O objetivo deste trabalho foi a construção de linhagens geneticamente modificadas de B. subtilis capazes de expressar porções de intimina, principal componente envolvido na capacidade de colonização de linhagens enteropatogênicas de Escherichia coli (EPEC), como estratégia vacinal de administração oral contra diarréias infecciosas. As vacinas desenvolvidas empregaram cinco regiões da intimina de EPEC e linhagens de B. subtilis capazes de expressar e acumular proteínas recombinantes no citoplasma. Além disso, avaliamos o uso de esporos e células vegetativas como veículos vacinais para a entrega de antígenos recombinantes a partir de sistema de expressão epissomal. A eficácia do modelo vacinal foi demonstrada pela: (i) produção de anticorpos sistêmicos (IgG) e secretados (sIgA) contra intimina, (ii) capacidade de neutralização das intiminas expressas por diferentes linhagens de EPEC pelos anticorpos específicos gerados nos animais imunizados; e (iii) proteção a desafio com linhagens de EPEC a partir de modelo experimental que emprega camundongos recém-nascidos. Os resultados representam uma etapa importante na validação de uma nova estratégia vacinal para o controle de patógenos entéricos. Além disto, propomos a utilização de um modelo animal como uma nova ferramenta para se avaliar o potencial protetor de vacinas contra EPEC. / The objective of this work was the construction of genetically modified strains of B. subtilis able to express portions of intimin, the main component involved in colonization by enteropathogenic Escherichia coli strains (EPEC) as a strategy of oral vaccination against infectious diarrhea. The vaccines employed five regions of EPEC intimin and B. subtilis strains expressing recombinant proteins in the cytoplasm. Furthermore, we evaluated the use of spores and vegetative cells as vaccine vehicles for the delivery of recombinant antigens based on an epissomal expression system. The efficacy of the vaccines was demonstrated by: (i) production of systemic (IgG) and mucosal (sIgA) antibody responses to intimin, (ii) neutralizing of intimin expressed by different strains of EPEC by the antibodies generated in immunized animals, and (iii) protection to lethal challenges carried out with EPEC strains using an experimental model based in newborn mice. The results represent an important step in the validation of a new vaccine strategy for the control of enteric pathogens. Moreover, we propose the use of an animal model as a new tool to evaluate the protective potential of vaccines against EPEC.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Reconhecida como agente de doença humana em 1982, E.coli enterohemorrágica (EHEC) pode causar diarréia sanguinolenta, colite hemorrágica e síndrome hemolítica urêmica (SHU). EHEC constitui um subgrupo especialmente virulento das E.coli produtoras de toxina de Shiga (Stx). O fator crítico da sua virulência é a toxina Shiga, capaz de interromper a síntese proteica da célula eucariótica. São conhecidos dois subgrupos de Stx, Stx1 e Stx2. Stx1 possui duas variantes Stx1c e Stx1d. Stx2 possui muitas variantes. Estudos epidemiológicos sugerem que cepas com os perfis toxigênicos Stx2 ou Stx2/Stx2c seriam mais frequentemente associadas a pacientes com SHU. Além da expressão de Stx, EHEC do sorotipo O157:H7 colonizam a mucosa intestinal induzindo a formação de lesões denominadas attaching/effacing (A/E). Para a produção da lesão A/E, é necessária a presença de uma ilha de patogenicidade cromossômica denominada LEE, composta por cinco operons, LEE 1 a LEE5. Em LEE 5 são codificadas a adesina intimina e o seu receptor Tir, o qual é translocado por um sistema de secreção tipo III (SSTT) e em LEE 4 são codificadas as proteínas secretadas EspA,B e D. Em EHEC O157:H7 são descritos muitos fatores de virulência, codificados em ilhas de patogenicidade, no cromossomo e no megaplasmídio pO157. Bovinos são o principal reservatório deste patógeno e alimentos de origem bovina e produtos contaminados com fezes de bovinos são causadores de surtos epidêmicos. Em nosso país EHEC O157:H7 é isolada do reservatório animal mas é muito rara a sua ocorrência em doença humana. Notamos que nas cepas bovinas predomina Stx2c, enquanto nas cepas humanas predomina o perfil toxigenico Stx2/Stx2c. Quanto a interação com enterocitos humanos cultivados in vitro (linhagem Caco-2), verificamos que tanto cepas bovinas quanto humanas mostram idêntica capacidade de invadir e persistir no compartimento intracelular das células Caco-2. No entanto, em comparação com as cepas humanas, as cepas bovinas mostram uma reduzida capacidade de produzir lesões A/E. Empregamos qPCR para aferir a transcrição de três diferentes locus (eae, espA e tir) situados nos operons LEE4 e LEE5 de cepas bovinas e humanas, durante a infecção de células Caco-2. Verificamos diferenças na expressão dos genes, especialmente espA, entre cepas bovinas e humanas com maior expressão para estas ultimas, em linha com os achados dos testes FAS. Através de clonagem e expressão de proteínas recombinantes, purificamos as proteínas Eae, EspA e Tir e obtivemos anticorpos específicos, empregados para acompanhar a sua expressão ao longo da infecção de células Caco-2, por imunofluorescencia. Verificamos que as três proteínas são detectadas tanto em cepas bovinas quanto humanas, mas nestas ultimas, a marcação é precoce e torna-se mais intensa com o avanço da infecção. Nossos resultados indicam que cepas EHEC O157:H7 isoladas do reservatório bovino em nosso país apresentam diferenças importantes em relação ao perfil toxigenico e a capacidade de indução de lesões A/E, características apontadas na literatura como relevantes para a virulência do micro-organismo. Por outro lado, nossos achados quanto a capacidade de invadir