Background: Bacterial contamination of surfaces with methicillin-resistant Staphylococcus aureus (MRSA) is a serious problem in the hospital environment and is responsible for significant nosocomial infections. The pathogenic contaminants form biofilms, which are difficult to treat with routine biocides. Thus, a continuous search for novel disinfection methods is essential for effective infection control measures. This demonstration of a novel technique for the control of virulent pathogens in planktonic form as well as in established biofilms may provide a progressive alternative to standard methodology. Methods: We evaluated a novel technique of normal atmospheric nonthermal plasma known as floating-electrode dielectric-barrier discharge (FE-DBD) plasma against a control of planktonic and biofilm forms of Escherichia coli, S aureus, multidrug-resistant methicillin-resistant S aureus (MRSA) -95 (clinical isolate), -USA300, and -USA400, using widely accepted techniques such as colony count assay, LIVE/DEAD BacLight Bacterial Viability assay, and XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay. Results: Exposure of free living planktonic forms of E coli, S aureus, and MRSA were rapidly inactivated by DBD plasma. Approximately 107 bacterial cells were completely (100%) killed, whereas 108 and 109 were reduced by approximately 90% to 95% and 40% to 45%, respectively, in less than 60 seconds (7.8 J/cm2) and completely disinfected in ≤120 seconds. In established biofilms, the susceptibility of MRSA USA400 was comparable with USA300 but less susceptible than MRSA95 (clinical isolate), S aureus, and E coli (P < .05) to FE-DBD plasma, and plasma was able to kill MRSA more than 60% within 15 seconds (1.95 J/cm2). The killing responses were plasma exposure-time dependent, and cell density dependent. The plasma was able disinfect surfaces in a less than 120 seconds. Conclusion: Application of DBD plasma can be a valuable decontamination technique for the removal of planktonic and biofilm-embedded bacteria such as MRSA -USA 300, -USA 400, methicillin-sensitive S aureus (MSSA), and E coli, the more common hospital contaminants. Of interest, E coli was more resistant than S aureus phenotypes.

Bacteria are known to adhere to surfaces, which allows for the formation of biofilms, possibly causing a surge in hospital-offset infections, perilous diseases, and in some cases, death. Although certain bacteria are present in the natural flora of the human skin, some present extreme clinical significance due to the ability to transmit and adhere, and can be resistant to antibiotics. They also evolve over time to survive in harsh environmental conditions. Current research reveals that design of plastic surfaces containing submicron structures, is becoming a popular approach to tackle issues concerning infection transmission, with inspiration being derived from biomimetics and self-cleaning surfaces, such as the surface of a gecko skin, and the hydrophobic wax layer of forest leaves. Main barriers to adoption include that these surfaces alone are difficult to manufacture on 3D products, expensive to fabricate on a large scale and do not last long when subjected to environmental wear. Replication of nano-scale ridges was carried out using micro-injection, and the various samples were characterised using a range of tools to determine physical and biomechanical parameters. The sample surfaces were then cultured with the pathogenic bacterium Staphylococcus aureus under several environmental conditions, and the results were statistically analysed to reveal that anti-fouling LIPSS (laser induced periodic surface structures) ridges perform better to reduce bacteria cell-substrate adhesion, when compared to flat surfaces, or surfaces containing dual structures (anti-fouling ridges combined with anti-wear walls). It was therefore demonstrated that nanotextured polymeric surfaces with hydrophobic characteristics have exceptional non-fouling properties, preventing S. aureus, a very significant bacterial strain, from initial adhesion, a critical primary mechanism in its ability to proliferate. Collectively, the findings of this study strongly support the literature, suggesting that the bacteria struggle to adhere onto polymeric topography with increased water contact angles and simple nanostructures. However, the addition of certain anti-wear micro-features increased bacterial adhesion, reducing the efficacy of the non-fouling nanostructures from preventing biofilm formation.

