Les gastroentérites aiguës (GEA) représentent un lourd fardeau pour la population pédiatrique. Elles sont responsables d’une mortalité importante, particulièrement chez les enfants de moins de 5 ans dans les pays à faibles revenus, et ont un impact socio-économique non négligeable dans les pays à hauts revenus. La prévalence des différents entéropathogènes potentiellement impliqués dans les GEA varie selon les études, dépendamment de la technique diagnostique utilisée, de la population étudiée – âge des patients, co-morbidités, situation géographique et socio-économique – et du moment où l’étude a été réalisée.Campylobacter est l’un des pathogènes entériques majeurs dans les pays à hauts revenus. Les espèces Campylobacter jejuni et coli sont les plus fréquemment retrouvées par les méthodes de culture sur des milieux sélectifs utilisées en routine dans la plupart des laboratoires de microbiologie. Cependant l’utilisation d’autres méthodes, comme la technique « de filtration » ou les techniques de PCR, permet de mettre en évidence d’autres campylobacters tels que Campylobacter concisus dont le rôle dans les GEA est sujet à controverse.Dans ce contexte, l’objectif général de ce travail de thèse est l’amélioration de la prise en charge diagnostique des GEA en pédiatrie à Bruxelles. Cet objectif se détaille en deux sousobjectifs: d’abord une étude de prévalence des entéropathogènes - et potentiels entéropathogènes -, ensuite une amélioration de techniques diagnostiques. Ces éléments sont détaillés ci-dessous.La première partie de ce travail nous a permis de recruter deux groupes de patients :l’un atteint de GEA (185 cas) et l’autre asymptomatique (179 témoins), mais comparables notamment en termes d’âge, de fréquentation de la crèche ou de l’école, de vaccination contre rotavirus, et de traitement par antibiotique. Au vu des techniques diagnostiques utilisées dans notre étude, Campylobacter jejuni-coli était le principal reponsable de GEA dans notre population (14% des cas), suivi de rotavirus (11% des cas). Seules 6 souches de C. concisus ont pu être retrouvées parmi les cas et 4 parmi les contrôles, ceci ne nous permettant pas de tirer de conclusions quant à un éventuel rôle de ce germe.Malgré une couverture vaccinale satisfaisante de plus de 80% parmi les cas et les témoins recrutés, rotavirus reste le deuxième entéropathogène en termes d’importance avec 11% des cas infectés dans la population étudiée. Si le calcul de l’efficacité vaccinale réalisé dans la seconde partie de ce travail n’a pas montré de résultat statistiquement significatif, les enfants de moins de 12 mois comptaient significativement plus de cas de GEA à rotavirus chez les non vaccinés que chez les vaccinés. La couverture vaccinale pourrait donc encore être améliorée afin de mieux couvrir cet âge à risque de GEA compliquée.Le faible nombre de souches de C. concisus retrouvé dans la première partie du travail nous a poussé à tenter d’améliorer la technique de culture dite « de filtration » utilisée au LHUB-ULB pour la mise en évidence des campylobacters, particulièrement des « non jejuni-coli ». En trois étapes, nous avons pu montrer la supériorité de la combinaison comprenant la gélose Columbia contenant 5% de sang de mouton, avec des filtres en polycarbonate comportant des pores de 0,60 μm de diamètre et une mise en culture dans une atmosphère microaérophile enrichie en hydrogène (7%) afin d’obtenir une meilleure sensibilité de la technique de filtration. Ces améliorations ont fait passer C. concisus en première position en termes de fréquence de Campylobacter, devant C. jejuni.Cette proposition de standardisation de la méthode permettra de faciliter la comparaison de futures études sur le sujet et d’augmenter le nombre de souches de C. concisus isolées afin de tester les hypothèses proposées de génotypes potentiellement pathogènes et de facteurs devirulence sur un échantillon plus large de souches.Parmi les techniques diagnostiques actuelles en microbiologie, les méthodes basées sur l’amplification d’acides nucléiques sont passées sur le devant de la scène, attrayantes par leur rapidité de résultats et par leur haut taux de réponse positive. L’une de ces techniques, le Luminex xTAG GPP a pu être testé sur les échantillons des cas et des témoins. Les résultats soulèvent quelques questions concernant l’utilité