e multiplicar-se no interior de enterócitos pode explicar a persistência do patógeno no reservatório animal e a sua capacidade de transmissão horizontal. / Recognized in 1982 as a human pathogen, enterohemorrhagic Escherichia coli (EHEC) causes bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). EHEC belonging to serotype O157:H7 are mostly important in North America, United Kingdom and Japan. Shiga toxin (Stx) is the critical factor of STEC. Stx is capable to interrupt the protein synthesis of the eukaryotic cell. Two subgroups of Stx are known, Stx1 and Stx2. Two variants of Stx1 are known (Stx1c and Stx1d), but several Stx2 variants have been described. Epidemiological studies suggest that STEC/EHEC strains carrying the toxigenic profiles Stx2 or Stx2/Stx2c are more frequently associated to HUS. Besides the expression of Stx, EHEC O157:H7 colonize the intestinal mucosa inducing the formation of characteristic histopathological lesions denominated attaching/effacing (A/E). To the production of A/E lesions, it is necessary the presence of a pathogenicity island called LEE (locus of enterocyte effacement), composed by five operons, LEE 1 to LEE5. An outer membrane adhesin (intimin) and its receptor Tir, which is translocated by a type three secretion sytem (TTSS), are both codified in LEE5 while the secreted proteins EspA, B and D, that constitute part of the SSTT, are codified in LEE4. Cattle are the main reservoir of this pathogen and foods of bovine origin and products contamined with bovine feces are common causes of epidemic outbreaks. In Brazil, EHEC O157:H7 can be isolated from the animal reservoir . Stx2c prevails among the bovine strains, while the toxigenic profiles Stx2 or Stx2/Stx2c are found among the human strains. Concerning the bacterial interaction with human enterocytes cultivated in vitro (Caco-2) we verified that both bovine and human strains showed almost identical ability to invade and to persist in the intracellular compartment of the Caco-2 cells. However, in comparison with the human strains, the bovine strains showed a reduced capacity to produce A/E lesions according to the FAS test. A quantitative FAS test confirmed the relative inefficiency of bovine strains to induce A/E lesions. We also used qPCR to follow the transcription of three genes (eae, espA and tir) of selected bovine and human strains, during the infection of Caco-2 cells. We verified differences in the gene expression, especially for espA, between bovine and human strains and these latter showed a larger expression, in line with the findings of the actin-aggregation tests. Through cloning and expression of recombinant proteins, we purified the Eae, EspA and Tir proteins and obtained specific antibodies, employed to follow the expression of those proteins, by immunofluorescence, along the infection of Caco-2 cells. We found that all proteins are detected both in bovine and human strains, but on these protein labeling occurs early and becomes more intense with the progress of the infection. Our results indicate that EHEC O157:H7 strains isolated from the bovine reservoir in Brazil shows, in comparison to strains isolated from human disease, important differences in relation to the toxigenic profile and the ability to induce A/E lesions. Our findings concerning the ability of the microorganism to invade and to multiply inside enterocytes can explain the persistence of the pathogen in the animal reservoir and its ability of horizontal transmission.

E. coli O157:H7 é um patógeno de importância em alimentos, tendo sido envolvido, nos últimos anos, em surtos de grandes proporções, principalmente por produtos cárneos. Entretanto sua ocorrência em alimentos, particularmente em carne crua, é baixa e poderia, eventualmente, ser atribuída à atividade antagônica expressa por outros microrganismos presentes. Assim sendo, foi avaliada a interferência de bactérias que fazem parte da microbiota normal de carne sobre a multiplicação de E. coli O157:H7 em carne bovina moída mantida em refrigeração e em temperatura ambiente. Com essa finalidade, foram realizados testes de desafio (\"challenge tests\") em porções de 25 g de carne bovina moída inoculadas com diferentes concentrações de E. coli O157:H7 (101, 103 e 106 CFC/g), desafiadas com diferentes inóculos de E. coli não patogênica, Pseudomonas putida e Leuconostoc spp. As cepas de Pseudamonas putida e de Leuconostoc spp., isoladas de carne, foram selecionadas em função de atividade inibitória contra E. calí O157:H7 observada \"in vitro\". Para o monitoramento de E. coli O157:H7, foram utilizados o método convencional, ou seja, plaqueamento em ágar Mac Conkey-sorbitol e identificação de colônias (testes bioquímicos e sorológicos), bem como um método considerado rápido, empregando o Petrifilm™ Kit-HEC. De maneira geral, não foram observadas interferências significativas da presença de diferentes inóculos de E. coli não patogênica, P. putida e Leuconostoc spp., sobre a multiplicação de diferentes inóculos de E. coli O157:H7 à temperatura ambiente e à temperatura de refrigeração. Paralelamente, o Petrifilm™ Kit-HEC revelou um alto índice de correlação com o ágar Mac Conkey-sorbitol (97,2%), com contagens da mesma ordem de grandeza. Os experimentos à temperatura ambiente revelaram um maior índice de correlação (99,0%), quando comparados àqueles à temperatura de refrigeração (94,9%). Aparentemente, a baixa ocorrência de E. coli O157:H7 em alimentos, particularmente em carne bovina crua, não pode ser atribuída à atividade antagônica de alguns microrganismos presentes. / Escherichia coli O157:H7 is a foodborne pathogen of increasing importance, since it has been involved in several threatening outbreaks, most of them associated with meat products. Though, it