A chemically defined medium for Salmonella growth was developed and optimised using supplements of amino acids, nucleosides, vitamins and different carbon sources. The medium developed was compared to commercially available pre-enrichment media BPW and Salmosyst. Growth of Salmonella was significantly higher in BPW and Salmosyst than the medium developed. The amino acid and nucleoside supplement was directly compared with peptone. The results show peptone to be nutritionally superior and promoting better growth of Salmonella. Three different ELISA assays were used to detect Salmonella growing in four different media. The ELISA assay sensitivity was determined and a degree of media interference with the immunoassays was established. Salmonella culture viability was investigated using three different procedures: differential culturing on selective and non-selective media; fluorescence microscopy with BACLIGHT stained cells and flow cytometry analysis of BACLIGHT and BEP stained cells. Flow cytometry was found to be the most consistent, sensitive and rapid procedure for cell viability measurement. Clusters of viable cells unable to grow on solid media and therefore remaining undetectable by cultural methods were identified using flow cytometry. Severely heat injured Salmonella was used to determine media recoverability. The results indicate that media which contain peptone recover injured Salmonella better than chemically defined or other media. Detection of Salmonella was performed using PCR assay after sample pre-enrichment. The amplification of Salmonella DNA extracted using a crude method resulted in an assay sensitivity of 20 Salmonella cells in pure cultures. The specificity of the oligonucleotide primers employed in the PCR assay was confirmed. Non-salmonella organisms present in high numbers interfered with PCR detection of Salmonella. Food components also interfered with PCR amplification and reduced the assay sensitivity. Interference by food components and non-salmonella DNA was eliminated by the use of a 24 hour pre-enrichment followed by a 3 hour secondary enrichment, a rapid DNA extraction and template preparation. Using this system it was possible to detect 3 Salmonella cells per gram of food in the presence of 106 non-salmonella cells within 28 hours.

A resistência de cinco microrganismos presentes na microbiota da área de produção estéril (Cristalização Estéril), frente a ação do gás de peróxido de hidrogênio foi determinada e o valor O obtido para cada microrganismo foi comparado ao valor D do Bacillus stearothermophilus ATCC 12980 exposto ao mesmo agente. Os microrganismos testados foram Bacillus sp, M. luteus, Corynebacterium, Staphylococcus sp e Penicillium sp. Este teste tinha a finalidade de comprovar que a resistência do Bacillus stearothermophilus é maior quando da exposição ao peróxido de hidrogênio se comparada a outros microrganismos presentes na área produtiva. A metodologia consistiu da inoculação de 0,01 mL da suspensão de cada microrganismo na contagem de 102UFC/0,01 mL em cupons de aço inoxidável, previamente esterilizados por calor seco e posterior exposição ao gás de peróxido de hidrogênio. O experimento demonstrou que o valor D obtido para o Bacillus stearothermophilus ésuperior aos obtidos para os outros microrganismos em teste comprovando que a escolha deste microrganismo para o desafio contra o peróxido de hidrogênio é apropriada. Também executou-se o teste que visava garantir que o aço inoxidável é o material de suporte mais recomendado para este fim, utilizando-se suportes de diversos materiais normalmente encontrados no interior dos isoladores (PVC, aço inoxidável, CKC, teflon, polipropileno, látex, silicone, Hypalon, vidro, nylon, saco de alumínio) com 0,01 mL de inóculo de Bacillus stearothermophilus na contagem de 102UFC/O,01 mL, o que foi devidamente comprovado. / The resistance of tive microrganisms found in the sterile production area (Crystallization Area) flora was tested against the hydrogen peroxide gas and the D value of each microrganism was compared to the Bacillus stearothermophilus D Value ATCC 12980 exposed to the same agent. The microrganisms tested were Bacillus sp, M. luteus, Corynebacterium, Staphylococcus sp e Penicillium sp. The purpose of this test was to prove that the resistance of Bacillus stearothermophilus against the exposition the hydrogen peroxide is higher when compared to others microrganisms found in the production area. The methodology consisted in inoculating 0.01 mL of microrganisms suspension with 102UFC/0,01 mL count in stainless steel coupons, treated previously with dry heat and further exposition to the hydrogen peroxide. The experiment demonstrated that the Bacillus stearothermophilus D value is higher against all others microrganisms tested proving that the use of this microrganism for the challenge is appropriate. It was also pertormed a test to guarantee that the stainless steel support is the most recommended one for this purpose, using supports of different materials normally found in the interior of the isolators (PVC, stainless steel, CKC, Teflon, polypropylene, latex, silicon, Hypalon, glass, nylon, aluminum foil) with 0,01 mL Bacillus stearothermophilus inoculum with the count of 102UFC/O,01 mL, that was properly veritied.

