Orientador: Tomomasa Yano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T11:52:53Z (GMT). No. of bitstreams: 1 Bernedo-Navarro_RobertAlvin_D.pdf: 2646572 bytes, checksum: 02b50bc28d5be6cddb78abfe621a047c (MD5) Previous issue date: 2013 / Resumo: Escherichia coli produtora de toxina de Shiga (STEC) é um grupo de importantes patógenos para humanos. Essas bactérias são relacionadas a várias doenças, como por exemplo, Síndrome Urêmica Hemolítica e produzem potentes toxinas denominadas toxinas de Shiga. Essas toxinas, tanto Stx1 quanto Stx2, compartilham um receptor celular comum, a globotriaosilceramida (Gb3) e exibem a mesma atividade biológica intracelular. O desenvolvimento de novos agentes neutralizantes dos danos induzidos por Stx pode representar uma estratégia promissora para o tratamento das doenças causadas por STEC em humanos. No presente estudo, nós desenvolvemos peptídeos sintéticos que exibem atividade neutralizante contra a citotoxicidade induzida por Stx tanto in vitro quanto in vivo e, além disso, que se ligam eficientemente ao receptor Gb3. O peptídeo P12-26 compete eficientemente com Stx2 para a ligação ao Gb3 in vitro. Além disso, os peptídeos PC7-12, P12-26 e PC7-30 inibiram a citotoxicidade de Stx1 e Stx2 em células Vero. Nós observamos que o peptídeo PC7-30 em forma de loop e o peptídeo P12-26 que é linear produziram as maiores porcentagens de inibição de Stx1 e Stx2 em células Vero, respectivamente. No entanto, o peptídeo P12-26 não inibiu a letalidade em camundongos, enquanto que o peptídeo PC7-30 inibiu a letalidade causada pela toxina Stx1. Nossos resultados indicam que os peptídeos P12-26 e PC7-30 são candidatos promissores para o desenvolvimento de agentes terapêuticos contra as doenças em seres humanos causadas por STEC / Abstract: Shiga toxin-producing Escherichia coli strains are important pathogens for humans. These bacteria are linked with severe diseases such as hemolytic uremic syndrome and produce potent known as Shiga toxins. These toxins, Stx1 and Stx2, share a common cellular receptor called globotriaosylceramide (Gb3) and exhibit the same intracellular biological activity. The development of new neutralizing agents for Stx-induced damage may represent a promising strategy for the treatment of diseases caused by STEC infections. In this study, we developed synthetic peptides that exhibit neutralizing activity against Stxinduced cytotoxicity both in vitro and in vivo and that bind efficiently to the Gb3 receptor. The peptide P12-26 competed efficiently with Stx2 for binding to Gb3 in vitro. Moreover, the peptides PC7-12, P12-26 and PC7-30 inhibited the cytotoxicity of Stx1 and Stx2 in Vero cells. We observed that the loop-constrained peptide PC7-30 and linear peptide P12-26 produced higher percentages of inhibition of Stx1 and Stx2 in Vero cells, respectively. However, the peptide P12-26 did not inhibit lethality in mice, whereas the loopconstrained peptide PC7-30 inhibited the lethality caused by Stx1. Our results indicate that the peptides P12-26 and PC7-30 are promising candidates for the development of therapeutic agents against diseases caused by STEC in humans / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Low water activity foods (LWAF), specifically nuts and dried fruits, have been generally considered safe because they do not support the growth of foodborne pathogens. However, many pathogens have been noted to survive in LWAF for considerable periods of time, and a number of recent outbreaks and recalls have implicated various types of nuts and dried fruits. The Food Safety Modernization Act requires food processors to develop preventive control plans that make ready-to-eat LWAF safer for consumers. The presented research was designed to investigate several aspects of LWAF safety by evaluating a steam process as a strategy to remove pathogen contamination from LWAF, modeling the inactivation of such treatments, and studying the thermal resistances of two E. coli strains in low-water activity solutions. Low-temperature, vacuum-assisted steam (vacuum-steam) was evaluated as a potential intervention and preventive control to remove pathogens from the surface of LWAF without using high-heat treatments that could damage product quality. The presented work examined the efficacy of vacuum-steam (<85°C) as a means to decontaminate the surface of whole macadamia nuts, dried apricot halves, and raisins from Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) contamination. The low-temperature steam treatments successfully reduced all pathogens by >4 log CFU/g from the surfaces of the foods. Additionally, Pediococcus acidilactici, proved to be a surrogate organism for these pathogens and could be used to challenge and validate similar treatments within processing plants. The data were fit to models, which showed that food type significantly impacted the fit, with the Weibull model best describing bacterial inactivation kinetics on raisins and macadamia nuts, and the Gompertz model best describing reductions on the apricot halves. The models were challenged for validation of their abilities to predict times required for 3-log reductions using internal and external datasets, determining the usefulness to industry members who wish to design similar thermal treatments for LWAF. Comparing predicted values from internally constructed models to observed