is possible that the low occurrence af E. coli O157:H7 in food, particularly in meat, may be due to antagonistic effects af other microorganisms present. Therefore, the influence of some bacteria isolated from meat, over E. coli O157:H7 in meat samples stored at chill and room temperatures was evaluated. For that purpose, studies were performed on 25 g of ground beef inoculated with different spiking levels of E. coli O157:H7 (101, 103 and 106 CFC/g), challenged with different spiking levels of non pathogenic E. coli, Pseudomonas putida or Leuconostoc spp. The Ps. putida and Leuconostoc spp. strains were selected based on deferred antagonism observed against E. coli O157:H7. Multiplication was monitored by means of cultural methods, employing sorbitol Mac Conkey agar and additional identification tests, and the rapid method Petrifilm™ Kit-HEC. No significant influence of non pathogenic E. coli, Pseudomonas putida and Leuconostoc spp. over the multiplication of E. coli O157:H7 was observed. Results on Petrifilm™ Kit-HEC showed high correlation with results on sorbitol Mac Conkey agar (97,2%). Experiments performed with meat kept at room temperatures resulted in higher correlation values (99,0%), when compared to those of meat kept at chill temperatures (94,9%). Apparently, the low occurrence of E. coli O157:H7 in food, particularly in raw meat, can\'t be attributed to antagonistic effects of other bacteria from natural microflora.

Les Escherichia coli entérohémorragiques (EHEC) sont des pathogènes zoonotiques responsables de toxi-infectionsalimentaires pouvant évoluer vers des atteintes potentiellement mortelles chez l’Homme. La survie des EHECet l’expression des gènes de virulence dans l’environnement digestif humain sont des facteurs essentiels dans laphysiopathologie de ces infections mais sont mal connus, essentiellement par manque de modèles d’études adaptés.L’absence de traitement spécifique a conduit à s’intéresser à des moyens préventifs et/ou curatifs alternatifs, commel’utilisation de probiotiques. L’objectif de ce travail de thèse est d’étudier le comportement de souches EHEC dansl’ensemble du tractus digestif et l’influence de souches probiotiques, en utilisant des approches in vitro et in vivocomplémentaires.In vitro, dans le tractus gastro-intestinal supérieur, on observe une mortalité bactérienne dans l’estomac, suivie d’unereprise de croissance dans les parties distales de l’intestin grêle. De plus, la survie des EHEC dépend à la fois de lasouche/sérotype étudié et de la matrice alimentaire dans laquelle les bactéries sont ingérées. En conditions coliqueshumaines simulées, les EHEC sont progressivement éliminés du milieu colique et leurs principaux gènes de virulence(stx1 codant la Shiga-toxine 1 et eae codant l’intimine) sont surexprimés dans les heures suivant l’inoculation dupathogène. L’ajout de levures probiotiques du genre Saccharomyces ne modifie pas la survie du pathogène dansl’environnement colique, que celles-ci soient administrées en traitement « curatif » ou « prophylactique ». Parcontre, l’administration de S. cerevisiae CNCM I-3856 permet (i) de moduler favorablement l’activité fermentairedu microbiote intestinal, en augmentant la production d’acétate et en réduisant celle du butyrate et (ii) de diminuersignificativement l’expression de stx1. Par ailleurs, l’effet du pathogène et des probiotiques sur le microbiote coliqueest individu dépendant, confortant l’hypothèse que des facteurs associés à l’hôte, comme le microbiote, pourraientconditionner l’évolution clinique des infections à EHEC et l’efficacité d’une stratégie probiotique. Enfin, dans unmodèle murin d’anses iléales, l’administration préventive de S. cerevisiae CNCM I-3856 limite significativementl’interaction d’O157:H7 avec les plaques de Peyer et les lésions hémorragiques associées.Ces résultats confirment donc l’intérêt d’une stratégie probiotique dans le contrôle des infections à EHEC. Uneétude plus approfondie du transcriptome du pathogène dans l’environnement digestif humain, en présence ou non deprobiotiques, permettrait de mieux comprendre la physiopathologie des infections à EHEC et les mécanismes associés / The enterohaemorrhagic Escherichia coli (EHEC) are zoonotic pathogens that cause food-borne infection withwhich leads to life-threatening damage in humans. EHEC survival and expression of virulence genes in the humandigestive track are key factors in the pathogenesis of these infections, but little is known, mainly due to lack ofappropriate study models. The absence of specific treatment has led to an interest in preventive and/or alternativemeasures healing, such as the use of probiotics. The objective of this study is the behavior of EHEC strains in theentire digestive tract and the influence of probiotic strains, using in vitro and in vivo complementary approaches.In vitro, in the upper gastrointestinal tract, a bacterial mortality was observed in the stomach, followed bya bacterial resumption in the distal segment of the small intestine. Moreover, survival depends on both theEHEC strain/serotype studied and the food matrix in which the bacteria are ingested. In simulated humancolon conditions, EHEC was progressively eliminated from the bioreactor and the major virulence genes (stx1encoding Shiga-toxin 1 and eae encoding intimin) are overexpressed in the hours following the inoculation ofpathogen. Probiotic yeasts Saccharomyces genus does not modify the survival of the pathogen in the in vitro colonicenvironment, that they be administered in treatment "curative" or "prophylactic". Still, the administration ofS. cerevisiae CNCM I-3856 allows (i) to favorably modulate fermentation activity of the intestinal microbiota, byincreasing the production of acetate and reducing