Limestone built heritage is at risk from the effects of biofilms, a microbial community encapsulated in a matrix of sugars, protein and extracellular DNA. Although biofilm research has been carried out in Mediterranean regions, few studies cover temperate Northern Europe climates, or the UK. This study concentrates on bacterial colonisation of Lincoln limestone, a highly vulnerable building material, and identifies the species, their role in biodeterioration and the efficacy of biocides against them. As part of this study the core species which comprise the bacterial component of the limestone microbiome have been characterised for the first time; this has allowed the identification of non-core species which are significantly associated with damaged and undamaged surfaces. Four mechanisms of biodeterioration have been identified, one previously unidentified, and isolated species have been characterised as to whether they are biodeteriorative and the mechanisms of biodeterioration that they employ. Two species, Curtobacterium flaccumfaciens and Solibacillus silvestris, have been characterised as producing biofilm matrix which actively causes biomechanical damage to the oolitic limestone structure as opposed to the passive enhancement of physical weathering which has been previously associated with biofilm matrix. Species capable of biodeterioration have also been shown to be present on both damaged and undamaged surfaces, something which has not been previously investigated. Environmental sampling, species identification and characterisation of species for biodeterioration have all combined to identify markers of biodeterioration, ie both physical markers and biomarkers. Specifically, a surface pH of 5.5 or lower and the presence of B. licheniformis is indicative of biodeterioration with a proportionally higher level of M. luteus when comparing damaged and undamaged stone. Finally this study brings the literature on conservation methods up to date by testing biocides which are in current usage, as many biocides in the literature are discontinued. This study is also the first in the field to show their efficacy against biofilm encapsulated bacteria and their propensity for chemically disrupting the biofilm matrix.

A resistência de cinco microrganismos presentes na microbiota da área de produção estéril (Cristalização Estéril), frente a ação do gás de peróxido de hidrogênio foi determinada e o valor O obtido para cada microrganismo foi comparado ao valor D do Bacillus stearothermophilus ATCC 12980 exposto ao mesmo agente. Os microrganismos testados foram Bacillus sp, M. luteus, Corynebacterium, Staphylococcus sp e Penicillium sp. Este teste tinha a finalidade de comprovar que a resistência do Bacillus stearothermophilus é maior quando da exposição ao peróxido de hidrogênio se comparada a outros microrganismos presentes na área produtiva. A metodologia consistiu da inoculação de 0,01 mL da suspensão de cada microrganismo na contagem de 102UFC/0,01 mL em cupons de aço inoxidável, previamente esterilizados por calor seco e posterior exposição ao gás de peróxido de hidrogênio. O experimento demonstrou que o valor D obtido para o Bacillus stearothermophilus ésuperior aos obtidos para os outros microrganismos em teste comprovando que a escolha deste microrganismo para o desafio contra o peróxido de hidrogênio é apropriada. Também executou-se o teste que visava garantir que o aço inoxidável é o material de suporte mais recomendado para este fim, utilizando-se suportes de diversos materiais normalmente encontrados no interior dos isoladores (PVC, aço inoxidável, CKC, teflon, polipropileno, látex, silicone, Hypalon, vidro, nylon, saco de alumínio) com 0,01 mL de inóculo de Bacillus stearothermophilus na contagem de 102UFC/O,01 mL, o que foi devidamente comprovado. / The resistance of tive microrganisms found in the sterile production area (Crystallization Area) flora was tested against the hydrogen peroxide gas and the D value of each microrganism was compared to the Bacillus stearothermophilus D Value ATCC 12980 exposed to the same agent. The microrganisms tested were Bacillus sp, M. luteus, Corynebacterium, Staphylococcus sp e Penicillium sp. The purpose of this test was to prove that the resistance of Bacillus stearothermophilus against the exposition the hydrogen peroxide is higher when compared to others microrganisms found in the production area. The methodology consisted in inoculating 0.01 mL of microrganisms suspension with 102UFC/0,01 mL count in stainless steel coupons, treated previously with dry heat and further exposition to the hydrogen peroxide. The experiment demonstrated that the Bacillus stearothermophilus D value is higher against all others microrganisms tested proving that the use of this microrganism for the challenge is appropriate. It was also pertormed a test to guarantee that the stainless steel support is the most recommended one for this purpose, using supports of different materials normally found in the interior of the isolators (PVC, stainless steel, CKC, Teflon, polypropylene, latex, silicon, Hypalon, glass, nylon, aluminum foil) with 0,01 mL Bacillus stearothermophilus inoculum with the count of 102UFC/O,01 mL, that was properly veritied.

