Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-29Bitstream added on 2014-06-13T20:44:10Z : No. of bitstreams: 1 pigatto_cp_dr_jabo.pdf: 841661 bytes, checksum: 288fcbc74e176eac0befc1827d1bc15e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Escherichia coli produtoras de toxina Shiga (STEC) são reconhecidas como agentes causadores de infecções em humanos em todo o mundo. O principal reservatório é o bovino. Neste trabalho, cepas de STEC previamente isoladas de fezes bovinas foram caracterizadas usando PCR multiplex para determinar os genes de virulência (stx1, stx2, ehxA, eaeA e saa), soroaglutinação passiva reversa em látex (RPLA-VTEC screen) para avaliar a expressão da toxina Shiga, PCR-RFLP e sequenciamento para obter os subtipos e a variabilidade dos genes stx2, respectivamente. Foram determinados também os sorotipos, o perfil de sensibilidade e a viabilidade das cepas de STEC em queijo minas frescal. A freqüência de STEC nas amostras de fezes bovinas foi de 37%. Foram encontrados trinta e quatro sorotipos de STEC sendo os mais freqüentes o ONT:H7 (10%), O22:H8, O22:H16 e ONT:H21 (7% cada). Onze sorotipos encontrados não tinham sido associados com STEC até o momento. A maioria das STEC (96%) foi susceptível a todos os antimicrobianos testados. A produção de toxina Shiga determinada pelo ensaio RPLA foi de 89%. Os marcadores de virulência foram encontrados em 11 diferentes combinações, a mais freqüente foi stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% cada). Foram detectados 8 subtipos de stx2: stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc e stx2O48. Os genes que apresentaram maior freqüência foram: stx2 e stx2c. As seqüências parciais obtidas sugerem a presença de elevada variabilidade nos genes do tipo stx2 nas STEC analisadas. A viabilidade de STEC não-O157 em queijo minas revelou que diferentes cepas de STEC podem ser detectadas nos queijos após 10 dias de armazenamento sob refrigeração. Os dados encontrados neste trabalho sugerem isolados com alto potencial de patogenicidade oferecendo risco de desencadear graves infecções à população. / Shiga toxin-producing Escherichia coli (STEC) is recognize worldwide as an organism capable to cause human diseases. Cattle are the main source of STEC. In this research, STEC strains previously isolated were analyzed using multiplex-PCR for virulence genes, the RPLA assay to detect the Shiga toxin production and serotyping. PCR-RFLP and nucleotide sequence were analyzed to detect stx2 genes subtypes and their variability. Moreover tests for antimicrobial susceptibility and the vialbility of STEC in Minas Frescal cheese were done. The frequency of cattle shedding STEC was 37%. Thirty-four serotypes of STEC were found, the most frequent being ONT:H7 (10%), O22:H8, O22:H16 and ONT:H21 (7% each). Eleven serotypes had not been associate with STEC until the moment. Most of the strains (96%) were susceptible to all antimicrobial agents tested. Production of Shiga toxin by the RPLA assay was detected in most (89%) of the STEC strains. The frequency of virulence markers were found in 11 diferent combinations: stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% each). Eigth stx2 subtypes were detect (stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc; stx2O48) and the most frequent were: stx2; stx2c. The partial sequences of stx2 genes suggested a high variability of stx2 types in the STEC analyzed. The STEC viability in cheese could be detected after 10 days of storage under refrigeration. The results found in this work suggest strains with high potential of pathogenicity offering risk to lead serious infections to the population.

Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.

Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.