that of butyrate and (ii) reduce significantly the expression ofstx1. Furthermore, the effect of pathogenic and probiotic on colonic microbiota is donor-dependent, supporting thehypothesis that factors associated with the host, as the microbiota could condition the clinical course of EHEC andefficiency a probiotic strategy. Finally, in a murine model of ileal loops, preventive administration of S. cerevisiaeCNCM I-3856 significantly limits the interaction of O157:H7 with the Peyer’s patches and results hemorrhagic lesions.These results confirms the interest of probiotic strategy in controlling EHEC infections. Further transcriptomestudies are warranted for the pathogen in the human digestive environment, with or without probiotics for the betterunderstanding of the pathophysiology of EHEC and so on the mechanisms involved in the antagonistic effect ofprobiotics.

Intimina é o principal fator de virulência envolvido na patogênese de Escherichia coli enteropatogênica (EPEC) e de Escherichia coli enterohemorrágica (EHEC). A detecção de EHEC e EPEC típica ou atípica é de fundamental importância na definição da conduta terapêutica das infecções promovidas por E. coli, que ainda são a principal causa de diarreia aguda em crianças e adultos em muitos países desenvolvidos e em desenvolvimento. Anticorpos são ferramentas importantes na detecção de diversos patógenos. Neste trabalho avaliou-se a sensibilidade e especificidade dos anticorpos policlonal e monoclonal anti-intimina frente a isolados de EPEC e EHEC por immunoblotting. Os anticorpos apresentaram 100% de especificidade e a sensibilidade foi de 97%, 92% e 78%, quando se utilizou a fração enriquecida em IgG do soro de coelho, antissoro de rato e anticorpo monoclonal, respectivamente. Esse anticorpo monoclonal anti-intimina foi caracterizado como IgG2b e 1 µg desse anticorpo reconheceu 0,6 µg de intimina purificada com uma constante de dissociação de 1.3 x 10-8 M. A menor reatividade do anticorpo monoclonal em relação aos anticorpos policlonais levou-nos à clonagem e expressão do fragmento variável de cadeia simples desse anticorpo (scFv). Para isso, o mRNA do hibridoma anti-intimina foi extraído, reversamente transcrito para cDNA e amplificadas as cadeias leve e pesada da fração variável do anticorpo, utilizando iniciadores aleatórios comerciais. As cadeias amplificadas foram ligadas ao vetor pGEM-T Easy e sequenciadas. Iniciadores específicos foram desenhados e utilizados em uma estratégia de amplificação e união das cadeias, formando o scFv, que por sua vez foi clonado no vetor de expressão pAE. Linhagem de E. coli BL21(DE3)pLys foi transformada com o plasmídeo pAE-scFv antiintimina e submetida à indução protéica. O scFv anti-intimina foi expresso de forma insolúvel, solubilizado, purificado e submetido ao ensaio de refolding. O rendimento obtido foi de 1 mg de proteína por 100 mL de cultivo bacteriano. Para testar a funcionalidade do scFv, foram realizados ensaios de ELISA de captura e imunofluorescência. Os resultados mostraram que 275 ng de scFv reagiram com 2 µg de intimina purificada a uma absorbância de aproximadamente 0,75 e por imunofluorescência mostrou uma forte reatividade ao isolado de EPEC típica E2348/69. Este estudo demonstrou que o anticorpo recombinante anti-intimina obtido foi capaz de reconhecer a região conservada de intimina (Int388-667) na forma purificada e a intimina α no isolado de EPEC típica, e se mostrou mais eficiente que o anticorpo monoclonal nativo. / Intimin is the major virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). The detection of EHEC and typical or atypical EPEC has fundamental importance in defining the therapeutic management of infections caused by E. coli, which are still the leading cause of acute diarrhea in children and adults in many developed and developing countries. Antibodies are important tools in the detection of several pathogens. In this study it was evaluated the sensitivity and specificity of polyclonal and monoclonal antibodies against intimin in the detection of EPEC and EHEC by immunoblotting. All employed antibodies showed 100% specificity and the sensitivity was 97%, 92% and 78% for rabbit anti-intimin IgG-enriched fraction, rat antisera and monoclonal antibody, respectively. This anti-intimin monoclonal was characterized as IgG2b and 1 mg recognized 0.6 µg of purified intimin with a dissociation constant of 1.3 x 10-8 M. The less extent reactivity of monoclonal led us to clone and express the single chain fragment variable of this antibody (scFv). Thus, the anti-intimin hybridoma mRNA was extracted, reverse transcribed to cDNA and the light and heavy chains of variable fragment of the antibody were amplified using commercial random primers. The chains were amplified, ligated to the pGEM-T Easy vector and the insert was sequenced. Specific primers were designed and used in a strategy to amplify and link the chains, obtaining the scFv, which was cloned into the pAE expression vector. E. coli BL21(DE3)plys was transformed with the pAE-scFv anti-intimin plasmid and subjected to induction of protein expression. The scFv anti-intimin, expressed in the insoluble fraction, was purified and submitted to refolding. The yield was 1 mg of protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed. The results showed that 275 ng of scFv reacted with 2 µg of purified intimin resulting in an absorbance of 0.75. By immunofluorescence it was observed a strong reactivity to the typical EPEC isolate E2348/69. This study demonstrated that the recombinant anti-intimin antibody obtained was able to recognize the conserved region of intimin (Int388-667) in its purified form and α intimin in a typical EPEC isolate, and was more efficient than the native monoclonal antibody.

