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Summary The group of E. coli strains is a highly heterogeneous group of strains, including pathogenic and non-pathogenic strains. The classification of these strains is made upon specific virulence factors of these bacteria. Some of these pathogenic strains are not capable to cross over the intestinal border and are responsible for intestinal disorders associated with diarrhoea. Other strains can cross the intestinal border and produce septicaemia and other complications depending on the infected organs. These pathogenic strains of E. coli interact with the host in a typical manner and produce specific lesions. These specific interactions are observed after the colonisation of the gut by pathogenic bacteria and are the results of the presence of specific virulence factors. The colonisation of the gut is mediated by specific adhesins, which are specific for each group of pathogenic E. coli. It seems that these adhesins are not only responsible to initiate the interaction between the pathogen and the host but are also responsible for the host specificity shown by some pathogenic strains. Its for that that a better understanding of these adhesins would permit a better understanding of the host specificity and a better evaluation of the zoonotic potential of these strains. The aim of this work was to identify bacterial structures involved in the intestinal adhesion step and in the intestinal colonisation of the enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) and verotoxigenic E. coli (VTEC) bovine isolates, focusing mainly (but not exclusively) on strains of serogroup O26. In order to achieve this objective, two different approaches have been followed: i) the immunologic approach and ii) the genetic approach. The immunologic approach is using the benefits of the 2F3 monoclonal antibody (MAb), which was obtained against an outer membrane protein extract from a human O26 EHEC strain. The work on this approach focused on the study of this MAb as epidemiological tool and on the identification of the epitope recognised by this antibody and of its genetic basis. The work on the genetic approach involves the screening by PCR and by colony hybridization of a collection of EHEC, VTEC and EPEC of human and bovine isolates for the presence of homologues for gene clusters coding for fimbriae of type I, II, III and IV: (i) putative adhesins of human EPEC and EHEC strains like LifA, Iha, LpfA, BfpA; (ii) fimbriaires adhesins (CS31A et F17) and afimbriaire adhesins of Afa family (Afa III, Afa VII, Afa VIII and F1845) described for other groups of bovine pathogenic E. coli. The accomplished work allowed, first of all, to confirm that the 2F3 MAb is specific for the O26 EPEC and O26 EHEC strains without being capable to distinguish between human and animal isolates, that means without being capable to distinguish them depending on theirs host. Moreover, it has been shown that: i) the epitope recognised by the 2F3 MAb is component of the O antigen from the O26 EPEC and O26 EHEC strains; ii) the genetic basis of this epitope is located inside the O antigen gene cluster of O26 EPEC and O26 EHEC strains; and iii) the 2F3 + and O26 + characters are two characters that can be dissociated by random insertional mutagenesis in a bovine EHEC strain. Nevertheless, the results of this work not allowed us to explain the specificity of the 2F3 MAb for the O26 EPEC and O26 EHEC strains. Actually, by sequencing the O-antigen gene cluster (the rfb locus) of an O26 non-EPEC and non-EHEC strain and comparing the obtained sequence with the already published sequence of the O-antigen gene cluster of an O26 EHEC strain no major differences (which could explain the specificity of the 2F3 MAb for the O26 EPEC or EHEC strains) were observed. The obtained results in the genetic approach have been shown that: i) all O145 and O157 EPEC and EHEC strains were positive for the LpfA probe (lpfA gene is coding for the type IV pili) and all other human and bovine strains belonging to other serogroups (O5, O26, O103, O111 and O118) have no homologue sequences for this probe; ii) a big majority of EPEC and EHEC strains belonging to O5, O26, O103, O111 or O118 serogroups were positives for the LifA probe (the lifA gene is involved in the synthesis of adhesins not very well characterised) but none of those belonging to the O145 et O157 serogroups; iii) different proportions of these strains belonging to various serogroups were positives for the Iha probe (the iha gene is involved in the synthesis of an adhesin not very well characterised); and iv) ten out of twelve bovine O26 EPEC strains were positive for the ClpE probe and all other strains (human EPEC and EHEC