Grape seed extract (GSE) and peanut skin extract (PSE) are waste products in the wine and peanut industries. Both extracts have high concentrations of polyphenols, known to possess antioxidant and antimicrobial properties. A subcategory of polyphenol is procyanidin, which can be divided into two types, type A and type B. Type A (PSE), contains two single bonds connecting the phenolic groups while type B (GSE), contains one single bond connecting the phenolic groups. The minimum inhibitory concentration (MIC) of the two extracts was evaluated for their antimicrobial effect on Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium using the pour plate method. GSE was found to have a significantly lower MIC (p ≤ 0.05) than PSE for L. monocytogenes (GSE=60.60ppm, PSE=not found), S. aureus (GSE=38.63ppm, PSE=51.36ppm), and S. Typhimurium (GSE=45.73ppm, PSE=60.60ppm). There was no significant difference in inhibition of E. coli O157:H7 (GSE=47.44ppm, PSE=51.13ppm). Since GSE, contributed to greater pathogen inhibition, its extract was fractionated into monomer and oligomers components. Growth curves of all four pathogens inoculated in the monomer and oligomer fractions were compared using the BioScreen method. Oligomers inhibited growth of L. monocytogenes, S. aureus, and E. coli O157:H7 while monomers inhibited growth of S. Typhimurium. These results indicate that an extract with type B procyanidins that are high in oligomers may be more effective as antimicrobials. Type B procyanidins have also been shown to prevent bacterial adhesion, as is the case with urinary tract infections, and may aid in the prevention of biofilms. / Master of Science in Life Sciences

Dentre tantas complicações do diabetes mellitus (DM), a infecção por bactérias comuns da microbiota superficial da pele como, por exemplo, a bactéria Gram-positiva Staphylococcus aureus, causadora de infecções como a peritonite, com altos índices de hospitalização e morte. A hipótese deste trabalho é a que o efeito da insulina na ativação das vias de sinalização MAPK, PKC e PI3K em peritonite induzida por S. aureus em animais diabéticos e não diabéticos possa regular a produção de citocinas. Foram utilizadas amostras de fígado, rim, linfonodos peritoniais e baço de animais oriundos de estudo anterior (Projeto FCF/USP-375), no qual animais diabéticos (aloxana, 42 mg/kg, i.v., 10 dias) e não-diabéticos com peritonite decorrente da infecção por S. aureus receberam uma dose de 4 UI e 1 UI de insulina NPH, respectivamente, por via subcutânea, 2 horas antes da infecção com S. aureus, e outras 3 doses de 2 UI e 0,5 UI às 17h00\', respectivamente. A glicemia foi determinada no dia anterior, 10 dias após a injeção de aloxana e após os tratamentos com insulina. Em amostras de fígado, rim, linfonodo e baço dos animais supra citados foram avaliados a dosagem de citocinas (IL-1&#946;, IL-4, IL-10, TNF-&#945;, CINC-1, CINC-2 e CINC-3) por ensaios de enzima-imunoensaio (ELISA); em homogenato de fígado foram avaliadas a expressão das moléculas das vias MAPK (fosfo P-38, fosfo ERK p42/44), PKC (fosfo PKC-&#945;, fosfo PKC-&#948;) e PI3K (fosfo-AKT) pelo método de Western Blotting. Na avaliação do fígado, a insulina foi capaz de aumentar a concentração das citocinas IL-4 e TNF-&#945; que apresentavam-se diminuídas em animais não diabéticos, em relação aos animais não diabéticos e não infectados, mas nos animais diabéticos, na infecção pela cepa N315, a insulina diminuiu a concentração de IL-4, que não estava alterada pela infecção, e não foi capaz de aumentar a concentração de IL-1&#946; que estava diminuída na infecção, em relação aos animais diabéticos e não infectados. Em linfonodos peritoniais de animais não diabéticos infectados pela cepa N315, a insulina diminuiu a produção de IL-1&#946; e IL-10, que não estavam alteradas na infecção, e diminuiu a concentração de IL-4, que estava aumentada na infecção, em relação aos animais não diabéticos e não infectados; em animais diabéticos, a insulina diminuiu a produção das citocinas IL-1&#946; e CINC-1, que estavam aumentadas, e aumentou a concentração de IL-10, que estava diminuída na infecção com a cepa N315, mas baixou a concentração de IL-4, em relação aos animais infectados, e na infecção pela cepa ATCC, a insulina aumentou a produção de IL-1&#946;, CINC-1 e CINC-3 dos animais tratados, em relação aos infectados e não tratados. Em baço, a insulina diminuiu a produção de IL-10 na infecção pela cepa ATCC tanto em animais não diabéticos quanto em animais diabéticos e, nesse último grupo, também aumentou a produção de CINC-3 em relação aos animais diabéticos não infectados; na infecção com a cepa N315, a insulina