Shiga Toxin (Stx)-konvertierende Bakteriophagen besitzen eine konservierte lambdo-ide Genomstruktur, weisen aber ein hohes Maß an genetischer Diversität auf. Aus diesem Grund wurden die bereits vorhandenen Sequenzdaten der Stx1-Phagen CP-1639 des Escherichia coli O111:H- Stammes 1639/77 und BP-4795 des E. coli O84:H4 Stammes 4795/97 vervollständigt und analysiert. Im Gegensatz zu dem induzierbaren Bakteriophagen BP-4795 fehlen CP-1639 einige Gene, die für den lytischen Lebenszyklus eines Bakteriophagen notwendig sind. Daher handelt es sich bei CP-1639 um einen kryptischen Prophagen, der nicht mehr in der Lage ist das Wirtschromosom zu verlassen und intakte Phagenpartikel zu bilden. Die Integra-tionsstellen wurden innerhalb des Gens yehV für BP-4795 und ssrA für CP-1639 bestimmt. In unmittelbarer Umgebung des Integrationsortes von CP-1639 befindet sich ein eigenständiges integratives Element. Dieses besteht aus drei offenen Lese-rahmen unbekannter Herkunft, sowie einem Integrasegen und kann auch ohne Assoziation mit Phagen-DNA auftreten. Phagen können zusätzlich zu ihrem Genom Gene bakteriellen Ursprungs tragen. Diese können unter anderem infolge von Transpositionen oder einer unkorrekten Exzision der Phagen-DNA aus dem Wirtschromosom während des lytischen Lebenszyklus in das betreffende Phagengenom eingebaut werden. Eines solches Gen des Bakteriophagen BP-4795 kodiert das Typ III Effektorprotein NleA4795, dessen Funktionalität nach dem C-terminalen Einbau von neun Codons des Hämagglutinin (HA)-Epitopes des humanen Influenzavirus in die Sequenz des Gens nleA4795 überprüft wurde. Dies erfolgte unter Verwendung der Western Blot Analyse durch die Expression dieses Fusionsproteins in dem Wildtyp-Stamm 4795/97 und in der Deletionsmutante 4795escN des Stammes 4795/97. Die Typ III Sekretionssys-tem (T3SS)-inaktive Mutante 4795escN wurde im Verlauf dieser Arbeit hergestellt. Diese Untersuchungen zeigten ebenfalls, dass zur Sekretion von NleA4795 ein in-taktes T3SS notwendig ist. Weiterhin zeigte die Infektion einer HeLa-Zelllinie mit NleA4795-HA exprimierenden Bakterienstämmen und die anschließende Analyse mit Hilfe der Immunfluoreszenz, die Translokation des Proteins in eukaryotische Wirts-zellen. Darüber hinaus deuten die Ergebnisse dieser Untersuchung auf eine Lokali-sation von NleA4795-HA innerhalb des Trans-Golgi Netzwerkes hin. Die Verbreitung des Gens nleA wurde in insgesamt 170 Shiga Toxin-produzierenden E. coli und enteropathogenen E. coli Stämmen untersucht. Dies führte zur Identifika-tion von 14 verschiedenen Varianten des Gens nleA in 149 der überprüften Stämme, wobei mit Ausnahme von zwei Isolaten ebenfalls ein Markergen des T3SS nachge-wiesen werden konnte. Neben drei bereits bekannten Varianten konnten elf neue Varianten identifiziert werden, deren abgeleitete Aminosäuresequenzen sich zu 71% bis 96% glichen. Sequenzunterschiede traten insbesondere aufgrund der Deletion oder Insertion von vier bis 51 Aminosäuren im Mittelteil der potentiellen Proteine auf. Weiterhin deuten die Ergebnisse dieser Untersuchungen eine Assoziation bestimm-ter Varianten des Gens nleA mit spezifischen E. coli Serogruppen an. Southern-Blot Hybridisierungen zeigten, dass etwa ein Viertel der nleA-positiven Stämme zwei Ko-pien dieses Gens in ihrem Genom tragen. Hierbei handelt es sich meist um zwei verschiedene Varianten. In fast allen Fällen kodiert eine dieser Varianten, aufgrund einer Punktmutation oder Insertion eines IS-Elementes, ein verkürztes und vermut-lich nicht funktionelles Protein. Mit Hilfe von Transduktionsexperimenten konnten verschiedene Varianten des Gens nleA im Genom induzierbarer Phagen nachge-wiesen werden, was auf eine Verbreitung des Gens nleA durch den horizontalen Gentransfer hinweist.

