Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-24T17:22:23Z No. of bitstreams: 1 Deutsch, Gabriela [Dissertação, 2014].pdf: 1124546 bytes, checksum: 4c2efe5d127b5965bea25e458da2afc4 (MD5) / Made available in DSpace on 2017-03-24T17:22:23Z (GMT). No. of bitstreams: 1 Deutsch, Gabriela [Dissertação, 2014].pdf: 1124546 bytes, checksum: 4c2efe5d127b5965bea25e458da2afc4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A balneoterapia é um importante procedimento realizado diariamente em pacientes queimados. Esta prática consiste na limpeza mecânica com fricção manual sobre as áreas lesionadas pela queimadura utilizando antisséptico. Poucas são as evidências de que sua prática seja efetiva na higienização das feridas e na prevenção de infecção cruzada entre pacientes que utilizam o mesmo tanque. Neste projeto, buscou-se estudar a influência da balneoterapia na descolonização da superfície corporal queimada (SCQ) de pacientes internados em um centro de tratamento de queimaduras (CTQ), quanto à presença de cepas de Staphylococcus aureus e Pseudomonas aeruginosa. Swabs foram coletados durante a realização da balneoterapia de 18 pacientes por 14 semanas. A água utilizada também foi avaliada. Testes fenotípicos e genotípicos foram utilizados para identificação de S. aureus e P. aeruginosa. A susceptibilidade a antimicrobianos e biocidas foi verificada segundo os critérios do Clinical and Laboratory Standards Institute (CLSI). Pulsed-field gel electrophoresis (PFGE) foi utilizado para avaliar a diversidade genômica. Análise exploratória das variáveis envolvidas no processo da balneoterapia foi determinado pela estatística descritiva e testes estatísticos não paramétricos foram utilizados na análise dos fatores de risco. Trezentos e cinquenta e dois swabs foram coletados dos quais 214 (61%) da SCQ, 60 (17%) da cavidade nasal e 78 (22%) da mesa onde ocorreu a balneoterapia. Detectou-se 13 cepas de S. aureus e 39 de P. aeruginosa. A concentração mínima inibitória (CMI) para sulfadiazina de prata foi ≥32μg/mL para as cepas de S. aureus enquanto que para a P. aeruginosa a maioria das cepas apresentou CMI de 128μg/mL. Com relação a clorexidina, as cepas de S.aureus apresentaram um CMI variando de 2 a 8g/mL enquanto para P. aeruginosa a variação foi de 16 a 64μg/mL. Cinco amostras foram identificadas como S. aureus resistentes a meticilina (MRSA) e nove como P. aeruginosa resistentes a carbapenêmicos. A análise do perfil de fragmentação do DNA total (PFGE) nas cepas de P. aeruginosa demonstrou a existência de 10 clones entre 35 cepas analisadas. O tipo A foi o mais prevalente, com 23 cepas distribuídas em 8 subtipos. Estes estavam presentes na SCQ coletada antes e após o banho e nas superfícies da mesa de banho, sugerindo que há contaminação cruzada de um indivíduo para o outro, de uma área queimada para outra no mesmo indivíduo, da mesa da balneoterapia para indivíduos e finalmente do indivíduo para mesa. Os resultados não se mostraram estatisticamente significativos, no entanto, quatro pacientes que não apresentaram contaminação antes do banho se tornaram positivos após este procedimento, e 10 pacientes que apresentaram contaminação antes do banho, assim permaneceram. Conclui-se que o procedimento de descontaminação não está sendo eficaz uma vez que houve similaridade clonal entre as cepas de P. aeruginosa coletadas em vários pontos e momentos / Balneotherapy is an important procedure usually performed in burn patients. This practice consists on a mechanical cleaning with manual friction on the damaged areas using an antiseptic. There is little evidence that this practice is effective to clean the wounds and avoid cross infection between patients using the same table. In this project, we study the influence of hydrotherapy in the decolonization of burned body surface area (BSA) of patients admitted to a burn center (BC) for the presence of Staphylococcus aureus and Pseudomonas aeruginosa isolates. Thus, swabs were used to collect bacteria from 18 patients submitted to balneotherapy during 14 weeks. The material from bath table and the water used were also evaluated. Genotypic and phenotypic tests were used to identify S. aureus and P. aeruginosa. Susceptibility to antimicrobials and biocides has been verified according to the criteria of the Clinical and Laboratory Standards Institute (CLSI). Genomic diversity was assessed by pulsed-field gel electrophoresis (PFGE). Descriptive statistics were used in the exploratory analysis of the variables involved in the balneotherapy and non-parametric statistical tests were used in process analysis of risk factors. Three hundred fifty-two swabs were collected of which 214 (61%) were from BSA, 60 (17%) from nasal cavity and 78 (22%) from table where balneotherapy occurred. Thirteen