Onemocnění z potravin (alimentární onemocnění) vyvolaná bakteriemi jsou stále aktuálním tématem v celosvětovém měřítku. Abychom zajistili výrobu zdravotně nezávadných potravin, je potřeba nových poznatků o virulenci patogenů, které by doplnily již známé skutečnosti o jejich růstu a přeživání v potravinách. Také potřebujeme vyvíjet rychlé a citlivé metody na detekci těchto patogenů. Dizertační práce popisuje metodu na detekci S. aureus v potravinách, která je založená na PCR v reálném čase ve spojení s namnožením v selektivním médium. Dále pojednává o vlivu environmentálních faktorů na růst S. aureus a tvorbu enterotoxinů v mléce a sýrech. Vyvinuli jsme rychlou a citlivou metodu na detekci S. aureus v potravinách s použitím selektivního namnožení a PCR v reálném čase. Nově vyvinutá metoda umožnila detekci S. aureus na druhý den od přijetí vzorku. Tato metoda může být použita jako rychlejší, citlivějsí a vysoce specifická alternativní metoda ke konvenční mikrobiologické metodě. Zkoumali jsme vliv tří různých teplot, 8°C, 12°C a 20°C na růst S. aureus a tvorbu enterotoxinu D v pasterizovaném mléce a na růst, expresi genu sed a tvorbu enterotoxinu D v tekutém médiu s extraktem z mozku a srdce (BHI). Experimenty byly prováděny v malých skleněných fermentorech po 6 dní. Genová exprese byla sledována pomocí qRT-PCR a tvorba enterotoxinu D byla měřena pomocí imunologické metody ELISA. Růstová křivka v BHI měla stejný průběh při 20°C a 12°C, ale v při 12°C začal růst se spožděním. Při 8°C nebyl pozorován žádný růst. Růst S. aureus v mléce byl ve srovnání s BHI menší. sed mRNA byla detekována při 20°C po 4 hodinách a při 12°C po 7 hodinách a produkce enterotoxinu se objevila v exponenciální fázi růstu. V mléce se produkce SED při 20°C a při 12°C objevila dříve, ale celkové množství vyprodukovaného SED bylo nižší než v BHI. Při 8°C nebyla pozorována žádná produkce SED stejně jako v BHI. Dále byl zkoumán společný vliv nízké teploty 12°C a přítomnosti kompetitivní doprovodné mikroflóry pocházející ze surového mléka na růst S. aureus a produkci enterotoxinu v pasterizovaném mléce. Byl pozorován inhibiční účinek na růst a produkci enterotoxinů a vliv kompetice byl výraznější než vliv nízké teploty. Produkce enterotoxinu byla nízká a odpovídala růstu. Snížením množství doprovodné mikroflóry a zvýšením inokula došlo pouze k nepatrnému zvýšení produkce enterotoxinu. V další fázi byly dva různé typy sýrů zaočkovány S. aureus za účelem simulace sekundární kontaminace při výrobě sýrů. Vzorky byly odebírány v průběhu 4 týdnů. Kritické faktory jako jsou kompetitivní mikrofóra nebo pH, které jsou zodpovědné za regulaci virulence S. aureus byly sledovány. Snažili jsem se rozlišit situace při kterých: (i) není pozorován růst, ale objevuje se produkce enterotoxinu a (ii) dochází k růstu ale bez produkce enterotoxinu.