Intimina é o principal fator de virulência envolvido na patogênese de Escherichia coli enteropatogênica (EPEC) e de Escherichia coli enterohemorrágica (EHEC). A detecção de EHEC e EPEC típica ou atípica é de fundamental importância na definição da conduta terapêutica das infecções promovidas por E. coli, que ainda são a principal causa de diarreia aguda em crianças e adultos em muitos países desenvolvidos e em desenvolvimento. Anticorpos são ferramentas importantes na detecção de diversos patógenos. Neste trabalho avaliou-se a sensibilidade e especificidade dos anticorpos policlonal e monoclonal anti-intimina frente a isolados de EPEC e EHEC por immunoblotting. Os anticorpos apresentaram 100% de especificidade e a sensibilidade foi de 97%, 92% e 78%, quando se utilizou a fração enriquecida em IgG do soro de coelho, antissoro de rato e anticorpo monoclonal, respectivamente. Esse anticorpo monoclonal anti-intimina foi caracterizado como IgG2b e 1 µg desse anticorpo reconheceu 0,6 µg de intimina purificada com uma constante de dissociação de 1.3 x 10-8 M. A menor reatividade do anticorpo monoclonal em relação aos anticorpos policlonais levou-nos à clonagem e expressão do fragmento variável de cadeia simples desse anticorpo (scFv). Para isso, o mRNA do hibridoma anti-intimina foi extraído, reversamente transcrito para cDNA e amplificadas as cadeias leve e pesada da fração variável do anticorpo, utilizando iniciadores aleatórios comerciais. As cadeias amplificadas foram ligadas ao vetor pGEM-T Easy e sequenciadas. Iniciadores específicos foram desenhados e utilizados em uma estratégia de amplificação e união das cadeias, formando o scFv, que por sua vez foi clonado no vetor de expressão pAE. Linhagem de E. coli BL21(DE3)pLys foi transformada com o plasmídeo pAE-scFv antiintimina e submetida à indução protéica. O scFv anti-intimina foi expresso de forma insolúvel, solubilizado, purificado e submetido ao ensaio de refolding. O rendimento obtido foi de 1 mg de proteína por 100 mL de cultivo bacteriano. Para testar a funcionalidade do scFv, foram realizados ensaios de ELISA de captura e imunofluorescência. Os resultados mostraram que 275 ng de scFv reagiram com 2 µg de intimina purificada a uma absorbância de aproximadamente 0,75 e por imunofluorescência mostrou uma forte reatividade ao isolado de EPEC típica E2348/69. Este estudo demonstrou que o anticorpo recombinante anti-intimina obtido foi capaz de reconhecer a região conservada de intimina (Int388-667) na forma purificada e a intimina α no isolado de EPEC típica, e se mostrou mais eficiente que o anticorpo monoclonal nativo. / Intimin is the major virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). The detection of EHEC and typical or atypical EPEC has fundamental importance in defining the therapeutic management of infections caused by E. coli, which are still the leading cause of acute diarrhea in children and adults in many developed and developing countries. Antibodies are important tools in the detection of several pathogens. In this study it was evaluated the sensitivity and specificity of polyclonal and monoclonal antibodies against intimin in the detection of EPEC and EHEC by immunoblotting. All employed antibodies showed 100% specificity and the sensitivity was 97%, 92% and 78% for rabbit anti-intimin IgG-enriched fraction, rat antisera and monoclonal antibody, respectively. This anti-intimin monoclonal was characterized as IgG2b and 1 mg recognized 0.6 µg of purified intimin with a dissociation constant of 1.3 x 10-8 M. The less extent reactivity of monoclonal led us to clone and express the single chain fragment variable of this antibody (scFv). Thus, the anti-intimin hybridoma mRNA was extracted, reverse transcribed to cDNA and the light and heavy chains of variable fragment of the antibody were amplified using commercial random primers. The chains were amplified, ligated to the pGEM-T Easy vector and the insert was sequenced. Specific primers were designed and used in a strategy to amplify and link the chains, obtaining the scFv, which was cloned into the pAE expression vector. E. coli BL21(DE3)plys was transformed with the pAE-scFv anti-intimin plasmid and subjected to induction of protein expression. The scFv anti-intimin, expressed in the insoluble fraction, was purified and submitted to refolding. The yield was 1 mg of protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed. The results showed that 275 ng of scFv reacted with 2 µg of purified intimin resulting in an absorbance of 0.75. By immunofluorescence it was observed a strong reactivity to the typical EPEC isolate E2348/69. This study demonstrated that the recombinant anti-intimin antibody obtained was able to recognize the conserved region of intimin (Int388-667) in its purified form and α intimin in a typical EPEC isolate, and was more efficient than the native monoclonal antibody.