isolates were identified as S. aureus and 39 as P. aeruginosa. Minimum inhibitory concentration (MIC) for silver sulfadiazine was ≥ 32μg/mL for S. aureus isolates and 128μg/mL for P. aeruginosa. It is possible that the increased MIC to silver sulfadiazine has occurred by the constant use of this antimicrobial in balneotherapy. However, MIC to chlorhexidine for S. aureus isolates range from 2 to 8mg/mL and for P. aeruginosa range from 16 to 64μg/mL. Furthermore, five S. aureus isolates were identified as methicillin-resistant Staphylococcus aureus (MRSA) and nine as P. aeruginosa resistant to carbapenems. A profile analysis of total P. aeruginosa DNA fragmentation showed 10 clones among 35 strains analyzed. Type A was the most prevalent, with 23 isolates distributed into eight subtypes. These subtypes were present in BSA collected before and after the bath and on the surfaces of the bath table, suggesting that there is cross-contamination from one individual to another, from a burned area to another in the same individual, from the balneotherapy table to an individual and finally from the individual to the table. The results were not statistically significant, however, four patients who were not contaminated before bathing became positive after this procedure, and 10 patients who were contaminated before bathing, remained so. Thus, it is possible to conclude that the procedure is not efficient for the decontamination because there was similarity between the clonal isolates of P. aeruginosa collected at various points and times

La phospholipase A2 sécrétée de type IIA (sPLA2-IIA) est une protéine possédant une activité antibactérienne très élevée envers les bactéries à Gram positif. La mucoviscidose (MV) est une maladie génétique résultant de la mutation du gène Cftr. L'altération de la fonction du CFTR dans les poumons favorise la colonisation par des pathogènes bactériens, dont les plus retrouvés, S. aureus et P. aeruginosa, colonisent les voies aériennes de manière séquentielle: S. aureus est principalement retrouvé chez les jeunes patients et P. aeruginosa chez les adultes. Les mécanismes impliqués dans ce changement de prévalence restent cependant mal connus. Dans ce travail, nous démontrons que l'expression de la sPLA2-IIA est plus importante dans les poumons de patients MV que dans les poumons de patients non MV et augmente avec l'âge des patients. La sPLA2-IIA présente dans les expectorations de patients MV est capable de tuer spécifiquement S. aureus. L'utilisation de modèles animaux nous a permis de démontrer sa spécificité d'action contre S. aureus et l'induction de son expression par P. aeruginosa contribuant à l'élimination de S. aureus des voies respiratoires. Enfin, nous avons identifié les cellules épithéliales comme une source majeure de sPLA2-IIA chez les patients MV. Dans ces cellules, P. aeruginosa induit l'expression de la sPLA2-IIA par un mécanisme dépendant de l'injection de la toxine ExoS et du facteur de transcription KLF2. L'ensemble de ces résultats indique i) que la sPLA2-IIA induite par P. aeruginosa, participe à l'élimination de S. aureus dans les voies aériennes des patients MV et ii) qu'une bactérie élimine une autre bactérie en utilisant la défense de l'hôte. / The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host defense protein endowed with antibacterial activity, especially against Gram positive bacteria. Cystic fibrosis (CF) is a genetic disease due to mutations of Cftr gene. In the lungs, CFTR mutation favored bacterial colonization by bacterial pathogens, of which S. aureus and P. aeruginosa are the most isolated. These two bacterial species sequentially colonized airways of CF patients: S. aureus is predominant in young patients and P. aeruginosa in adults. But the mechanisms involved in this switch of bacterial prevalence are still unknown. In this work we showed that sPLA2-IIA levels were increased in lungs of CF patients compared to lungs of non-CF patients and that sPLA2-IIA levels increased with age of patients. sPLA2-IIA recovered from CF patients expectorations was active and killed specifically S. aureus. Using animal models of lung infection, we demonstrated the selectivity of sPLA2-IIA against S. aureus and that P. aeruginosa induced sPLA2-IIA expression, the latter contributed to S . aureus elimination from the airways. Finally, we identified epithelial cells as a major source of sPLA2-IIA in CF airways. In these cells, P. aeruginosa induced sPLA2-IIA expression through injection of ExoS toxin and activation of KLF2 transcription factor. Taken together, these results indicate that i) P. aeruginosa-induced sPLA2-IIA expression in CF airways participated to S. aureus elimination and ii) a bacteria eliminate another bacteria by manipulating host innate immunity.