Le gène de l’entérotoxine thermostable b (estB) d’Escherichia coli a été fusionné au gène de la protéine liant le maltose (malE) dans le vecteur pMAL-p via PCR. Par la suite, deux constructions plasmidiques ont été realisées à partir de ce nouveau vecteur, nommé pMAL-STb. Dans un premier temps, un marqueur hexahistidine (His6) a été ajouté entre malE et estB, et dans un deuxième temps, un marqueur décahistidine (His10) a été placé en amont de malE. La séquence signal de la protéine liant le maltose (MBP) dirige l’exportation de la protéine de fusion du cytoplasme vers le périplasme, où l’entérotoxine STb acquière sa conformation active. MBP est également reconnue pour améliorer le rendement et la solubilité de la protéine passagère tandis que le marqueur histidine, connu comme étant le meilleur marqueur d’affinité pour la purification protéique, facilite sa purification jusqu’à homogénéité. De plus, les gènes fusionnés sont sous le contrôle du promoteur tac (Ptac), un promoteur fort et inductible. Suite à l’induction par l’IPTG, la souche recombinante exprime une protéine d’environ 48 kDa, qui est facilement identifiable par électrophorèse à partir du surnageant obtenu via choc osmotique. Une séquence encodant un site de clivage spécifique au facteur Xa est présente dans le plasmide afin de séparer les marqueurs MBP et histidine de STb. Le clivage de la protéine de fusion avec le facteur Xa libère MBP (42 kDa) attachée au marqueur histidine et un polypeptide de 5.2 kDa, correspondant au poids moléculaire de STb mature. Avec cette méthode, nous visons à obtenir une méthode plus efficace pour la production et la purification de STb. / The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE) into the pMAL-p vector using PCR. Afterward, two plasmid constructs were realized from this new vector, named pMAL-STb. Firstly, a hexahistidine tag (His6) was added between malE and estB and secondly, a decahistidine tag (His10) was placed upstream of malE. The signal sequence of maltose-binding protein (MBP) directs the export of the fusion protein from the cytoplasm to the periplasm, where the enterotoxin STb acquires its active conformation. MBP is also known to improve the yield and solubility of the passenger protein while the histidine tag, viewed as the best affinity tag for protein purification, facilitates its purification to homogeneity. Furthermore, the fused genes are controlled by the tac promoter (Ptac), a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a protein of approximately 48 kDa, which is easily identified from osmotic shock fluid following electrophoresis. A sequence encoding a factor Xa cleavage site is present in the plasmid to separate MBP and histidine tags from STb. The cleavage of the fusion protein with factor Xa generates the maltose-binding protein (42 kDa) attached to the histidine tag and a polypeptide of 5.2 kDa, corresponding to the molecular mass of mature STb. With this method, we aim at obtaining a more efficient way to produce and purify STb.

Les E. coli entérotoxinogènes (ETEC) sont souvent la cause de diarrhée post-sevrage chez le porc. Deux types d’entérotoxines sont retrouvées chez les ETEC, soit les thermolabiles, comme la toxine LT, et les thermostables, comme EAST-1, STa et STb. Cette dernière est composée de 48 acides aminés et est impliquée dans la pathologie causée par les ETEC. Pour la première fois un variant de la toxine STb fut découvert dans une étude. Nous avons alors émis l’hypothèse qu’il y a présence de variants dans la population de souches ETEC du Québec. Dans les 100 souches STb+ analysées, 23 possédaient le gène de la toxine avec une variation dans la séquence génétique : l’asparagine était présente en position 12 remplaçant ainsi l’histidine. Une corrélation entre la présence du variant et la présence de facteurs de virulence retrouvés dans ces 100 souches ETEC étudiées a été effectuée. Ce variant semble fortement associé à la toxine STa puisque toutes les souches variantes ont hybridé avec le gène codant pour cette dernière. Étant donné sa présence répandue dans la population de souches ETEC du Québec, nous avons de plus émis l’hypothèse que ce variant a des caractéristiques biologiques altérées par rapport à la toxine sauvage. L’analyse par dichroïsme circulaire a montré que le variant et la toxine sauvage ont une structure secondaire ainsi qu’une stabilité similaires. Par la suite, l’attachement au récepteur de la toxine, le sulfatide, a été étudié par résonnance plasmonique de surface (biacore). Le variant a une affinité au sulfatide légèrement réduite comparativement à la toxine sauvage. Puisque l’internalisation de la toxine fut observée dans une étude précédente et qu’elle semble liée à la toxicité, nous avons comparé l’internalisation du variant et de la toxine sauvage à l’intérieur des cellules IPEC-J2. L’internalisation du variant dans les cellules est légèrement supérieure à l’internalisation de la toxine sauvage. Ces résultats suggèrent que le variant est biochimiquement et structurellement comparable à la toxine sauvage. / Enterotoxigenic Escherichia coli (ETEC) are a major cause of post-weaning diarrhea. STb is one of two heat-stable toxins produced by ETEC and is mostly associated with pathogenic porcine isolates. For the first time, a variant of the toxin was observed in a study in 2003. Our hypothesis is that STb variants are present in ETEC strains from Quebec. To screen for alterations at the gene level, a collection of 100 STb+ ETEC strains isolated from diseased pigs was randomly selected and analyzed. A total of 23 strains had a change from His12 to Asn. An association between the presence of the variant and virulence factors present in those strains was done. These strains were also positive for STa. Since this variant seems to be widely distributed in Quebec, we hypothesize that the variant has different biological properties compared to the wild-type STb. First, the secondary structure of the variant and wild-type toxin and their thermal stability was determined by circular dichroism. Both show similar structures and thermal stability. In addition, the binding affinity with the toxin receptor, the sulfatide, was determined by surface plasmon resonance. The affinity of the wild-type for the sulfatide is slightly superior to the variant. Finally, the internalization inside IPEC-J2 cells of the variant was compared to the wild-type. The variant is able to internalize more cells than the wild-type. Altogether, these results suggest that both the variant and the wild-type toxin are biochemically and structurally similar.

