Made available in DSpace on 2016-08-15T20:31:14Z (GMT). No. of bitstreams: 1 TeresaEPG_DISSERT.pdf: 731676 bytes, checksum: f024d9272492149e6edd0a0a8f38a28f (MD5) Previous issue date: 2010-12-22 / It evaluated the quality of carne-de-sol in the period from June to September of 2010 sold in five cities of Rio Grande do Norte - Mossoró, Apodi, Areia Branca, Baraúna and Grossos. It was collect 80 samples of carne-de-sol, of which 44 were from small establishments and 36 purchased in supermarkets and stores. The samples were subjected to microbiological testing - Salmonella, determining the most probable number of fecal coliform, halophilic bacteria and Staphylococcus aureus, the samples were positive for this bacteria were analyzed for resistance to antibiotics, and physical-chemical properties (pH, water activity, minerals and humidity). There was an interaction between intrinsic pH value (&#956; = 5.702) and Aa (&#956; = 0.868), , indicating a favorable environment for their multiplication, resulting in values that stood out significantly in what is expected legislation counting S. aureus (&#956; = 4.81 log UFC at 78.75%), Salmonella (25%) and fecal coliform (&#956; = 2.36 LogNMP / g, 63.75%). A significant correlation (p <0.05) between the number of halophilic bacteria (&#956; = 4.87 log UFC) and S. aureus, such conditions may be used as an indicator of contamination in the sun-dried meat. To the antibiotics, 22.5% of strains were resistant to gentamicin, whereas for penicillin G, chloramphenicol, tetraciclina were 100%, 67.5% and 46.26% respectively. It was found that the meat produced and marketed in the sun studied region is improper (96.25%) for consumption in accordance with Brazilian law / Foram avaliadas a qualidade da carne-de-sol no período de junho a setembro de 2010 comercializada em cinco cidades do Rio Grande do Norte Mossoró, Apodi, Areia Branca, Baraúna e Grossos. Sendo coletadas 80 amostras de carne-de-sol, as quais 44 foram provenientes de estabelecimentos de pequeno porte e 36 adquiridas em supermercados e frigoríficos. As amostras foram submetidas à análises microbiológicas Salmonella, determinação do número mais provável de coliformes termotolerantes, bactérias halofílicas e Staphylococcus aureus, as amostras positivas para esta bactéria foram submetidas à análise de resistência à antibióticos; e físico-químicas (pH, atividade de água, cinzas e umidade). Verificou-se interação dos fatores intrínsecos, pH (µ=5,702) e Aa (µ=0,868), do produto com a contagem bacteriana, indicando ambiente favorável para a sua multiplicação, resultando assim em valores que sobressaíram de maneira significativa o que está previsto na legislação para contagem de S. aureus (µ=4,81LogUFC, em 78,75%), Salmonella (presença em 25%) e coliformes termotolerantes (µ=2,36LogNMP/g, em 63,75%). Houve correlação significativa (p<0,05) entre a contagem de bactérias halofílicas (µ=4,87LogUFC) e a de S. aureus, podendo aquelas serem utilizadas como indicador de contaminação na carne-de-sol. Quanto à sensibilidade das cepas S. aureus aos antibióticos, 22,5% das cepas apresentaram resistência à gentamicina, enquanto para penicilina G, tetraciclina e cloranfenicol foram 100%, 67,5% e 46,26% respectivamente. Ficou constatado que a carne de sol produzida e comercializada na região estudada apresenta-se imprópria (96,25%) para o consumo de acordo com a legislação brasileira