strains and bovines EHEC strains) were negative for the ClpE probe. The clpE gene is involved in the synthesis of the chaperon protein of the CS31A fimbria, which was described for the enterotoxigenic and septicaemic bovine and porcine E. coli isolates. Since the O145 and O157 EHEC strains show the same pathotype, it looks like that the presence of the lpfA gene and the absence of the lifA gene is linked more to a specific pathotype than to a specific serotype. Concerning the results obtained results with the ClpE probe for the O26 EPEC strains, it is possible that these strains have not an entire clp gene cluster since all the strains positive for the ClpE probe were negative for the ClpG (clpG gene is involved in the biosynthesis of the major unit of the CS31A fimbriae). Nevertheless, it is possible that the clpE-like gene of these strains belongs to an entire gene cluster for which the major unit is different from the ClpG. Finally, the screening of a collection of O8 and O20 VTEC bovine isolates for the presence of sequences homologues to the genes involved in the biosynthesis of adhesins of the Afa family (Afa III, Afa VII, Afa VIII and F1845), adhésines produced mainly by human uropathogenic isolates and by some septicaemic strains isolated from animals, allowed the identification of two O8 VTEC bovine strains harbouring the afa-VIII D/afa-VIII E genes and expressing the Afa VIII E adhesin. During this work, a lot of progresses, that could be useful for a better understanding of the pathogenesis of the EPEC, EHEC and VTEC strains, have been done. Despite that, the general objective of this project description of new factors involved in the initial adhesion stage and/or in the intestinal colonisation by the EPEC, EHEC and VTEC bovine strains in order to allow an easy and reliable diagnostic of these strains have been not accomplished. In perspective of this work, complementary works should focus: i) to identify the epitope recognised by the 2F3 MAb; and ii) to investigate which is the role of the adhesins, for which homologues sequences have been described in the human or bovine EPEC and EHEC isolates of different serogroups, in the adhesion to eukaryotic cells like bovine and human enterocytes. Résumé Lespèce Escherichia coli (E. coli) est hétérogène, contenant des souches pathogènes et des souches inoffensives. La classification des différentes souches pathogènes est basée sur leurs propriétés spécifiques de virulence. Les souches pathogènes dE. coli sont en effet associées à des pathologies variées. Certaines souches ne franchisent pas la barrière intestinale produisant des lésions dentérite, associées à de la diarrhée et dautres souches pathogènes dE. coli peuvent franchir la barrière intestinale, produisant de la septicémie, avec des complications variables selon les organes infectés. Ces souches interagissent avec lhôte dune manière particulière produisant des lésions typiques au niveau cellulaire et tissulaire. Ces interactions spécifiques sont dues aux propriétés de virulence spécifiques après la colonisation de lintestin. Létape de colonisation de lintestin se fait grâce à des adhésines particulières, spécifiques pour chaque groupe de souches pathogènes dE. coli. Il semble que ces adhésines nont pas seulement le rôle dinitialiser linteraction entre lhôte et le pathogène mais quelles soient responsables aussi de la spécificité dhôte présentée par certaines souches pathogènes. Ainsi la connaissance de ces adhésines permet la reconnaissance de la véritable spécificité dhôte de ces souches et lévaluation de leur potentiel zoonotique. Lobjectif général du travail était didentifier des structures bactériennes impliquées dans ladhérence aux entérocytes et, donc, dans la colonisation intestinale par des souches entérohémorragiques (EHEC), entéropathogènes (EPEC) et vérotoxinogènes (VTEC) bovines, et qui représenteraient une base de la spécificité dhôte de ces souches, en ciblant plus particulièrement le sérogroupe somatique O26. Pour accomplir lobjectif de ce travail, deux approches ont été suivies : immunologique et génétique. Lapproche immunologique a utilisé lanticorps monoclonal 2F3 qui a été dérivé contre des extraits protéiques de membrane dune souche EPEC humaine du sérogroupe O26. Le travail a consisté à identifier l'antigène reconnu par l'anticorps monoclonal 2F3, à déterminer sa base génétique et à lutiliser en tant qu'outil d'épidémiologie moléculaire. Le travail dans lapproche génétique a consisté à rechercher, par des épreuves de PCR et d'hybridation sur colonies sur une collection de souches EHEC, EPEC et VTEC bovines et humaines, la présence de gènes dopérons qui codent pour différents fimbriae de type I, II, III et IV : (i) des adhésines potentielles