L’entérotoxine stable à la chaleur STb est produite par les Escherichia coli entérotoxinogènes (ETEC). Son rôle dans la diarrhée post-sevrage porcine est établi. L’internalisation de STb a été observée dans des cellules épithéliales intestinales humaines et de rat. Cependant, le mécanisme d’internalisation n’est pas totalement compris, particulièrement dans le jéjunum porcin, la cible in vivo de STb. Par la cytométrie en flux, nous avons examiné l’internalisation de STb couplée à un marqueur fluorescent dans les cellules épithéliales intestinales porcines IPEC-J2 et les fibroblastes murins NIH3T3. Nos résultats révèlent que l’internalisation de STb est températureindépendante dans les IPEC-J2 tandis qu’elle est température-dépendante dans les NIH3T3, où la réorganisation de l’actine est aussi nécessaire. Toutefois, les niveaux de sulfatide, le récepteur de STb, sont semblables à la surface des deux lignées. Le sulfatide est internalisé à 37°C de façon similaire entre les deux types cellulaires. La rupture des lipid rafts, les microdomaines membranaires contenant le sulfatide, par la méthyl-βcyclodextrine ou la génistéine, n’affecte pas l’internalisation de STb dans les deux lignées. Notre étude indique que le mécanisme d’internalisation de STb est dépendant du type cellulaire. L’activité de la cellule hôte peut être requise ou non. Le récepteur de STb, le sulfatide, n’est pas directement impliqué dans ces mécanismes. L’internalisation activité cellulaire-dépendante suggère une endocytose, nécessitant la réorganisation de l’actine mais pas les lipid rafts. L’internalisation de STb est donc un processus complexe dépendant du type cellulaire, qu’il apparait plus relevant d’étudier dans des modèles cellulaires représentatifs des conditions in vivo. / Heat-stable enterotoxin b (STb) is one of the toxins produced by enterotoxigenic Escherichia coli (ETEC) and its role in swine post-weaning diarrhea is well established. Internalization of STb in intestinal human and rat epithelial cells has been shown by previous studies. However, the uptake mechanism is still not fully understood, especially in porcine jejunum epithelium, the in vivo STb target. Using flow cytometry, we studied internalization of fluorescently-labelled STb in porcine epithelial intestinal IPEC-J2 and murine fibroblast NIH3T3 cell lines. Our results revealed that STb is internalized in both cell lines. Toxin uptake is not dependent on the temperature in IPEC-J2 cells, whereas it is in NIH3T3 fibroblasts. Actin reorganization is only required for STb internalization in NIH3T3 cells. However, membrane sulfatide, the toxin receptor, is similarly present in both cell lines and similarly internalized with time at 37°C. Disruption of lipid rafts, known to contain sulfatide, with inhibitors (methyl-βcyclodextrin or genistein), did not affect toxin uptake in both cell lines. Altogether, these data indicate that STb internalization mechanisms are cell-type dependent. Moreover, uptake can depend on host cell activity or not. Sulfatide, the toxin receptor, is not directly involved in these mechanisms. Uptake independent on cell activity occurs in porcine intestinal epithelium. The cell activity-dependent uptake suggests an endocytosis, which requires actin rearrangement and is not mediated by lipid rafts. STb internalization is therefore a complex process varying upon cell type, which should preferentially be studied in cellular models representative of in vivo conditions, such as porcine cell lines.