Made available in DSpace on 2015-04-01T14:16:02Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 578629 bytes, checksum: 0e1eef478991e7f411dddd815ac56a75 (MD5) Previous issue date: 2013-08-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Furocoumarins (FCs) are an important class of photoactive compounds which may potentially bind to DNA forming intermolecular complexes, and once excited by UVA light (~ 365 nm) they re able to form photoadducts, which may result in mutagenicity and lethality. However, when the 8-methoxypsoralen (8-MOP) is added to the post-irradiation plating medium increases the sensitization probably by inhibiting repairs in damaged DNA. The FCs also have a protective effect against UVC (~254 nm) attributed to the inhibition of pyrimidine dimers. FCs associated with UVB light (312 nm), remain few known. The aim of the present study was to investigate the lethal effect of UVB light alone, and combined with solutions of 8-MOP, 4,5',8-trimethylpsoralen (TMP) and 3-carbetoxypsoralen (3-CPs) at different concentrations on Staphylococcus aureus growth. We also evaluated the effect of these FCs in the plating medium. Treatment with 8-MOP-UVB and TMP-UVB were more effective in inducing lethality than the UVB treatment alone. Increasing the solution concentration of 8-MOP resulted in a higher mortality while the increase in the concentration of the TMP led to a reduction in the lethality. For other hand, 3-CPs displayed a photoprotective effect against UVB damage in all concentrations tested. The results of FCs in the plating medium showed that the 8-MOP induzed a higher lethal effect and also increased mortality from bacterial strain treated by FC-UVB. The different behaviors shown by FCs may be related with differences in the sequence specificity of binding and photoreaction, inhibition of pyrimidine dimers formation by intercalated molecules and efficacy of repair systems. These results emphasize the need for further studies to elucidate the participation of FCs as photosensitizing and photoprotective agents in biological systems, when combined with UVB. / As furocumarinas (FCs) são uma importante classe de compostos fotoativos que potencialmente podem se ligar ao DNA formando complexos intermoleculares, e uma vez excitados por luz UVA (~365 nm) são capazes de formar fotoadições, que podem resultar em mutagênese e letalidade. Porém, quando a 8-metoxipsoraleína (8-MOP) está adicionada ao meio de plaqueamento pós-irradiação aumenta a sensibilidade provavelmente por inibir reparo de lesões no DNA. As FCs também possuem efeito protetor contra UVC (~254 nm) atribuído à inibição da formação de dímeros de pirimidina. As FCs associadas à UVB (312 nm) permanecem pouco conhecidas. O objetivo do presente estudo foi investigar o efeito da luz UVB, e combinada com soluções de 8-MOP, 4,5 ,8-trimetilpsoraleína (TMP) e 3-carbetoxipsoraleína (3-CPs) em diferentes concentrações, sobre o crescimento de Staphylococcus aureus. Avaliamos também o efeito destas FCs em meio de plaqueamento. O tratamento com 8-MOP-UVB e TMP-UVB foram mais eficazes em induzir letalidade do que o tratamento apenas com UVB. O aumento da concentração de 8-MOP resultou em mortalidade mais elevada enquanto que o aumento na concentração de TMP levou a redução na mortalidade. Por outro lado, 3-CPs exibiu efeito fotoprotetor contra danos causados por UVB em todas as concentrações testadas. Os resultados com FCs no meio de plaqueamento mostraram que a 8-MOP induziu o maior efeito letal e também aumentou a mortalidade da cepa bacteriana tratada por FC-UVB. Os diferentes efeitos apresentados pelas FCs podem estar relacionados com diferenças na especificidade por seqüência de ligação e fotorreação, a inibição da formação de dímeros de pirimidina por moléculas intercaladas e eficácia de sistemas de reparo. Esses resultados reforçam a necessidade de mais estudos para elucidar a participação das FCs como agentes fotossensibilizantes e fotoprotetores em sistemas biológicos, quando combinadas com UVB.

