Made available in DSpace on 2014-06-12T16:26:39Z (GMT). No. of bitstreams: 2 arquivo1159_1.pdf: 618867 bytes, checksum: 7cd487fc5f601809f1f857894332f68c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente estudo teve como objetivo principal a determinação da atividade bacteriostática do óleo essencial de Lippia gracilis Schauer, planta nativa do semi-árido nordestino, conhecida vulgarmente por alecrim da chapada e do carvacrol metabólito secundário presente no gênero Lippia. Para esta avaliação foi utilizado o método de micro diluição em meio liquido e dez cepas de Pseudomonas aeruginosa ou de Staphylococcus aureus, ambas com perfis de resistência a pelo menos dois agentes antimicrobianos. O óleo essencial de Lippia gracilis e o carvacrol foram diluídos de forma a obter concentrações que variaram de 1,56% a 0,012%v/v. Os inocula bacterianos foram padronizados em 104 UFC/poço. Uma completa inibição do crescimento de P. aeruginosa e S. aureus foi observada em concentrações equivalentes a 0,78%v/v do óleo essencial de L. gracillis. O carvacrol na concentração de 0,012% v/v inibiu 70% das cepas de S. aureus. Esta atividade quando comparada ao óleo essencial de Lippia gracilis não foi estatisticamente diferente. O óleo essencial de Lippia gracilis mostrou-se mais ativo frente a P. aeruginosa que o carvacrol e estes resultados foram diferentes estatisticamente (p<0,05). O óleo essencial de Lippia gracilis é eficaz sobre microrganismos resistentes de patogenicidade reconhecida e de grande interesse nas infecções nosocomiais

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2007. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-25T19:06:21Z No. of bitstreams: 1 microsoft word - dissertao.final.pdf: 515857 bytes, checksum: afa0c33effce4f7fb4a24ce581f95202 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2013-06-18T18:32:39Z (GMT) No. of bitstreams: 1 microsoft word - dissertao.final.pdf: 515857 bytes, checksum: afa0c33effce4f7fb4a24ce581f95202 (MD5) / Made available in DSpace on 2013-06-18T18:32:39Z (GMT). No. of bitstreams: 1 microsoft word - dissertao.final.pdf: 515857 bytes, checksum: afa0c33effce4f7fb4a24ce581f95202 (MD5) Previous issue date: 2007 / Os biossurfactantes compreendem uma classe importante de compostos anfipáticos de origem microbiana. A bactéria gram negativa Pseudomonas aeruginosa é conhecida por sua habilidade de produzir biossurfactante do tipo ramnolipídio a partir de diversas fontes de carbono. A investigação da produção por novas linhagens pode resultar em processos mais eficientes, maiores rendimentos e biossurfactantes com características distintas, sendo aplicados em várias indústrias, tais como as indústrias farmacêutica, de cosméticos, de petróleo e de alimentos. Por outro lado, com o maior consumo de petróleo e derivados, cresceu a exposição de ambientes a possíveis acidentes relacionados à produção, transporte, carga e descarga desses materiais, sendo cada vez mais necessário buscarem-se alternativas tecnológicas para a recuperação desses ambientes. A biorremediação pode ser definida como o uso das potencialidades dos microrganismos em degradar compostos poluentes, reduzindo o grau de contaminação ou mesmo descontaminando ambientes degradados. Entre as técnicas disponíveis, a bioestimulação é uma das mais usadas, em que a microbiota nativa é estimulada pela adição de nutrientes e coadjuvantes como os biossurfactantes. Este trabalho teve como objetivo estudar a produção de ramnolipídios de Pseudomonas aeruginosa isolada de resíduos de pescado capturado no extremo sul do Brasil, utilizando diversas fontes de carbono (óleo de soja, borra de óleo de soja, óleo de pescado, glicerol e glicose) e fontes de nitrogênio (nitrato de sódio, nitrato de amônio, uréia e sulfato de amônio), bem como diferentes relações C/N. O biossurfactante foi caracterizado quanto à influência de fatores físicoquímicos na formação de emulsões. A degradação de petróleo em solo retirado de local próximo a um oleoduto, por técnicas de bioestimulação, com e sem a utilização do biossurfactante, também foi estudada. Os cultivos microbianos foram realizados em triplicata, sendo