des souches EHEC et EPEC humaines, telle que LifA, Iha, LpfA, BfpA; (ii) des adhésines fimbriaires (CS31A et F17) et afimbriaires la famille Afa (Afa III, Afa VII, Afa VIII et F1845) présentes chez dautres catégories de souches dE. coli pathogènes pour le bovin. Les travaux effectués ont tout dabord permis de confirmer que lanticorps monoclonal 2F3 est spécifique pour les souches EPEC et EHEC du sérogroupe O26 et ne reconnaît pas les souches non-EHEC non-EPEC, sans cependant quil puisse faire la différence entre les souches EHEC et EPEC dorigine humaine et animale, cest-à-dire en fonction de leur hôte. Dans la partie des recherches consacrée à létude de cet anticorps, il a été aussi démontré que : i) lépitope reconnu par lanticorps monoclonal 2F3 est une composante du lipopolysaccharide (LPS) des souches EPEC et EHEC du sérogroupe O26 ; ii) la base génétique de cet épitope est situé dans lopéron codant pour lantigène O26 ; et iii) le caractère 2F3+ peut néanmoins être dissocié du caractère O26+ par mutagenèse par transposition aléatoire dans une souche EHEC bovine. Néanmoins, les résultats de ces travaux nont pas permis dexpliquer la raison pour laquelle lanticorps monoclonal 2F3 reconnaît les souches EPEC et EHEC O26, mais non les souches non-EPEC et non-EHEC O26. En effet, le séquençage de lopéron codant pour lantigène O26 (locus rfb) dune souche non-EHEC non-EPEC et sa comparaison avec la séquence publiée de lopéron rfb dune souche O26 EHEC na pas permis de reconnaître une différence pouvant expliquer la reconnaissance par lanticorps 2F3. Les résultats obtenus dans le cadre des travaux selon lapproche génétique ont montré que : i) toutes les souches EHEC bovines et humaines appartenant aux sérogroupes O145 et O157 étaient positives pour la sonde LpfA (les gènes lpfA codent pour des pili de type IV), mais aucune souche appartenant aux autres sérogroupes (O5, O26, O103, O111 et O118); ii) une grande majorité des souches EHEC et EPEC bovines et humaines aux sérogroupes O5, O26, O103, O111 et O118 étaient positives pour la sonde LifA (les gènes lifA codent pour des adhésines encore peu caractérisées), mais pas celles appartenant aux sérogroupes O145 et O157; iii) des proportions variables de souches appartenant à ces différents sérogroupes étaient positives pour la sonde Iha (les gènes iha codent aussi pour des adhésines peu caractérisées); et iv) dix souches EPEC bovines O26 sur les douze étudiées étaient positives pour la sonde ClpE (dérivée du gène codant pour la protéine chaperone du fimbria CS31A produit par des souches entérotoxinogènes et septicémiques bovines et porcines dE. coli), alors que les souches EHEC bovines et humaines du même sérogroupe, ainsi que toutes les souches des autres sérogroupes, étaient négatives pour cette sonde. Comme les souches EHEC O157 et O145 présentent le même pathotype, il semblerait que la présence des gènes lpfA et labsence des gènes lifA soient associées plus à un pathotype particulier quà un, ou quelques sérogroupes. En ce qui concerne les résultats avec la sonde ClpE sur les souches EPEC bovines O26, il est possible que ces souches ne possèdent pas lopéron clp complet puisque toutes les souches étaient négatives pour la sonde clpG (codant pour la sous-unité majeure du fimbria CS31A). Il est cependant aussi possible que le gène clpE-like de ces souches appartienne à un opéron complet dont la sous-unité majeure soit différente de ClpG. Enfin, la recherche des gènes intervenant dans la biosynthèse dadhésines de la famille Afa (Afa III, Afa VII, Afa VIII and F1845), surtout produites par des souches uropathogènes humaines et sur certaines souches septicémiques animales, dans la collection des souches VTEC dorigine bovine de sérogroupe O8 et O20 a permis la mise en évidence de deux souches VTEC de sérogroupe O8 qui possèdent les gènes afa-8D/afa-8E et qui expriment ladhésine Afa 8E. Au cours de nos travaux une série de résultats, qui pourront amener à une meilleure compréhension des mécanismes de pathogénicité et des souches EPEC, EHEC et VTEC ont été réalisés. Cependant, lobjectif général du projet - la description de facteurs spécifiques de lhôte impliqués dans le processus dadhérence initiale des souches EPEC, EHEC et VTEC bovines permettant la colonisation intestinale, afin de pouvoir les diagnostiquer et de les typer de manière spécifique - na pas été atteint. A court terme, les compléments de travaux à envisager sont la poursuite des essais didentification de lépitope reconnu par lanticorps monoclonal 2F3 et le rôle dans ladhérence aux cellules eucaryotes, comme les entérocytes bovines et humains, des adhésines dont les gènes ont été détectés dans les souches EHEC et EPEC bovines et humaines appartenant à différentes sérogroupes.