Les plaquettes sanguines sont des cellules anucléées qui jouent un rôle majeur dans l’hémostase. Au-delà de cette fonction, elles possèdent une composante inflammatoire multifacette ; recouvrant la détection du signal de danger, la libération de cytokines et la migration leucocytaire. Dans ce contexte, la première partie de ces travaux met en avant la capacité des plaquettes à mettre en place une activation de type inflammatoire en réponse à un pathogène. En effet, lors de l’infection à HIV les plaquettes sont dans un état d’hyperréactivité et libèrent des facteurs immunomodulateurs pouvant participer à l’inflammation observée chez les patients infectées. D’une manière parallèle, les plaquettes présentent une sensibilité aux bactéries, faisant intervenir les TLR2 et 4 mais aussi les exotoxines, voire les bactéries entières. Le profil de la réponse inflammatoire induite est assez conséquent et diversifié pour participer à la physiopathologie du sepsis. La participation des plaquettes à l’inflammation concerne également leur interconnexion avec les neutrophiles. La seconde partie des travaux traite d’ailleurs de cette coopération qui ne semble pas s’arrêter à la barrière endothéliale, car lors de leur extravasation les neutrophiles transportent avec eux les plaquettes ; qui sont encore capables d’entretenir l’inflammation au niveau du site inflammatoire (ici, modèle de l’alvéole pulmonaire). La diversité du répertoire moléculaire plaquettaire, mis en avant au cours de cette thèse, qui participe à l’inflammation ouvre plusieurs possibilités quant à l’élaboration d’anti-plaquettaires qui pourraient moduler une réponse inflammatoire exacerbée / Blood platelets are anucleate cells which play an key role in haemostasis. In addition to this function, they participate in a number of other functions related to the inflammatory response including danger detection, cytokine release, and leukocyte transmigration. In the first part of the study, we highlight the ability of platelets to undergo an inflammatory activation response to a pathogen. Indeed during HIV infection, platelets are hyperresponsive and release immunomodulatory factors that can be involved in the inflammatory state seen in the patients. In a parallel way, platelets are also sensitive to bacteria, involving TLRs 2 and 4, exotoxins, as well as whole live bacteria. The inflammatory profile induced is sufficient, and quite diversified to participate in sepsis physiopathology. Platelet inflammatory functions also apply to their ability to crosstalk with neutrophils. Thus in the second part of our study, we focus on this interconnection, which does not appear to be stopping at the endothelial barrier, and can be seen during extravasation where neutrophils carry surface bound platelets in order to maintain inflammation directly onsite (alveolar inflammation model here).The diversity of platelet inflammatory activities highlighted in our work leads to several possibilities for the development of an antiplatelet therapeutic target which could modulate an exacerbated inflammatory response

As condições de produção de queijos frescos são favoráveis à ocorrência e à produção de enterotoxinas produzidas por estafilococos, sendo estes os principais micro-organismos relacionados aos casos de intoxicação alimentar no mundo, tornando-se necessários estudos sobre a rastreabilidade das fontes de contaminação durante a fabricação, identificação genotípica e habilidade de cepas em produzir enterotoxinas. Este trabalho teve como objetivo isolar e identificar estafilococos positivos para o teste de coagulase, potencialmente produtores de enterotoxinas em laticínios do estado de São Paulo, desde o leite recebido até o produto final. A técnica da mPCR foi utilizada para identificar três espécies de Staphylococcus coagulase-positiva (S. aureus, S. hyicus e S. intermedius) entre os isolados obtidos de diferentes pontos de processamento em laticínios do Estado de São Paulo. O perfil genético das cepas foi avaliado através da comparação de bandas pela técnica Enterobacteria Repetitive Intergenic Consensus (ERIC-PCR) e graficamente demonstrados por um dendrograma. O DNA de 102 cepas isoladas, de amostras de leite cru, leite pasteurizado, coágulo, manipulador, superfícies de equipamentos de laticínios do Estado de São Paulo e queijos foi extraído e submetido à reação em cadeia da polimerase (PCR), utilizando-se primers específicos para a detecção de fragmentos dos genes envolvidos na síntese das toxinas (SE) do tipo A, B, C, D, E, G, H, I e J. Das 102 cepas avaliadas, 5 apresentaram amplificação do fragmento do gene responsável pela codificação da toxina A, 3 para toxina C, 56 para toxina G, 59 para toxina H, 9 para toxina I, e nenhuma para toxinas B, D, E e J. Amostras de leite cru, leite pasteurizado e queijos produzidos nos laticínios e dos isolados de Staphylococcus spp. coagulase positiva que apresentaram ao menos