Probiotics have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The beneficial effects of probiotics in the gut are well described and roles including immunomodulation and colonisation resistance have been documented. Recent reports suggest that topical use of probiotic bacteria may be an effective strategy to promote skin health or inhibit disease. Therefore, in this thesis the potential of probiotics to protect skin from pathogenic bacteria was assessed using primary keratinocytes as a model system, and the skin pathogen, Staphylococcus aureus. The ability of three probiotics, L. reuteri ATCC 55730, L. rhamnosus AC413 and L. salivarius UCC118 to inhibit the growth of S. aureus was tested using well-diffusion assays and spot on the lawn assays. All three probiotics inhibited the growth of S. aureus in well-diffusion assays, though this property was dependent on growth medium. Inhibition of S. aureus growth was principally via the production of organic acids rather than bacteriocin production. Next, to determine whether probiotics could protect keratinocytes, confluent normal human epidermal keratinocytes (NHEK) were infected with S. aureus (106 CFU/ml) in the presence or absence of the probiotic (108 CFU/ml). NHEK viability was measured using trypan blue exclusion assays. L. reuteri had a significant protective effect on NHEK when applied 1h prior to (P=0.0003), or simultaneously with S. aureus (P=0.002). L. reuteri did not however protect NHEK when applied 1h after S. aureus addition. There was no change in the number of viable S. aureus in cell culture assays. To determine whether the protective effect was due to the inhibition of adhesion, NHEK were either pre-exposed to the probiotic for 1h, simultaneously exposed to the probiotic and S. aureus for 1h, or exposed to the probiotic 30 minutes after S. aureus addition for 1h. Pre-exposure of NHEK to L. reuteri (exclusion) and simultaneous exposure to L. reuteri and S. aureus (competition) resulted in significantly less staphylococci adhering to NHEK (P=0.03 and P=0.008 respectively). However when L. reuteri was added after S. aureus (displacement), the number of adherent staphylococci was not reduced. The necessity of S. aureus adherence for the inactivation of NHEK was demonstrated using a α5β1 integrin blocking antibody. Finally, to compare the innate response of NHEK to probiotics with S. capitis and S. aureus, TLR-2, antimicrobial peptide (AMP) expression and IL-8 production were measured. TLR-2 protein (but not mRNA) expression was reduced in the presence of S. aureus (P=0.018). NHEK pre-exposed to S. capitis prior to S. aureus infection however, exhibited elevated TLR-2 protein and mRNA expression (P<0.0001 and P=0.009 respectively). NHEK pre-exposed to L. reuteri prior to S. aureus had no significant change in TLR-2 expression compared to untreated controls. ELISAs demonstrated that IL-8 production was significantly increased in NHEK pre-exposed to L. reuteri prior to S. aureus infection (P=0.0001). In conclusion, L. reuteri protected NHEK from the toxic effects of S. aureus at least partly through competitive exclusion of binding sites on NHEK. Finally, NHEK innate responses to probiotic bacteria were akin to those to the skin commensal, S. capitis. L. reuteri induced expression of a neutrophil chemoattractant, suggesting it could be of importance in priming the innate immune response against S. aureus infections. Taken together, these results suggest that probiotic bacteria could be used prophylactically within skin creams and soaps to prevent S. aureus colonisation and infection in skin.

