Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T06:30:19Z (GMT). No. of bitstreams: 1 Figueiredo_CamilaAlexandrinaVianade_D.pdf: 9514972 bytes, checksum: 602b147a415d93d261e4b82f68a9f117 (MD5) Previous issue date: 2005 / Resumo: Esse trabalho investiga o efeito protetor do tratamento com diferentes doses (50, 100 e 250 mg/Kg) do extrato padronizado de Chá Verde (GTE) em camundongos infectados com Listeria monocytogenes (LM). A eficácia terapêutica foi avaliada para uma dose letal de LM por sete dias consecutivos com diferentes doses de GTE. A proteção significativa do extrato foi verificada durante a fase aguda da infecção e um mecanismo de ação foi proposto a partir da análise dos efeitos do GTE sobre o crescimento e a diferenciação de progenitores hematopoéticos para granulócitos e macrófagos (CFU-GM) da medula óssea e do baço, sobre a atividade estimuladora de colônias (CSA) do soro, sobre a atividade lítica de células NK, sobre a proliferação de linfócitos e sobre a produção de citocinas de padrão Thl (IFN-y) / Th2 (IL-10) em animais normais e infectados com LM. Os resultados demonstraram que a resistência dos animais infectados com uma dose letal de LM tratados com GTE está relacionada com a modulação da mielossupressão e diminuição da hematopoese extramedular produzidas pela infecção. Também foi verificado um aumento da atividade lítica de células NK, da capacidade proliferativa de linfócitos e da polarização da resposta imunológica para Thl. O GTE não apresentou toxicidade sobre o estroma medular e estimulou a geração de progenitores para granulócitos-macrófagos em sistema de cultura líquida de longa duração (LTBMC). O mecanismo de ação da atividade imunomoduladora do GTE ainda não está completamente definido. No entanto, os mecanismos de ação apresentados neste trabalho são de fundamental importância para a atividade antibacteriana in vivo do GTE / Abstract: In the present study we investigated the protective effects of the oral administration of Green tea (GTE) (50, 100 and 250mg/Kg) in mice infected with Listeria monocytogenes (LM). We demonstrated that GTE protects mice from a lethal dose of LM when administered prophylactically for seven consecutive days. To determine the mechanism of action of GTE, we examined the effect of GTE on the number of granulocyte and macrophage colony-forming units (CFU-GM) from bone marrow and spleen, on the colony-stimulating activity (CSA) of serum, on natural killer (NK) cell activity, on lymphocyte proliferation and on Thl/Th2-based cytokine production in normal and LM infected mice. Myelosuppression and extramedular hematopoiesis/splenomegaly were the major features of LM infection, in association with NK cell activation and alteration of lymphocyte proliferation. The resistance of mice infected with a lethal dose of LM but treated prophylactically with 100 and 250mg/Kg of GTE could be related to the modulation of myelosuppression and extramedular hematopoesis induced by the infection, and to an increase in NK cell activity, lymphocyte proliferation and polarization of the Thl-based immune response. The GTE extract was not toxic to either the stromal cell layer or to GM progenitors generated from long-term bone marrow culture (LTBMC). In this way, the mechanism of action through GTE exerts its pharmacological effect is not completely elucidated, although the parameters mentioned here are essential for its antibacterial effect in vivo / Doutorado / Doutor em Farmacologia

Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T02:48:18Z (GMT). No. of bitstreams: 1 Rocha_JoaoAndersonKeiti_M.PDF: 6430386 bytes, checksum: 2a5815761af437dc46567523865e2440 (MD5) Previous issue date: 2004 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Tecnologia de Alimentos / Mestre em Tecnologia de Alimentos