algum dos genes relacionados à produção de toxinas clássicas (A, B, C, D e E) foram submetidos a um teste de detecção de enterotoxinas pelo sistema VIDAS® Staph Enterotoxin (SET2). Os dados obtidos para a identificação molecular das cepas, da ocorrência de cepas portadoras dos genes relacionados à produção de enterotoxinas e da produção fenotípica das enterotoxinas foram submetidos ao teste qui-quadrado. O presente trabalhou confirmou que o risco de intoxicação estafilocócica é real, pois foi encontrada enterotoxina em amostras de leite pasteurizado e queijos. / The production conditions of fresh cheese are favorable to the occurrence and production of enterotoxin produced by Staphylococci, which are the main microorganisms related to food poisoning cases in the world, requiring studies to trace the sources of contamination during manufacturing and genotypic identification ability of strains to produce enterotoxin. This study aimed to isolate and identify staphylococci positive for coagulase test, potentially producing enterotoxins in dairy products in the state of São Paulo, from milk receiving to final product. In three dairy producers of Minas fresh cheese, samples were collected from various points in the manufacturing process. the technique of mPCR was used to identify three coagulase-positive species (S. aureus, S. hyicus and S. intermedius) between isolates from different points of a processing dairy in the state of São Paulo. The genetic profile of the strains were evaluated by comparing the bands through the technique Enterobacteria Repetitive Intergenic Consensus (ERIC-PCR) and were graphically displayed by a dendrogram. The DNA of 102 strains isolated from samples of raw milk , pasteurized milk , curd, handler and equipment surface from dairies in São Paulo State and cheese were subjected to the polymerase chain reaction (PCR), using primers for the detection of specific fragments of the genes involved in the synthesis of toxins (SE) of type A, B , C , D, E, G , H, I and J. Of the 102 strains tested, five showed amplification of the gene fragment encoding toxin A, three for toxin C , 56 to toxin G, 59 to toxin H, 9 to toxin I, and none for toxins B D, E and J. For confirmation, each isolated strain carrying at least some of the genes related to the production of classical enterotoxinas, cheese and milk samples was subjected to detection by enterotoxigenic VIDAS® Staph Enterotoxin II (VIDAS SET2) bioMérieux. This identification reinforces the need to adopt proper hygiene practices in the dairy, avoiding thus the spread of these microorganisms and the possible production of enterotoxin . The data obtained for the molecular identification of the strains, the occurrence of strains carrying the genes related to the production of enterotoxin production and phenotypic enterotoxins were subjected to the Chi-squared test. This work confirmed that the risk of staphylococcal food poisoning is real, because enterotoxin was found in samples of pasteurized milk and cheeses.

Les brûlures thermiques affectent plusieurs millions de personnes chaque année dans le monde. Même si le taux de mortalité à diminué, la prise en charge clinique des brûlures sévères a peu évolué depuis plus de 30 ans. De plus, le traitement des grands brûlés demeure une source de séquelles physiques, pathophysiologiques et psychologiques non négligeables.Les plasmas froids (CAP) sont des gaz partiellement ionisés qui ont démontré des propriétés bioactives grâce aux espèces réactives qu’ils produisent. En effet, ils permettent d’améliorer la cicatrisation de plaie cutanée et ont des propriétés bactéricides et fongicides.Dans le cadre de cette thèse, nous avons évalué l’effet thérapeutique des CAP pour le traitement de brûlures cutanées.Nous avons démontré pour la première fois que le CAP favorise la cicatrisation et la prise de greffe dans un modèle murin de brûlure cutanée. In vivo, CAP stimule la cicatrisation en promouvant l’angiogenèse et la sécrétion de la matrice extracellulaire du derme et de la jonction derme-épiderme. Des études approfondies in vitro ont premièrement montré que CAP stimule la migration de cellules cutanée. Il a aussi été démontré que les effets pro-angiogéniques du plasma sont liés à des mécanismes impliquant l’oxyde nitrique. De même, la formation du collagène par les fibroblastes est activée par le plasma via la voie du TGF-β1. Nous nous sommes dans un deuxième temps intéressés aux effets bactéricides du CAP. En effet, les infections sont la première source de morbidité et de mortalité des brûlures. Nous avons pu démontrer que les CAP inactivent des Staphylocoques dorées en suspension in vitro mais aussi in vivo dans le cas de plaies infectées. Dans le cadre de cette étude, nous avons pu également montré que les CAP peuvent activer l’activité phagocytaire de macrophages murins.Ces résultats semblent donc appuyer l’intérêt des traitements plasmas