conduzidos em incubadora rotatória a 30°C e 180rpm, por um período de 96 horas. Pelo teste de Tukey, a 95% de confiança, o óleo de soja (40g/L) e o nitrato de sódio (8g/L) foram as melhores fontes de carbono e nitrogênio, respectivamente, obtendose, com uma relação C/N 22, 0,94g/L de ramnose e índice de emulsificação de 62,1%. Com uma relação C/N 100 foram produzidos 1,42g/L de ramnose e índice de emulsificação de 60,7%, indicando que a produção é favorecida em condição nitrogêniolimitante. A formação de emulsões estáveis foi melhor em concentrações salinas menores que 0,5%, pH na faixa de 6-9 e temperaturas na faixa de 35-40°C, mantendo cerca de 80% de sua atividade original para salinidade de até 3% e 120 min de exposição a 100°C. Quanto à biorremediação, os melhores resultados foram obtidos nos tratamentos com adição de nutrientes e adição de nutrientes mais biossurfactante, com 90,4% e 80,9% de remoção de hidrocarbonetos totais de petróleo, respectivamente, demonstrando efetiva melhoria em relação ao controle (42,2%), havendo uma clara relação entre a dinâmica da microbiota local e a biodegradação. / Biosurfactants consist of an important class of amphipathic compounds of microbial origin. The Gram-negative bacteria Pseudomonas aeruginosa is known for its ability to produce rhamnolipid-type biosurfactant from diverse carbon sources. The investigation of the production by new strains can result in more efficient processes, greater yields and biosurfactants with distinct characteristics, being applied in various industries, such as pharmaceuticals, cosmetics, oils and foods. On the other hand, with the huge consumption of petroleum and its derivatives, the exposure of environments to possible accidents related to the production, transport, loading and unloading of these materials has increased, making it necessary to search for technological alternatives to restore of these environments. Bioremediation can be defined as the use of the potential of microrganisms in degrading pollutant compounds, reducing the degree of their contamination or decontaminating degraded environments. Among the available techniques, biostimulation is one of most commonly used, where native microbiota is stimulated by the addition of nutrients and amendments as surfactants. This work had as its objective to study the production of biosurfactant from a strain of Pseudomonas aeruginosa isolated from fish samples captured in the extreme south region of Brazil, using diverse carbon sources (soybean oil, soybean oil soapstock, fish oil and glycerol) and nitrogen sources (sodium nitrate, ammonium nitrate, urea and ammonium sulphate), as well as different C/N ratios. The biosurfactant was characterized taking into account the influence of physio-chemical factors in emulsion formation. The degradation of oil from the soil removed from a place next to a pipe-line in the region by different biostimulation techniques, with and without the use of the biosurfactant, also was studied. The microbial culture assays, carried out in triplicate, were conducted in a rotary shaker at 30°C and 180rpm, for a period of 96 hours, and the results analyzed by the Tukey test. The soybean oil (40g/L) and the sodium nitrate (8g/L) were the best sources of carbon and nitrogen, respectively, obtaining with a C/N 22, 0.94 g/L of rhamnose and an emulsification index of 62.1%. With a C/N ratio 100, 1.42 g/L of rhamnose and an emulsification index of 60.7% were produced, indicating that the production is favored in a nitrogen-limiting condition. The formation of stable emulsions was better in saline concentrations less than 0.5%, pH in the range of 6-9 and temperatures in the range of 35-40°C, maintaining about 80% of its original activity for salinity up to 3% and 120 min of exposure at 100°C. For bioremediation, the best results were obtained in the treatments with addition of nutrients and addition of nutrients with more biosurfactant, with 90.4% and 80.9% of TPH removal, respectively, demonstrating effective improvement in relation to the control (42.2%), having a clear relation between the dynamics of local microbiota and the biodegradation.