Shigatoxin-producing Escherichia coli (STEC) can cause infections in humans which can beserious and sometimes fatal. There is a great need for methods that are able to detect differentserogroups of STEC. In this project, conventional and real-time PCR assays for detection ofSTEC O104:H4 and O121, as recommended by the European Union Reference Laboratory(EU-RL) for STEC, were validated. The specificity, limit of detection, repeatability,efficiency and robustness were determined for three real-time PCR assays. The validationshowed that the real-time PCR reactions were specific and sensitive although some additionaltests are required.

Escherichia coli produtora de toxina Shiga (STEC) está entre os patógenos envolvidos em surtos de doenças transmitidas por alimentos devido ao consumo de vegetais. No entanto, até agora, os relatos sobre a presença de STEC em vegetais no Brasil são escassos. Esse microrganismo é veiculado por alimentos, contaminados direta ou indiretamente por fezes animais, sendo responsável por um amplo espectro de doenças que compreende desde diarréia leve que pode evoluir para colite hemorrágica (CH), até síndrome hemolítico-urêmica (SHU) e púrpura trombocitopênica trombótica (PTT). O presente estudo teve como objetivo investigar a presença de STEC em vegetais orgânicos cultivados na região metropolitana da cidade de São Paulo, Brasil, caracterizando os fatores de virulência stx1, stx2, eae e ehx, bem como o sorotipo. Um total de 200 amostras de vegetais orgânicos (folhas verdes), obtido a partir de três produtores foi analisado quanto à presença de cepas de STEC. Caldo triptona de soja (TSB) suplementado com vancomicina (8 mg / L), cefixima (50 µg / L) e telurito de potássio (2,5 mg / L) foi utilizado na etapa de pré-enriquecimento, com incubação a 37ºC / 24 h, seguido por semeadura em MacConkey Sorbitol (SMAC) e CHROMagar STEC (CHROM). Após incubação a 37ºC / 24 h, as colônias suspeitas foram confirmadas por testes bioquímicos e submetidas a PCR objetivando a detecção dos genes de virulência stx1, stx2, eae, ehx, e os genes fliCH7 e rfbO157. Entre as 200 amostras de vegetais orgânicos analisadas, 30 (15%) foram positivas para E. coli, mas nenhum isolado apresentou os genes de virulência pesquisados. Nossos resultados indicam baixo risco de infecção devido ao consumo destes produtos frescos em São Paulo, Brasil. No entanto, são necessárias mais pesquisas, abrangendo um maior número de amostras e área pesquisada, uma vez que este patógeno já foi encontrado no meio ambiente em estudos anteriores e poucas pesquisas investigaram a presença de STEC em vegetais no Brasil. / Shiga toxin producing Escherichia coli (STEC) strains are among the pathogens involved in foodborne disease outbreaks due to consumption of vegetables. However, reports on the presence of STEC in vegetables in Brazil are lacking. STEC is an important pathogen transmitted by food, directly or indirectly contaminated with animal feces, responsible for a broad spectrum of diseases varying from mild diarrhea to hemorrhagic colitis (HC), syndrome hemolytic uremic (HUS) and thrombotic thrombocytopenic purpura (TTP). This study aimed at investigating the presence of STEC in organic vegetables in the metropolitan region of São Paulo city, Brazil, characterizing the virulence factors stx1, stx2, eae and ehx as well as identifying the serotype. A total of 200 samples of organic vegetables (green leafy), obtained from three organic producers was analyzed for the presence of STEC strains. Tryptic Soy Broth (TSB) supplemented with vancomycin (8mg/L), cefixim (50µg/L) and potassium telurite (2.5mg/L) was used in the pre enrichment step with incubation at 37°C/24 h, followed by plating onto Sorbitol-MacConkey (SMAC) agar and CHROMagar STEC (CHROM). After incubation at 37°C/24 h, presumptive colonies were confirmed by biochemical tests and submitted to PCR targeting the detection of stx1, stx2, eae and ehx virulence genes, as well as fliCH7 and rfbO157. Among the 200 organic vegetable samples analyzed for STEC strains, 30 (15%) were positive for E. coli, but none of them showed the virulence genes studied. These findings indicate low risk of infection due to the consumption of these fresh produce in Sao Paulo, Brazil. However, more research is required, covering a larger number of samples and area, since this pathogen has already been found in the environment in previous studies, and few research investigating the presence of STEC in vegetables has been reported in Brazil.