Staphylococcus aureus is a gram-positive opportunistic pathogen responsible for more deaths every year than HIV/AIDS. Its formidable repertoire of virulence factors, ubiquitous nature, and ability to acquire antibiotic resistance quickly allow S. aureus to colonize and persist in nearly any body site if given the opportunity. S. aureus is the leading cause of many common and severe skin diseases, i.e. atopic dermatitis and surgical site infections, which can result in significant morbidity and mortality due to lack of available treatments and chronic non-healing nature of each infection. The human body is capable of producing many antimicrobial factors, such as defensins in the epidermis, in conjunction with providing a seamless barrier to many environmental threats, i.e. the skin, yet when given the opportunity, S. aureus can overtake these innate defenses, colonize, and cause disease. Despite S. aureus being a prominent organism in skin infections, little has been done to identify critical factors of S. aureus to cause skin infections. This document demonstrates the capacity of specific S. aureus virulence factors, superantigens and cytotoxins, to alter re-epithelialization and wound healing, as indicated by altered keratinocyte migration and proliferation. In an attempt to harness natural occurring host defenses, we have also identified and generated novel antimicrobial peptides capable of ablating toxin production independent of bacterial growth inhibition. Evidence presented should convince the reader that S. aureus exotoxin production is critical in perpetuating chronic wounds through local keratinocyte interaction. This suggests targeting production of these toxins to prevent cell toxicity and inflammatory responses, could allow the host to repair damaged tissue effectively.

This master thesis is focusing on analysis of two kinds of confectionary products: nutty and lemon balls. The samples were analysed and they determinate the numbers of total count of bacteria, number of S. aureus, yeast and microscopic fungi, coliforms bacteria and E. coli, detection of Salmonella spp. and Listeria monocytogenes. Furthermore, S. aureus (strain CCM 5974) was inoculated in the samples. These samples determinate the number of S. aureus and possibility of the production an enterotoxins of S. aureus. Staphylococcal enterotoxin was detected with the reverse passive latex agglutination (RPLA). The result of determination is that the numbers of total count of bacteria influence of storage period the number was reduced (p<0,05). Statistical differences were not found between the nutty and the lemon balls (p>0,05). During the first and the second analysis was found growth of yeast and microscopic fungi in the nutty balls. In the lemon balls was found a growth of yeast and microscopically fungi during the first analyses and in one sample only. Also the number of bacteria was reduced with S. aureus (p<0,05). There were not found any statistical differences between the nutty and the lemon balls (p>0,05). The number of E. coli was not found in any our samples. The coliform bacterium was found only with the first and the second analysis in the nutty samples. In the lemon samples the coliform bacterium was found only in the first analysis. During the first detection of Salmonella spp. were found four colonies of this bacterium in the nutty samples. Colonies of Listeria monocytogenes were not detected in any of these samples. Microbiological analysis of inoculated sample confirms that our confectionary products are not suitable for growth of pathogenic microorganism S. aureus. It also proves that between the nutty and the lemon samples were no statistical difference (p>0,05). During the inoculation was proven that S. aureus (CCM 5974) with concentration 1,2.105 CFU.g-1 is capable to form in products enterotoxins, which stays in products in contrast with S. aureus, after a certain period of time.

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Pathogens in drinking water are the cause of over 1.5 million deaths around the world every year, mostly in developing countries. Practical, cheap, and effective tools for detection of these pathogens are critical to advance public health in many areas around the globe. Micro electro-mechanical systems (MEMS) are miniaturized structures that can be used for a variety of purposes, including, but not limited to, small scale sensors. Therefore, MEMS can be used in place of expensive laboratory equipment and offer a cheap and practical tool for pathogen detection. The presented work's research objective is to introduce a new technique called embedded passivated-electrode insulator-based dielectrophoresis (EπDEP) for preconcentration, separation, or enrichment of bioparticles, including living cells. This new method combines traditional electrode-based DEP and insulator-based DEP with the objective of enhancing the electric field strength and capture efficiency within the microfluidic channel while alleviating direct contact between the electrode and the fluid. The EπDEP chip contains embedded electrodes within the microfluidic channel covered by a thin passivation layer of only 4 μm. The channel was designed with two nonaligned vertical columns of insulated microposts (200 μm diameter, 50 μm spacing) located between the electrodes (600 μm wide, 600 μm horizontal spacing) to generate the nonuniform electric field lines to concentrate cells while maintaining steady flow in the channel. The performance of the chip was demonstrated using Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial pathogens in aqueous media. Trapping efficiencies of 100% were obtained for both pathogens at an applied AC voltage of 50 V peak-to-peak and flow rates as high as 10 uL/min. / Master of Science