The majority of surfactants commercially available is synthesized from petroleum, thus representing an important source of pollution, causing adverse biological effects to the aquatics organisms. In the detergent industries, despite of many trades available in the market are called as biodegradable and under the legislation actual, it's known that in real the actives components are tensoactives obtained by chemical and not by biochemical route. In other words, what occurred was only a change of the main active component alkylbenzene sodium sulphonate branched chain for one of linear chain, what in fact facilitated the molecular degradation by microorganisms, but not as much as compared to the natural surfactants. Arming to salve those problems, in this work we will show a development process of two biodetergents from biosurfactants that follow environmental appeal and available new alternatives products in the market, using a new technology which may be inserted promise of sustainable industrial development that give attention, specially, to the use of clean technologies. The purpose of this invention is to combine the main soap and synthetic detergent’s features providing an alternative to using of the latter, therefore it adds from the soap its higher biodegradability’s features and from the synthetic detergent its advantage on acting in a still efficient way, even when utilized in hard waters. Initially, were produced two biosurfactants called as liposan and rhamnolipid obtained from aerobic fermentation, using a Yarrowia lipolytica IMUFRJ 50682 yeast strain and another Pseudomonas aeruginosa INCQS 0588092 bacterium straim, respectively. After the analysis of the different experimental conditions, it was concluded that the pH 7.0, at temperature of 35°C with rotation of 150 rpm, were the main factor that influenced on the production of both biosurfactants. The response variables used were surface tension, the index E24, the production of biomass, biosurfactant production and consumption of substrate. After the separation and extraction of the liposan and the rhamnolipid, it was held the modification of the two molecules by a chemical reaction and the biodetergents were formulated adding the adjuvant agents and completing the final volume with distilled water. The efficiency of the biodetergents was evaluated comparing the viscosities from a sample of crude oil with a produced water emulsion/oil containing the biodetergents, where it was verified a reduction on the viscosity, about 8% for the biodetergent 1 derived from the liposan and 36% for the biodetergent 2 derived from the rhamnolipid. It was observed from the analysis of the DSC that the developed biodetergents didn’t show any physico-chemical change when dissolved in samples of distilled water and compared to the produced water, concluding that both show good thermal stability and it wasn’t detected any chemical interaction, on the studied thermal range, between the biodetergents and the present salts in great quantity in the produced water, showing also a good tolerance to ionic strength. Regarding the capacity to produce foam and remove dirt in tissues and dishes both produced biodetergents showed foaming power and detergent action similar when compared to the synthetic detergent commercial. Therefore, it’s concluded that the produced biodetergents show good surfactant and emulsification capacity compared to the chemical synthetic surfactants, maybe being used in substitution themselves for the presented advantages. / A maioria dos surfactantes disponíveis comercialmente é sintetizada a partir de derivados do petróleo, representando assim uma importante fonte de poluição, causando efeitos biológicos adversos a organismos aquáticos. Na indústria de detergentes, apesar das várias marcas disponíveis no mercado serem consideradas biodegradáveis e amparadas pela legislação em vigor, sabe-se que na verdade os componentes ativos são tensoativos obtidos por via química e não bioquímica, ou seja, o que houve foi apenas a mudança do principal componente ativo alquilbenzeno sulfonato de sódio de cadeia ramificada pelo de cadeia linear, o que de fato facilitou a degradação da molécula por microrganismos, mas não tanto quanto ao comparado com os surfactantes naturais. Com intuito de solucionar tais inconvenientes, neste trabalho apresentaremos um processo de desenvolvimento de dois biodetergentes, a partir de biossurfactantes que atendam ao apelo ambiental e que disponibilize no mercado novos produtos alternativos aos já existentes, utilizando uma nova tecnologia que possa estar inserida na promessa de desenvolvimento industrial sustentável que prima, sobretudo, pelo