As condições de produção de queijos frescos são favoráveis à ocorrência e à produção de enterotoxinas produzidas por estafilococos, sendo estes os principais micro-organismos relacionados aos casos de intoxicação alimentar no mundo, tornando-se necessários estudos sobre a rastreabilidade das fontes de contaminação durante a fabricação, identificação genotípica e habilidade de cepas em produzir enterotoxinas. Este trabalho teve como objetivo isolar e identificar estafilococos positivos para o teste de coagulase, potencialmente produtores de enterotoxinas em laticínios do estado de São Paulo, desde o leite recebido até o produto final. A técnica da mPCR foi utilizada para identificar três espécies de Staphylococcus coagulase-positiva (S. aureus, S. hyicus e S. intermedius) entre os isolados obtidos de diferentes pontos de processamento em laticínios do Estado de São Paulo. O perfil genético das cepas foi avaliado através da comparação de bandas pela técnica Enterobacteria Repetitive Intergenic Consensus (ERIC-PCR) e graficamente demonstrados por um dendrograma. O DNA de 102 cepas isoladas, de amostras de leite cru, leite pasteurizado, coágulo, manipulador, superfícies de equipamentos de laticínios do Estado de São Paulo e queijos foi extraído e submetido à reação em cadeia da polimerase (PCR), utilizando-se primers específicos para a detecção de fragmentos dos genes envolvidos na síntese das toxinas (SE) do tipo A, B, C, D, E, G, H, I e J. Das 102 cepas avaliadas, 5 apresentaram amplificação do fragmento do gene responsável pela codificação da toxina A, 3 para toxina C, 56 para toxina G, 59 para toxina H, 9 para toxina I, e nenhuma para toxinas B, D, E e J. Amostras de leite cru, leite pasteurizado e queijos produzidos nos laticínios e dos isolados de Staphylococcus spp. coagulase positiva que apresentaram ao menos algum dos genes relacionados à produção de toxinas clássicas (A, B, C, D e E) foram submetidos a um teste de detecção de enterotoxinas pelo sistema VIDAS® Staph Enterotoxin (SET2). Os dados obtidos para a identificação molecular das cepas, da ocorrência de cepas portadoras dos genes relacionados à produção de enterotoxinas e da produção fenotípica das enterotoxinas foram submetidos ao teste qui-quadrado. O presente trabalhou confirmou que o risco de intoxicação estafilocócica é real, pois foi encontrada enterotoxina em amostras de leite pasteurizado e queijos. / The production conditions of fresh cheese are favorable to the occurrence and production of enterotoxin produced by Staphylococci, which are the main microorganisms related to food poisoning cases in the world, requiring studies to trace the sources of contamination during manufacturing and genotypic identification ability of strains to produce enterotoxin. This study aimed to isolate and identify staphylococci positive for coagulase test, potentially producing enterotoxins in dairy products in the state of São Paulo, from milk receiving to final product. In three dairy producers of Minas fresh cheese, samples were collected from various points in the manufacturing process. the technique of mPCR was used to identify three coagulase-positive species (S. aureus, S. hyicus and S. intermedius) between isolates from different points of a processing dairy in the state of São Paulo. The genetic profile of the strains were evaluated by comparing the bands through the technique Enterobacteria Repetitive Intergenic Consensus (ERIC-PCR) and were graphically displayed by a dendrogram. The DNA of 102 strains isolated from samples of raw milk , pasteurized milk , curd, handler and equipment surface from dairies in São Paulo State and cheese were subjected to the polymerase chain reaction (PCR), using primers for the detection of specific fragments of the genes involved in the synthesis of toxins (SE) of type A, B , C , D, E, G , H, I and J. Of the 102 strains tested, five showed amplification of the gene fragment encoding toxin A, three for toxin C , 56 to toxin G, 59 to toxin H, 9 to toxin I, and none for toxins B D, E and J. For confirmation, each isolated strain carrying at least some of the genes related to the production of classical enterotoxinas, cheese and milk samples was subjected to detection by enterotoxigenic VIDAS® Staph Enterotoxin II (VIDAS SET2) bioMérieux. This identification reinforces the need to adopt proper hygiene practices in the dairy, avoiding thus the spread of these microorganisms and the possible production of enterotoxin . The data obtained for the molecular identification of the strains, the occurrence of strains carrying the genes related to the production of enterotoxin production and phenotypic enterotoxins were subjected to the Chi-squared test. This work confirmed that the risk of staphylococcal food poisoning is real, because enterotoxin was found in samples of pasteurized milk and cheeses.

