purpose of this workshop was to promote interdisciplinary learning and collaborations between UK and India in the area of bacteria and wounds, particularly when bacteria are increasingly antibiotic resistant. To this end we invited colleagues from India with clinical experience ofmanaging bacterially infected wounds in patients, particularly when antibiotic resistant (Dr Prashant Garg) and colleagues from India with expertise in developing new drug delivery systems (Prof Nikhil Singh and Dr Vamsi Venuganti) and together with UK delegates, spanning those with international experience in investigating bacterial infections (Prof Simon Foster), investigating how biofilms communicate (Prof Paul Williams), international expertise in discovering materials for antimicrobial resistance and for instruction of macrophages (Prof Morgan Alexander), expertise in developing responsive films for detecting infection in wounds using a visible signal (Prof Toby Jenkins) and other scientists working on the axis of detecting and treating infection in wounds. / MRC/DBT

The rise of antimicrobial resistance (AMR) has culminated in the most pressing problem in modern medicine. The situation is most acute with regards to the management of multi- drug resistant Gram-negative infections (MDRGNB) with common infections increasingly untreatable due to rapidly dwindling therapeutic options. A solution to the problem of AMR is unlikely to be easily found, but revisiting and re-purposing existing antimicrobials is a viable approach in the medium term. This study investigated the use of unorthodox antimicrobial combination therapies for the treatment of MDRGNB, with particular focus on agents of last resort. A systematic review of clinical studies highlighted the potential for polymyxin (colistin) combination therapies (e.g. colistin-rifampicin, colistin-carbapenems), although this could not be supported in a formal meta-analysis. A systematic approach for screening MDRAB for susceptibility to novel colistin combinations using multiple methods was employed and uncovered a number that were more potent than those previously identfied. The most potent combination that was consistently identified was colistin when combined with fusidic acid, despite this drug having no useful activity against MDRGNB on its own. The combination was further evaluated in static time-kill assays against a range of Gram-negative pathogens with defined resistance mechanisms, including to polymyxins and using invertebrate (Galleria mellonella) and murine models of MDRGNB infection. Colistin and fusidic acid combination therapy was subsequently used to successfully treat a case of ventilator-associated pneumonia due to MDR A. baumannii. This work highlights how older drugs can be re-purposed to tackle the problem of AMR using a precision medicine approach. Further studies to elucidate the mechanism of action of the colistin- fusidic acid combination and a formal clinical trial are warranted to investigate the potential utility of this combination in the treatment of MDRGNB with the expressed goal of bridging the current antimicrobial development gap.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-04T11:59:28Z No. of bitstreams: 2 Dissertação - Carolina Oliveira Matos - 2015.pdf: 5441476 bytes, checksum: 86d4c7bb5073b76109534a172cf579e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-04T12:01:47Z (GMT) No. of bitstreams: 2 Dissertação - Carolina Oliveira Matos - 2015.pdf: 5441476 bytes, checksum: 86d4c7bb5073b76109534a172cf579e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-04T12:01:47Z (GMT). No. of bitstreams: 2 Dissertação - Carolina Oliveira Matos - 2015.pdf: 5441476 bytes, checksum: 86d4c7bb5073b76109534a172cf579e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Antimicrobial peptides are part of the innate defense of several organisms, and represent candidate drugs in their natural form, allowing for the development of modified peptides with improved pharmacological profiles. In this context, this study aimed of the synthesis of Cn-AMP1, an antimicrobial peptide naturally isolated from green coconut water, which has activity against fungi, gram-positive and