Memoria para optar al título de Ingeniero en Alimentos / No autorizada por el autor para ser publicada a texto completo en el Portal de Tesis Electrónicas / En los mercados de muchos países, incluido Chile, son ofrecidos una amplia variedad de vegetales mínimamente procesados o de cuarta gama. Sin embargo, estudios recientes han demostrado que algunos de estos productos podrían ser un riesgo potencial para la salud pública, debido a contaminación microbiológica. Como un tratamiento factible para resolver este problema, se propone la irradiación de esta clase de productos vegetales. Fue determinada la contaminación microbiológica natural de dos ensaladas surtidas mínimamente procesadas, Toscana y Cuatro Estaciones, controlando el recuento de aerobios mesófilos (RAM), el recuento de enterobacterias y la presencia o ausencia de Listeria spp. en 25 g. Se determinó el valor D10 inoculando ambas ensaladas con Listeria innocua, como microorganismo marcador para una posible contaminación con Listeria monocytogenes. Se determinó el efecto de la irradiación de vegetales con dosis de 5D10 en la microflora inicial y en la evaluación de posibles cambios en la calidad microbiológica y sensorial durante 7 días de almacenamiento refrigerados. El efecto de la calidad sensorial fue evaluada por un panel de 10 jueces entrenados, a través de un test triangular a 0 y 7 días, en orden de determinar posibles diferencias significativas entre la muestra no irradiada y la muestra irradiada. Se llevó a cabo un test para descriptores de calidad a 0, 3 y 7 días, para comparar las ensaladas irradiadas con el control no irradiado. Los atributos sensoriales evaluados fueron apariencia, color, olor, sabor, gusto dulzón, gusto amargo, textura y calidad total. Los niveles de la microflora inicial encontrados para RAM están entre 106 ufc/g y 108 ufc/g en ambas ensaladas, Toscana y Cuatro Estaciones. El nivel inicial de enterobacterias fue entre 104 ufc/g y 107 ufc/g en ensalada Toscana y 105 ufc/g y 107 ufc/g en ensalada Cuatro Estaciones. En ambos tipos de ensaladas no se detectó presencia de Listeria spp. en 25 g. Los valores de D10 calculados fueron 0,19 kGy y 0,21 kGy para la ensalada Toscana y Cuatro Estaciones, respectivamente. La dosis de 5D10 calculada, fue 1,00 kGy, considerando la media de los valores de D10. Después de irradiar la ensalada Toscana con 1,00 kGy, se observó una reducción de 3,46 y 3,5 ciclos logarítmicos en RAM y enterobacterias, respectivamente. En la ensalada Cuatro Estaciones, se presentó inicialmente en RAM y en enterobacterias, una reducción en 3,5 y 4,0 ciclos logarítmicos, respectivamente, después de la irradiación. No se detectó presencia de Listeria spp en 25 g. Al final del almacenamiento de las muestras refrigeradas, no irradiadas el aumento de el RAM fue 2,8 y 1,6 ciclos logarítmicos para ensalada Toscana y Cuatro Estaciones, respectivamente. Fue observado un aumento mayor en la enterobacteria, alcanzando al día 7 valores de 3,0 y 2,7 ciclos logarítmicos en ensalada Toscana y Cuatro Estaciones, respectivamente. En las muestras irradiadas, se observó, sólo un pequeño cambio en el RAM y enterobacteria. Los resultados del test triangular, no mostraron diferencias significativas a 0 y 7 días entre las ensaladas no irradiadas y las irradiadas. Durante el almacenamiento refrigerado, sólo en algunos parámetros se observó diferencias significativas entre los días. El promedio de los resultados obtenidos para la ensalada Cuatro Estaciones, fue similar al valor central (5,0) correspondiente al control no irradiado. De acuerdo a los resultados del estudio, la irradiación es un tratamiento factible para ensaladas surtidas mínimamente procesadas. Cuando se irradia con 1,00 kGy, se obtiene una reducción de la microflora inicial, sin afectar la calidad sensorial

