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.

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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) are globally relevant bacterial pathogens responsible for epidemic outbreaks of hemorrhagic diarrhea with variable progression to potentially fatal systemic Shiga toxicosis. Predictive clinical biomarkers and targeted therapeutic interventions for systemic Shiga toxicosis in diagnosed STEC patients are not available, and the impact of Shiga toxin production on STEC colonization and survival remain unclear. Improved murine models of STEC infection are needed to address knowledge gaps surrounding the gastrointestinal effects of Shiga toxins, as previously published models utilize ablation of host defense responses or microbiota depletion to facilitate colonization and are poorly suited for study of the effects of Shiga toxins on host responses. Dextran sulfate sodium (DSS) colitis in rodents has been associated with outgrowths of commensal E. coli in the literature, suggesting that DSS colitis could open a gastrointestinal niche usable by pathogenic STEC. This DSS colitis-based approach successfully induced susceptibility to robust colonization by two clinical isolate STEC strains in standard C57BL/6 mice. Studies using a Shiga-like toxin 2 (STX2)-producing clinical isolate STEC strain and its paired isogenic STX2 deletion strain (STEC(ΔSTX2)) revealed that STX2 was associated with delayed gastrointestinal clearance of STEC and concurrent reduction in colonic interleukin 23 (IL-23) axis transcripts known to be critical for pathogen clearance in other gastrointestinal pathogen models. In vivo reductions in IL-23 axis transcripts in the DSS+STEC model were supported by decreased IL-23 protein secretion by human macrophage-like cells during Shiga intoxication in vitro. Increased morbidity during STX2-producing STEC infection was associated with renal injury consistent with murine systemic Shiga toxicosis characterized by elevations in renal transcripts of molecular injury markers and histologically apparent renal tubular injury in a subset of mice. The dissertation research establishes a novel model of DSS colitis-facilitated murine STEC infection that recapitulates progression to systemic Shiga toxicosis in a subset of infected mice and demonstrates a clear STEC survival benefit associated with STX2 production. Shiga toxin-induced suppression of IL-23 axis signaling is a novel finding facilitated by the DSS+STEC model, demonstrating its utility for future delineation of the impacts of Shiga toxins on gastrointestinal host responses to STEC.