<p>Silicon-based electric chip arrays were developed for characterization of Bacillus</p><p>cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all</p><p>chemical reactions until and including the enzymatic reaction took place in an</p><p>eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control</p><p>cells of B. subtilis. The 16 positions of the silicon chip array were activated by</p><p>immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation.</p>

Les E. coli entérotoxinogènes (ETEC) sont souvent la cause de diarrhée post-sevrage chez le porc. Deux types d’entérotoxines sont retrouvées chez les ETEC, soit les thermolabiles, comme la toxine LT, et les thermostables, comme EAST-1, STa et STb. Cette dernière est composée de 48 acides aminés et est impliquée dans la pathologie causée par les ETEC. Pour la première fois un variant de la toxine STb fut découvert dans une étude. Nous avons alors émis l’hypothèse qu’il y a présence de variants dans la population de souches ETEC du Québec. Dans les 100 souches STb+ analysées, 23 possédaient le gène de la toxine avec une variation dans la séquence génétique : l’asparagine était présente en position 12 remplaçant ainsi l’histidine. Une corrélation entre la présence du variant et la présence de facteurs de virulence retrouvés dans ces 100 souches ETEC étudiées a été effectuée. Ce variant semble fortement associé à la toxine STa puisque toutes les souches variantes ont hybridé avec le gène codant pour cette dernière. Étant donné sa présence répandue dans la population de souches ETEC du Québec, nous avons de plus émis l’hypothèse que ce variant a des caractéristiques biologiques altérées par rapport à la toxine sauvage. L’analyse par dichroïsme circulaire a montré que le variant et la toxine sauvage ont une structure secondaire ainsi qu’une stabilité similaires. Par la suite, l’attachement au récepteur de la toxine, le sulfatide, a été étudié par résonnance plasmonique de surface (biacore). Le variant a une affinité au sulfatide légèrement réduite comparativement à la toxine sauvage. Puisque l’internalisation de la toxine fut observée dans une étude précédente et qu’elle semble liée à la toxicité, nous avons comparé l’internalisation du variant et de la toxine sauvage à l’intérieur des cellules IPEC-J2. L’internalisation du variant dans les cellules est légèrement supérieure à l’internalisation de la toxine sauvage. Ces résultats suggèrent que le variant est biochimiquement et structurellement comparable à la toxine sauvage. / Enterotoxigenic Escherichia coli (ETEC) are a major cause of post-weaning diarrhea. STb is one of two heat-stable toxins produced by ETEC and is mostly associated with pathogenic porcine isolates. For the first time, a variant of the toxin was observed in a study in 2003. Our hypothesis is that STb variants are present in ETEC strains from Quebec. To screen for alterations at the gene level, a collection of 100 STb+ ETEC strains isolated from diseased pigs was randomly selected and analyzed. A total of 23 strains had a change from His12 to Asn. An association between the presence of the variant and virulence factors present in those strains was done. These strains were also positive for STa. Since this variant seems to be widely distributed in Quebec, we hypothesize that the variant has different biological properties compared to the wild-type STb. First, the secondary structure of the variant and wild-type toxin and their thermal stability was determined by circular dichroism. Both show similar structures and thermal stability. In addition, the binding affinity with the toxin receptor, the sulfatide, was determined by surface plasmon resonance. The affinity of the wild-type for the sulfatide is slightly superior to the variant. Finally, the internalization inside IPEC-J2 cells of the variant was compared to the wild-type. The variant is able to internalize more cells than the wild-type. Altogether, these results suggest that both the variant and the wild-type toxin are biochemically and structurally similar.