de cette technique pour la prise en charge clinique des patients au vu des hauts taux de positivité chez cas et témoins impliquant une réserve dans l’interprétation des résultats, particulièrement pour Salmonella. Certains faux négatifs gênent également l’implémentation en routine de ce test :certaines bactéries retrouvées en culture (Shigella, Yersinia, Campylobacter) ne sont pas détectées par le Luminex. Son intérêt est donc faible en clinique dans l’état actuel de la méthode.Ce travail permet de poser des balises pour l’interprétation des tests microbiologiques effectués et d’attirer l’attention des cliniciens sur l’importance de rester critique en ce qui concerne les résutats obtenus :un résultat positif n’indique pas systématiquement que l’entéropathogène détecté est responsable de la clinique présentée et un négatif ne l’absout pas pour autant. Pareillement, les microbiologistes doivent connaître les besoins des cliniciens afin de proposer des tests qui peuvent y répondre, tant en réduisant le délai de réponse qu’en améliorant la pertinence de celle-ci, selon le contexte de la demande :un individu malade, une prise en charge d’épidémie, une étude épidémiologique. Autant de situations où une discussion et une réflexion sont nécessaires afin d’améliorer la prise en charge des patients et d’éliminer les tests inadéquats générant des coûts inutiles pour le patient, l’hôpital et la société. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Resumo: Para verificar os fatores de risco e a viabilidade de métodos diagnósticos para a campilobacteriose genital bovina (CGB), amostras prepuciais de touros de 102 rebanhos leiteiros de Presidente Prudente, São Paulo, foram avaliadas por testes bacteriológicos, reação da polimerase em cadeia (PCR) e imunofluorescência direta (IFD). Durante a colheita de lavados prepuciais, um questionário sobre desordens reprodutivas nas fêmeas e condições sanitárias foi aplicado aos proprietários das fazendas. As amostras foram plaqueadas em ágar seletivo de Skirrow, ágar Brucella verde brilhante e ágar tioglicolato com 20% do sangue. As características seletivas dos três sistemas da cultura foram comparadas pela avaliação de escores de densidade de contaminantes bacterianos nas placas e a capacidade de suportar diferentes fontes de contaminantes. A PCR foi conduzida usando seqüências dos nucleotídeos que amplificam um fragmento com 835 pares das bases do DNA ribossomal bacteriano. A IFD foi executada usando o conjugado anti-C. fetus subesp. venerealis marcado com isotiocianato de fluoresceína. Resultados positivos foram obtidos para culturas microbianas, PCR e IFD de 7,8% , 5,8% e 1,9% das fazendas, respectivamente. Detectou-se associação significativa entre touros positivos e rebanhos cujas vacas apresentavam taxas de concepção abaixo de 85% (OR=7,6). A concordância entre a PCR e as culturas bacteriológicas foi considerada ótima (Kappa = 0,847), mas foi pobre entre a IFD e os outros testes. Apenas o ágar seletivo de Skirrow não foi influenciado por contaminantes do lavado prepucial. Conclui-se que a CGB está presente em rebanhos leiteiros de Presidente Prudente, onde é associada com os distúrbios reprodutivos. / Abstract: To verify the risk factors and viability of diagnostic methods for genital bovine campylobacteriosis (GBC), preputial samples from bulls from 102 dairy herds from Presidente Prudente, São Paulo State, were evaluated using bacteriological tests, polymerase chain reation (PCR) and direct immunofluorescence (DFA). During the preputial sampling, some questions were made about female reproductive disorders and sanitary conditions of the farms. The preputial samples were plated in Skirrowþs agar, Brucella Brilliant green agar and Thioglycolate agar with 20% of blood. The selective characteristic of the three systems of culture was compared by the evaluation of bacterial density scores in the plates and for the capacity to support bacterial contaminants of different sources. The PCR was conducted using sequences of nucleotides that amplify a fragment with 835 pairs of bases of the ribossomal DNA. Direct immunofluorescence was performed using anti-C. fetus subesp. venerealis conjugated with fluorescein isothiocyanate. Positive results were obtained by microbial cultures, PCR and DFA results of 7,8%, 5,8% and 1,9% of the farms, respectively. Statistical