Escherichia coli produtora de toxina Shiga (STEC) está entre os patógenos envolvidos em surtos de doenças transmitidas por alimentos devido ao consumo de vegetais. No entanto, até agora, os relatos sobre a presença de STEC em vegetais no Brasil são escassos. Esse microrganismo é veiculado por alimentos, contaminados direta ou indiretamente por fezes animais, sendo responsável por um amplo espectro de doenças que compreende desde diarréia leve que pode evoluir para colite hemorrágica (CH), até síndrome hemolítico-urêmica (SHU) e púrpura trombocitopênica trombótica (PTT). O presente estudo teve como objetivo investigar a presença de STEC em vegetais orgânicos cultivados na região metropolitana da cidade de São Paulo, Brasil, caracterizando os fatores de virulência stx1, stx2, eae e ehx, bem como o sorotipo. Um total de 200 amostras de vegetais orgânicos (folhas verdes), obtido a partir de três produtores foi analisado quanto à presença de cepas de STEC. Caldo triptona de soja (TSB) suplementado com vancomicina (8 mg / L), cefixima (50 µg / L) e telurito de potássio (2,5 mg / L) foi utilizado na etapa de pré-enriquecimento, com incubação a 37ºC / 24 h, seguido por semeadura em MacConkey Sorbitol (SMAC) e CHROMagar STEC (CHROM). Após incubação a 37ºC / 24 h, as colônias suspeitas foram confirmadas por testes bioquímicos e submetidas a PCR objetivando a detecção dos genes de virulência stx1, stx2, eae, ehx, e os genes fliCH7 e rfbO157. Entre as 200 amostras de vegetais orgânicos analisadas, 30 (15%) foram positivas para E. coli, mas nenhum isolado apresentou os genes de virulência pesquisados. Nossos resultados indicam baixo risco de infecção devido ao consumo destes produtos frescos em São Paulo, Brasil. No entanto, são necessárias mais pesquisas, abrangendo um maior número de amostras e área pesquisada, uma vez que este patógeno já foi encontrado no meio ambiente em estudos anteriores e poucas pesquisas investigaram a presença de STEC em vegetais no Brasil. / Shiga toxin producing Escherichia coli (STEC) strains are among the pathogens involved in foodborne disease outbreaks due to consumption of vegetables. However, reports on the presence of STEC in vegetables in Brazil are lacking. STEC is an important pathogen transmitted by food, directly or indirectly contaminated with animal feces, responsible for a broad spectrum of diseases varying from mild diarrhea to hemorrhagic colitis (HC), syndrome hemolytic uremic (HUS) and thrombotic thrombocytopenic purpura (TTP). This study aimed at investigating the presence of STEC in organic vegetables in the metropolitan region of São Paulo city, Brazil, characterizing the virulence factors stx1, stx2, eae and ehx as well as identifying the serotype. A total of 200 samples of organic vegetables (green leafy), obtained from three organic producers was analyzed for the presence of STEC strains. Tryptic Soy Broth (TSB) supplemented with vancomycin (8mg/L), cefixim (50µg/L) and potassium telurite (2.5mg/L) was used in the pre enrichment step with incubation at 37°C/24 h, followed by plating onto Sorbitol-MacConkey (SMAC) agar and CHROMagar STEC (CHROM). After incubation at 37°C/24 h, presumptive colonies were confirmed by biochemical tests and submitted to PCR targeting the detection of stx1, stx2, eae and ehx virulence genes, as well as fliCH7 and rfbO157. Among the 200 organic vegetable samples analyzed for STEC strains, 30 (15%) were positive for E. coli, but none of them showed the virulence genes studied. These findings indicate low risk of infection due to the consumption of these fresh produce in Sao Paulo, Brazil. However, more research is required, covering a larger number of samples and area, since this pathogen has already been found in the environment in previous studies, and few research investigating the presence of STEC in vegetables has been reported in Brazil.