uso de tecnologias limpas. A presente invenção conjuga as principais propriedades do sabão e do detergente sintético proporcionando uma alternativa ao uso destes últimos, pois, agrega do sabão as características de maior biodegradabilidade e do detergente sintético a vantagem de agir de forma ainda eficiente mesmo quando utilizado em águas duras. Inicialmente produziram-se dois biossurfactantes denominados de liposan e ramnolipídeo obtidos a partir da fermentação aeróbia, utilizando-se uma cepa da levedura Yarrowia lipolytica IMUFRJ 50682 e outra cepa da bactéria Pseudomonas aeruginosa INCQS 0588092, respectivamente. Após a análise exploratória das diferentes condições experimentais, concluiu-se que o pH 7,0, a temperatura de 35°C e agitação de 150 rpm, foram os fatores que mais influenciaram na produção dos dois biossurfactantes. As condições experimentais foram analisadas quanto à tensão superficial, o índice E24, a produção de biomassa, a produção do biossurfactante e o consumo do substrato. Após a separação e extração do liposan e do ramnolipídeo, realizou-se a modificação das duas moléculas através de uma reação química e formulou-se os biodetergentes adicionando-se os agentes coadjuvantes e completando-se o volume final com água destilada. A eficiência dos biodetergentes foi avaliada comparando as viscosidades de uma amostra de óleo bruto com uma emulsão água produzida/óleo contendo os biodetergentes, onde se verificou uma redução da viscosidade em torno de 8% para o biodetergente 1 derivado do liposan e 36% para o biodetergente 2 derivado do ramnolipídeo. Observou-se através da análise de DSC que os biodetergentes desenvolvidos não apresentaram transformações físico-químicas quando dissolvidos em amostras de água destilada e comparadas com água produzida, concluindo-se que ambos apresentaram boa estabilidade térmica e que não foi detectada nenhuma interação química, na faixa de temperatura estudada, entre os biodetergentes e os sais presentes em grande quantidade na água produzida, mostrando assim também uma boa tolerância à força iônica. Em relação à capacidade de produzir espuma e de remover sujidades em tecidos e em louças, os dois biodetergentes produzidos apresentaram poder espumante e ação detergente semelhante quando comparado ao sintético comercial. Desta forma, pode-se concluir que os biodetergentes produzidos apresentaram boa capacidade tensoativa e de emulsificação comparado aos surfactantes químicos sintéticos, podendo ser utilizados em substituição aos mesmos pelas vantagens apresentadas.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-06-13T12:50:57Z No. of bitstreams: 1 texto completo.pdf: 739948 bytes, checksum: e22b7ad0fc203f73756f4e4f2c810e59 (MD5) / Made available in DSpace on 2018-06-13T12:50:57Z (GMT). No. of bitstreams: 1 texto completo.pdf: 739948 bytes, checksum: e22b7ad0fc203f73756f4e4f2c810e59 (MD5) Previous issue date: 2018-03-14 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Biofilmes bacterianos formados em áreas de processamento de alimentos podem se tornar fonte de contaminação cruzada. Dessa forma, torna-se importante a correta manipulação das matérias-primas que chegam até a indústria, além do uso adequado de sanitizantes no processo de higienização de equipamentos e superfícies. Objetivou-se com este estudo avaliar a formação de biofilme e potencial de biotransferência de Pseudomonas aeruginosa em aço inoxidável, em contato com leite integral UHT e caldo de vegetais. E o efeito de três surfactantes, em concentrações abaixo, próxima e acima da concentração crítica de micelas, na redução de biofilmes. Cada meio de cultivo foi inoculado com P. aeruginosa ATCC 15442, distribuídos em poços de microplaca contendo cupons de aço inoxidável AISI 304 # 4 e incubados a 15 °C. O número de células aderidas foi determinado após 24, 48, 72, 96 e 120 h e o potencial de biotransferência a partir de 48 h. Soluções de cloreto de benzalcônio (CB), dodecilbenzeno sulfonato de sódio (DBSS) e Tween 80 foram avaliados sobre os biofilmes formados por 72 h. Cupons de aço, antes e após os tratamentos, foram observados por microscopia confocal de varredura a laser. Verificou-se que, apesar do leite integral conter maior concentração de nutrientes em relação ao caldo de vegetais, não houve diferença na formação de biofilme de P. aeruginosa. O potencial de biotransferência do micro-organismo aos meios de cultivo foi observado durante todo o período avaliado. As soluções surfactantes mostraram-se eficientes na redução do número de células aderidas com o aumento da concentração, principalmente o CB. Tween 80 e DBSS reduziram menos de 2 log UFC∙cm -2 de células viáveis, enquanto