association between positive bulls and low fertility of the herd cows was detected (OR= 7,6). The overall agreement between the PCR and bacteriological cultures was excellent (Kappa = 0,847), but poor between DFA and the other tests. The Skirrowþs media was the only that was not influenced by contaminant characteristics of preputial fluid samples. It is concluded that GBC is present in dairy herds from Presidente Prudente, where was associated with reproductive disturbances. / Orientador: José Rafael Modolo / Coorientador: Andrey Pereira Lage / Banca: Marcelo George Mungai Chacur / Banca: Antônio Carlos Paes / Banca: Ary Fernandes Junior / Banca: Luis Carlos de Souza / Doutor

Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes microorganismos em diferentes habitats tais como: água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de C. jejuni de origem avícolas para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino, equino e bovino, sendo cada sangue testado isoladamente. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2 , MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de Campylobacter jejuni em placas de TSA – sangue, foi possível observar que houve hemólises em 48,89% das amostras quando utilizado sangue eqüino, em 40% em sangue de bovino e em 31,11% quando de ovino. Quanto à influência de agentes quelantes e íons em caldo TSB na atividade de hemolisinas em amostras de Campylobacter jejuni semeadas em placas de TSA – sangue ovino, foi observada atividade hemolítica em 26,67% quando utilizado CaCl2, 15,55% (FeCl3), 22,22% (EDTA), 11,11% (MgCl2) e apenas 2,22% (ácido acético). No tocante à atividade hemolítica, o TSA - sangue bovino apresentou 15,55% (CaCl2), 24,44% (FeCl3), 26,26% (EDTA), 20% (MgCl2) e 11,11% (ácido acético). A atividade hemolítica para o sangue equino foi de 24,44% (CaCl2), 22,22% (FeCl3), 28,89% (EDTA), 28,89% (MgCl2) e 8,89% (ácido acético). Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação da Polimerase em Cadeia (PCR), foram utilizadas 119 amostras de C. jejuni de origem avícolas. Foi possível observar que 38% possuíam os três genes, e foram identificados somente os genes cdtA e cdtC em 19% do total de amostras, sendo que o gene cdtB foi encontrado em 14%, o gene cdtC foi observado em 12%, os genes cdtA e cdtB em somente 1%, os genes cdtB e cdtC em 1% e para cdtA em 1%. Observou-se que os resultados são dignos de atenção, pois demonstraram em amostras avícolas a presença de estirpes de C. jejuni com potencial virulento. A atividade hemolítica apresentou significativo aumento quando utilizado sangue de origem equina. A mesma foi diminuída quando utilizados agentes quelantes ou íons, nos três tipos de sangue. / Thermophilic members of the Campylobacter genus are recognized as important enteropathogenics for humans and also for other animals. The great diversity of ecological habitats in different organisms such as water, food, and animals may promote new virulence factors. This study aimed at detecting the distending cytolethal toxin (CDT) encoding genes by PCR, studying the activity of hemolysin and also the influence of chelation solutions and ions. A total of 45 samples of C. jejuni from poultry origin, grown in Tryptone Soy Broth (TSB) were used for investigating hemolytic activity. After bacterial growth, samples were plated on Tryptic Soy Agar (TSA) containing 5% sheep, equine or bovine blood, being each blood tested individually. In order to check the influence of chelation agents and ions solution on the hemolytic activity, samples of C. jejuni strains were grown in TSB containing chelation agents individually: EDTA, acetic acid, CaCl2 ion, MgCl2 and FeCl3 solutions, all in microaerophilic atmosphere. Regarding the detection of Campylobacter jejuni hemolysin activity on TSA plates, blood hemolysis were observed in 48.89 % of samples when equine blood was used; in 40% of samples when bovine blood was used and in 31.11 % when the blood used was of sheep origin. The influence of ions and chelation agents in hemolysin activity in TSB when Campylobacter jejuni was plated on TSA with sheep blood can be described as: hemolytic activity was observed at 26.67% of samples when CaCl2 was used, at 15.55 % for FeCl3, 22 22 % for EDTA, 11.11 % for MgCl2 and only 2.22% when acetic acid was used. The hemolytic activity