This thesis focuses on the effects of lactobacilli and their end-products, lactic acid and acetic acid, on 'Saccharomyces cerevisiae' growth and fermentation, and on antimicrobials used to manage such contaminants. To assess the effects of the bacteria, normal gravity (22-24 g/100 ml dissolved solids) wheat mashes inoculated with yeast at ~106 colony forming units (CFU)/ml were deliberately infected (coinoculated) with each of five industrially important strains of lactobacilli at ~10 5, ~106, ~107, ~10 8, and ~109 CFU/ml. Controls with yeast alone or with bacteria alone (~107 CFU/ml) were included. End-products, yeast growth and fermentation rates were monitored. Results indicated that production of lactic acid by lactobacilli and suspected competition of the bacteria with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and decreases in final ethanol yield. A chemically defined minimal medium was used to determine the effects of added acetic and lactic acid, and their mode of action on two strains of ' S. cerevisiae'. The effects of these two acids on yeast intracellular pH (pHi), plasma membrane H+-ATPase activity and on the plasma membrane lipid composition were studied. It was found that the specific growth rates ([mu]) of the two yeast strains decreased exponentially (R2 > 0.9) as the concentrations of acetic or lactic acid were increased. Acetic and lactic acids synergistically reduced the specific growth rate of yeast. Acetic acid caused the yeast cell to expend ATP to pump out excess protons that result from the passive diffusion of the acid into the cell at medium pH (pHe) followed by its dissociation within the cell as a result of higher pHi. Lactic acid (0.5 % w/v) caused intracellular acidification (which could lead to arrest in glycolytic flux) as a result of a significant decrease (P = 0.05) in the plasma membrane H +-ATPase activity. Moreover, the plasma membrane fluidity was reduced due to decrease in unsaturated fatty acyl residues. Among the antimicrobials studied, urea hydrogen peroxide (UHP) was superior compared to stabilized chlorine dioxide and nisin, but its bactericidal activity was greatly affected by the presence of particulate matter. When used near 30 mmoles/L (in unclarified mash), in addition to its bactericidal effect, UHP provided near optimum levels of assimilable nitrogen and oxygen that aided in vigorous yeast fermentation. This process was patented.

A micologia marinha é uma ciência relativamente recente e pouco se conhece sobre a diversidade das suas comunidades. Assim, o isolamento, triagem e preservação de fungos derivados do mar podem levar à descoberta de novas tecnologias. O objetivo deste estudo foi conhecer a diversidade de fungos filamentosos derivados marinhos e selecionar isolados capazes de produzir enzimas celulolíticas. Para tanto, foram isolados seletivamente fungos filamentosos a partir de amostras de macro-organismos marinhos coletados em 2007 e 2008. Os resultados demonstraram uma ampla diversidade de fungos potencialmente celulolíticos, pertencentes ao filo Basidiomycota e Ascomycota. Nos experimentos de produção de celulases, 17 apresentaram resultados satisfatórios de CMCase e FPase e foram selecionados para a avaliação da Celobiase. Os experimentos de cinética enzimática apresentaram os melhores resultados de produção de celulases em meio contendo farelo de trigo. O trabalho demonstra o potencial para aplicação biotecnológica dos fungos e estimula novos estudos com as celulases. / The Marine mycology is a relatively recent and little is known about the diversity of its communities. Thus, the isolation, separation and preservation of fungi derived from the sea can lead to the discovery of new technologies. The aim of this study was the diversity of filamentous fungi isolates derived marine and select capable of producing cellulolytic enzymes. It had been selectively isolated filamentous fungi from samples of marine macro-organisms collected in 2007 and 2008. The results showed a wide range of potential cellulolytic fungi, belonging to the phylum Basidiomycota and Ascomycota. In the experiments to produce cellulases, 17 had satisfactory results of CMCase and FPase and were selected for evaluation of cellobiase. The enzyme kinetics experiments showed better results for the production of cellulases in a medium containing wheat bran. The work demonstrates the potential for biotechnological application of fungi and stimulate further research with cellulases.

