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

No description available.

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.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Cattle are a reservoir for major Shiga toxin-producing Escherichia coli (STEC), which includes STEC O157 and the top six non-O157 serogroups (STEC-6; O26, O45, O103, O111, O121, O145). Collectively known as the STEC-7, these organisms are harbored in the hindgut and shed in the feces of cattle, which can contaminate hides. The de-hiding step during beef cattle processing can introduce fecal contaminants from the hide onto the carcass surface, creating the potential for contaminated beef products. The STEC-7 have been declared by the USDA-Food Safety and Inspection Service as adulterants in ground beef and non-intact beef products, and are monitored during beef cattle processing. However, many of the culture- and PCR-based tests for detection and/or quantification of the STEC, particularly of the STEC-6, are not established or require improvement and also virulence characteristics of STEC strains from cattle have not been fully analyzed. Therefore, the following studies were conducted: 1. Immunomagnetic separation (IMS)-based culture-method for detection of STEC-6 in cattle feces was developed and compared to a PCR-based method; 2. Detection sensitivity of pooled vs. individual IMS beads for isolation STEC-6 from cattle feces was evaluated; 3. Real-time PCR assay, based on the clustered regularly interspaced short palindromic repeat sequence polymorphisms (CRISPR), was developed and validated for serotype-specific detection and quantification of STEC O157:H7 in cattle feces; 4. Virulence gene profiles of bovine enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype E. coli O103 strains were examined with whole genome sequence (WGS)-based comparative analysis; 5. Prevalence and concentration of STEC-7 of fed-beef, cull beef and cull dairy cattle were determined. The culture and PCR methods detected all six serogroups in samples negative by the other method. Based on noninferiority tests, detection with pooled IMS beads was not inferior to detection with individual beads. Detection limits of the CRISPR-based qPCR assay for cattle feces spiked with pure cultures were 2.1 x 10³ and 2.3 x 10⁰ colony-forming units/g before and after enrichment, respectively. WGS-based analysis of E. coli O103 strains revealed key differences in the virulomes and mobilomes of EHEC, EPEC, and putative non-pathotype strains. The prevalence study revealed that a significantly higher (P < 0.01) proportion of hide samples from fed beef cattle (4.8%) were positive for STEC O157:H7, compared to samples from cull beef (1.6%) or cull dairy (0.2%); the majority of quantifiable STEC O157:H7 from each cattle type was at concentrations between 3 to 4 log CFU/100 cm². These data contribute to a knowledge gap on prevalence and concentration of STEC-7 and surrogate bacteria on cattle hides and carcasses, respectively. Furthermore, the development and refinement of culture- and PCR-based screening assays may lead to increased surveillance of major STEC serogroups, especially if the potential of WGS-based comparative genomics in identifying novel gene targets can be harnessed.

Shiga toxin-producing Escherichia coli (STEC) are extensive contributors to foodborne illness, causing renal and central nervous system damage due to production of Shiga toxin (Stx). Rapid Stx detection is important to distinguish STEC from other enteric pathogens. Current detection techniques are time consuming, expensive, and lack sensitivity. We have developed and evaluated a novel targeted mass spectrometry-based assay for detection of Stx using parallel reaction monitoring (PRM). The PRM assay used 11 target tryptic peptides and was validated using STEC and non-STEC bacterial cultures. Stx was detected in 56 of 62 STEC isolates and did not detect Stx in any of the 29 non-STEC isolates. The PRM assay successfully determined the Stx2 subtype in 32 of 46 Stx2-positive isolates. By applying a targeted proteomics assay, we were able to simultaneously detect Stx toxins 1 and 2 and subtype Stx2 into six toxin subgroups in Stx2-positive isolates. / February 2017

Shiga toxin-producing E. coli (STEC) cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). All STEC produce one or both of two Shiga toxins, Stx1 and Stx2. STEC strains that produce Stx2 are more strongly associated with HUS than strains that produce Stx1 or both Stx1 and Stx2. Epidemiologic evidence indicates a recent increase in the rate of HUS among STEC outbreaks. The increasing rate of HUS could be explained by a shift in the toxin profiles of STEC strains. The purpose of this study was to examine trends in toxin profiles of human STEC O157 isolates from 1996 to 2008 and to assess whether an increase in the number of Stx2-only-producing strains could be correlated with a recent increase in HUS cases. Data from three independent datasets, collected from PulseNet, eFORS and NARMS, were used. Additionally, trends such as seasonal variations, geographical variations, gender differences, and age differences were examined for each toxin profile. Results from this study show a shift in the toxin profile of human STEC O157 strains in the United States, in that the proportion of Stx2-only producing strains has increased dramatically since 1996.

No description available.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Natalia Cernicchiaro / David G. Renter / Cattle shed Shiga toxin-producing Escherichia coli (STEC) in their feces. Therefore, cattle pose a risk to contaminate produce, water, and beef products intended for human consumption. The United States Department of Agriculture Food Safety and Inspection Service consider seven STEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, non-intact beef products. Contrary to O157, the frequency and distribution of non-O157 serogroups and virulence genes have not been well-established in cattle. Therefore, the objectives of my thesis research were: 1) to appraise and synthesize data from peer-reviewed literature on non-O157 serogroup and virulence gene prevalence, and 2) to determine the prevalence of seven STEC in feedlot cattle feces across seasons. A systematic review and meta-analysis of published literature were conducted to gather, summarize, and interpret the existent data regarding non-O157 serogroup and virulence gene prevalence in cattle. Random-effects meta-analyses were used to obtain pooled non-O157 fecal prevalence estimates for continents worldwide and meta-regression analyses were conducted to evaluate effects of specific factors on between-study heterogeneity. Results indicated that non-O157 serogroup and virulence gene fecal prevalence significantly differed (P < 0.05) by geographic region, with North America yielding the highest pooled prevalence estimate worldwide. While previous research has demonstrated a strong seasonal shedding pattern of STEC O157, data regarding the seasonality of non-O157 STEC shedding in cattle is very limited. A repeated cross-sectional study was conducted to obtain serogroup and virulence gene prevalence data for the seven STEC in pre-harvest cattle feces, in summer and winter. We found that non-O157 serogroups were recovered in fecal samples collected in both seasons but virulence genes, thus STEC, were rarely detected in summer and undetected in winter. In conclusion, non-O157 STEC are present in cattle feces at very low frequencies, but STEC O103 and O157 significantly differed (P < 0.05) between seasons. Overall, the research described in this thesis greatly contributes to the limited body of data regarding non-O157 serogroup and virulence gene distribution in cattle and provides a better understanding of two major risk factors, season and geographic distribution, associated with STEC fecal shedding in cattle.