detected when bovine blood - TSA was used indicated 15.55% for CaCl2, 24.44% for FeCl3, 26.26 % for EDTA, 20 % for MgCl2 and 11.11% for acetic acid. In terms of the hemolytic activity when equine blood was used, the results indicated 24.44% for CaCl2, 22.22 % for FeCl3, 28.89 % for EDTA, 28.89 % for MgCl2 and 8.89% for acetic acid. Finally, regarding the detection of cdtA, cdtB and cdtC through PCR, 119 samples of C. jejuni from poultry origin were used. The results indicated that all three genes were present in 38 % of the samples, whereas only two genes were identified in 19 % of samples, while the cdtB gene was singly found in 14%, the cdtC gene was independently observed in 12%, cdtA and cdtB genes together were found in 1% of the samples; the cdtB and cdtC genes associated were detected in 1%, while cdtA alone answered for 1% of detections. The results also showed the presence of C. jejuni strains with virulence potential. The hemolytic activity increased significantly when blood of equine origin was used, and that this activity was reduced when ions or chelating agents were used in combination with the three types of blood cells.

<p>Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.</p>

Campylobacter is the major bacterial cause of human gastroenteritis in the United States and other developed countries. Poultry are considered a main source of human Campylobacter infections. Thus, reduction of Campylobacter load in poultry is significant in food safety and public health. However, no effective measure is commercially available to prevent Campylobacter colonization in poultry to date. Antimicrobial peptides (AMPs) are short and bactericidal peptides widely present in intestine to limit bacterial infections. Recently, AMPs have been increasingly recognized as a novel class of antibiotics (peptide antibiotics) to control foodborne pathogens. Notably, several potent anti-Campylobacter bacteriocins, a group of AMPs produced by commensal bacteria, dramatically reduced C. jejuni colonization in chickens and are being directed toward on-farm control of this pathogen to protect public health. As an important strategy to evade killing by potential peptide antibiotics and by host innate defense, AMP resistance mechanisms in C. jejuni are critical to understand, but are still unknown. In this dissertation, molecular basis of Campylobacter resistance to polymyxin B, the anti-Campylobacter bacteriocins (BCNs), and a chicken host defense AMP (fowlicidin-1) was comprehensively examined using both in vitro and in vivo systems. Although polymyxin B has been successfully used as a model peptide to study AMP resistance in other Gram-negative bacteria, functional genomics examination in this study suggested that polymyxin B is not a good surrogate to study Campylobacter resistance to physiologically relevant AMPs. Campylobacter only developed low-level BCN resistance with low frequency in vitro and in vivo; the acquired BCN resistance was not stable in Campylobacter. Genomic examination of two BCN resistant mutants using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic and acquired resistance of Campylobacter to the BCNs. Random transposon mutagenesis and targeted site-directed mutagenesis identified four genes (cbrR, tig, cjaB, and cj1583c) involved in Campylobacter resistance to fowlicidin-1. These genes were also required for optimal colonization of Campylobacter in chickens. Together, the findings from this dissertation revealed uniqueness and complexity of AMP resistance in Campylobacter and will enable us to develop more sustainable peptide antibiotics and novel intervention strategies to prevent and control Campylobacter infections in humans and animal reservoirs. Key words: Campylobacter, antimicrobial peptide resistance, polymyxin B, bacteriocins, fowlicidins