values generated from external data, models were shown to be limited in scope and application and could only be applied to pathogen inactivation on different LWAF or thermal processes in certain circumstances. First-order and Weibull model predictions of bacterial reductions on dried apricots had varied success in predicting times for 3-log reductions on other thermally treated LWAF. However, the models of bacterial reductions on thermally treated macadamia nuts frequently overestimated the times required for 3-log bacterial reductions for other LWAF. In an effort to understand the effect that reduced water activity has specifically on STEC, two strains were investigated for induced thermal resistance due to osmotic stress. Thermal resistance of STEC strains (O121:H19 and O157:H7) were evaluated on the basis of strain variation, culture preparation, and water activity (D- and z-values). At the lowest treatment temperature (56°C), O121 displayed greater heat resistance than O157, and the broth-grown samples exhibited greater heat resistance than the lawn-grown cells, but significant differences were not observed at higher temperatures. Samples in reduced-water activity solutions displayed reduced thermal resistance at 56°C, but the z-values were 29-43% higher than those of high-water activity samples. While water activity has been shown to impact thermal resistance of pathogens, comparisons of STEC thermal resistance according to the D- and z-values revealed that other factors also play roles in pathogen thermal resistance on LWAF. Results from the collection of experiments conclude that efficacy of thermal treatments is impacted by the physiological state of the cells, stress experienced in the food matrix, and characteristics of the food, including water activity and composition. / Doctor of Philosophy / Consumers expect foods they purchase to be safe to consume by themselves and family members, particularly those that are ready-to-eat with no additional cooking requirements. Many of these foods are low-water activity foods (LWAF), like nuts and dried fruits, with very little water content that could be used by bacteria. These foods may be preferred snack foods due to their affordability, long shelf lives, and health benefits over other types of snack foods. Until recently, LWAF were generally considered safe because they do not support the growth of foodborne pathogens due to the lack of moisture or water within the food. However, a number of recent outbreaks related to various types of nuts and dried fruits have proven that many pathogens can survive in dried foods, even if not actively growing, for considerable amounts of time. Designed to address these types of food safety issues, the Food Safety Modernization Act recognizes risks associated with foods and responded with regulations requiring food processors to take steps to make ready-to-eat LWAF, like nuts and dried fruits, safer for consumers. A popular strategy is to treat foods with heat to destroy pathogens, however the quality attributes of some nuts and dried fruits could be damaged by high-heat treatments like roasting. An alternative process uses a vacuum to form steam at lower temperatures, allowing for efficient heat transfer through water droplets to the surface of the foods, thus causing less damage to the foods without introducing too much moisture. This research evaluated how this process could be used by food processors to remove harmful bacteria from the surfaces of whole macadamia nuts, dried apricot halves, and raisins. Results indicated that the low-temperature steam treatments successfully reduced Salmonella, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) by >4 log CFU/g (>99.99%) from the surfaces of the foods. Additionally, a nonpathogenic lactic acid bacterium, Pediococcus acidilactici, exhibited similar or greater heat tolerance, which would allow food processors to use it as a substitute, or surrogate, for in-plant studies without introducing harmful bacteria into the food processing environment. Mathematical models were used to describe the trends of bacterial death due to the steam treatments, and the results indicated that the type of food significantly impacted the reduction of bacteria. The models were tested using additional data collected within our own laboratory, as well as others. Results indicated that some of the models could be used as predictors of bacterial death for similar LWAF but can only be applied with caution and consideration for the type of food and process. Additionally, two different E. coli strains associated with outbreaks (O121:H19 and O157:H7) were investigated to understand impacts of strain variation, growth method, and water activity on thermal resistance. Some differences in heat resistance were observed between the strains and between the growth methods. Additionally, the reduced water activity seemed to decrease the bacteria's ability to withstand some heat treatments. Overall, thermal resistance studies indicated that several factors, in addition to water activity, impact pathogens' development of resistance to heat treatments. The experiments' results show that there are complex relationships between bacteria and the food they inhabit. Food processors must consider these relationships in order to design the best thermal processes to make LWAF safe for consumers.