o CB reduziu até 6 log UFC∙cm -2 . A partir dos resultados obtidos verificou-se que P. aeruginosa pode produzir biofilmes em superfície utilizada no processamento de alimentos, independente do tipo de resíduo alimentar presente. Os surfactantes aplicados apresentaram melhor eficiência em concentrações mais elevadas, acima da crítica de micelas, e a classe desses agentes também contribuiu para a redução das células aderidas. / Bacterial biofilms formed in food processing areas can become a source of cross-contamination. Thus, the correct handling of the raw materials coming into the industry becomes important, as well as the proper use of sanitizing in the cleaning process of equipment and surfaces. The aim of this study was to evaluate the biofilm formation and biotransferential potential of Pseudomonas aeruginosa in stainless steel, in contact with UHT whole milk and vegetable broth. Also, it was evaluated the effect of three surfactants at concentrations below, above and near the critical micelle concentration, in the reduction of biofilms. Each culture medium was inoculated with P. aeruginosa ATCC 15442, distributed in microplate wells containing stainless steel coupons AISI 304 # 4 and incubated at 15 °C. The number of adhered cells was determined after 24, 48, 72, 96 and 120 h and the potential of biotransference after 48 h. Benzalkonium chloride (BAC), sodium dodecylbenzene sulfonate (SDBS) and Tween 80 solutions were evaluated on biofilms formed for 72 h. Steel coupons, before and after treatments, were observed by confocal laser scanning microscopy. It was verified that, although the milk had higher concentration of nutrients in relation to the vegetable broth, there was no difference in the formation of P. aeruginosa biofilm. The biotransfer potential of the microorganism to the culture media was observed throughout the evaluated period. The surfactant solutions were efficient in reducing the number of cells adhered with the concentration increase, mainly of the BAC. Tween 80 and SDBS reduced less than 2 log CFU∙cm -2 of viable cells, while BAC reduced up to 6 log CFU∙cm -2 . From the results obtained it was verified that P. aeruginosa can produce biofilms on the surface used in food processing, regardless of the type of food residue present. The applied surfactants presented better efficiency at higher concentrations, above the critical micelle, and the class of these agents also contributed to the reduction of the adhered cells.

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

Current smoking cessation guidelines recommend nicotine replacement therapy (NRT) to assist pregnant smokers to quit, but this is without strong evidence for effectiveness and safety. Nicotine, the main addictive component of tobacco, is known to exert physiological effects by binding to its receptor, the nicotinic acetylcholine receptor (nAChR). Recent studies have identified the presence of nAChRs in non-neuronal cells, and in macrophages, functional alteration upon stimulation with nicotine has been documented. To understand the impact of in utero nicotine exposure on various immune cell disposition and function, we designed preliminary studies using an in vivo model of P. aeruginosa infection. In this model, pregnant mice were exposed to nicotine and after weaning, offspring were infected intra-tracheally and humanely killed 5 days later. Nicotine-exposed mice had a greater weight reduction post-infection. This was accompanied by a decreased number of neutrophil, resident macrophages, and B lymphocytes in the lungs, while the number of B lymphocytes in the lymph nodes were greater than that of the control group. In the lung lavage fluids, IL-6, MCP-1, and TNFα concentrations were elevated in nicotine-exposed mice. In an in vitro system using bone marrow-derived macrophages, a significantly reduced production of IFNγ was observed in nicotine-exposed mice when cells were stimulated with LPS. To characterize and compare gene expression in macrophages isolated from neonates developmentally exposed to nicotine, we designed a clinical study to recruit pregnant mothers who 1) did not smoke during pregnancy, 2) smoked throughout pregnancy, or 3) used NRT during pregnancy. We found that successful RNA isolation can be achieved from neonatal tracheal aspirate samples and cell number and reagent volumes were important determinants of acceptable RNA quality and quantity. Together, these preliminary findings demonstrate a possible alteration in immune response as a result of in utero nicotine exposure and sets a groundwork for future studies in identifying mechanisms underlying the impact of developmental nicotine exposure.