This work was focused on the characterization of microparticles with particular emphasis on waterborne pathogens which pose a great health risk to human lives. The goal of this study was to develop microfluidic systems for enhanced characterization and isolation of bioparticles. Insulator-based dielectrophoresis (iDEP) is a promising technique for analyzing, characterizing and isolation of microparticles based on their electrical properties. By employing insulator-based constrictions within the microchannel in combination with microelectrodes within the vicinity of the electrodes, dielectrophoretic performance is enhanced. In this study, three dimensional insulator-based dielectrophoresis devices are fabricated using our in-house developed 3D micromachining technique. This technology combines the benefits of electrode-based DEP, insulator-based DEP, and three dimensional insulating features with the goal of improving trapping efficiency of biological species at low applied signals and fostering wide frequency range operation of the microfluidic device. The dielectric properties of bacteria as well as submicron polystyrene beads are discussed and the impact of these results on the future development of iDEP microfluidic systems is explored. / Master of Science

In this project, we synthesized a new homologous series of five long-chain, two-headed amphiphiles [2CAm13, 2CAm15, 2CAm17, 2CAm19, 2CAm21; CH3(CH2)n-1CONHC(CH3)(CH2CH2COOH)2, n = 13, 15, 17, 19, 21]. The synthesis of the 2CAmn series was accomplished in four steps. The first step involves a reaction of nitroethane and two equivalents of tert-butyl acrylate to create the nitrodiester synthon [O2NC(CH3)(CH2CH2COOtBu)2] by successive Michael additions. The second step in the synthesis consists of a reduction of nitrodiester with H2 and Raney nickel to give the diesteramine [H2NC(CH3)(CH2CH2COOtBu)2]. The third step is the condensation of an acid chloride with diesteramine to give an alkanamido diester [2EAmn; CH3(CH2)n-1CONHC(CH3)(CH2CH2COOtBu)2, n = 13, 15, 17, 19, 21]. The final step is the removal of the tert-butyl protecting groups to give 2CAmn. Critical micelle concentration measurements were collected by the pendant drop method for measuring surface tension for a series of triethanolamine/2CAmn solutions to establish the concentration required for detergency. The CMCs for the 2CAmn series were found to decrease in value from 3.0 Ã 10â 2 M (2CAm13) to 1.7 Ã 10â 4 M (2CAm21) in a linear fashion [log CMC = (â 0.28 ± 0.01)n + (2.2 ± 0.1)]. The CMCs for the 2CAmn series falls in between the CMCs for three series of homologues three-headed amphiphiles (3CAmn, 3CCbn, 3CUrn) and the CMCs for fatty acids, with fatty acids having the lowest CMCs. Antibacterial activity (minimal inhibitory concentrations, MICs) for a series of homologous dendritic two-headed amphiphiles and three series of homologous, three-headed amphiphiles against Staphylococcus aureus and methicillin-resistent S. aureus (MRSA) were measured by broth microdilution to compare the effect of chain length and, hence, hydrophobicity. Inoculum density affected antibacterial activity of the 2CAmn series against both S. aureus and MRSA. MIC measurements at different cell densities showed that activity decreased with higher cell densities. For all four series, the MICs were relatively flat at low inoculum densities. This flat region defines the intrinsic activity, MIC0. The MIC0 results revealed that inoculum density, chain-length, and hydrophobicity all influenced antibacterial activity and that activity correlates strongly with clogp, an established measure of hydrophobicity. The most hydrophobic members from each homologous series exhibited antibacterial activity. The most active homologue of the 2CAmn series was 2CAm21 with MIC0 of 2.0 ± 1.0 and 3.2 ± 1.0 μM against S. aureus and MRSA, respectively. The CMCs and MIC0s of the two- and three-headed amphiphiles were compared for both S. aureus and MRSA to gauge the effect that micelles may have on activity. Amphiphile 2CAm19 has the largest ratio between CMC and MIC0 (CMC/MIC0 = 205) against S. aureus and 3CUr20 has the largest ratio (CMC/MIC0 = 339) against MRSA. These ratios suggest that micelle formation is not a mechanism of action for anti-Staphylococcal activity. / Master of Science