froids pour le traitement de plaie cutanée infectée. Des études pré-cliniques sont encore néanmoins requises pour vérifier la plus value d’une telle thérapie dans le contexte de la brûlure. / Thermal injuries affect millions of adults and children worldwide. In the last 30 years little progress has been made in the management of major burns and despite improved rates of survival, patients continue to suffer debilitating consequences such as infections, hypertrophic scars, contractures, post-traumatic stress disorder and immunometabolic dysfunctions.Cold-atmospheric plasmas (CAP) are partially ionized gases which showed ability to promote wound closure and possess bactericidal properties. They safely deliver to the wounds a therapeutic mixture of reactive oxygen and nitrogen species at body temperature.This PhD study aimed to assess the therapeutic potential of CAP to improve healing of burn wounds under aseptic and septic conditions.First, we designed a plasma source that could be used for in vitro and in vivo studies. Although the first in vitro experiments showed that CAP treatment can improve cellular migration, we couldn’t see any beneficial of CAP on the healing of full-thickness excisional wounds in mice. Second, using a model of third degree burn reconstructed with an allogeneic graft, we assessed both effectiveness and mechanism of action of CAP compared to a placebo control (helium). Indeed, it was shown that through the promotion of pro-angiogenic activities as well as stimulation of the extracellular matrix formation, CAP improved engraftment and healing of the burn wounds. These effects are at least partly mediated by the TGF-β signaling pathway and eNOS modulation. Third, the bactericidal activity of CAP was evaluated in vitro using S. aureus strains and macrophages and in vivo using an infected burn wound model. We showed that CAP had the ability to significantly inactivate S. aureus compared to the helium control. Capacities of CAP treatment to activate macrophages were also evident.This proof-of-concept study demonstrated the therapeutic potential of CAP on tissue repair and burn wound healing. Given that mouse skin does not perfectly mimic wound healing patterns observed in human skin, further investigations are warranted using large animal models with greater similarity to human skin.

Efter upptäckten av penicillinet observerades en ökad resistens hos S. aureus vilket ledde till att alla stammar ansågs vara resistenta. Dock har många stammar enligt senare studier visat sig vara känsliga vilket har öppnat upp möjlighet att åter använda benzylpenicillin som ett behandlingsalternativ eftersom det kan ges i lägre koncentration jämfört med andra alternativ. Metoden för att upptäcka penicillinresistens hos S. aureus enligt EUCAST har bedömts svår att tillämpa på grund av subjektivitet vid bedömning av zonkanten. Studiens syfte var att utvärdera metoden för senare implementering på Klinisk Mikrobiologi Unilabs i Skövde. Metodens användbarhet utvärderades genom resistensbestämning av en referensstam samt tio stammar från ett referenslaboratorium för EUCAST. Bedömningen av de tio stammarna från referenslaboratoriet stämde vid jämförelse överens med deras resultat förutom för en stam som bedömdes som resistent i stället för känslig. Repeterbarheten var hög då ingen skillnad (p=0,156) uppvisades för referensstammen.  Resultatet tyder på att det kan vara svårt att göra en korrekt tolkning av zonkantens utseende då det är en subjektiv bedömning. För att uppnå en högre säkerhet kan det vara fördelaktigt att mer än en person gör en bedömning. Slutsatsen var att metoden kan implementeras på laboratoriet. / Following the discovery of penicillin, resistance rates increased until all strains of S. aureus were considered resistant. Recent studies have shown that many strains are susceptible, making it possible to again use penicillin as an alternative for treatment since it can be used in lower concentrations compared to alternatives. The EUCAST method for discovering penicillin resistance in S. aureus has been judged difficult due to the subjectiveness of assessing the zone edge. The aim of this study was to evaluate the method to implement it in the Section for Clinical Microbiology Unilabs Skövde. The useability of the method was evaluated by resistance testing of a reference strain and ten strains from a EUCAST reference laboratory. The assessment of the strains from the reference laboratory equaled their results except for one strain that was assessed resistant rather than sensitive. The repeatability was high since no difference (p=0,156) was found for the reference strain. The results show how difficult it is to correctly assess the zone edge due to its subjectiveness. It could be beneficial with more than one person assessing the zone edge to achieve a higher security. In conclusion the method is possible to implement in the laboratory.