não aumentou a concentração de IL-1&#946; e TNF-&#945;, que estavam diminuídas na infecção. Em rim, não houveram alterações significativas na produção de citocinas na infecção com nenhuma das cepas estudadas, nem para os grupos diabéticos, nem para os não diabéticos. Verificou-se que os animais diabéticos apresentam maior alteração tanto nas vias de sinalização estudadas quando na produção de citocinas pró-inflamatórias, quando comparados aos animais não diabéticos, na infecção por ambas as cepas de S. aureus estudadas. Assim, os resultados obtidos sugerem que o tratamento com insulina possa modular parcialmente a produção das citocinas IL-1&#946;, TNF-&#945; e IL-10 no fígado e nos linfonodos peritoniais dos animais infectados principalmente pela cepa N315 de S.aureus, modulando parcialmente a expressão das moléculas da via de sinalização (MAPK e PKC), envolvidas na produção dessas citocinas. / Among so many complications of diabetes mellitus (DM), infection by common bacteria of the superficial microbiota of the skin, for example, a gram-positive bacteria Staphylococcus aureus, causing infections like peritonitis, with high rates of hospitalization and death. The hypothesis of this study is that the effect of insulin on the activation of MAPK, PKC and PI3K signaling pathways in peritonitis induced by S. aureus in diabetic and non-diabetic animals may regulate the production of proinflammatory cytokines. Liver, kidney, peritonial lymph nodes and spleen samples of animals from the previous study (FCF / USP-375 Project) were used in this project; diabetic animals (alloxan, 42 mg / kg, iv, 10 days) and non-diabetic animals with peritonitis due to S. aureus infection received one dose of 4 IU and 1 IU of NPH insulin, respectively, subcutaneously, 2 hours before infection with S. aureus, and another 3 doses of 2 IU and 0.5 IU at 5:00 p.m., respectively. Blood glucose was determined the day before, 10 days after alloxan injection and after insulin treatments. In the liver, kidney, lymph node and spleen samples of the above-mentioned animals the cytokines (IL-1&#946;, IL-4, IL-10, TNF-&#945;, CINC- 2 and CINC-3) by enzyme-immunoassay (ELISA) assays; we avaliated, by Western Blotting, the signaling pathways MAPK (phospho-P-38, phospho ERK p42 / 44), PKC (phospho PKC-&#945; and phospho PKC-&#948;) and PI3K (phospho AKT) in liver, insulin was able to increase the concentration of cytokines IL-4 and TNF-&#945; that were decreased in non-diabetic animals, in relation to non-diabetic and non-infected animals, but in diabetic animals, in strain N315, insulin decreased the concentration of IL-4, which was not altered by the infection, and was not able to increase the concentration of IL-1&#946; that was decreased in infection, relative to diabetic and uninfected animals. In peritonial lymph nodes from non-diabetic animals infected with the N315 strain, insulin decreased the production of IL-1&#946; and IL-10, which were not altered in the infection, and decreased the concentration of IL-4, which was increased in infection, in relation to non-diabetic and non-infected animals; in diabetic animals, insulin decreased IL-1&#946; and CINC-1 which were increased, and increased the concentration of IL-10, which was decreased in infection with strain N315, but decreased the concentration of IL-4 in Infected animals, and in infection by the ATCC strain, insulin increased the production of IL-1&#946;, CINC-1 and CINC-3 of treated animals over infected and untreated animals. In spleen, insulin decreased IL-10 production on infection by the ATCC strain in both non-diabetic and diabetic animals and, in this last group, also increased the production of CINC-3 in relation to uninfected diabetic animals; in infection with the N315 strain, insulin did not increased the concentration of IL-1&#946; and TNF-&#945;, which were decreased in infection. In kidney, there were no significant changes in cytokine production in infection with any of the strains studied, neither for diabetic groups nor for non-diabetics. It was verified that diabetic animals present a greater alteration both in the signaling pathways studied and in the production of pro-inflammatory cytokines, when compared to non-diabetic animals, in the infection by both strains of S. aureus studied. Thus, the results suggest that insulin treatment may partially modulate the production of IL-1&#946;, TNF-&#945; and IL-10 cytokines in the liver and in the peritonial lymph nodes of animals infected mainly with S. aureus strain N315, since they partially modulating the expression of signaling pathway molecules (MAPK and PKC), involved in the production of these cytokines.