gram-negative bacterias well as effects on the proliferation of cancer cells and low toxicity to mammalian cells, the analogs [Trp9] Cn-AMP1 and [Gly9] Cn-AMP1 were also obtained by solid phase peptide synthesis using the Fmoc strategy. The characterization and purification of the peptides were performed by mass spectrometry and high-performance liquid chromatography. Structural studies of the peptides were performed by circular dichroism (CD) and nuclear magnetic resonance (NMR) in the presence of biomimetic enviroments. CD and NMR results indicated that the peptides do not present preferential conformations in aqueous solutions, however adopt helical conformations in membrane mimetic environments. CD studies have shown that Cn-AMP1 and [Gly9] Cn-AMP1 do not adopt show defined conformation in the presence of DPC micelles at different pH values, however the peptide [Trp9] Cn- AMP1 showed a small a-helical content in the presence of 100mM DPC. In the presence of SDS the spectra of all peptides showed helical profiles at both pH 4, pH 7 as well as in the absence of buffer. NMR experiments indicated the interaction of the peptides [Trp9] Cn-AMP1 and [Gly9] Cn-AMP1 with SDS micelles at pH 4 (25 °C), and structural calculations indicated that [Trp9]Cn- AMP1 adopts an α-helical conformation between Val2-Gly8, and that [Gly9] Cn- AMP1 adopts an α -helical conformation between Val2-Arg5. Dynamic light scattering and zeta potential experiments were performed to investigate the effect of addition of peptides into phospholipid vesicles. All of the peptides caused variations on the POPC:POPG (3:1) and POPC, LUVs hydrodynamic radios, however major changes were observed for the anionic vesicles due to the strong interaction between the arginine residue of the peptides and the negativelly charge of membrane. / Os peptídeos antimicrobianos fazem parte da defesa inata de vários organismos e representam candidatos a fármacos em sua forma natural, o que possibilita o desenvolvimento de peptídeos modificados com perfis farmacológicos melhorados. Neste contexto, o trabalho teve como objetivo a síntese do peptídeo antimicrobiano Cn-AMP1, originalmente isolado da água de coco verde, o qual apresenta atividade contra fungos, bactérias grampositivas e gram-negativas, efeitos sobre a proliferação de células cancerígenas e baixa toxicidade em células de mamíferos. Foram também sintetizados seus análogos [Trp9]Cn-AMP1 e [Gly9]Cn-AMP1 por síntese de peptídeos em fase sólida utilizando a estratégia Fmoc. A caracterização e purificação dos peptídeos foram realizadas por espectrometria de massas e cromatografia líquida de alta eficiência. Estudos estruturais dos peptídeos foram avaliados por Dicroísmo Circular (CD) e Ressonância Magnética Nuclear (RMN) na presença de meios biomiméticos. Os resultados de CD e RMN evidenciaram que os peptídeos se apresentam de forma desordenada em solução aquosa, porém adotam conformações helicoidais na presença de meios que mimetizam a membrana celular. Estudos detalhados de CD mostraram que os peptídeos Cn-AMP1 e [Gly9]Cn-AMP1 não apresentam estrutura definida em DPC a diferentes valores de pH, e o peptídeo [Trp9]Cn- AMP1 mostrou uma pequena estruturação em a-hélice na presença de 100mM de DPC. Em SDS todos os peptídeos apresentam um perfil de estrutura helicoidal em pH 4, pH 7 e sem a adição de tampão. Experimentos de RMN mostraram a interação dos peptídeos [Trp9]Cn-AMP1 e [Gly9]Cn-AMP1 com micelas de SDS em pH 4 a 25ºC. Cálculos estruturais demonstraram que o peptídeo [Trp9]Cn-AMP1 assume conformação em α-hélice entre os resíduos Val2-Gly8, ao passo que o peptídeo [Gly9]Cn-AMP1 assume conformação em α- hélice entre os resíduos Val2-Arg5. Experimentos de espalhamento de luz dinâmico e potencial zeta mostraram ainda o efeito da adição de peptídeos no tamanho e nas cargas superficiais de vesículas fosfolipídicas. Todos os peptídeos causaram variação no Dh de LUVs de POPC:POPG (3:1) e LUVs de POPC, tendo-se observado maiores alterações em vesículas predominantemente aniônicas de POPC:POPG, sendo favorecido pela maior aproximação e atração do resíduo de arginina presente nos peptídeos com a carga negativa extra das unidades de POPG.