Dans le but de mieux comprendre le mode d’action et de nous permettre de déterminer la cible de la tomatidine, nous avons dans un premier temps tenté de mieux circonscrire le spectre d’activité de la tomatidine. Grâce à ces travaux, nous sommes, en effet, maintenant en mesure de dire que la tomatidine possède une activité antibactérienne contre les espèces de la division des Firmicutes et plus précisément contre les bactéries de l’ordre des Bacillales dont font partie les genres Bacillus, Staphylococcus et Listeria. Nous avons également découvert, grâce à des expériences en collaboration avec le laboratoire d’Éric Marsault, qu’un analogue de la tomatidine (FC04-100) avait non seulement des propriétés similaires à la molécule naturelle, mais démontrait une activité par lui-même contre S. aureus à phénotype normal alors que la tomatidine possède uniquement une activité contre les « small colony variants ». De plus, alors que la tomatidine possède plutôt une activité bactériostatique contre la forme SCV de L. monocytogenes, le nouveau composé (FC04-100) démontre quant à lui, une forte activité bactéricide contre cette souche, tout comme contre la forme SCV des autres Bacillales. Parallèlement, et toujours dans le but de rechercher le mode d’action et la cible de la tomatidine, nous avons obtenu, par passages successifs dans un milieu avec antibiotiques, des mutants de S. aureus à phénotype normal et des SCV résistants à la tomatidine ou à la combinaison tomatidine et gentamicine. Après le séquençage de ces mutants, l’étude de la position de ces mutations, à l’aide de différents logiciels de bio-informatique, nous a permis d’émettre un modèle-hypothèse quant au mode d’action et à la cible de la tomatidine. Selon les résultats que nous avons à ce stade-ci, la cible de la tomatidine chez S. aureus serait la sous-unité c de l’ATP synthase. Cependant, son mode d’action serait également dépendant de la fonctionnalité de la chaine de transport des électrons et donc de la polarisation membranaire et de la production de ROS intracellulaire, ce qui expliquerait la différence d’activité entre les souches à phénotype normal et les SCV.

Atopic Dermatitis (AD) is a common chronic relapsing inflammatory skin disease affecting 15 - 20% of children and 2 - 10% of adults worldwide, with significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics and infections. Another typical feature of AD is skin infections, especially from Staphylococcus aureus (S. aureus), which closely relates with the disease severity. Although not a normal flora, S. aureus is found on 75-100% of AD lesions (< 30% on healthy skin). S. aureus secrete a range of virulence factors, including extracellular toxins and proteases which contribute to disease pathogenesis. S. aureus serine protease A (SspA/V8) is a well-characterised extracellular protease widely expressed among different S. aureus strains. The pathogenic effect of V8 protease has been demonstrated in vivo, damaging murine skin integrity via effects on the stratum corneum (SC), but the targets for this V8-mediated damage remains unclear. The capacity of proteases to induce barrier dysfunction has been proposed as a key driving force in the initiation and exacerbation of AD. Thus, understanding the host factors that maintain barrier function is a priority in developing novel therapeutic approaches. This thesis therefore aimed at detecting host factors which can combat the barrier dysfunction caused by pathogenic proteases, assessing their relevance in vitro and ex vivo and elucidating the underlying mechanisms. Firstly, an in vitro skin barrier integrity model was developed, using both immortalized and primary keratinocytes, to evaluate the barrier damage mediated by pathogenic proteases. The results revealed that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment. In addition, studies demonstrated that V8 protease itself was sufficient to induce barrier disruption, and this phenotype was not dependent on cell death, but rather on breaking down of cell-cell junctions. Key tight junction proteins including claudin-1 and occludin were found to be degraded by V8 protease. Next, a wide range of host and bacterial factors were investigated to determine whether they could promote protection of keratinocytes against V8 damage. Several factors, including IL-1β, TNF-α, heat-killed Staphylococcus epidermidis (which is the main skin normal flora), were found to induce protection against V8 protease, with IL-1β having the strongest effect. In addition, data indicated that this IL-1β-mediated protection was independent of effects on claudin-1 but occurred via secretion of a transferrable host factor. Induction of keratinocyte expression of the antimicrobial/host defence peptide human beta-defensin 2 (hBD2) was found to be the mechanism underpinning this IL-1β- induced protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was also protective. An ex vivo model using human skin tissue was also established to address this novel function of hBD2, and preliminary data indicated that exogenous hBD2 protected against V8-mediated damage in this system. Overall, my data reveal a novel function for the antimicrobial/host defence peptide hBD2. This modulatory property of hBD2, independent of its antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential to prevent S. aureus-mediated aggravation of skin barrier dysfunction in AD.

Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of respiratory disease in humans. One of the main complications of primary IAV infection is increased susceptibility to secondary bacterial co-infection, with Staphylococcus aureus being the most common co-infecting species. Previous work identified secreted proteases from S. aureus as a pro-viral factor, leading to specific cleavage of the IAV surface hemagglutinin and increase in infectious viral titre. The aim of this study was to investigate the effect of bacterial proteases, and other secreted bacterial proteins, on IAV replication. Supernatants from the S. aureus community-associated epidemic clone USA300 were separated by size exclusion chromatography and each fraction was tested for an impact on IAV replication in primary chicken embryo fibroblast (CEF) cells. A fraction that increased viral titre by at least 10-fold was identified, but this effect was independent of known secreted proteases. Through the use of mass spectrometry fingerprinting and bacterial mutagenesis, a single protein, S. aureus lipase 1, was identified to be responsible for the pro-viral effect. Lipase 1 is expressed by an array of diverse S. aureus strains of distinct clonal origins. Both the native and recombinant form of lipase 1 were pro-viral only during the infection of primary cells, including primary human lung fibroblasts. Further validation of this interaction indicated lipase 1 was pro-viral in a concentration dependant manner and for a range of IAV strains. Investigation into the mechanism of action of lipase 1 revealed the protein acts during a single infectious cycle in a manner dependent on its active site. Time of addition studies and western blot analysis showed lipase 1 affects the later stages of virus replication, but there is no direct interaction with the virus particle; rather, the protein manipulates the cell, resulting in an increased number of infectious particles being produced. This work has identified and validated a single S. aureus protein, which affects IAV replication. Thus, it has elucidated some of the complex interactions that occur between the virus and bacteria during co-infection. It has also demonstrated a novel role for a bacterial enzyme in IAV replication, the study of which can further our understanding of both IAV and cell biology.

Biofilm phenotypes were studied in 32 Staphylococcus aureus strains isolated from store-bought meats and 22 from diseased patients in hospitals. Of the meat-associated strains, 21 were methicillin-resistant Staphylococcus aureus (MRSA) and 11 were methicillin-susceptible Staphylococcus aureus (MSSA). The hospital-associated strains included 15 MRSAs and 7 MSSAs. We studied the robustness and composition of the biofilms produced by these strains. We found that on average hospital-associated strains form more robust biofilms than meat associated strains. The model often used to describe S. aureus biofilm composition includes two biofilm types defined by the presence or absence of polysaccharide intercellular adhesin (PIA), PIA-dependent and PIA-independent respectively. In this model, PIA-independent biofilms are structurally reliant on proteins and extracellular DNA (eDNA) and PIA-dependent are structurally reliant on polysaccharides. Enzymatic degradation of the extracellular matrix can reveal which compounds are essential for the structural integrity of the biofilm, and by this model PIA-independent biofilms should be susceptible to both DNase and proteinase K. We found that hospital-associated strains are, on average, more susceptible to degradation by proteinase K. Interestingly, hospital-associated strains are less susceptible to degradation by DNase than meat-associated strains. Finding that proteinase K and DNase susceptibility for these strains are not linked gives evidence to support the idea that S. aureus biofilm composition can vary greatly from strain to strain and that the PIA-dependent and PIA-independent dichotomy of the standard model may be insufficient to describe the variety of S. aureus biofilm composition and may only apply to the extremes of the spectrum. Additionally, we saw no relationship between MRSA or MSSA strains and biofilm robustness, proteinase K degradation, or DNase degradation. Differences in biofilm characteristics between hospital-associated and meat-associated strains reinforce previous findings that these populations are genetically distinct.