O objetivo deste estudo foi verificar a ocorrência de S. aureus e E. coli O157: H7 no leite de vacas com mastite subclínica e no leite de mistura de 42 propriedades leiteiras localizadas em duas regiões do Estado de São Paulo: São Carlos e Ribeirão Preto. Paralelamente, entre os isolados de S. aureus foi objetivo identificar os produtores de toxinas e determinar sua origem epidemiológica. O isolamento de S. aureus foi realizado em agar Baird-Parker (35ºC, 48h) e a confirmação bioquímica através da catalase, coagulase, termonuclease, produção de acetoína e fermentação aeróbia da maltose. O isolamento de E. coli O157: H7 foi realizado em agar Sorbitol MacConkey MUG (35ºC, 24h). Para confirmação utilizaram-se as provas do IMVC e sorologia através do kit Soro Anti E. coli O157. Para a detecção da TSST-1 e das enterotoxinas A, B, C e D utilizou-se aglutinação reversa passiva em látex (RPLA). A identificação epidemiológica dos isolados de S. aureus foi realizada por eletroforese em gel de campo pulsado (PFGE). A ocorrência de S. aureus no leite individual nas regiões 1 (São Carlos) e 2 (Ribeirão Preto) foram 3,9% e 6,7%, respectivamente. Animais pertencentes às propriedades leiteiras com produção entre 400 L e 1.000 L/dia apresentaram maior risco de veiculação de S. aureus através do leite quando comparadas com propriedades com produção 1.000 L/dia. Quanto ao leite de mistura, verificou-se que a ocorrência de S. aureus foi a mesma em ambas as regiões (19%). A produção simultânea das enterotoxinas B e C foi observada em 4,7% dos isolados de leite individual, enquanto que 4,7% produziram enterotoxina A e toxina TSST-1. A produção de TSST-1, isoladamente, foi constatada em 14,3% dos isolados de leite individual e em 25% do leite de mistura. Houve similaridade genética entre os isolados de S. aureus, evidenciando sua dispersão epidemiológica entre as propriedades avaliadas. A ocorrência de E. coli O157: H7 no leite individual foi 1% na região 1 e 1,6% na região 2. No leite de mistura não foi detectada E. coli O157: H7. Ressalte-se a importância de medidas preventivas para assegurar a qualidade do leite durante a ordenha, a fim de evitar a ocorrência de microrganismos patogênicos, principalmente S. aureus, e conseqüentemente prevenir riscos de veiculação de toxinfecções através deste alimento. / The aim of this study was to verify the occurrence of S. aureus and E. coli O157: H7 in the milk from dairy cows with subclinical mastitis and in the bulk milk from 42 dairy farms located in two regions of São Paulo State (Region 1: São Carlos, Region 2: Ribeirão Preto). Among the S. aureus strains isolated, the aim was to identify the toxin producers and their epidemiological origin. The isolation of S. aureus was conducted using Baird-Parker agar, and the strains were confirmed by catalase, coagulase, thermonuclease, maltose aerobic fermentation and acetoin production. The isolation of E. coli O157: H7 was conducted using Sorbitol MacConkey MUG agar. The strains were confirmed by IMVC and serology using anti E. coli O157 sera. Rapid passive latex agglutination was used for detection of TSST-1 and enterotoxigenic strains of S. aureus. The epidemiological identification was performed using pulsed field gel electrophoresis (PFGE). S. aureus was isolated from 3.9% and 6.7% of the individual milk samples from regions 1 and 2, respectively. Dairy cows belonging to farms with milk production ranging from 400 to 1.000 L/day showed higher risk of S. aureus carrying-over, when compared with dairy farms with milk production < 400 L/day and > 1.000 L/day. In bulk milk samples, the occurrence of S. aureus was the same in both regions evaluated (19%). The simultaneous production of enterotoxin B and C was observed in 4.7% of strains isolated from individual milk samples, while 4.7% produced both enterotoxin A and TSST-1. TSST-1 production alone was observed in 14.3% of S. aureus strains isolated from individual milk and 25% of bulk milk samples. S. aureus strains tested by PFGE demonstrated genetic similarity, showing the dispersion patterns of this microorganism among dairy farms. E. coli O157: H7 was isolated from 1% and 1.6% of individual milk samples from regions 1 and 2, respectively, although it was not detected in bulk milk samples. The importance of preventive measures to ensure milk quality during milking extraction is stressed, aiming to avoid pathogenic agents, mainly S. aureus, and therefore, to prevent the carry-over of food borne diseases to humans through milk.

S. aureus peut produire de très nombreux facteurs de virulence. Les exotoxines superantigéniques sont responsables de la réponse pro-inflammatoire observée au cours du choc toxique staphylococcique et sont suspectées d'être impliquées dans le choc septique à S. aureus. En se fixant sur la partie variable Vbeta du récepteur T des lymphocytes T, ces toxines induisent une prolifération lymphocytaire T Vbeta dépendante et chacune de ces toxines a théoriquement une « signature Vbeta » spécifique.<br />Chez des patients présentant un choc toxique menstruel ou non-menstruel, nous avons respectivement retrouvé la signature des toxines TSST-1 ou SEB à la phase aiguë de la maladie, après avoir identifié in vitro la signature Vbeta des principales exotoxines superantigéniques. Au cours de bactériémies à S. aureus, les isolats responsables de choc septique possédaient plus fréquemment le gène codant la toxine SEA et le clone de S. aureus résistant à la méticilline prédominant en France (dénommé clone Lyon) possédait également ce gène. De plus, SEA a des propriétés pro-inflammatoires particulières que nous avons documentées in vitro. Cela renforcait l'hypothèse que SEA est impliqué dans le choc septique à S. aureus. Cependant, la signature Vbeta de SEA n'a pas été retrouvée chez des patients présentant un choc septique à S. aureus (sensible ou résistant à la méticilline) et dans un modèle animal de choc septique, le clone Lyon induisait certes une plus grande mortalité par rapport à des isolats sensibles à la méticilline, mais celle-ci n'était pas directement attribuable à SEA.<br />La mise en évidence in vivo de la signature Vbeta des exotoxines superantigéniques au cours du choc toxique staphylococcique permet de préciser quelle toxine est en cause, permet de faire le diagnostic plus précocement et facilite l'optimisation du traitement de cette pathologie. L'implication des exotoxines superantigéniques et notamment de SEA, dans le choc septique qui résulte de l'expression de multiples facteurs de virulence, reste incertaine.