Tendo em vista a importância do processo esterilizante aplicado a produtos farmacêuticos e aos produtos médico-hospitalares, distintos métodos foram desenvolvidos de forma a possibilitar sua aplicação, inclusive a materiais termossensíveis. Neste contexto, o processo que se tornou mais amplamente empregado consiste naquele utilizando o óxido de etileno. Este agente, devido às características de inflamabilidade e explosividade, tem sido usado diluído em gases inertes, predominantemente os clorofluorcarbonos (CFCs), que contornam tais problemas. Conhecimentos recentemente adquiridos e consenso internacional quanto ao risco da depleção da camada de ozônio da estratosfera ocasionaram a busca de alternativas, dentre as quais a adoção dos hidroclorofluorcarbonos (HCFCs). O delineamento do presente trabalho objetivou a comparação da eficácia esterilizante de misturas de óxido de etileno em CFC 12 (Oxyfume 12R), e em HCFCs 22 e 124 (Oxyfume 2002R), quando aplicadas em diferentes concentrações (450 mg/L e 600mg/L) e sob distintas temperaturas (45°C, 55°C e 65°C). Procedeu-se a desafios subletais (3, 6 ,9, 12 e 15 minutos de exposição), empregando a cada ciclo seis indicadores biológicos laboratorialmente preparados, com esporos de Bacillus subtilis var. niger ATCC 9372 obtidos em garrafas de Roux contendo meio de esporulação, sendo a seguir padronizados, inoculados em suportes celulósicos e acondicionados em embalagens filme-papel, perfazendo um total de 1080 monitores. Paralelamente foram também submetidos a desafios indicadores biológicos de aquisição comercial (AttestR 1264, 3M), dois a cada ciclo, perfazendo 360 unidades. Os resultados de resistência obtidos (valor D) nos 180 ciclos privilegiaram estes últimos, levando tal resultado a considerações quanto ao procedimento de purificação da suspensão de esporos e diferenças existentes entre suportes e acondicionamentos empregados. A eficácia esterilizante de ambas as misturas, Oxyfume 12R e Oxyfume 2002R, revelou-se equivalente, mesmo em diferentes concentrações. Além de observações que evidenciaram os efeitos significantes quanto à preparação dos indicadores biológicos, foi evidente e estatisticamente significante ( p=1% ) o efeito do aumento da temperatura, promovendo efeito mais intenso sobre a letalidade dos esporos. Numa outra abordagem, de conotação ocupacional, procedeu-se também no decorrer do trabalho experimental à monitoração do ambiente quanto a resíduos de óxido de etileno, usando bomba e tubos de detecção da DragüerR: os resultados obtidos nas diferentes posições e momentos, inferiores a 1 ppm, proporcionaram a conclusão de que, respeitando a adoção dos conceitos de engenharia indicados na Portaria Interministerial nº 482 de 16 de abril de 1999, publicada no Diário Oficial da União do dia 19 do mesmo mês e ano, é obtida condição compatível com a presença humana. É portanto gratificante que se possa concluir o trabalho com o sentimento de que efetivamente se tenha constituído em contribuição no sentido de permitir a manutenção de emprego do processo esterilizante em pauta, conferindo segurança ao paciente no uso de produtos, sem entretanto comprometer a vida humana em nosso planeta. / Taking into account the importance of the sterilization process applied to medicines and medical devices, different kinds of methods have been developed, also applicable to heat sensitive materiais. Ethylene oxide is the process most widely applied to medical devices. Due its explosiveness and inflamability, it has been used associated to non active gases, wich inhibit these properties, mainly the chlorofluorocarbons (CFCs). Recent knowledge about ozone-depleting gases and an international consensus on the need of reducing their effects are promoting a search for alternative chemicals. From these, one of the most interesting are the hydrochlorofluorocarbons (HCFCs) which, besides having this role, can also be used as transitional compounds while more environmentally suitable compounds are not available. This paper aims to compare two ethylene oxide sterilization mixtures: Oxyfume 12R (using CFC 12) and Oxyfume 2002R (using HCFCs 22 and 124), under different concentrations (450mg/L and 600mg/L) and different temperatures (45°C, 55°C and 65°C). To accomplish this procedure, sub-Iethal challenges were performed (3, 6, 9, 12 and 15 minutes of exposition), in a total of 180 cycles, using six biological indicators per cycle prepared in a laboratory, in