Resistant bacteria are becoming more and more of a problem but treatment with antibiotics in combination may overcome and prevent resistance development. The combination of meropenem and colistin against Pseudomonas aeruginosa has been proposed as a promising treatment to increase the bactericidal effect and a synergistic effect has been proposed. A Pharmacokinetic-Parmacodynamic (PKPD) model that describes the dynamics of bacteria kill could be used to evaluate if the effects are additive or not. The model could later also be used to find optimal dosing for both of the antibiotics used alone or in combination with each other. The aim of the present study was to develop a PKPD model that describes the bactericidal activity of the two antibiotics, both in mono-therapy and in combination. The data were from in vitro static time kill-curve experiments that had been conducted on two strains of Pseudomonas aeruginosa; the wild-type (ATCC 27853) and the resistant-type (PL0603761). Resistance was observed in the experimental data and thus it had to be taken into account in the modelling. PKPD models were fitted to the bacterial counts in NONMEM with pharmacodynamic compartments for susceptible and resting bacteria. In the resting compartment the bacteria could not be killed. The bacteria moved into the resting compartment from the susceptible compartment when a certain concentration of bacteria was obtained. A pharmacokinetic compartment characterized changes in drug concentrations and the drug degradation during the experimental time was considered. Two different drug effects were tried on the susceptible bacteria, linear effect and Emax models.. The resistance development occurring during the experiments was described by two compartments where the parameter kon determined the rate of onset of resistance development. In the final model, kon was found to either be concentration-independent or dependent, depending on antibiotics and bacteria. The degree of resistance development produced an overall inhibitory effect on the drug effect. The growth rate was estimated to be lower and the EC50 to be higher for the resistant compared to the wild-type bacteria. The model was used to predict the expected time-kill curve if the effect of the two drugs are additive when combining the two drugs. The observed  bacteria kill was lower than the model predicted for the wild-type bacteria. For the resistant bacteria the assumption of additive bacteria kill for the two drugs-seemed adequate.

Multidrug resistant bacteria (MDR) have the potential to push back society to the pre-antibiotic era. Although discovered before penicillin, the inexorable rise in antibiotic resistance has revitalised interest in bacteriocins as treatments for bacterial infections. Bacteriocins are protein antibiotics principal to competition amongst pathogens and commensals, but the mechanisms by which they translocate across the Gram-negative cell envelope are poorly understood. The work presented in this thesis demonstrates how the endonuclease bacteriocin pyocin S2 (pyoS2) exploits the iron transporter FpvAI to translocate across the outer membrane (OM) of Pseudomonas aeruginosa. FpvAI is a 22-strand &beta;-barrel and virulence factor in P. aeruginosa that transports iron into the cell in the form of a small siderophore, ferripyoverdine (Fe-Pvd). Uptake of Fe-Pvd requires the proton motive force (PMF), which is transduced to the ligand-bound receptor by TonB1 and its partner proteins ExbB-ExbD in the inner membrane (IM). The crystal structure of the high affinity complex (Kd = 240 pM) formed between the N-terminal domain of pyoS2 (pyoS2^NTD) and FpvAI is presented, which shows pyoS2^NTD mimics Fe-Pvd, and induces the same conformational changes in the receptor. Fluorescently-labelled pyoS2^NTD was actively imported into P. aeruginosa PAO1 cells and this import was dependent on the PMF, TonB1 and a TonB1-box motif at the N-terminus of pyoS2^NTD. Finally, photo-activated crosslinking of stalled translocation intermediates demonstrated pyoS2^NTD translocates through the FpvAI &beta;-barrel lumen by a process analogous to that of Fe-Pvd. Following binding to FpvAI, translocation begins by the unfolding of a force-labile portion of the plug domain, opening a narrow channel through FpvAI. This enables pyoS2 to deliver its own TonB1-box to the periplasm where contact with TonB1 activates its import through the same channel, most likely as an unfolded polypeptide. Hence, this study demonstrates that bacteria possess a rudimentary protein import system that exploits energised nutrient transporters in the OM.