Individual and environmental risk factors in hand eczema have been investigated in a prospective cohort study of 2452 newly employed hospital workers with a follow-up time of 20 months. Current hand eczema was analyzed in 142 wet hospital workers from this cohort with respect to the etiologic importance of irritants, allergens and contact urticants. The density of the microflora and the effect on the microflora of topical treatment with a potent corticosteroid were studied in 20 patients with hand eczema. ’Wet’ hospital work was found to increase the odds of developing hand eczema only twice compared to 'dry' office work. Nursing children under four years old and the lack of a dish-washing machine significantly increased the risk of contracting hand eczema. Unfavourable combinations of these domestic factors increased the risk as much as wet work. A history of atopic dermatitis approximately tripled the odds both in wet as well as in dry work. Histories of earlier hand eczema (HHE), metal dermatitis (HMD) and of atopy were analyzed as risk factors for hand eczema in 1857 women in wet work. HHE increased the odds by a factor of 12.9 and created a subdivision of the population into high risk individuals and normal risk individuals. HHE was found in half of the subjects with atopic dermatitis, in one quarter of the subjects with atopic mucosal symptoms and in one fifth of the non-atopics. A HMD increased the odds by a factor of 1.8. This increase was seen as a high risk level in subjects with HHE and as a normal risk level in subjects with no HHE. A history of atopic disease as a complement to information about HHE and HMD increased the odds by another 1.3 times. The predicted probability of developing hand eczema ranged from 91 % in subjects with a combination of HHE, HMD and atopy to 24% in subjects with none of these risk factors. Subjects with AD were found to suffer a more severe form of hand eczema with significantly higher figures for medical consultation, sick- leave, termination due to hand eczema, early debut, permanent symtoms and vesicular lesions. Amongst the patients investigated for current hand eczema high risk individuals were overrepresented. It was claimed in 92.3% of the cases that trivial irritant factors had elicited the current episodes of hand eczema. In 35% of the cases the exposure to the irritant took place largely at home. Although contact sensitivity and contact urticaria were fairly common, they mostly seemed to be of minor importance in the etiology of the current hand eczema. Staphylococcus aureus colonized eczematous lesions of the hands in 18/20 patients. The density exceeded 105 colony forming units/cm2 in 15/20 patients. Only three of these patients showed signs of clinical infection. Successful topical treatment with a potent corticosteroid significantly reduced the colonization of S. aureus. / <p>Härtill 4 uppsatser</p> / digitalisering@umu

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-08-14T14:53:58Z No. of bitstreams: 2 Dissertação - Jéssika Vieira dos Reis - 2018.pdf: 3710658 bytes, checksum: de2614771619ec9c58bbd1e7d57942c3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-16T11:28:50Z (GMT) No. of bitstreams: 2 Dissertação - Jéssika Vieira dos Reis - 2018.pdf: 3710658 bytes, checksum: de2614771619ec9c58bbd1e7d57942c3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-16T11:28:50Z (GMT). No. of bitstreams: 2 Dissertação - Jéssika Vieira dos Reis - 2018.pdf: 3710658 bytes, checksum: de2614771619ec9c58bbd1e7d57942c3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / With the increasing resistance of microrganisms to available drugs, mainly in hospital environments, the pharmaceutical industries have concentrated efforts in the research of new versatility of their molecular structures and their biological effects achieved. The hydrazones belong to an important class as they present a broad pharmacological profile whose properties have been extensively studied in medicinal chemistry because of their chelating capacity and the role of coordination in their biochemical mechanism of action. In this work a chemical ligand called J3 was synthesized which was complexed with copper nitrate (II), nickel nitrate, cadmium nitrate, cobalt perchlorate and diphenyltin dichloride, being called: J3Cu, J3Ni, J3Cd, J3Co and J3Sn, respectively. From the formation of the crystals, they were subjected to spectroscopic analysis of single crystal X-ray diffraction and infrared spectroscopy, which confirmed the chemical structures obtained from the ligand and its complexes. The biological activity of the six synthesized compounds was evaluated through the microdilution method on 96 wells plates, and the determination of the minimum inhibitory concentration (MIC) was determined by the density measurement in ELISA reader after incubations of 24, 48, 72 and 96 hours in bacteria of the species Escherichia