Antimicrobial peptides (AMPs) and photosensitizers (PS) have gained attention as potential alternatives to traditional antibiotics for the treatment of microbial infection due to the decreased likelihood for acquired resistance. However, many AMPs and PS suffer from insufficient activity, specificity, or a combination thereof. AMPs can require high concentrations for effective activity, leading to non-specific side effects and increased costs. PS, on the other hand, are quite active, but are typically hydrophobic and suffer from non-specific binding and damage to host tissues. To solve these problems, we report a novel PS-AMP construct of the soluble PS eosin Y conjugated to the selective AMP (KLAKLAK)_(2). Eosin Y has a high singlet oxygen quantum yield, which is suitable for photodynamic activity, although the solubility of eosin Y results in poor binding and activity toward membranes on its own. On the other hand, the specificity of (KLAKLAK)_(2) is high for an AMP, but could still benefit from enhanced activity at lower concentrations. The killing activity and binding specificity of eosin-(KLAKLAK)_(2) toward both bacteria and mammalian cells was assessed using microbiology, biochemistry, and fluorescence microscopy techniques. Additionally, the mechanism of eosin-(KLAKLAK)_(2) activity was investigated using liposome models to determine factors involved in binding and membrane disruption. Furthermore, novel applications of transmission electron microscopy (TEM) methods were employed to observe the photodynamic effects of eosin-(KLAKLAK)_(2) against bacteria. The PS-AMP conjugate eosin-(KLAKLAK)_(2) displays synergistic activity between PS and AMP in model liposome systems, and is capable of killing several clinically relevant bacteria, including the multi-drug resistant Acinetobacter baumannii AYE strain. Furthermore, bacterial killing is achieved in the presence of red blood cells (RBCs) and other mammalian cell lines without significant toxicity. Liposome models reveal that the lipid composition of bacteria is a potential factor responsible for the observed binding specificity and corresponding activity. Additionally, TEM methods show that eosin-(KLAKLAK)_(2) causes extensive membrane damage to both Gram positive Staph aureus and Gram negative Escherichia coli, indicating a primary cause of cell death. A model is proposed where the activities of the PS and AMP, respectively, facilitate the activity of one another, leading to enhanced membrane disruption, and effective antibacterial activity while maintaining cell selectivity.

La résistance aux antibiotiques est une menace sérieuse pour la santé publique en Europe, entrainant une augmentation des coûts de la santé, des échecs thérapeutiques, et de la mortalité (ECDC, 2011). Le développement de l'antibiorésistance est principalement lié à la consommation d'antibiotiques chez l'Homme et les animaux. Depuis le début des années 2000, les pays européens ont limité cette consommation et en 2006, l'UE a interdit l'utilisation d'antibiotiques comme promoteurs de croissance. Ceci a favorisé le développement de diverses alternatives à l'utilisation d'antibiotiques. L'objectif principal de ce projet de thèse est d'évaluer l'utilisation d'alternatives spécifiques et non spécifiques à l'utilisation d'antibiotiques dans les élevages de porcs européens. L'étude sera organisée en trois parties: i) une évaluation technique, visant à quantifier le lien entre l'utilisation d'antibiotiques et les performances techniques des élevages, ii) une évaluation économique, basée sur une analyse coût-efficacité et coûts-bénéfices des stratégies alternatives aux antibiotiques et iii) une évaluation psychosociologique, décrivant les attitudes et les comportements des éleveurs, vétérinaires et scientifiques vis-à-vis de l'utilisation d'antibiotiques en élevage porcin. Ce projet impliquera à la fois la réalisation de visites d'élevages français, l'utilisation d'outils statistiques variés et de méthodes de recherche qualitative et d'évaluation des risques. Ainsi, ce projet fournira les bases d'une compréhension globale des facteurs techniques, économiques et psychosociologiques qui orientent les décisions des éleveurs et des vétérinaires au sujet de la santé et de la production porcine et qui, par conséquent, définissent les possibles interventions sur l'utilisation d'antibiotiques. Cette étude fait partie du projet de recherche du Consortium MINAPIG financé par le programme Emida Era-Net. Un financement supplémentaire est fourni par l'Office vétérinaire fédéral suisse. / Antimicrobial resistance is a serious threat to public health in Europe, leading to mounting healthcare costs, treatment failure, and deaths (ECDC, 