Resumo: Escherichia coli produtoras de toxina Shiga (STEC) são reconhecidas como agentes causadores de infecções em humanos em todo o mundo. O principal reservatório é o bovino. Neste trabalho, cepas de STEC previamente isoladas de fezes bovinas foram caracterizadas usando PCR multiplex para determinar os genes de virulência (stx1, stx2, ehxA, eaeA e saa), soroaglutinação passiva reversa em látex (RPLA-VTEC screen) para avaliar a expressão da toxina Shiga, PCR-RFLP e sequenciamento para obter os subtipos e a variabilidade dos genes stx2, respectivamente. Foram determinados também os sorotipos, o perfil de sensibilidade e a viabilidade das cepas de STEC em queijo minas frescal. A freqüência de STEC nas amostras de fezes bovinas foi de 37%. Foram encontrados trinta e quatro sorotipos de STEC sendo os mais freqüentes o ONT:H7 (10%), O22:H8, O22:H16 e ONT:H21 (7% cada). Onze sorotipos encontrados não tinham sido associados com STEC até o momento. A maioria das STEC (96%) foi susceptível a todos os antimicrobianos testados. A produção de toxina Shiga determinada pelo ensaio RPLA foi de 89%. Os marcadores de virulência foram encontrados em 11 diferentes combinações, a mais freqüente foi stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% cada). Foram detectados 8 subtipos de stx2: stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc e stx2O48. Os genes que apresentaram maior freqüência foram: stx2 e stx2c. As seqüências parciais obtidas sugerem a presença de elevada variabilidade nos genes do tipo stx2 nas STEC analisadas. A viabilidade de STEC não-O157 em queijo minas revelou que diferentes cepas de STEC podem ser detectadas nos queijos após 10 dias de armazenamento sob refrigeração. Os dados encontrados neste trabalho sugerem isolados com alto potencial de patogenicidade oferecendo risco de desencadear graves infecções à população. / Abstract: Shiga toxin-producing Escherichia coli (STEC) is recognize worldwide as an organism capable to cause human diseases. Cattle are the main source of STEC. In this research, STEC strains previously isolated were analyzed using multiplex-PCR for virulence genes, the RPLA assay to detect the Shiga toxin production and serotyping. PCR-RFLP and nucleotide sequence were analyzed to detect stx2 genes subtypes and their variability. Moreover tests for antimicrobial susceptibility and the vialbility of STEC in Minas Frescal cheese were done. The frequency of cattle shedding STEC was 37%. Thirty-four serotypes of STEC were found, the most frequent being ONT:H7 (10%), O22:H8, O22:H16 and ONT:H21 (7% each). Eleven serotypes had not been associate with STEC until the moment. Most of the strains (96%) were susceptible to all antimicrobial agents tested. Production of Shiga toxin by the RPLA assay was detected in most (89%) of the STEC strains. The frequency of virulence markers were found in 11 diferent combinations: stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% each). Eigth stx2 subtypes were detect (stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc; stx2O48) and the most frequent were: stx2; stx2c. The partial sequences of stx2 genes suggested a high variability of stx2 types in the STEC analyzed. The STEC viability in cheese could be detected after 10 days of storage under refrigeration. The results found in this work suggest strains with high potential of pathogenicity offering risk to lead serious infections to the population. / Orientador: Ruben Pablo Schocken-Iturrino / Coorientador: José Moacir Marin / Coorientadora: Cyntia Maria Telles Fadel-Pichetch / Banca: Luiz Augusto do Amaral / Banca: Hélio José Montassier / Banca: Alessandra Aparecida Medeiros / Banca: Elaine Cristina Pereira de Martins / Doutor