coli and Staphylococcus aureus. The J3Cu, J3Cd and J3Sn compounds presented moderate activity for the tested bacteria, whereas the J3 ligand and its J3Co and J3Ni complexes did not inhibit 100% of the microrganisms in the tested concentrations. Through the tests of determination of the minimum bactericidal concentration (MBC), it was concluded that the complexes J3Sn and J3Cd presented bacteriostatic and bactericidal activity and J3Cu complex presented activity bacteriostatic. / Com o crescente aumento da resistência de microrganismos aos fármacos disponíveis, principalmente em ambiente hospitalar, as indústrias farmacêuticas têm concentrado esforços na pesquisa de novos fármacos. Diversas classes de compostos orgânicos despertam o interesse de pesquisadores devido à versatilidade de suas estruturas moleculares e seus efeitos biológicos alcançados. As hidrazonas pertencem a uma classe importante pois apresentam um amplo perfil farmacológico cujas propriedades têm sido extensivamente estudadas na Química Medicinal em razão de sua capacidade quelante e do papel da coordenação no seu mecanismo bioquímico de ação. Neste trabalho foi sintetizado um ligante químico denominado J3 o qual foi complexado com Nitrato de Cobre (II), Nitrato de Níquel, Nitrato de Cádmio, Perclorato de Cobalto e Dicloreto de difenilestanho, sendo denominados de: J3Cu, J3Ni, J3Cd, J3Co e J3Sn, respectivamente. A partir da formação dos cristais, os mesmos foram submetidos a análises espectroscópicas de difração de raios x em monocristal e espectroscopia na região do infravermelho, que confirmaram as estruturas químicas obtidas do ligante e seus complexos. Foi avaliada a atividade biológica dos seis compostos sintetizados através do método de microdiluição em placas de 96 poços, e a determinação da concentração inibitória mínima (CIM) foi determinada através da medida de densidade em leitor de ELISA após incubações de 24, 48, 72 e 96 horas em bactérias das espécies Escherichia coli e Staphylococcus aureus. Foi determinada a concentração bactericida mínima para os compostos que apresentaram CIM contra as bactérias testadas. Os compostos J3Cu, J3Cd e J3Sn apresentaram atividade moderada para as bactérias testadas, ao passo que o ligante J3 e seus complexos J3Co e J3Ni não inibiram 100% dos microrganismos nas concentrações testadas. Através dos testes de determinação da concentração bactericida mínima, concluiu-se que os complexos J3Sn e J3Cd apresentaram atividade bacteriostática e bactericida, o complexo J3Cu apresentou atividade bacteriostática.

O objetivo deste estudo foi verificar a ocorrência de S. aureus e E. coli O157: H7 no leite de vacas com mastite subclínica e no leite de mistura de 42 propriedades leiteiras localizadas em duas regiões do Estado de São Paulo: São Carlos e Ribeirão Preto. Paralelamente, entre os isolados de S. aureus foi objetivo identificar os produtores de toxinas e determinar sua origem epidemiológica. O isolamento de S. aureus foi realizado em agar Baird-Parker (35ºC, 48h) e a confirmação bioquímica através da catalase, coagulase, termonuclease, produção de acetoína e fermentação aeróbia da maltose. O isolamento de E. coli O157: H7 foi realizado em agar Sorbitol MacConkey MUG (35ºC, 24h). Para confirmação utilizaram-se as provas do IMVC e sorologia através do kit Soro Anti E. coli O157. Para a detecção da TSST-1 e das enterotoxinas A, B, C e D utilizou-se aglutinação reversa passiva em látex (RPLA). A identificação epidemiológica dos isolados de S. aureus foi realizada por eletroforese em gel de campo pulsado (PFGE). A ocorrência de S. aureus no leite individual nas regiões 1 (São Carlos) e 2 (Ribeirão Preto) foram 3,9% e 6,7%, respectivamente. Animais pertencentes às propriedades leiteiras com produção entre 400 L e 1.000 L/dia apresentaram maior risco de veiculação de S. aureus através do leite quando comparadas com propriedades com produção 1.000 L/dia. Quanto ao leite de mistura, verificou-se que a ocorrência de S. aureus foi a mesma em ambas as regiões (19%). A produção simultânea das enterotoxinas B e C foi observada em 4,7% dos isolados de leite individual, enquanto que 4,7% produziram enterotoxina A e toxina TSST-1. A produção de TSST-1, isoladamente, foi constatada em 14,3% dos isolados de leite individual e em 25% do leite de mistura. Houve similaridade genética entre os isolados de S. aureus, evidenciando sua dispersão epidemiológica entre as propriedades avaliadas. A ocorrência de E. coli O157: H7 no leite individual foi 1% na região 1 e 1,6% na região 2. No leite de mistura não foi detectada E. coli O157: H7. Ressalte-se a importância de medidas preventivas para assegurar a qualidade do leite durante a ordenha, a fim de evitar a ocorrência de microrganismos patogênicos, principalmente S. aureus, e conseqüentemente prevenir riscos de veiculação de toxinfecções através deste alimento. / The aim of this study was to verify