Determina la presencia de bacterias patógenas y en particular de Listeria monocytogenes en las ensaladas de pollerías del distrito de Los Olivos, Lima, Perú. Utiliza muestras de ensalada obtenidas de 83 pollerías. El análisis microbiológico se realiza en base a la Norma Técnica Peruana Nº 071 (MINSA/DIGESA, 2003). Utiliza el método de ICMSF- 2000 para el recuento de bacterias aerobias mesófilas. Emplea los métodos de FDA-BAM para la numeración de Coliformes totales y Escherichia coli, así como el recuento de Staphylococcus aureus, detección de Listeria monocytogenes y Salmonella sp. Encuentra que el 96,39% (80) de las muestras de ensaladas eran no aptas para el consumo humano, el 84,34% (70) presenta más de 105 UFC/g de Aerobios Mesófilos, el 95,18% (79) más de 102 NMP/g de Coliformes totales, el 24,10% (20) más de 10 NMP/g de E. coli, y el 21,69% (18) más de 10 UFC/g de S. aureus. Respecto a las bacterias patógenas, el 7,23% (06) presentó Listeria spp., el 3,61% (03) Listeria monocytogenes y el 14,46% (12) Salmonella sp. Los resultados obtenidos demuestran que un elevado porcentaje de ensaladas frescas que se expenden en las pollerías del distrito Los Olivos es no apto para el consumo humano, y un porcentaje significativo está contaminado con microorganismos patógenos, constituyendo un serio riesgo para la salud de los comensales. / Tesis

Listeria monocytogenes (L. monocytogenes) are Gram-positive, facultatively anaerobic and intracellular bacilli, occupying a wide range of ecological niches and are responsible for a number of serious infections in man. Primarily transmitted to humans through contaminated food stocks, L. monocytogenes invade mammalian cells in a phagosome, escaping and growing in the cell cytoplasm. Currently, there is a great deal of information about pathogenesis of L. monocytogenes, however, much less is known about the physiology of the bacteria. In particular, very little is known about the physiology during intracellular growth and even less about host cell physiology and changes in response to infection. The focus of this research was to address these issues using a multidisciplinary approach, utilising multiple biological techniques. The catabolic metabolism of L.monocytogenes was elucidated using mutagenesis and protein purification studies. The results are not completely conclusive; however, it was shown that unlike in Escherichia coli, L.moncytogenes may not be dependent on fermentation enzymes Ldh and Pflb during anaerobic growth. Instead anaerobic respiration is hypothesised, utilising a putative fumarate reductase with fumarate as a terminal electron acceptor. The putative fumarate reductase gene was purified and confirmed to have enzymatic activity.External and internal metabolism of HeLa cells, and the effect of L.monocytogenes infection was elucidated by mass spectrometry. The external metabolomic studies proved inconclusive. The internal metabolomic studies show that a number of key amino acids are being sequestered by L.monocytogenes during the course of an infection. Also, the studies show that a large number of carbon compounds are being sequestered by L.monocytogenes, pointing to a complex carbon metabolism for L.monocytogenes during intracellular growth. A targeted analysis of the nitrogen metabolism of L.monocytogenes has shown that L.monocytogenes may utilise a number of nitrogen compounds with glutamine and glutamate being particularly important. The ability to synthesise glutamine de novo is shown to be essential for normal intracellular growth.