<p>The purpose of this study was to investigate the occurrence<strong> </strong>of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant<strong> </strong>in Lysekil.</p><p>Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of <em>vtx1</em> and <em>vtx2</em> genes and enrichment of bacteriophages on non Verotoxin-producing <em>Escherichia coli</em> O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by <em>E. coli</em> bacteria</p><p>The levels of Verotoxin-encoding phages and <em>E.coli</em> outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.</p><p> </p><p> </p>

The purpose of this study was to investigate the occurrence of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant in Lysekil. Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of vtx1 and vtx2 genes and enrichment of bacteriophages on non Verotoxin-producing Escherichia coli O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by E. coli bacteria The levels of Verotoxin-encoding phages and E.coli outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.

Shiga toxin-producing E. coli (STEC) have important public health and food safety implications. Cattle are the primary reservoir for STEC, which are transmitted to humans via contact with cattle or related food products. Dairy farms in particular have been incriminated as an important source of STEC. The broad aim of this study was to examine in depth the epidemiology of STEC on the dairy farm. The presence of STEC on three Australian dairy farms was surveyed. This aimed to provide data on the prevalence and nature of STEC on Australian dairy farms, as well as to examine in more detail the pre-harvest/slaughter ecology of STEC. STEC, E. coli O157:H7 and E. coli O26:H11 prevalences were similar to those from dairy farms in other countries. Replacement heifers were the most important source of STEC on the farms. Calves excreted STEC from an early age, with faecal prevalence peaking at weaning. Higher STEC prevalence was also associated with group housing of calves during weaning. Calf isolates were potential human pathogens based on serotype and virulence markers. Clonal relationships between isolates were analysed. Calf isolates were diverse and had a high clonal turnover. STEC isolated from within the same farm had a higher genetic similarity than those from different farms. Vertical and horizontal transmission were both identified among cattle. The farm environment was also identified as an important source of STEC. Reasons for increased levels of STEC excretion by calves were investigated. Two broad hypotheses for higher faecal shedding were proposed and examined individually. The first was that an animal is more likely to excrete STEC when its exposure to STEC is greater, thus promoting inoculation of the gastrointestinal tract. Calves were experimentally inoculated with a traceable STEC strain to examine the infection dynamics of STEC within cattle groups, and explore the effect of calf management procedures. Calves which were housed in groups and co-jointly fed and managed had a higher prevalence of the inoculation strain than animals housed individually. The test strain was readily isolated from the hides and saliva of inoculated calves, as well as their immediate environments. Calves become infected with STEC via the faecal-oral route, iv either by direct contact with other calves, or indirectly through contact with faecally contaminated materials. The second hypothesis was that individual animals are variably susceptible to intestinal colonisation by STEC, which leads to differing magnitudes and durations of STEC carriage. Factors influencing colonisation susceptibility to STEC and the mechanisms behind these factors were also examined. In order to compare enteric colonisation under a range of different conditions, a suitable experimental system was developed. In vitro organ culture of explanted ruminant colonic tissues provided a laboratory model that was representative of in vivo bacterial-mucosal attachment. The degree of STEC colonisation was enumerated using an immunofluorescent filtration technique. The quantitative colonisation assay was applied to determine the effects of host-dependant variables on STEC colonisation. Colonic tissues from weaning calves and adult cattle did not differ significantly in