the occurrence of S. aureus and E. coli O157: H7 in the milk from dairy cows with subclinical mastitis and in the bulk milk from 42 dairy farms located in two regions of São Paulo State (Region 1: São Carlos, Region 2: Ribeirão Preto). Among the S. aureus strains isolated, the aim was to identify the toxin producers and their epidemiological origin. The isolation of S. aureus was conducted using Baird-Parker agar, and the strains were confirmed by catalase, coagulase, thermonuclease, maltose aerobic fermentation and acetoin production. The isolation of E. coli O157: H7 was conducted using Sorbitol MacConkey MUG agar. The strains were confirmed by IMVC and serology using anti E. coli O157 sera. Rapid passive latex agglutination was used for detection of TSST-1 and enterotoxigenic strains of S. aureus. The epidemiological identification was performed using pulsed field gel electrophoresis (PFGE). S. aureus was isolated from 3.9% and 6.7% of the individual milk samples from regions 1 and 2, respectively. Dairy cows belonging to farms with milk production ranging from 400 to 1.000 L/day showed higher risk of S. aureus carrying-over, when compared with dairy farms with milk production < 400 L/day and > 1.000 L/day. In bulk milk samples, the occurrence of S. aureus was the same in both regions evaluated (19%). The simultaneous production of enterotoxin B and C was observed in 4.7% of strains isolated from individual milk samples, while 4.7% produced both enterotoxin A and TSST-1. TSST-1 production alone was observed in 14.3% of S. aureus strains isolated from individual milk and 25% of bulk milk samples. S. aureus strains tested by PFGE demonstrated genetic similarity, showing the dispersion patterns of this microorganism among dairy farms. E. coli O157: H7 was isolated from 1% and 1.6% of individual milk samples from regions 1 and 2, respectively, although it was not detected in bulk milk samples. The importance of preventive measures to ensure milk quality during milking extraction is stressed, aiming to avoid pathogenic agents, mainly S. aureus, and therefore, to prevent the carry-over of food borne diseases to humans through milk.

Dentre tantas complicações do diabetes mellitus (DM), a infecção por bactérias comuns da microbiota superficial da pele como, por exemplo, a bactéria Gram-positiva Staphylococcus aureus, causadora de infecções como a peritonite, com altos índices de hospitalização e morte. A hipótese deste trabalho é a que o efeito da insulina na ativação das vias de sinalização MAPK, PKC e PI3K em peritonite induzida por S. aureus em animais diabéticos e não diabéticos possa regular a produção de citocinas. Foram utilizadas amostras de fígado, rim, linfonodos peritoniais e baço de animais oriundos de estudo anterior (Projeto FCF/USP-375), no qual animais diabéticos (aloxana, 42 mg/kg, i.v., 10 dias) e não-diabéticos com peritonite decorrente da infecção por S. aureus receberam uma dose de 4 UI e 1 UI de insulina NPH, respectivamente, por via subcutânea, 2 horas antes da infecção com S. aureus, e outras 3 doses de 2 UI e 0,5 UI às 17h00\', respectivamente. A glicemia foi determinada no dia anterior, 10 dias após a injeção de aloxana e após os tratamentos com insulina. Em amostras de fígado, rim, linfonodo e baço dos animais supra citados foram avaliados a dosagem de citocinas (IL-1&#946;, IL-4, IL-10, TNF-&#945;, CINC-1, CINC-2 e CINC-3) por ensaios de enzima-imunoensaio (ELISA); em homogenato de fígado foram avaliadas a expressão das moléculas das vias MAPK (fosfo P-38, fosfo ERK p42/44), PKC (fosfo PKC-&#945;, fosfo PKC-&#948;) e PI3K (fosfo-AKT) pelo método de Western Blotting. Na avaliação do fígado, a insulina foi capaz de aumentar a concentração das citocinas IL-4 e TNF-&#945; que apresentavam-se diminuídas em animais não diabéticos, em relação aos animais não diabéticos e não infectados, mas nos animais diabéticos, na infecção pela cepa N315, a insulina diminuiu a concentração de IL-4, que não estava alterada pela infecção, e não foi capaz de aumentar a concentração de IL-1&#946; que estava diminuída na infecção, em relação aos animais diabéticos e não infectados. Em linfonodos peritoniais de animais não diabéticos infectados pela cepa N315, a insulina diminuiu a produção de IL-1&#946; e IL-10, que não estavam alteradas na infecção, e diminuiu a concentração de IL-4, que estava aumentada na infecção, em relação aos animais não diabéticos e não infectados; em animais diabéticos, a insulina diminuiu a produção das citocinas IL-1&#946; e CINC-1, que estavam aumentadas, e aumentou a concentração de IL-10, que estava diminuída na infecção com a cepa N315, mas baixou a concentração de IL-4, em relação aos animais infectados, e na infecção pela cepa ATCC, a insulina aumentou a produção de IL-1&#946;, CINC-1 e CINC-3 dos animais tratados, em relação aos infectados e não tratados. Em baço, a insulina diminuiu a produção de