Memoria para optar al Título Profesional de Médico Veterinario / Escherichia coli productora de toxina Shiga (STEC) es un patógeno zoonótico emergente, que puede causar enfermedades severas en humanos, como colitis hemorrágica (CH) y síndrome hemolítico urémico (SHU). Se han reportado infecciones con STEC en personas producto del contacto directo con animales de compañía, y de estos se han aislado cepas de este patógeno con virulotipo asociado a enfermedad severa. Sin embargo, en Chile a la fecha no se encuentra información disponible al respecto. Por esta razón, este proyecto busca aportar con información sobre la caracterización de cepas de STEC circulantes en parte de la población canina y felina nacional. Para cumplir con lo anterior, se recolectaron torulados rectales de 300 perros y 300 gatos mascotas, entre octubre y diciembre del año 2018. Se realizaron dos rondas de cultivo, en caldo tripticasa de soya y en agar MacConkey; y mediante PCR se buscó determinar la presencia de los genes stx1 y stx2 con el fin de identificar cepas de STEC en las muestras obtenidas. Además, se contempló la detección de los genes eae, lpf, saa y hlyA en las cepas de STEC aisladas. Sin embargo, con esta metodología no fue posible el aislamiento de cepas de STEC en las muestras procesadas. En vista de los resultados obtenidos podemos concluir que los perros y gatos con dueño en algunas comunas de la Región Metropolitana representarían un riesgo bajo en la transmisión de cepas de STEC con determinantes de virulencia asociados a CH y SHU en las personas. Sin embargo, es necesaria la realización de otros estudios, con un mayor tamaño muestreal y que analicen las distintas variables que puedan afectar la colonización de STEC en estos animales. / Shiga toxin-producing Escherichia coli (STEC) is an emerging zoonotic pathogen, which can cause severe diseases in humans, such as hemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Infections have been reported in people as a result of direct contact with companion animals, and from these STEC strains with virulotype associated with severe disease have been isolated. However, to date in Chile there is no information available about it. For this reason, this project seeks to provide information on the characterization of circulating STEC strains in part of the national dog and cat population. In order to comply with the above, rectal swabs from 300 dogs and 300 pet cats were collected between October and December of 2018. Two rounds of culture were carried out in tripticase soy broth and on MacConkey agar; and by means of PCR, the presence of the stx1 and stx2 genes was attempted in order to identify STEC strains in the samples obtained. In addition, the detection of eae, lpf, saa and hlyA genes in the isolated STEC strains was contemplated. However, with this methodology it was not possible to isolate STEC strains in the processed samples. In view of the results obtained, we can conclude that household dogs and cats in some communes of the Metropolitan Region would represent a low risk in the transmission of STEC strains with virulence determinants associated with HC and HUS in people. However, it is necessary to carry out further studies, with a larger sample size and that address the different variables that may affect the colonization of STEC in these animals. / Financiamiento: Proyecto Fondecyt Iniciación N° 11170363

No description available.

Shiga-like toxin 1 (SLT-1) is produced by Escherichia coli strains like the pathogenic strain O157:H7. These bacterial strains are responsible for worldwide cases of food poisoning and water contamination, and the toxin is a major cause of hemorrhagic colitis and the hemolytic uremia syndrome. SLT-1 is defined as a type II ribosome-inactivating protein (RIP) and belongs to a family of plant and bacterial AB toxins. The A1 chain blocks protein synthesis in eukaryotic cells by depurinating a single adenine base in 28S rRNA. The mechanisms by which the A chain of SLT-1 interacts with the host components to route itself to the cytosol remains largely unknown. This thesis project identified a list of putative cellular proteins that interact with the SLT-1 A1 chain by the use of yeast-2-hybrid (Y2H) screens and HeLa lysate pull down/mass spectrometry analyses. Further assessment of the top 8 host interactors did not yield true interactions.

Shiga-like toxin 1 (SLT-1) is produced by Escherichia coli strains like the pathogenic strain O157:H7. These bacterial strains are responsible for worldwide cases of food poisoning and water contamination, and the toxin is a major cause of hemorrhagic colitis and the hemolytic uremia syndrome. SLT-1 is defined as a type II ribosome-inactivating protein (RIP) and belongs to a family of plant and bacterial AB toxins. The A1 chain blocks protein synthesis in eukaryotic cells by depurinating a single adenine base in 28S rRNA. The mechanisms by which the A chain of SLT-1 interacts with the host components to route itself to the cytosol remains largely unknown. This thesis project identified a list of putative cellular proteins that interact with the SLT-1 A1 chain by the use of yeast-2-hybrid (Y2H) screens and HeLa lysate pull down/mass spectrometry analyses. Further assessment of the top 8 host interactors did not yield true interactions.