L’entérotoxine stable à la chaleur STb est produite par les Escherichia coli entérotoxinogènes (ETEC). Son rôle dans la diarrhée post-sevrage porcine est établi. L’internalisation de STb a été observée dans des cellules épithéliales intestinales humaines et de rat. Cependant, le mécanisme d’internalisation n’est pas totalement compris, particulièrement dans le jéjunum porcin, la cible in vivo de STb. Par la cytométrie en flux, nous avons examiné l’internalisation de STb couplée à un marqueur fluorescent dans les cellules épithéliales intestinales porcines IPEC-J2 et les fibroblastes murins NIH3T3. Nos résultats révèlent que l’internalisation de STb est températureindépendante dans les IPEC-J2 tandis qu’elle est température-dépendante dans les NIH3T3, où la réorganisation de l’actine est aussi nécessaire. Toutefois, les niveaux de sulfatide, le récepteur de STb, sont semblables à la surface des deux lignées. Le sulfatide est internalisé à 37°C de façon similaire entre les deux types cellulaires. La rupture des lipid rafts, les microdomaines membranaires contenant le sulfatide, par la méthyl-βcyclodextrine ou la génistéine, n’affecte pas l’internalisation de STb dans les deux lignées. Notre étude indique que le mécanisme d’internalisation de STb est dépendant du type cellulaire. L’activité de la cellule hôte peut être requise ou non. Le récepteur de STb, le sulfatide, n’est pas directement impliqué dans ces mécanismes. L’internalisation activité cellulaire-dépendante suggère une endocytose, nécessitant la réorganisation de l’actine mais pas les lipid rafts. L’internalisation de STb est donc un processus complexe dépendant du type cellulaire, qu’il apparait plus relevant d’étudier dans des modèles cellulaires représentatifs des conditions in vivo. / Heat-stable enterotoxin b (STb) is one of the toxins produced by enterotoxigenic Escherichia coli (ETEC) and its role in swine post-weaning diarrhea is well established. Internalization of STb in intestinal human and rat epithelial cells has been shown by previous studies. However, the uptake mechanism is still not fully understood, especially in porcine jejunum epithelium, the in vivo STb target. Using flow cytometry, we studied internalization of fluorescently-labelled STb in porcine epithelial intestinal IPEC-J2 and murine fibroblast NIH3T3 cell lines. Our results revealed that STb is internalized in both cell lines. Toxin uptake is not dependent on the temperature in IPEC-J2 cells, whereas it is in NIH3T3 fibroblasts. Actin reorganization is only required for STb internalization in NIH3T3 cells. However, membrane sulfatide, the toxin receptor, is similarly present in both cell lines and similarly internalized with time at 37°C. Disruption of lipid rafts, known to contain sulfatide, with inhibitors (methyl-βcyclodextrin or genistein), did not affect toxin uptake in both cell lines. Altogether, these data indicate that STb internalization mechanisms are cell-type dependent. Moreover, uptake can depend on host cell activity or not. Sulfatide, the toxin receptor, is not directly involved in these mechanisms. Uptake independent on cell activity occurs in porcine intestinal epithelium. The cell activity-dependent uptake suggests an endocytosis, which requires actin rearrangement and is not mediated by lipid rafts. STb internalization is therefore a complex process varying upon cell type, which should preferentially be studied in cellular models representative of in vivo conditions, such as porcine cell lines.