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the United States. Increasing resistance of Campylobacter to clinical antibiotics raises an urgent need for novel strategies to prevent and control infections in humans and animal reservoirs, which necessitates a better understanding of Campylobacter pathogenesis. We hypothesize that multidrug efflux pump CmeABC and ferric enterobactin (FeEnt) iron acquisition systems, which play a critical role in Campylobacter pathogenesis, are novel targets for developing effective measures against Campylobacter. To test this, the molecular, antigenic, functional, and protective characteristics of two outer membrane proteins, CmeC (an essential component of CmeABC drug efflux pump) and CfrA (a FeEnt receptor), were examined. Both CmeC and CfrA are highly conserved and widely produced in C. jejuni strains. Anti-CmeC and Anti-CfrA antibodies inhibited the function of CmeABC efflux pump and CfrA, resulting enhanced susceptibility to bile salts and reduced utilization of FeEnt of C. jejuni, respectively. Immunoblotting analysis also indicated that CfrA is expressed and immunogenic in vivo. Amino acid substitution mutagenesis demonstrated that a highly conserved basic amino acid R327 in CfrA plays a critical role in FeEnt acquisition. The purified recombinant CmeC and a Salmonella live vaccine expressing the protective epitope of CfrA were evaluated as subunit vaccines against Campylobacter infection in the chicken model. CmeC vaccination elicited immune response but failed to reduce C. jejuni colonization in the intestine. However, Salmonella-vectored vaccine conferred significant protection against C. jejuni challenge. To further elucidate the role of iron acquisition in the pathogenesis of Campylobacter, whole genome sequence of a unique C. jejuni strain was determined using a 454 GS FLX sequencer with Titanium series reagents. Comparative genomics analysis led to the identification of a novel Campylobacter Enterobactin Esterase (Cee) that is essential in the CfrB-dependent FeEnt utilization pathway. Extensive genetic manipulation revealed molecular pathways and mechanistic features of the two orchestrated FeEnt acquisition systems in Campylobacter. This project provides critical information about the feasibility of targeting CmeC and CfrA for immune protection against Campylobacter colonization in the intestine, and increases our understanding of the critical role of FeEnt acquisition in the pathophysiology of Campylobacter.

Les toxi-infections dorigine alimentaire ont un impact important sur la santé humaine. Cette étude cible les principaux agents bactériens pathogènes pour lhomme transmis par les denrées alimentaires dorigine animale en Europe et aux USA, à savoir Salmonella et Campylobacter, ainsi que leurs éventuels germes index. Globalement, trois types de surveillance permettent de vérifier lefficacité des mesures prises dans le but de diminuer les zoonoses. La première est le suivi de lhygiène et dindicateurs par le dénombrement des flores indicatrices, surtout de contamination fécale. La deuxième est la surveillance dindex pour certains pathogènes tels que Salmonella et Campylobacter. La troisième est la recherche ou le dénombrement direct des flores pathogènes. Le type de surveillance est déterminé par les objectifs visés. Lobjectif principal de ce travail était de déterminer la pertinence de lutilisation dindicateurs de contamination fécale pour surveiller et maîtriser la contamination des filières belges de production de produits carnés par Salmonella et Campylobacter. Dans un premier temps, un plan de surveillance des filières de production et de transformation des viandes a dû être mis en place et optimisé. Les différents paramètres à préciser étaient le choix des agents pathogènes devant faire lobjet dun monitoring dans chacune des filières, et le choix de la méthodologie déchantillonnage et danalyse. Cette étude a montré la qualité et la représentativité des plans de surveillance mis en place en Belgique pour la production carnée. Ils permettent en effet de nombreux types dinterprétation des résultats et cadrent parfaitement avec les programmes de contrôle intégrés pluriannuels imposés récemment par la Commission européenne. Dans un deuxième temps, une étude spécifique a évalué la pertinence de la méthode belge de prélèvement des carcasses de porc (par écouvillonnage) par rapport à la méthode de référence européenne (destructive), qui a fait lobjet dun nouveau règlement européen fin 2005. Une comparaison entre les deux méthodes pour le dénombrement dE. coli et de germes aérobies totaux ainsi que la recherche de Salmonella et de Campylobacter a montré lefficacité de la méthode belge de prélèvement sur les carcasses de porc. Dans un troisième temps, le plan de surveillance mis en place a permis de suivre lévolution de la