Les bovins sont porteurs de souches d'Escherichia coli entérohémorragiques (EHEC), pathogènes pour l'homme et également de souches d'E. coli résistantes aux antibiotiques. Dans un premier temps, nous avons évalué la fréquence de ces souches dans les effluents de station d'épuration des eaux usées de deux abattoirs, l'un de bovins adultes et l'autre de veaux de boucherie. Les pourcentages d'E. coli antibiorésistantes et porteuses d'intégrons de résistance de classe 1 étaient significativement plus élevés dans les effluents et boues d'abattoirs de veaux (87,5%, 56,2%) par rapport aux bovins adultes (5,0%, 0%). Ces pourcentages n'étaient pas modifiés par le traitement épuratoire. Le traitement épuratoire n'a également eu aucun impact sur les pourcentages de souches d'E. coli productrices de shigatoxines (STEC). Une souche STEC O157:H7 hautement pathogène a été isolée des boues destinées à l'épandage agricole dans la station d'épuration d'abattoir de bovins adultes. Ces résultats ont confirmé qu'il existe des risques de dissémination environnementale d'E. coli antibiorésistantes et/ou pathogènes via les effluents d'abattoir de bovins et ont mis en évidence que ce risque était différent selon la catégorie de bovins abattus. Dans un deuxième temps, nous avons évalué la résistance de souches STEC du top 5 (O26:H11, O103:H2, O111:H8, O145:H28 et O157:H7) isolées de fèces de bovins adultes. Sept des 39 souches STEC testées étaient résistantes, dont 6 à au moins 3 classes d'antibiotiques. Les souches non-STEC et aEPEC du top 5 d'E. coli de la flore fécale de ces mêmes bovins étaient toutes sensibles, indiquant un possible lien génétique entre gènes de résistance et virulence. Nous avons mis en évidence que le gène ehxA, marqueur fiable du plasmide de virulence des EHEC, et les gènes de résistance blaTEM, strA-strB, tet(A), sulII étaient localisés sur un même plasmide de grande taille pour 4 souches STEC (1 O26:H11, 1 O103:H2 et 2 O111:H8). Cette association génétique soulève la problématique de la sélection clonale de ces souches pathogènes lors du traitement des bovins porteurs avec des antibiotiques. / Cattle are known to be reservoir of enterohemorrhagic Escherichia coli (EHEC), pathogenic for humans and antibiotic resistant E. coli as well. In a first stage, we assessed frequencies of these strains in two bovine slaughterhouse wastewater treatment plants, one slaughtered only adult cattle and the other only veal calves. Percentages of resistant and class 1 integron-bearing E. coli were significantly higher in veal calves effluents and thickened sludges (87.5%, 56.2%) compared to those of adult cattle (5.0%, 0%). These percentages were not impacted by treatment process. The treatment had no impact on percentages of Shiga toxin-producing E. coli (STEC) either. A STEC O157:H7 highly pathogenic for humans was isolated from the thickened sludge of the adult cattle slaughterhouse, intended to be spread on agricultural lands. These results confirmed that bovine slaughterhouse effluents might contribute to the environmental dissemination of antibiotic resistant and/or pathogenic E. coli and underlined that the risks of dissemination differ according to slaughtered bovine category. In a second stage, we assessed the antibiotic resistance of top 5 STEC (O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7) isolated from adult cattle fecal samples. Seven of the 39 top 5 STEC were resistant, of which 6 resistant to at least 3 classes of tested antibiotics. Non-top 5 STEC and aEPEC E. coli strains from fecal flora of the same bovine carriers were all susceptible to the tested antibiotics, indicating a possible link between EHEC-associated virulence genes and antibiotic resistance genes. We showed that ehxA gene, which is a reliable marker of the EHEC virulence plasmid, and antibiotic-resistance genes blaTEM, strA-strB, tet(A), sulII were located on a same large plasmid in 4 antibiotic-resistant top 5 STEC strains (1 O26:H11, 1 O103:H2 and 2 O111:H8). This genetic association raises the concern about the clonal selection of such pathogenic strains by antibiotic use in bovine carriers.

Der zeitnahe und möglichst sichere Nachweis bakterieller Krankheitserreger und deren Empfindlichkeit gegenüber verfügbaren antibakteriell wirksamen Chemotherapeutika (Antibiotika) stellt einen Hauptaufgabenbereich der medizinischen mikrobiologischen Routinediagnostik dar. Hierzu wurden im Laufe der Jahre unterschiedliche Methoden entwickelt, womit von der genauen Beschreibung der Kolonie- und mikroskopischen Morphologie, Anfärbbarkeit und Formation über die Charakterisierung der biochemischen Leistungsfähigkeit bis hin zur genauen Sequenzierung des gesamten Genoms ein enormer Fortschritt zu verzeichnen war. Seit Mitte der 1990er Jahre etablierte sich die Massenspektrometrie als phänotypisches Nachweisverfahren und gewann zunehmend an Bedeutung. Ebenso konnten Erfolge beim Nachweis Antibiotika resistenter Bakterien verzeichnet werden. Um das Potential dieser noch jungen Nachweismethode weiter zu erforschen, wurden in dieser Arbeit Spezies der Familie Enterobacteriaceae und der Nonfermenter in eine eigene massenspektrometrische Datenbank aufgenommen, um diese als Grundlage zur Validierung des Identifizierungspotentials der Methode mittels Blindstudie zu nutzen. Im selben Arbeitsschritt wurde der Versuch unternommen, Antibiotika resistente Stämme im Zuge der Speziesidentifizierung zu detektieren, um so Aussagen über eine mögliche Einschränkung der therapeutischen Möglichkeiten und gegebenenfalls notwendigen Hygienemaßnahmen treffen zu können.