Made available in DSpace on 2016-08-15T20:31:33Z (GMT). No. of bitstreams: 1 GermanaGR_DISSERT.pdf: 1130619 bytes, checksum: 8a9b56b38e3290f0902f54c5111e97b8 (MD5) Previous issue date: 2015-09-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to investigate the presence of enterotoxigenic and methicillin-resistant genes in Staphylococcus aureus isolated from goat milk obtained from Rio Grande do Norte state producers. To achieve such goal, 49 samples of goat milk and searched for S. aureus. The colonies were isolated in Petrifilm plates, Staph Express 3M model and biochemically identified. Afterwards, the isolated strains underwent an assessment to evaluate their antibiotic resistance profile, through the use of disk diffusion technique on Mueller-Hinton agar. Following we carried out a polymerase chain reaction (PCR) to search for the following selected genes: 16S rRNA (for molecular identification of S. aureus), mecA (characterizes the methicillin-resistant S.aureus) and enterotoxigenic genes (sea, seb-sec, sec, sed, see, seg, seh, sei, sej and tsst-1). Results from morphological and biochemical analyses revealed that all isolated strains belonged to the Staphylococcus aureus species. We also observed, through the antibiogram, high percentage of resistance to the antibiotics penicillin G (87.75%) and oxacillin (75.51%). Multidrug resistance was detected in 73.46% of the isolated strains. Analyzing the PCR, we confirmed that 100% of the isolated strains were S. aureus because of the amplification of the 16S rRNA gene. The mecA gene was found in 4.08% of the samples. In samples confirmed as S. aureus, concerning the research for staphylococcal enterotoxins (SE), there was amplification of the sej gene fragments in 79.5% of the samples, followed by the sei gene in 48.9%, sed gene in 22.4%, sea gene in 12, 2%, seh and sec genes in 8.1%, and sec gene in 2% for the samples. We did not observe any amplification for the seb-sec, see and tsst-1 genes. The strains of S. aureus isolated from goat milk have shown the presence of genes responsible for the production of toxins, a fact that requires greater care when processing this milk in the dairy industry. In addition, to antibiotic resistance and detection of mecA gene also leads to concern, can pose risks to consumer health / O objetivo do presente trabalho foi investigar a presença de genes enterotoxigênicos e de resistência à meticilina em Staphylococcus aureus isolados de leite caprino obtido de produtores do estado do Rio Grande do Norte. Para a realização do estudo, realizou-se a pesquisa de S.aureus em 49 amostras de leite caprino. O isolamento foi realizado em placas Petrifilm , modelo Staph Express 3M e identificadas bioquimicamente. Em seguida, os isolados foram submetidos a avaliação quanto ao perfil de resistência a antibióticos, utilizando a técnica de difusão em discos em Agar Mueller-Hinton. Seguiu-se com a realização da reação em cadeia pela polimerase (PCR) para pesquisa dos seguintes genes selecionados: 16S rRNA (para identificação molecular dos S. aureus), mecA (que caracteriza S. aureus resistente à meticilina) e genes enterotoxigênicos (sea, seb-sec, sec, sed, see, seg, seh, sei, sej e tsst-1). Os resultados das análises morfológicas e bioquímicas revelaram que todos os isolados pertenciam a espécie Staphylococcus aureus. Observou-se através do antibiograma realizado um maior percentual de resistência para os antimicrobianos penicilina G (87,75%) e oxacilina (75,51%). Multirresistência foi verificada em 73,46% dos isolados. Com a análise da PCR, confirmou-se que 100% dos isolados eram S. aureus, pela amplificação do gene 16S rRNA. O gene mecA foi detectado em 4,08% das amostras. Na pesquisa de enterotoxinas estafilocócicas (SE), nas amostras confirmadas para S. aureus, houve amplificação em 79,5 % dos fragmentos do gene sej, seguido de 48,9% do gene sei, 22,4% do gene sed, 12,2% para o gene sea, 8,1% para os genes seh e seg e 2% para o gene sec. Não foi observada nenhuma amplificação para os genes seb-sec, see e Tsst-1. As amostras de S. aureus isoladas a partir do leite caprino demostraram presença de genes responsáveis pela produção de toxinas, fato que exige um maior cuidado no tratamento deste leite pela indústria de alimentos. Além disso a resistência aos antibióticos e detecção do gene mecA também leva a preocupação, podendo representar riscos à saúde do consumidor