a total of 1080 units, as well as two others purchased from an American supplier (AttestR 1264, 3M), in a total of 360 units. The former indicators were obtained using Bacillus subtilis var. niger ATCC 9372 in Roux bottles, innoculated on paper carriers and wrapped up on protective packaging. The posterior lethality study and D value calculation highlighted the highest resistance of AttestR indicators in comparison with the laboratory ones. This can be explained by different leveIs of spores purification, alongside with the differences between the carriers and packaging used in both cases. The sterilizing efficacy of Oxyfume 12R and Oxyfume 2002R revealed equivalent results, even in different concentrations. The influence of higher temperatures was efficient (p=1,0). Aiming at occupational safety, environmental monitoration was also performed related to ethylene oxide, using DragüerR detection tubes and foley pump. The results, obtained in different positions and moments under 1 ppm, confirmed that the engineering concepts indicated by the Portaria Interministerial nº 482 from 16 april 1999, published on the 19th of the same month in the Diário Oficial, offer residual safe levels compatible with human presence. It is therefore gratifying to conclude that the sterilization process using Oxyfume 2002R is both an efficient contribution to a safe utilization of products and, at the same time, to preserve the animallife on Earth.

Burkholderia sacchari é uma nova espécie bacteriana do solo brasileiro que tem a capacidade de crescer em sacarose e acumular grânulos intracelulares de poliésteres pertencentes à família dos polihidroxiaIcanoatos (PHA). Quando cultivado em sacarose, o homopolímero poli-3¬hidroxibutirato é acumulado por esta bactéria, que é usado como um plástico biodegradável e biocompatível. Quando sacarose e ácido propiônico são fornecidos como fontes de carbono, as células de B. sacchari acumulam o copolímero poli-3-hidroxibutirato-co-3-hidroxivalerato (P3HB-co-3HV). Entretanto, uma pequena porcentagem do ácido propiônico fornecido é convertido a unidades 3HV devido à eficientes vias catabólicas que convertem este substrato preferencialmente a biomassa, CO2 e água, reduzindo portanto a eficiência da produção do polímero. Ao menos duas vias do catabolismo de propionato foram previamente propostas em B. sacchari: a-oxidação e ciclo do 2-metilcitrato (2MCC), sendo somente a última confilmada no nível molecular. Mutantes UV, obtidos anteriormente, foram incapazes de crescer em propionato (prp) e também apresentaram fenótipo afetado no crescimento em intermediários da a-oxidação. No presente trabalho, após uma busca em bibliotecas genômicas de B. sacchari, uma delas construída também no presente trabalho, três diferentes fragmentos de DNA presentes nos clones AI, PI e P2 foram capazes de restaurar o fenótipo prp+ aos mutantes. Experimentos quantitativos revelaram que AI somente restaurou parcialmente a conversão de propionato a unidades 3HV aos mutantes. PI foi capaz de restaurar a capacidade de crescimento em propionato, e em outros intermediários da a-oxidação, a um dos mutantes. Um DNA de 1.2 Kb, subfragmento de PI, ainda capaz de complementar mutantes prp, foi subclonado e seqüenciado, demonstrando similaridade a seqüências de DNA codificadoras de reguladores transcricionais do tipo LysR de várias bactérias, incluindo espécies de Bllrkholderia. Regiões adjacentes a LysR em diferentes genomas de Burkholderia são anotados como codificadores de acil-CoA desidrogenases, ao lado de proposta acil-CoA transferases/carnitina desidrogenases e de uma permease do facilitador maior da superfamília MFS-l. Após confirmação das mesmas regiões adjacentes em B. sacchari e também a sua específica deleção, será possível provar a presença da via do catabolismo de propionato indicada neste trabalho. / Burkholderia sacchari is a new bacterial species from brazilian soil, able to grow in sucrose, accumulating intracellular granules of polyester belonging to the polyhydroxyalkanoate family (PHA). When cultivated on sucrose, the homopolymer poly-3-hydroxybutyrate is accumulated by this bacterium, which is used as biodegradable and biocompatible plastic. When sucrose and propionic acid are supplied as carbon sources, B. sacchari cells accumulate the copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co-3HV). However, a small percentage ofthe propionic acid supplied is converted to 3HV units, because