2011). The development of antimicrobial resistance is mainly due to antimicrobial consumption in humans and animals. From early 2000s, European countries have implemented restriction measures and in 2006, EU banned the use of antibiotics as growth promoters in animal feed. This has promoted the development of various alternatives to antimicrobial. The main objective of this PhD project is to assess and evaluate specific and unspecific alternatives to antimicrobials in the European pig industry. The study will be organised in three main parts: i) a technical assessment, quantifying the link between antimicrobial use and technical performances of the pig farms, ii) an economic evaluation, conducting cost-effectiveness and cost-benefit analyses of alternative strategies in comparison with antimicrobial usage, and iii) a psycho-sociological evaluation, describing farmers, veterinarians and pig experts attitudes, beliefs and behaviours regarding the use of antimicrobials in pig farming. The project will involve field work in France, statistical analysis using a range of methods, qualitative research methods, conceptual work and the use of risk assessment methods. We expect this PhD project to provide the foundation for an integrated understanding of technical, economical and psychological factors driving decisions of farmers and veterinarians about pig health and production and the consequential interventions, particularly the use of antimicrobials. This study is part of the MINAPIG Consortium Research project funded by the Era-Net programme Emida. Additional funding is available through the Federal Veterinary Office of Switzerland.

Rapid development of antimicrobial resistance (AMR) among bacteria, combined with diminished new antibiotic discovery rates, is an increasing threat to human health. Bacterially derived antimicrobial peptides (AMP) hold excellent potential as potent novel therapeutics. This study embraces traditional natural AMP discovery methods and the newer in silico genome mining tool BAGEL 3 to facilitate identification of novel antimicrobial agents. The traditional screening efforts led to the discovery of two promising antimicrobial producer strains; Bacillus pumilus J1 producing two AMPs, peptides NI03 and NI04, and Klebsiella pneumoniae A7, which produced peptide NI05. In silico mining of the B. pumilus J1 and K. pneumoniae A7 genomes and those from under exploited anaerobic bacteria using BAGEL 3 yielded 18 putative bacteriocin structures that were associated with multiple known and relevant bacteriocin accessory genes and/or carried significant homologies to known bacteriocins. Peptide NI04 proved to be active against Gram positive species only, including meticillin resistant Staphylococcus aureus and vancomycin resistant enterococci and peptide NI03, in addition to these pathogens, showed activity against E. coli. Peptide NI05 was active against Gram-negative pathogens including extended spectrum β-lactamase producing E. coli. All isolated peptides were observed to be proteinaceous in nature and highly heat stable. Peptides were purified or partially purified using solid phase extraction followed by RP-HPLC. The mass of the peptides was determined using ESI or MALDI-TOF mass spectrometry. Tandem MS fragmentation of peptide NI04 generated several sequence tags. Draft genome sequences of the B. pumilus J1 and K. pneumoniae A7 producer strains were obtained using the Illumina MiSeq platform. This allowed identification of the genes encoding peptide NI04, which was confirmed to be novel and was named pumicin NI04. Further characterisation of pumicin NI04 demonstrated it was non-toxic to keratinocytes, Vero cells and non-haemolytic up to at least 18x the minimum inhibitory concentration. The discovery revealed that pumicin NI04 belongs to the WXG-100 peptide superfamily, having homology with the mycobacterial and staphylococcal virulence factor EsxA. This represents the first report of antimicrobial activity in a WXG-100 peptide and has intriguing evolutionary implications. Although it was not possible to fully characterise peptides NI03 and NI05, when BAGEL 3 was used to mine the B. pumilus J1 genome, a promising putative bacteriocin candidate was identified that was homologous to Enterocin AS-45, which also confers anti Gram-negative activity and may be related to the activity observed for NI03, however more evidence is required. Investigations of the K. pneumoniae A7 bacteriocin on the other hand helped establish that the K. pneumoniae microcin E492 pathway was present and highly conserved in strain A7, and is likely to be responsible for the activity observed indicating that NI05 was not a novel peptide.

No description available.