Shiga Toxin (Stx)-konvertierende Bakteriophagen besitzen eine konservierte lambdo-ide Genomstruktur, weisen aber ein hohes Maß an genetischer Diversität auf. Aus diesem Grund wurden die bereits vorhandenen Sequenzdaten der Stx1-Phagen CP-1639 des Escherichia coli O111:H- Stammes 1639/77 und BP-4795 des E. coli O84:H4 Stammes 4795/97 vervollständigt und analysiert. Im Gegensatz zu dem induzierbaren Bakteriophagen BP-4795 fehlen CP-1639 einige Gene, die für den lytischen Lebenszyklus eines Bakteriophagen notwendig sind. Daher handelt es sich bei CP-1639 um einen kryptischen Prophagen, der nicht mehr in der Lage ist das Wirtschromosom zu verlassen und intakte Phagenpartikel zu bilden. Die Integra-tionsstellen wurden innerhalb des Gens yehV für BP-4795 und ssrA für CP-1639 bestimmt. In unmittelbarer Umgebung des Integrationsortes von CP-1639 befindet sich ein eigenständiges integratives Element. Dieses besteht aus drei offenen Lese-rahmen unbekannter Herkunft, sowie einem Integrasegen und kann auch ohne Assoziation mit Phagen-DNA auftreten. Phagen können zusätzlich zu ihrem Genom Gene bakteriellen Ursprungs tragen. Diese können unter anderem infolge von Transpositionen oder einer unkorrekten Exzision der Phagen-DNA aus dem Wirtschromosom während des lytischen Lebenszyklus in das betreffende Phagengenom eingebaut werden. Eines solches Gen des Bakteriophagen BP-4795 kodiert das Typ III Effektorprotein NleA4795, dessen Funktionalität nach dem C-terminalen Einbau von neun Codons des Hämagglutinin (HA)-Epitopes des humanen Influenzavirus in die Sequenz des Gens nleA4795 überprüft wurde. Dies erfolgte unter Verwendung der Western Blot Analyse durch die Expression dieses Fusionsproteins in dem Wildtyp-Stamm 4795/97 und in der Deletionsmutante 4795escN des Stammes 4795/97. Die Typ III Sekretionssys-tem (T3SS)-inaktive Mutante 4795escN wurde im Verlauf dieser Arbeit hergestellt. Diese Untersuchungen zeigten ebenfalls, dass zur Sekretion von NleA4795 ein in-taktes T3SS notwendig ist. Weiterhin zeigte die Infektion einer HeLa-Zelllinie mit NleA4795-HA exprimierenden Bakterienstämmen und die anschließende Analyse mit Hilfe der Immunfluoreszenz, die Translokation des Proteins in eukaryotische Wirts-zellen. Darüber hinaus deuten die Ergebnisse dieser Untersuchung auf eine Lokali-sation von NleA4795-HA innerhalb des Trans-Golgi Netzwerkes hin. Die Verbreitung des Gens nleA wurde in insgesamt 170 Shiga Toxin-produzierenden E. coli und enteropathogenen E. coli Stämmen untersucht. Dies führte zur Identifika-tion von 14 verschiedenen Varianten des Gens nleA in 149 der überprüften Stämme, wobei mit Ausnahme von zwei Isolaten ebenfalls ein Markergen des T3SS nachge-wiesen werden konnte. Neben drei bereits bekannten Varianten konnten elf neue Varianten identifiziert werden, deren abgeleitete Aminosäuresequenzen sich zu 71% bis 96% glichen. Sequenzunterschiede traten insbesondere aufgrund der Deletion oder Insertion von vier bis 51 Aminosäuren im Mittelteil der potentiellen Proteine auf. Weiterhin deuten die Ergebnisse dieser Untersuchungen eine Assoziation bestimm-ter Varianten des Gens nleA mit spezifischen E. coli Serogruppen an. Southern-Blot Hybridisierungen zeigten, dass etwa ein Viertel der nleA-positiven Stämme zwei Ko-pien dieses Gens in ihrem Genom tragen. Hierbei handelt es sich meist um zwei verschiedene Varianten. In fast allen Fällen kodiert eine dieser Varianten, aufgrund einer Punktmutation oder Insertion eines IS-Elementes, ein verkürztes und vermut-lich nicht funktionelles Protein. Mit Hilfe von Transduktionsexperimenten konnten verschiedene Varianten des Gens nleA im Genom induzierbarer Phagen nachge-wiesen werden, was auf eine Verbreitung des Gens nleA durch den horizontalen Gentransfer hinweist.