Memoria para optar al Título Profesional de Médico Veterinario / Escherichia coli productor de toxina Shiga (STEC) es un patógeno emergente que puede provocar casos esporádicos y brotes de diarreas con y sin sangre, colitis hemorrágica y/o síndrome hemolítico urémico en humanos, especialmente niños y ancianos. La bacteria es transmitida por alimentos, principalmente a través de productos cárnicos crudos o insuficientemente cocidos. El objetivo del presente estudio fue determinar la prevalencia de STEC en carne molida de vacuno obtenida en diferentes áreas del Gran Santiago y caracterizar los aislados obtenidos. Se recolectaron 430 muestras de carne molida de vacuno desde carnicerías y supermercados de la ciudad entre los meses de Marzo del 2016 a Enero de 2017, dividiendo en 4 zonas geográficas a la ciudad. Las muestras fueron sometidas a un tamizaje a través de PCR para los genes stx1 y stx2 y desde las muestras positivas se aislaron colonias sospechosas a E. coli. La confirmación de los aislados se realizó mediante PCR para los genes stx1 y stx2 y pruebas bioquímicas para E. coli, además de un PCR para E. coli. Los aislados se caracterizaron para la presencia de los factores de virulencia de la intimina (eae) y hemolisina (hlyA), junto a la determinación de serotipos O157 y Big Six (O26, O45, O103, O111, O121, O145). Un 9,3% (40/430) de las muestras resultaron positivas a STEC; 10,2% provenientes (22/215) de carnicerías y 8,4% (18/215) de supermercados. No se detectaron diferencias significativas entre las prevalencias de cada área de la ciudad. Desde las 40 muestras positivas se recuperaron 47 aislados de STEC, de las cuales 12,8% (6/47) contenía el gen stx1, 74,5% (35/47) stx2 y 12,8% (6/47) tenía ambos genes de virulencia. Ninguna de los aislados contenía el gen de la intimina (eae), y un 40% fue positivo al factor de virulencia hlyA. Ninguno de los aislados fue de los serogrupos de STEC O157 o “Big Six”. En conclusión, la carne molida ofrecida en Santiago es un vector de STEC, sin embargo, se requieren más estudios para determinar el potencial patogénico de las cepas y el riesgo que representa la carne molida para la población / Shiga toxin-producing Escherichia coli (STEC) is an emerging pathogen that can cause sporadic cases and outbreaks of diarrhea, hemorrhagic colitis and / or hemolytic uremic syndrome in humans, especially children and the elderly. The bacterium is transmitted by food, especially through raw or undercooked meat products. The objective of the study was to determine the prevalence of STEC in ground beef obtained in the city of Santiago, Chile, and to characterize the isolates. We collected 430 ground beef samples from butcher shops and supermarkets across Santiago, from March 2016 to January 2017. The samples were screened through a PCR for the genes stx1 and stx2, and presumptive E. coli colonies were isolated from each positive sample. STEC confirmation was carried out by PCR for the stx1 and stx2 genes and biochemical tests for E. coli. All the isolates were characterized for the presence of the virulence factors intimin (eae) and hemolysin (hlyA) and tested for the presence of serotypes O157 and Big Six (O26, O45, O103, O111, O121, O145). From the ground beef samples, 9.3% (40/430) were positive to STEC: 10.2% (22/215) were from butcher shops and 8.4% (18/215) from supermarkets. No significant differences were detected among the prevalence of each area. From the 40 positive samples, 47 STEC isolates were recovered. Among the isolates, stx1 gene was present in 12.8%, 74.5% carried stx2, and 12.8% had both virulence genes. None of the isolates contained the intimin (eae) gene, and 40% were positive for the hlyA virulence factor. None belonged to the serogroups STEC O157 or Big Six. In conclusion, ground meat sold in Santiago, Chile carries STEC; however, more studies are required to determine the pathogenic potential of the strains and the risk that ground beef represents for the population / Financiamiento: Proyecto Fondecyt 11150491

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