efficient catabolic pathways convert this substrate preferentially to biomass, CO2 and water, thus reducing the efficiency of polymer production. At least two propionate catabolic pathways have been previously indicated in B. sacchari: a-oxidation and the 2-methylcitric acid (2MCC), the latter confirmed at molecular leveI. UV mutants previously obtained were unable to grow in propionate (prp) and also showed the phenotype affected concerning grow on intermediates of propionate a-oxidation. In the present work, after a screening in B. sacchari genomic libraries, one ofthem constructed also in the present work, the prp + phenotype was restored to the mutants by three different DNA fragments harbored by dones A), PI and P2. Quantitative experiments revealed that AI restored only partially the quantitative conversion of propionate to 3HV units to the mutants. PI restored the ability to grow in propionate and in other intermediates of a-oxidation to one prp mutant. A DNA 1.2 Kb subfragment of PI, still able to complement prp mutants, was subcloned and sequenced, showing similarity to DNA sequences encoding to LysR-type transcriptional regulators of various bacteria, including BlIrkholderia species. Adjacent regions to LysR in different genomes of BlIrkholderia are annotated as encoding to acyl-CoA dehydrogenases, neighboring a predicted acyl-CoA transferases/carnitine dehydratase and a permease ofthe major facilitator superfamily MFS-1. After confirmation ofthe same adjacent regions in B. sacchari and also their especific deletion, it will be possible to prove the presence of the pathway indicated here in the catabolism of propionate.

Os tipos de cassete cromossômico estafilocócico mec (SCCmec) encontrados em cepas de Staphylococcus aureus resistente a oxacilina (ORSA) isoladas em ambiente hospitalar são denominados I, II e III. Um novo SCCmec, tipo IV, foi identificado em cepas isoladas de pacientes que não tinham conexão aparente com hospitais. Essas cepas, denominadas ORSA adquiridas na comunidade ou CA-ORSA, não costumam conter genes de resistência a antimicrobianos além do gene mecA, porém, são mais virulentas. Com a finalidade de conhecer a diversidade estrutural do SCCmec de cepas ORSA brasileiras, foram estudadas 50 cepas ORSA, entre elas algumas S. aureus com resistência intermediária a vancomicina (VISA) e S. aureus com resistência heterogênea intermediária a vancomicina (HVISA). As cepas foram isoladas entre 1995 e 2000, provenientes de hospitais de 13 Estados brasileiros, pertencentes a diversos clones de PFGE. As cepas foram analisadas quanto ao perfil de sensibilidade, RFLP do gene da coagulase, fagotipagem e tipagem do SCCmec. A maioria das cepas é pertencente ao clone endêmico brasileiro, que carrega o SCCmec tipo III, também presente em outros clones de PFGE. O SCCmec tipo IV, encontrado em 3 cepas (clones J, L e D), mostrou suscetibilidade a um maior número de antimicrobianos pertencentes a diferentes classes, em comparação aos outros tipos de SCCmec. O SCCmec tipo IV está presente entre os ORSA brasileiros e pode estar disseminando na comunidade e hospitais do país. / The types of SCCmec found in nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains can be designated type I, II or III. A novel type of SCCmec, designated type IV, was identified in strains isolated in outpatients. These strains, called community-acquired MRSA or CA-MRSA, do not contain antimicrobial resistance genes other than mecA, however, they are more virulent. In order to characterise the structural diversity of SCCmec in Brazilian MRSA strains, 50 MRSA strains were studied, including some vancomycin intermediate resistant S. aureus (VISA) and heterogeneous vancomycin intermediate S. aureus (HVISA) strains. All strains were isolated between 1995-2000, in hospitaIs in 13 Brazilian States, and belonged to different PFGE clones. Strains were analysed as to their susceptibility profile, RFLP of the coagulase gene, phagetype and SCCmec type. The majority of strains belonged to the Brazilian Endemic Clone, which carries an type IH SCCmec, which in turn, was also found in other PFGE clones. The type IV SCCmec was found in 3 strains (belonging to the J, L and D clones) and presented a susceptibility profile to a number of drugs of different antimicrobial classes. The type IV SCCmec is present among Brazilian MRSA strains and can be disseminated in the community and in hospitaIs throughout the country.

