The Shigella species are Gram-negative enteropathogens that produce severe diarrhea, cramping, and dehydration in millions of people annually. The pathogens most commonly infect children under the age of 5 years in developing nations, where the rise of multidrug-resistant species is increasingly problematic. Despite several attempts to develop a vaccine against these pathogens, no successful vaccine has been produced. In order to achieve this goal, several characteristics of Shigella must be further elucidated. Namely, we must better understand the mechanisms Shigella employs in order to circumvent the immune response. A key way in which Shigella circumvents the innate defenses of the host is through resistance to bile salts, the principal component of bile, a substance found in the small intestine that is required for digestion. One such bile salt resistance mechanism of Shigella involves lipopolysaccharide (LPS), an extracellular structure composed of three regions: a transmembrane lipid, a polysaccharide core, and an O-antigen. LPS and LPS modifications have been implicated in bile salts resistance in other enteropathogens. Thus, the goal of this study was to build from preliminary findings to understand the role of LPS in conferring bile salts resistance in Shigella. Two Shigella flexneri mutants were studied to understand the roles of the polysaccharide core and O-antigen on bacterial growth and LPS modifications during exposure to bile salts. Growth comparisons of the mutants relative to wild type bacteria in the presence of bile salts were performed, including analysis of growth with exposure to bile salts and with varying levels of environmental glucose. Additionally, LPS was extracted from wild type and mutant bacteria grown in these conditions for analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The growth curves demonstrated that both the O-antigen and polysaccharide core mutants exhibited slow growth with exposure to bile salts, while the SDS-PAGE analyses revealed changes in the LPS profile of wild type and both LPS mutants when grown in bile salts. These data indicate that the O-antigen likely has an important role in conferring bile salts resistance and that the polysaccharide core may also facilitate resistance. This study allows us to better understand how LPS contributes to bile salts resistance in S. flexneri, which may enhance efforts to develop an effective vaccine against this pathogen. / 2023-12-10T00:00:00Z

Submitted by ISAMERY AUXILIADORA MACHADO DE SARMIENTO null (isamerymachado@yahoo.com) on 2016-08-24T00:16:43Z No. of bitstreams: 1 Archivo combinado tese completa con link Isa.pdf: 3685365 bytes, checksum: 2df7162dfd0eb21c6e675c7966fce592 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-25T20:40:51Z (GMT) No. of bitstreams: 1 machadodesarmiento_ia_dr_bot.pdf: 3685365 bytes, checksum: 2df7162dfd0eb21c6e675c7966fce592 (MD5) / Made available in DSpace on 2016-08-25T20:40:51Z (GMT). No. of bitstreams: 1 machadodesarmiento_ia_dr_bot.pdf: 3685365 bytes, checksum: 2df7162dfd0eb21c6e675c7966fce592 (MD5) Previous issue date: 2016-06-30 / As doenças de transmissão alimentar (DTA) por bactérias constituem um problema de saúde pública no mundo. O objetivo do trabalho foi identificar por métodos microbiológicos e moleculares a presença de Campylobacter spp., Salmonella spp. e Escherichia coli em frangos de Botucatu-SP, Brasil. Sessenta (60) amostras de frangos resfriados de diferentes marcas foram coletadas aleatoriamente em diferentes estabelecimentos localizados tanto na periferia, quanto no centro da cidade de Botucatu, SP, no período de julho a outubro de 2015. Posteriormente, Campylobacter spp, Salmonella spp e E. coli foram isolados através de métodos microbiológicos convencionais. A confirmação das bactérias isoladas se realizou por provas bioquímicas e PCR. Além disso, as provas para determinar a resistência antimicrobiana e a existência de gene integron classe 1 foram realizadas. Os resultados demostraram a presença de três das principais bactérias que causam doenças de origem alimentar em carcaças de frango provenientes de supermercados e casas de carne de Botucatu. A prevalência de Campylobacter, Salmonella, e E. coli foi 38,3%, 13,3% e 60%, respectivamente. A resistência antimicrobiana dos isolados foi elevada e presença do gene íntegron classe 1 foi identificada em alguns agentes patogénicos. / The transmission of Foodborne Diseases (FBD) by bacteria constitutes a public health problem in the world. The objective of this study was to identify by microbiological and molecular methods the presence of Campylobacter spp., Salmonella spp. and Escherichia coli in broiler chickens at Botucatu, SP, Brazil. Sixty samples of chilled chickens of different brands were randomly collected from supermarkets and meat houses located in both the periphery and the center of the city, in the period from July to October 2015. Later, Campylobacter spp., Salmonella spp., and E. coli were isolated by conventional microbiological methods. Confirmation of the isolated bacteria was performed by biochemical and PCR tests. In addition, proofs to determine antimicrobial resistance and the existence of class 1 integron gene were carried out. Results confirmed the presence of the three assessed bacteria in poultry carcasses from selected establishments. The prevalence was 38.3%, 13.3% and 60% to Campylobacter, Salmonella and E. coli, respectively. Antimicrobial resistance of the isolates was high and the presence of integron class 1 gene has been identified in some pathogens.

Orientador: Noeme Sousa Rocha / Resumo: As doenças de transmissão alimentar (DTA) por bactérias constituem um problema de saúde pública no mundo. O objetivo do trabalho foi identificar por métodos microbiológicos e moleculares a presença de Campylobacter spp., Salmonella spp. e Escherichia coli em frangos de Botucatu-SP, Brasil. Sessenta (60) amostras de frangos resfriados de diferentes marcas foram coletadas aleatoriamente em diferentes estabelecimentos localizados tanto na periferia, quanto no centro da cidade de Botucatu, SP, no período de julho a outubro de 2015. Posteriormente, Campylobacter spp, Salmonella spp e E. coli foram isolados através de métodos microbiológicos convencionais. A confirmação das bactérias isoladas se realizou por provas bioquímicas e PCR. Além disso, as provas para determinar a resistência antimicrobiana e a existência de gene integron classe 1 foram realizadas. Os resultados demostraram a presença de três das principais bactérias que causam doenças de origem alimentar em carcaças de frango provenientes de supermercados e casas de carne de Botucatu. A prevalência de Campylobacter, Salmonella, e E. coli foi 38,3%, 13,3% e 60%, respectivamente. A resistência antimicrobiana dos isolados foi elevada e presença do gene íntegron classe 1 foi identificada em alguns agentes patogénicos. / Abstract: The transmission of Foodborne Diseases (FBD) by bacteria constitutes a public health problem in the world. The objective of this study was to identify by microbiological and molecular methods the presence of Campylobacter spp., Salmonella spp. and Escherichia coli in broiler chickens at Botucatu, SP, Brazil. Sixty samples of chilled chickens of different brands were randomly collected from supermarkets and meat houses located in both the periphery and the center of the city, in the period from July to October 2015. Later, Campylobacter spp., Salmonella spp., and E. coli were isolated by conventional microbiological methods. Confirmation of the isolated bacteria was performed by biochemical and PCR tests. In addition, proofs to determine antimicrobial resistance and the existence of class 1 integron gene were carried out. Results confirmed the presence of the three assessed bacteria in poultry carcasses from selected establishments. The prevalence was 38.3%, 13.3% and 60% to Campylobacter, Salmonella and E. coli, respectively. Antimicrobial resistance of the isolates was high and the presence of integron class 1 gene has been identified in some pathogens. / Doutor