Biofilms are surface-bound bacterial colonies that are held together by a self-produced extracellular polymeric matrix. They are highly resistant to antibiotics and host defence mechanisms, and are known to be the cause of persistent infections. Biofilm-degrading enzymes have been shown to prevent biofilm formation, remove mature biofilm, and enhance the efficacy of antibiotics. This study investigated the antibacterial and antibiofilm actions of the commercial enzyme Pectinex Ultra SP-L (Pectinex), alone and in combination with antibiotics, on standard and clinical cultures of Staphylococcus aureus and Pseudomonas aeruginosa. The cytotoxicity of Pectinex was determined on human cell cultures in vitro. Pectinex (7.42 – 950 PGU/ml) was not bactericidal, and had no effect on the antibacterial efficacy of amoxicillin-clavulanate and ciprofloxacin in cultures of S. aureus (ATCC 12600) and P. aeruginosa (ATCC 9027), respectively. However, in clinical cultures of P. aeruginosa, Pectinex caused an 89.0% (from 1.0 to 1.89 μg/ml) and 92.8% (from 1.67 to 3.22 μg/ml) increase in the MIC and MBC of ciprofloxacin, respectively. In clinical cultures of S. aureus, both bactericidal indices of amoxicillin-clavulanate were increased by 28.0% (from 2.0 to 2.56 μg/ml). In all bacterial cultures, low concentrations of Pectinex (≤ 118.75 PGU/ml) and prolonged incubation periods (≥ 6 h) were both associated with increased viability and biofilm biomass. Over a short incubation period (≤ 6 h), higher concentrations of Pectinex (237.5 – 950 PGU/ml) effectively inhibited biofilm formation in P. aeruginosa ATCC (237.5 – 950 PGU/ml) and clinical (950 PGU/ml) strains but not in S. aureus cultures. Pectinex (237.5 – 950 PGU/ml) was cytotoxic to HeLa cells, lymphocytes and neutrophils, and induced morphological features that included shrunken rounded cells, blebs, apoptotic bodies, cytoplasmic vacuoles and cell debris. The effects at 475 and 950 PGU/ml were comparable to mitomycin C 10 μg/ml and staurosporine 1 μg/ml. Pectinex was shown to either enhance or reduce biofilm biomass and cell viability in cultures of S. aureus and P. aeruginosa. The manifested effects depended on the concentration of the enzyme, the specific bacterial species and strain, and the maturity of the biofilms. Further studies are still needed in order to determine the actions of Pectinex on other clinical pathogens. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Pharmacology / PhD / Unrestricted

M.Cur / According to data from the Centers for Disease Control and Prevention’s(CDC) National Nosocomial Infections Surveillance System of 1996, Pseudomonas aeruginosa(P. aeruginosa) can be rated as the number two cause of nosocomial pneumonia(Chen & Rudoy,2006). Nosocomial pneumonia increases hospital cost and morbidity and mortality in patients. Most of the patients in the critical care unit are immune compromised because of underlying illnesses. Antibiotics eliminates the patient’s normal flora which causes opportunity for pathogens to colonise. Indwelling procedures like endotracheal intubation cause a point of entrance for pathogens like P.aeruginosa. The endotracheal tube bypasses the normal physiological processes and inhibits the cough reflex. It is the nurse’s responsibility to remove secretion through endotracheal suctioning. During the past ten years the closed suction method was increasingly implemented to remove secretions because studies showed closed suction caused less infection than open suction. In a spesific critical care unit in a private hospital in Pretoria the nurses are of the opinion that closed suctioning does not effectively remove secretion. Patients are therefore suctioned open which can cause air contamination because the colonised ventilator circuit is opened. The following question can be asked in view of the above arguments and problem statement: Is there a difference in aircontamintion between open and closed suctioning? The aim of the study is to determine whether any difference in air contamination exists between open and closed suctioning in a spesific critical care unit in Pretoria. v A comparitive contextual design with crossover methods was used. Patients are allocated to group 1 or group 2 through random sampling. An air exstractor is used to take airsamples before, during and after suctioning. There was no significant difference in terms of air contamination for open and closed suction. This is probably because of too small a sample. The null hypothesis is accepted and that is there is no significant difference in air contamination between open and closed suction.