their susceptibility to colonisation; nor did tissues from cattle fed either high forage or high grain diets. Colonic explants from sheep, however, demonstrated significantly higher numbers of adherent STEC than bovine explants. It was therefore concluded that while species-specific differences in host tissues may mediate STEC carriage differences, this did not explain in vivo variability in age and diet related excretion. Factors that indirectly affect the susceptibility of host tissues to colonisation were examined. E. coli O157:H7 cultured in media designed to represent the enteric contents of a well-fed ruminant colonised the colonic mucosa in reduced numbers, indicating that age and diet may be correlated with differences in STEC carriage and excretion because of differing physiological augmentation of the intra-enteric environment. In conclusion, while group dynamics and management practices may increase STEC shedding prevalences for cattle via increased STEC exposure, factors that modulate an individual ruminant’s gastrointestinal carriage of STEC have a significant role in mediating STEC excretion. Either directly or indirectly, species, age and diet can affect the numbers of STEC that colonise the bowel wall, thereby influencing the magnitude and duration of STEC excretion. Both of these features of ruminant STEC ecology should be addressed in order to reduce the pre-slaughter/harvest presence of STEC.

Background: Mastitis in dairy cattle is a common ailment worldwide. A cause of mastitis can be bacteria such as Escherichia coli. Mastitis is not a deadly ailment and sometimes the dairy cows show no symptoms but if certain virulence genes are present in the bacteria that cause the mastitis, the bacteria can be transmitted to humans and cause severe diseases. The potential presence of enterohemorrhagic Escherichia coli (EHEC) in particular would be a major concern for human health. Aim: The aim for this study was to analyze the presence of virulence genes known to be present in E.coli strains isolated from dairy cows with mastitis in Sweden. Method: A Qiagen BIO ROBOT EZ1 was used to purify DNA from 90 bacterial cultures. A panel of virulence genes were amplified and biotinylated from the purified DNA by PCR and an E.coli based DNA microarray was used to detect presumed virulence genes in E.coli. Result: There were no samples that had all the genes traditionally used to classify E.coli as EHEC or potential EHEC. 63 samples were analyzed without any problems but 27 samples were not fully analyzed. Conclusion: The DNA based microarray proved to be a reliable method to detect genes from pathogenic bacteria but it needed high concentration of purified DNA which was not always easy to obtain. There were some samples in this study that contained virulence genes.

Enterohemorrhagic E. coli, EHEC, is a verotoxin producing, zoonotic pathogen, which causes diseases in humans such as bloody or watery diarrhea. Microorganisms compete for limited living space, nutrients and other resources and therefore other microorganisms are EHECs biggest competitors. To avoid outbreaks and infections with EHEC, one possible approach is to use harmless but competitive bacteria as probiotics. Therefore, the aim of this study was to evaluate three probiotic E. coli strains and their ability to outcompete EHEC in bovine feces.     Ten different cattle fecal samples from three different farms were used to mix with the three probiotic and EHEC strains. The mixture was diluted and cultivated at 0 h as a control and then incubated for 48 h at 20°C and 37°C before dilution and cultivation on CT-SMaC. Colonies was counted and ratios between EHEC and probiotic E. coli before and after incubation were calculated. Kruskal-Wallis test with Dunn’s test as post hoc test were used to see if observed reductions of EHEC were significant or not.     In 37°C, strain 10 was the only strain producing a significant reduction of EHEC. In contrast, no significant reduction was observed at 20°C in any of the strains.     Future research studying other factors and performed on live cattle models are necessary to confirm the usefulness of the studied probiotic candidates. However, these results indicate probiotics can be a useful tool to avoid infections and big outbreaks of EHEC in the future.