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-24T13:23:39Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Jessica_Silveira_Vitoria.pdf: 6777460 bytes, checksum: a576eea96dc73d5ae068edefa155b085 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-24T13:57:09Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Jessica_Silveira_Vitoria.pdf: 6777460 bytes, checksum: a576eea96dc73d5ae068edefa155b085 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-24T13:57:18Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Jessica_Silveira_Vitoria.pdf: 6777460 bytes, checksum: a576eea96dc73d5ae068edefa155b085 (MD5) / Made available in DSpace on 2018-05-24T13:57:18Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Jessica_Silveira_Vitoria.pdf: 6777460 bytes, checksum: a576eea96dc73d5ae068edefa155b085 (MD5) Previous issue date: 2017-07-31 / Sem bolsa / A alimentação escolar deve ofertar alimentos seguros quanto à sua condição higienicossanitária buscando a proteção e promoção da saúde dos escolares. O objetivo deste estudo foi avaliar as condições higienicossanitárias de escolas municipais da cidade de Pelotas - RS por meio da aplicação de uma lista de verificação e de análises microbiológicas do ar ambiental, da superfície de manipulação de alimentos, da água, de preparações alimentícias e das mãos de manipuladores de alimentos. Além disso verificou-se a presença de genes produtores de enterotoxinas estafilocócicas clássicas (A, B, C, D e E) nos isolados de Staphylococcus spp. Foram realizadas visitas em 15 escolas municipais com aplicação da lista de verificação e coleta das amostras para as análises microbiológicas. Realizaram-se contagens de bactérias aeróbias mesófilas e bolores e leveduras nas amostras de ar ambiental, contagens de coliformes termotolerantes e bactérias aeróbias mesófilas em amostras de superfícies de manipulação e contagem de coliformes totais e Escherichia coli em amostras de água. Nas mãos dos manipuladores foram realizadas análises de coliformes termotolerantes e Staphylococcus spp. Nas preparações alimentícias foram realizadas análises de coliformes termotolerantes, Bacillus cereus, Staphylococcus spp. e Salmonella spp. Os isolados de Staphylococcus spp. foram submetidos à técnica de reação em cadeia da polimerase multiplex a fim de verificar a presença dos genes codificadores de enterotoxinas estafilocócicas clássicas. Os resultados obtidos a partir da lista de verificação apontaram alto percentual de não conformidades, sendo que duas das 15 escolas apresentaram "grau de risco sanitário alto" e todas as outras foram classificadas como “situação de risco sanitário regular”. Em relação às análises microbiológicas, a qualidade do ar ambiental foi satisfatória em todas as escolas avaliadas. Entretanto, foram encontradas contagens acima do permitido para bactérias aeróbias mesófilas em superfícies de manipulação de alimentos e coliformes totais e Escherichia coli em amostras de água. As mãos dos manipuladores e as preparações alimentícias apresentaram contagens acima do recomendado para Estafilococos coagulase positiva e coliformes termotolerantes. Em três isolados de Staphylococcus spp. verificou-se a presença de genes codificadores de enterotoxina estafilocócica B. Pode-se concluir que as escolas que fizeram parte da pesquisa não apresentam padrões higienicossanitários adequados, pois foram encontradas inadequações tanto na avaliação pela lista de verificação como nos resultados das análises microbiológicas. / School feeding should provide safe food for their hygienic and sanitary conditions, seeking to protect and promote the health of students. The aim of this study was to evaluate the sanitary conditions of municipal schools in the city of Pelotas - RS, through the application of a checklist and microbiological analyses of air, food handling surface, water, food preparations and hands of food handlers. In addition, the presence of classical EE genes (A, B, C, D and E) in Staphylococcus spp. was verified. Visits were carried out in 15 municipal schools with application of the checklist and sample collection for microbiological analyses. Counts of mesophilic aerobic bacteria and molds and yeasts in the air samples, counts of thermotolerant coliforms and aerobic mesophilic bacteria in samples of manipulation surfaces and counting of coliforms and Escherichia coli in water samples were carried out. In the food handlers’ hands were counts of thermotolerant coliforms and Staphylococcus spp. In the food preparations, counts of thermotolerant coliforms, Bacillus cereus, Staphylococcus spp. and Salmonella spp. were realized. Isolates from Staphylococcus spp. were submitted to the multiplex polymerase chain reaction technique in order to verify the presence of genes coding for classical staphylococcal enterotoxins. The results obtained from the checklist indicated a high percentage of nonconformities, and two of the 15 schools presented a "high sanitary risk situation" and all others were classified as "regular sanitary risk situation". Regarding the microbiological analyses, the air quality was satisfactory in all schools evaluated. However, counts above that allowed for counting of mesophilic aerobic bacteria in food and total coliform surfaces and Escherichia coli in water samples were found. Food handlers’ hands and the food preparations presented counts above the recommended for Staphylococcus coagulase positive and thermotolerant coliforms. In three isolates of Staphylococcus spp. the presence of staphylococcal enterotoxin B encoding genes was verified. It can be concluded that the schools that were part of the research did not present adequate hygienic and sanitary standards, since inadequacies were found both in the evaluation by the checklist and in the results of the microbiological analyses.