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont responsables d'infections humaines, allant d'une diarrhée aqueuse bénigne pouvant se compliquer en syndrome hémolytique et urémique (SHU), parfois mortel. La transmission de STEC à l'Homme s'effectue principalement par l'ingestion d'aliments contaminés. Le principal facteur de virulence des STEC est le gène stx (codant la Shiga-toxine), localisé dans le génome d'un prophage. La thèse a été centrée sur les STEC O26:H11, deuxième sérotype à l'origine de SHU dans le monde, et premier retrouvé dans les fromages au lait cru.Le premier objectif était de caractériser génétiquement les STEC O26:H11 et leurs phages Stx (variants du gène stx et sites d'insertion des phages Stx) afin d'identifier d'éventuelles différences entre ces souches selon leur origine. La majorité des souches alimentaires et bovines étudiées possèdent le variant stx1a et leurs phages Stx sont intégrés dans wrbA et yehV. Les souches humaines possèdent les variants stx1a et stx2a en proportions équivalentes. Leurs phages Stx sont aussi intégrés dans wrbA et yehV mais à la différence des souches alimentaires et bovines, le site yecE a été identifié comme site d'insertion. Toutes les souches humaines qui possédaient un phage Stx2a intégré dans wrbA et yecE ont causé des SHU ; ce qui pourrait être un indicateur de haute virulence. Des études ont montré que des souches E. coli Attachant/Effaçant (AEEC) O26:H11 sont isolées à partir d'aliments identifiés comme « stx+ » par PCR. En dehors de l'absence du gène stx, ces souches AEEC sont similaires aux STEC. Leur caractérisation a montré ici que la majorité d'entre elles ont leurs sites d'insertion intacts, caractéristique compatible avec une perte de phage Stx par excision spontanée. La stabilité des phages Stx a donc été évaluée chez les STEC O26:H11. La présence/absence de phages Stx a été quantifiée par PCRq pour chaque souche au niveau de la population bactérienne entière montrant que les STEC ont la capacité de perdre leurs phages Stx. En revanche, cette instabilité n'est pas liée aux sites d'insertion. Plusieurs essais visant à introduire des phages Stx dans les souches AEEC pour les convertir en STEC ont été réalisés mais les résultats ont montré qu'il était difficile d'infecter ces souches. L'étude du taux d'induction des phages Stx in vitro chez les STEC O26:H11 a montré, qu'en présence de mitomycine C, les phages Stx2 étaient plus inductibles que les phages Stx1. En revanche, il n'a pas été mis en évidence de différences en fonction de l'origine des souches testées et du site d'intégration des phages Stx. L'analyse morphologique de quelques phages Stx a montré que le type Stx1 ou Stx2 n'était pas lié à une forme spécifique de phage. L'étude des stress relatifs à la technologie fromagère a montré que le stress salin et le stress oxydatif, lié à la libération potentielle d'H2O2 par d'autres bactéries, entrainaient l'induction des phages Stx. Comme des souches AEEC sont fréquemment isolées à partir d'aliments qualifiés de « stx+» par PCR, le processus analytique d'isolement des STEC a aussi été étudié. La production de phages Stx lors de la phase d'enrichissement semble possible à partir d'un aliment contaminé. En revanche, aucun composant de cette méthode, testé individuellement, n'a pu être identifié comme inducteur des phages Stx. Ces travaux ont permis d'acquérir des connaissances sur la diversité des phages Stx issus de STEC O26:H11 isolées chez l'Homme et dans la filière laitière. Des différences au niveau des variants du gène stx mais aussi des sites d'insertion des phages Stx ont pu être observées en fonction de l'origine des souches. De plus les niveaux d'induction des phages Stx diffèrent selon le variant du gène stx. Ces différences pourraient refléter l'existence de clones distincts, aux potentiels de virulence différents, circulant dans les aliments, chez les bovins et les patients / Shiga toxin-producing Escherichia coli (STEC) are responsible for human infections, ranging from mild diarrhea to hemolytic-uremic syndrome (HUS), sometimes with fatal outcome. Transmission of STEC to humans occurs mainly through the ingestion of contaminated food. The main virulence factor of STEC is the stx gene (encodes Shiga-toxin) located in the genome of a prophage. The PhD thesis was focused on STEC O26:H11, which is the second serotype causing HUS in the world, and the first one found in raw milk cheeses. The first objective was to characterize genetically STEC O26:H11 strains and their Stx phages (stx gene subtypes and insertion sites of Stx phages) in order to identify any differences between these strains according to their origin. The majority of the investigated food and bovine strains possessed stx1a subtype and their Stx phages were integrated into wrbA and yehV. Human strains possessed the stx1a and stx2a subtypes in equivalent proportions. Their Stx phages were also integrated into wrbA and yehV but unlike food and bovine strains, yecE site were identified as insertion site. All the human strains carrying an Stx2a phage integrated into wrbA and yecE caused HUS, which could indicate a high virulence. Studies showed that Attaching/Effacing E. coli (AEEC) O26:H11 strains were isolated from foods identified as "stx +" by PCR. Except for the absence of stx gene, these AEEC strains are similar to STEC. Their characterization showed, in this study, that the majority of them had intact insertion sites, in agreement with a possible loss of Stx phage by spontaneous excision. The stability of Stx phages was evaluated in STEC O26:H11. Presence/absence of Stx phages was quantified for each strain in the total bacterial population, showing that STEC were capable of losing their Stx phages. However, this instability was not related to the insertion sites. Several attempts to introduce Stx phages in AEEC strains in order to convert them into STEC were conducted but the results showed that it was difficult to infect these strains. The induction rate of Stx phages in vitro in STEC O26:H11 showed that, with mitomycin C, the Stx2 phages were more inducible than Stx1 phages. However no difference was found with the origin of the strains tested and the Stx phage integration site used. The morphological analysis of some Stx phages showed that Stx1 or Stx2 type was not related to a specific phage shape. Study of various stress related to the cheese-making process showed that the osmotic and oxidative stress related to the potential release of H2O2 by other bacteria, led to the induction of Stx phages. Because AEEC strains are frequently isolated from food qualified as "stx+" by PCR, the analytical STEC isolation procedure was studied for its ability to induce Stx phages. Production of Stx phages during the enrichment phase seemed possible from contaminated food. However, none of the components of this method, tested individually, could be identified as an inducer of Stx phages. This work highlighted the diversity of Stx phages from STEC O26:H11 isolated from humans and dairy sector. Differences in stx subtypes and Stx phages insertion sites present among the STEC O26:H11 strains were observed depending on the origin of the strains. Moreover the induction levels of Stx phages differed according to the stx subtypes. These differences might reflect the existence of distinct clones, with varying virulence potential, circulating in foods, cattle and patients