IL-10 na infecção pela cepa ATCC tanto em animais não diabéticos quanto em animais diabéticos e, nesse último grupo, também aumentou a produção de CINC-3 em relação aos animais diabéticos não infectados; na infecção com a cepa N315, a insulina não aumentou a concentração de IL-1&#946; e TNF-&#945;, que estavam diminuídas na infecção. Em rim, não houveram alterações significativas na produção de citocinas na infecção com nenhuma das cepas estudadas, nem para os grupos diabéticos, nem para os não diabéticos. Verificou-se que os animais diabéticos apresentam maior alteração tanto nas vias de sinalização estudadas quando na produção de citocinas pró-inflamatórias, quando comparados aos animais não diabéticos, na infecção por ambas as cepas de S. aureus estudadas. Assim, os resultados obtidos sugerem que o tratamento com insulina possa modular parcialmente a produção das citocinas IL-1&#946;, TNF-&#945; e IL-10 no fígado e nos linfonodos peritoniais dos animais infectados principalmente pela cepa N315 de S.aureus, modulando parcialmente a expressão das moléculas da via de sinalização (MAPK e PKC), envolvidas na produção dessas citocinas. / Among so many complications of diabetes mellitus (DM), infection by common bacteria of the superficial microbiota of the skin, for example, a gram-positive bacteria Staphylococcus aureus, causing infections like peritonitis, with high rates of hospitalization and death. The hypothesis of this study is that the effect of insulin on the activation of MAPK, PKC and PI3K signaling pathways in peritonitis induced by S. aureus in diabetic and non-diabetic animals may regulate the production of proinflammatory cytokines. Liver, kidney, peritonial lymph nodes and spleen samples of animals from the previous study (FCF / USP-375 Project) were used in this project; diabetic animals (alloxan, 42 mg / kg, iv, 10 days) and non-diabetic animals with peritonitis due to S. aureus infection received one dose of 4 IU and 1 IU of NPH insulin, respectively, subcutaneously, 2 hours before infection with S. aureus, and another 3 doses of 2 IU and 0.5 IU at 5:00 p.m., respectively. Blood glucose was determined the day before, 10 days after alloxan injection and after insulin treatments. In the liver, kidney, lymph node and spleen samples of the above-mentioned animals the cytokines (IL-1&#946;, IL-4, IL-10, TNF-&#945;, CINC- 2 and CINC-3) by enzyme-immunoassay (ELISA) assays; we avaliated, by Western Blotting, the signaling pathways MAPK (phospho-P-38, phospho ERK p42 / 44), PKC (phospho PKC-&#945; and phospho PKC-&#948;) and PI3K (phospho AKT) in liver, insulin was able to increase the concentration of cytokines IL-4 and TNF-&#945; that were decreased in non-diabetic animals, in relation to non-diabetic and non-infected animals, but in diabetic animals, in strain N315, insulin decreased the concentration of IL-4, which was not altered by the infection, and was not able to increase the concentration of IL-1&#946; that was decreased in infection, relative to diabetic and uninfected animals. In peritonial lymph nodes from non-diabetic animals infected with the N315 strain, insulin decreased the production of IL-1&#946; and IL-10, which were not altered in the infection, and decreased the concentration of IL-4, which was increased in infection, in relation to non-diabetic and non-infected animals; in diabetic animals, insulin decreased IL-1&#946; and CINC-1 which were increased, and increased the concentration of IL-10, which was decreased in infection with strain N315, but decreased the concentration of IL-4 in Infected animals, and in infection by the ATCC strain, insulin increased the production of IL-1&#946;, CINC-1 and CINC-3 of treated animals over infected and untreated animals. In spleen, insulin decreased IL-10 production on infection by the ATCC strain in both non-diabetic and diabetic animals and, in this last group, also increased the production of CINC-3 in relation to uninfected diabetic animals; in infection with the N315 strain, insulin did not increased the concentration of IL-1&#946; and TNF-&#945;, which were decreased in infection. In kidney, there were no significant changes in cytokine production in infection with any of the strains studied, neither for diabetic groups nor for non-diabetics. It was verified that diabetic animals present a greater alteration both in the signaling pathways studied and in the production of pro-inflammatory cytokines, when compared to non-diabetic animals, in the infection by both strains of S. aureus studied. Thus, the results suggest that insulin treatment may partially modulate the production of IL-1&#946;, TNF-&#945; and IL-10 cytokines in the liver and in the peritonial lymph nodes of animals infected mainly with S. aureus strain N315, since they partially modulating the expression of signaling pathway molecules (MAPK and PKC), involved in the production of these cytokines.