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

The first aim of this study was to determine the effect of irrigation intervals on the survival of L. monocytogenes on spray irrigated broccoli under field trial conditions, and subsequent survival of the pathogen on broccoli during postharvest processing procedures. The nonpathogenic L. innocua was used as surrogate organism to L. monocytogenes. Broccoli in the field was treated with irrigation water inoculated with L. innocua, during intervals over a period of five weeks and the growth and survival of the organism was monitored weekly. L. innocua numbers remained similar over intervals that received consecutive inoculations and L. innocua numbers decreased by at least 2.3 log cfu/g after inoculation ceased, which showed an inoculation effect and that time had an influence on organism survival. Cessation of irrigation before harvest was found to effectively reduce pathogen contamination levels on the crop, whilst repeated irrigation with contaminated water contributes to maintenance of L. innocua as well as elevated total microbial counts on the broccoli. A lack of correlation between the L. innocua counts and the recorded environmental temperatures in the field, including temperature and relative humidity, suggested that survival is not solely dependent on and influenced by, nor can it be predicted by these parameters. It was found that the presence of high levels of contamination (with, in this case L. innocua) in irrigation water used for vegetable crops, can be associated with an increased microbial population on the crop surface. Secondly, the effect of processing on organism survival post-harvest was assessed. Washing with water caused a 1 log reduction of L. innocua, whilst washing with 200 ppm chlorinated water facilitated a further 1 log reduction. Cooking reduced L. innocua numbers on broccoli by an average of 1.1 log units and aerobic plate counts by between 1 and 2 log units. A combined treatment of washing with chlorine, storage in MAP (5% CO2, 5% O2) for two days at 4°C and final microwave heating resulted in the lowest pathogen numbers, causing a 5.13 log cfu/g log reduction. Therefore, even though chlorine isprocessing, it does not suffice alone to eliminate pathogens (with L. innocua being representative of L. monocytogenes) from vegetables, just as MAP storage is only effective as part of a hurdle procedure. Cooking is essential in destroying L. innocua present on broccoli and to ensure vegetables that are safe for consumption in terms of pathogenic exposure. With this knowledge on the behaviour of L. monocytogenes on broccoli, the risk associated with the application of contaminated irrigation water to fresh produce can be better understood and the hazard managed. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Food Science / unrestricted

Não consta resumo na publicação. / Abstract not available.

Dispersion is a natural part of a biofilm life cycle in many bacterial species. Dispersion occurs when bacteria revert from a stationary, sessile state to a free-swimming, planktonic state and are freed from a biofilm. Bacterial biofilms consist of proteins, polysaccharides, and extracellular DNA that together make up the extracellular polymeric substances. Surrounded by this mucus-like substance, sessile cells can be extremely difficult to eradicate as compared to the planktonic form of Listeria monocytogenes. Biofilms are robust due to increased surface adherence, inhibition of diffusion of harmful compounds, and increased genetic diversity that exists within a biofilm. As a result, traditional biofilm removal methods are often inadequate; and a novel method for the eradication of Listeria monocytogenes biofilms is needed. Here it is shown that two known biofilm dispersal agents, nitric oxide and cis-2-Decenoic acid, do not induce dispersion in Listeria monocytogenes strain LM23. Nitric oxide and cis-2-Decenoic acid do not influence planktonic cell numbers or biofilm biomass. Ten carbohydrates were screened for their influence on biofilm biomass for use in investigation into natural biofilm dispersion in Listeria monocytogenes strain LM23. Carbohydrate source can significantly increase or decrease biofilm biomass as compared to glucose. Natural biofilm dispersion in Listeria monocytogenes remains inconclusive, yet warrants further investigation. Changes in planktonic cells numbers, sessile cell numbers, and biofilm biomass were tracked under static growth conditions, and suggested a possible dispersion event. However, treatment of biofilms with spent media and observation using scanning electron microscopy did not clarify the results obtained. This research deems the nitric oxide donors, molsidomine (N- (ethoxycarbonyl)-3-(4-morpholinyl)-sydnone imine) and MAHMA NONOate (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), as well as cis-2-Decenoic acid as ineffective in inducing biofilm dispersion. It also brings about new research questions into natural biofilm dispersion in Listeria monocytogenes.

A single tube detection kit was designed as a rapid, easy-to use and reliable test to detect Listeria spp.. Various food samples (vegetables and raw catfish fillets) were used in order to validate the performance of the detection kit. L. grayi was detected in one ready-to-eat (RTE) vegetables with the detection kit while no Listeria spp. was detected using the modified FDA-BAM method. In addition, both the detection kit and modified FDA-BAM method indicated that twelve catfish fillets were Listeria positive. The detection kit had 100% sensitivity and specificity in less detection time (24 h) than the modified FDA-BAM method (60% specificity, >72 h). There was no difference (P<0.05) between the kit and the modified FDA-BAM method on MPN for Listeria spp.