A Síndrome Hemolítica Urêmica (SHU) é a principal doença associada à infecção com linhagens de Escherichia coli produtoras de toxina de Shiga (Stx), doença para qual não há uma vacina ou tratamento específico. A toxina Stx é formada por uma subunidade A enzimaticamente ativa e uma B pentamérica responsável pela ligação da toxina na célula hospedeira. Neste trabalho propomos o uso de Bacillus subtilis, uma bactéria não patogênica e formadora de esporos, como veículo vacinal para a expressão de formas atóxicas da Stx2, sob o controle de um promotor induzível por estresse (PgsiB). Camundongos BALB/c imunizados com células vegetativas ou esporos das linhagens vacinais de B. subtilis, por diferentes vias, induziram baixos níveis de anticorpos anti-Stx em soro (IgG) e fezes (IgA). Avaliamos também o potencial imunogênico da Stx gerada em linhagens recombinates de E. coli, mas os anticorpos gerados não foram capazes de neutralizar a toxina nativa. Os resultados indicam que formas alternativas de expressão e/ou o uso de adjuvantes são necessárias para gerar formulações vacinais eficazes contra a SHU. / The Hemolytic Uremic Syndrome (HUS) is the main disease associated with infections with Shiga toxin (Stx) - producing Escherichia coli strain and no effective vaccine or treatment exist. The Stx toxin consist of an enzymatically active A subunit and a pentameric B subunit responsible toxin binding to host cells. In this work we propose the use of Bacillus subtilis, a harmless spore form bacteria as a vaccine vehicle for the expression atoxic forms of Stx2, under the control of stress inducible (PgsiB) promoter. BALB/c mice immunized with vegetative cells and spores of the B. subtilis vaccine strain using different immunization routes elicited low specific antibody levels at serum (IgG) or fecal extracts (IgA). We also investigated the immunogenic potencial of StxB purified from recombinant E. coli strain, but the induced anti-StxB antibodies did not neutralize the native toxin. The results indicate that alternative expression system or the incorporation of the adjuvants are required for the generation of vaccine formulation active against HUS.

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.

A Síndrome Hemolítica Urêmica (SHU) é a principal doença associada à infecção com linhagens de Escherichia coli produtoras de toxina de Shiga (Stx), doença para qual não há uma vacina ou tratamento específico. A toxina Stx é formada por uma subunidade A enzimaticamente ativa e uma B pentamérica responsável pela ligação da toxina na célula hospedeira. Neste trabalho propomos o uso de Bacillus subtilis, uma bactéria não patogênica e formadora de esporos, como veículo vacinal para a expressão de formas atóxicas da Stx2, sob o controle de um promotor induzível por estresse (PgsiB). Camundongos BALB/c imunizados com células vegetativas ou esporos das linhagens vacinais de B. subtilis, por diferentes vias, induziram baixos níveis de anticorpos anti-Stx em soro (IgG) e fezes (IgA). Avaliamos também o potencial imunogênico da Stx gerada em linhagens recombinates de E. coli, mas os anticorpos gerados não foram capazes de neutralizar a toxina nativa. Os resultados indicam que formas alternativas de expressão e/ou o uso de adjuvantes são necessárias para gerar formulações vacinais eficazes contra a SHU. / The Hemolytic Uremic Syndrome (HUS) is the main disease associated with infections with Shiga toxin (Stx) - producing Escherichia coli strain and no effective vaccine or treatment exist. The Stx toxin consist of an enzymatically active A subunit and a pentameric B subunit responsible toxin binding to host cells. In this work we propose the use of Bacillus subtilis, a harmless spore form bacteria as a vaccine vehicle for the expression atoxic forms of Stx2, under the control of stress inducible (PgsiB) promoter. BALB/c mice immunized with vegetative cells and spores of the B. subtilis vaccine strain using different immunization routes elicited low specific antibody levels at serum (IgG) or fecal extracts (IgA). We also investigated the immunogenic potencial of StxB purified from recombinant E. coli strain, but the induced anti-StxB antibodies did not neutralize the native toxin. The results indicate that alternative expression system or the incorporation of the adjuvants are required for the generation of vaccine formulation active against HUS.

No description available.