contamination par Salmonella et Campylobacter des viandes de bovin, de porc et de volaille depuis 2000. Cette étude a confirmé la forte contamination par Salmonella et Campylobacter des carcasses et viandes de volaille. Elle a également montré la plus faible contamination par Campylobacter et, dans une moindre mesure, par Salmonella, des échantillons issus de bovins et de porcs. Une diminution significative de la contamination par Salmonella de la viande de porc et de la prévalence de Campylobacter sur certains échantillons de volaille a été observée entre 2000 et 2003. Cette étude a également souligné limportance de disposer de données nationales de contamination par les microorganismes pathogènes des aliments. Elles permettent en effet de suivre lévolution et de la comparer dun point de vue international. Ces données pertinentes peuvent être utilisées pour une évaluation quantitative des risques et pour la détermination de critères microbiologiques adaptés. Dans un quatrième temps, cette étude a évalué la pertinence du suivi des principaux indicateurs dhygiène et des bonnes pratiques lors des processus de transformation (les germes aérobies totaux, les entérobactéries et les E. coli ), ainsi que de leur qualité dindex des principaux agents pathogènes non sporulés dorigine digestive. Ce chapitre propose une procédure pour fixer des critères dhygiène des procédés adaptés à la Belgique pour les carcasses et la viande de buf, de porc et de volaille. Cette étude a montré que la situation belge en matière de microorganismes indicateurs est comparable à la situation décrite par plusieurs autres études publiées, et que la méthode de détermination de critères dhygiène des procédés en se basant sur les résultats des plans de surveillance est à privilégier pour aider le secteur de production à améliorer la qualité microbiologique de ses produits. Cette approche peut aider à la diminution de la contamination par les agents pathogènes dorigine digestive mais ne peut, à elle seule, donner des garanties de maîtrise de ces dangers. Enfin, il peut être conclu que le dénombrement dE. coli est très utile pour la détermination de lhygiène des procédés de production de viande en tant quindicateur de contamination fécale et en tant quindex dagents zoonotiques dorigine intestinale tels que Salmonella et Campylobacter. Cependant, la recherche, voire le dénombrement direct des agents pathogènes reste nécessaire pour évaluer le risque et sassurer de lefficacité des mesures de maîtrise. Lensemble des résultats de cette étude et les stratégies appliquées sont également très utiles aux autorités belges dans le cadre de négociations au niveau européen pour la détermination de critères microbiologiques. Il est donc important que des plans de surveillance adaptés aux exigences réglementaires européennes continuent de suivre lévolution des agents pathogènes en Belgique en respectant les objectifs fixés au niveau européen. De plus, les critères dhygiène des procédés doivent être régulièrement revus, sur base des résultats des plans de surveillance. Il faut également tenir compte des indicateurs les plus pertinents pour surveiller et maîtriser la contamination par des microorganismes pathogènes émergents, ou dautres agents pathogènes. Ces critères dhygiène des procédés devraient être repris dans les guides dautocontrôle sectoriels. La méthodologie de détermination de critères de sécurité des aliments en se basant sur une évaluation quantitative du risque, conformément aux directives du Codex Alimentarius, devrait être développée, standardisée et appliquée aux niveaux européen et belge. Dans ce cadre, disposer de plans de surveillance performants et quantitatifs sera essentiel pour alimenter les modèles à léchelle de la Belgique.

The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

<p>Species of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals / and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42&ordm / C as well as at ambient room temperature (&plusmn / 22&ordm / C). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4oC for up to 297 days.</p>

Thesis (M.S.)--University of Tennessee, Knoxville, 2003. / Title from title page screen (viewed Mar., 19, 2004). Thesis advisor: Robert T. Burns. Document formatted into pages (xiv, 148 p. : ill. (some col.)). Vita. Includes bibliographical references.