The effects of inhaled air pollution particles on lung clearance mechanisms is an important factor in understanding how the mammalian lung deals with such pollutants and, as such, how exposure to these pollutants can be regulated. The nanoparticle(diameter S lOOnm) and transition metal components of PMIO (particulate matter with a diameter less than lO~m) have been implicated as playing major roles in the impairment of alveolar macrophage function and the subsequent retention of particles in the respiratory system. The aim of this study was to investigate the effects of components of PMIO on macrophage functions both directly, by examining macrophage phagocytosis and migration, and indirectly, by studying peripheral factors affecting macrophagefunction such as recruitment by type II cells and complement based mechanisms. We hypothesised that the alveolar epithelial type II cell line would release leukocyte chemoattractants in response to particle exposure and that this could be measured by use of a macrophage migration assay. A sub-toxic dose (125 ~g/ml)of surrogate air pollutionparticles (fine and nanoparticle carbon black and titanium dioxide) was established by measuring LOH release from a murine alveolar macrophage cell line (1774.2) and an alveolar epithelial type II cell line (L-2) in response to particle exposure. Optimisation ofa chemotaxis assay and measurement of macrophage migration towards conditioned medium obtained from the particle-exposed type II cells was conducted and it was determined that carbon black nanoparticles induced type II cells to secrete a chemoattractant that resulted in significant increases in macrophage migration compared to the negative control. This was in contrast to other particle types tested in this study which did not induce any increases in macrophage migration. It was also hypothesised that complement proteins could be involved in macrophage recruitment to sites of particle deposition and, as such, the migration of macrophages towards particle exposed blood serum was examined in vitro. Foetal bovine serum (FBS) was exposed to fine and nanoparticle caroon black and titanium dioxide (l-Smg/ml) for 2 hours. It was found, in accord with the previous study involving type II cells, that carbon black nanoparticles could activate the generation of chemotactic factors in serum that could subsequently induce significant increases (p < 0.001) in macrophage migration when serum was diluted to 10% using serum-free RPMI 1640 culture medium. This effect could be ameliorated by co-incubating the particle-treated serum in the presence of the antioxidant Trolox suggesting that oxidative stress played a role in the generation of the chemoattractant molecules. However, incubation of the serum with a pure oxidant at a range of doses did not result in the generation of chemotactic molecules suggesting that another factor could be involved in the chemoattractant generation. Further investigation to determine the exact molecular mechanism behind the chemoattractant generation is warranted. In contrast to the previous studies, we have also found evidence that components of PM₁₀ can cause decreased efficacy of macrophage clearance mechanisms in vivo and in vitro. It was hypothesised that PM₁₀ instillation would result in a decrease in macrophage phagocytic potential and an increase in chemotactic potential ex vivo. Rats were instilled with 125 and 250μg of PM₁₀ collected from North Kensington, London or sterile saline (negative control). Post-instillation (18 hours), significantly elevated concentrations of TNFa were detected in the BAL fluid together with a significant increase in the number of BAL neutrophils. Phagocytosis and chemotaxis assays conducted with BAL macrophages ex vivo showed that macrophage migration towards a positive chemoattractant, Zymosan Activated Serum (ZAS), was significantly lower than the macrophages obtained from the negative control rats. Macrophage phagocytosis of latex beads ex vivo was also found to be significantly decreased when PM₁₀ was visible inside the cell. An in vitro study where a macrophage cell line (J774.Al) was exposed to a low dose of nanoparticle carbon black (31.25μg) together with varying concentrations (100μM - 100nM) of zinc chloride (ZnCl₂) was also conducted. Exposure of macrophages to nanoparticle carbon black and zinc chloride alone induced a decrease in macrophage phagocytosis. It was found that when macrophages were co-exposed to nanoparticle carbon black and ZnCl₂, there was an additive decrease in macrophage phagocytic potential. The results contained within this manuscript demonstrate that the components of PM₁₀ can induce adverse effects on specific aspects of macrophage clearance mechanisms, but that nanoparticles can also stimulate the production of chemoattractants to aid in the recruitment of phagocytes and subsequent particle clearance. Although a contrary relationship appears to exist between these findings, the recruitment of leukocytes in response to particulate exposure is a mechanism that supports particle clearance. However, the retardation of phagocytic and chemotactic mechanisms in particle exposed macrophages may help to explain the increased toxicity, inflammation and retention time observed with nanoparticle inhalation.