Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-06-26T12:47:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Camile_Milan.pdf: 974433 bytes, checksum: de53680fe197bd4ba3ac70ba2bac3df5 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-26T13:50:13Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Camile_Milan.pdf: 974433 bytes, checksum: de53680fe197bd4ba3ac70ba2bac3df5 (MD5) / Made available in DSpace on 2017-06-26T13:50:13Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Camile_Milan.pdf: 974433 bytes, checksum: de53680fe197bd4ba3ac70ba2bac3df5 (MD5) Previous issue date: 2016-02-18 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / As doenças transmitidas por alimentos são causadas pela ingestão de alimentos contaminados, sendo que em 2013 os principais agentes causadores destas doenças foram Salmonella spp. e Campylobacter spp. No homem, Campylobacter spp. provoca sintomatologia gastrointestinal, que pode variar de uma forma mais suave até formas mais severas. Suínos podem ser portadores destes micro-organismos e produtos de suínos têm sido implicados em casos e surtos de campilobacteriose em humanos. Logo, o objetivo deste trabalho foi rastrear Campylobacter spp. no fluxograma de abate de suínos para compreender o comportamento destes patógenos na linha de produção. Foi acompanhado um total de 40 suínos oriundos de 10 baias de uma granja no Rio Grande do Sul durante o abate. Amostras de fezes foram coletadas no reto do animal logo após a insensibilização. Amostras da superfície da carcaça foram coletadas após a saída dos animais da depiladeira, após a evisceração, momentos antes da carcaça entrar na câmara de resfriamento e da papada. Amostras da água do tanque de escaldagem foram coletadas antes de iniciar o abate de cada lote e após a passagem dos animais também. Os isolados foram obtidos através de culturas microbiológicas e a identificação das espécies foi realizada pela técnica de PCR. Os perfis de bandas das cepas foram determinados por rep-PCR e comparados entre si. Das 200 amostras coletadas, 19 (9,5%) foram positivas para Campylobacter, sendo todas caracterizadas como C. coli. Destes isolados, sete (36,8%) foram obtidos das amostras do reto, sete (36,8%) da pós-evisceração e cinco (26,3%) antes do ingresso na câmara de resfriamento. Através da análise por rep-PCR, observou-se que os animais, uma vez contaminados por C. coli na granja, podem carrear o micro-organismo durante as etapas do fluxograma de abate e que contaminações cruzadas também têm papel importante na contaminação final da carcaça. O manejo higiênico-sanitário adotado nas boas práticas de fabricação é fundamental no controle da contaminação por C. coli. / The foodborne illnesses are caused by contaminated food, and in 2013 the main causative agents of these diseases were Salmonella spp. and Campylobacter spp. In man, Campylobacter spp. causes gastrointestinal symptoms, which can vary more smoothly even more severe forms. Pigs can be carriers of microorganisms and pork products have been implicated in cases of campylobacteriosis outbreaks in humans. Therefore, the aim of this study was to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line. A total of 40 pigs originating from 10 bays a farm in Rio Grande do Sul was accompanied during slaughter. Stool samples were collected in the animal's rectum immediately after stunning. Surface samples were collected after removal of the animals from scrap machine, after evisceration, before the refrigeration chamber and from jowls. Samples from the scalding tank water before and after the passage of animals were collected too. The isolates were obtained for microbiological analysis and the confirmation of species was performed by PCR. Strains bands profiles were determined by rep-PCR and compared. Of the 200 samples 19 (9.5%) were positive for Campylobacter, which are all characterized as C.coli. In these isolates, 7 (36.8%) were the rectum, 7 (36.8%) after evisceration and 5 (26.3%) before the refrigeration chamber. Through rep-PCR analysis, it was observed that the animals contaminated by C. coli in the farm may carry the micro-organism through the steps of the flowchart slaughter and the cross contamination also has an important role on the final contamination. The hygienic-sanitary management of care adopted in good manufacturing practices are key in controlling the contamination by C. coli.

Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines. / text

Campylobacter jejuni is the leading bacterial cause of gastrointestinal diarrheal disease in humans worldwide. This zoonotic pathogen has a complex epidemiology due to its presence in many different host organisms. The overall aim of this thesis was to explore the role of amoebae of the genus Acanthamoeba as an intermediate host and vector for survival and dissemination of C. jejuni. Earlier studies have shown that C. jejuni can enter, survive and replicate within Acanthamoebae spp. In this thesis, I have shown that C. jejuni actively invades Acanthamoeba polyphaga. Once inside, C. jejuni could survive within the amoebae by avoiding localization to degradative lysosomes. We also found that A. polyphaga could protect C. jejuni in acid environments with pH levels far below the range in which the bacterium normally survives. Furthermore, low pH triggered C. jejuni motility and invasion of A. polyphaga. In an applied study I found that A. polyphaga also could increase the survival of C. jejuni in milk and juice both at room temperature and at +4ºC, but not during heating to recommended pasteurization temperatures. In the last study we found that forty environmental C. jejuni isolates with low bacterial concentrations could be successfully enriched using the Acanthamoeba-Campylobacter coculture (ACC) method. Molecular genetic analysis using multilocus sequence typing (MLST) and sequencing of the flaA gene, showed no genetic changes during coculture. The results of this thesis have increased our knowledge on the mechanisms behind C. jejuni invasion and intracellular survival in amoebae of the genus Acanthamoeba. By protecting C. jejuni from acid environments, Acanthamoebae could serve as important reservoirs for C. jejuni e.g. during acid sanitation of chicken stables and possibly as vectors during passage through the stomach of host animals. Furthermore, Acanthamoeba spp. could serve as a vehicle and reservoir introducing and protecting C. jejuni in beverages such as milk and juice. Validation of the ACC method suggests that it is robust and could be used even in outbreak investigations where genetic fingerprints are compared between isolates. In conclusion, Acanthamoeba spp. are good candidates for being natural hosts and vectors of C. jejuni.

This thesis is concerned with the molecular epidemiology of Campylobacter jejuni and Salmonella in New Zealand and the development of source attribution tools for these pathogens. Although campylobacteriosis is the leading enteric zoonosis worldwide, the pathogen's complex epidemiology and di culties with existing typing schemes, have posed challenges for the control of this disease. The rst study of this thesis gives an overview of existing approaches to microbial risk assessment and source attribution, with particular respect to campylobacteriosis, and describes their advantages and shortcomings. Further, the chapter discusses phenoand genotyping techniques for Campylobacter spp. and the value of including microbial typing data in risk assessments. In the second study, data from a sentinel surveillance site in the Manawatu region was used to investigate the molecular epidemiology of human campylobacteriosis cases. This analysis revealed the presence of a dominant C. jejuni clone, namely sequence type (ST) 474, which accounted for 30.7 % of human cases in the study and identi ed risk factors for infection with ruminant and poultry associated STs. The third study investigated the link between C. jejuni in human cases and samples taken from poultry. By applying epidemiological and population genetic techniques this part of the thesis provided further evidence that poultry is a major contributor to human infection. In the fourth study an existing Bayesian source attribution model was modi ed and consecutively applied to New Zealand's major foodborne zoonoses: campylobacteriosis and salmonellosis. The majority (80 %) of human campylobacteriosis cases attributable to C. jejuni were estimated to have been acquired from poultry sources, whereas wildlife source were estimated to contribute only a minor proportion of cases. In the fth study the Salmonella dataset was descriptively analysed and a large proportion of human cases was found to be caused by `exotic' Salmonella types. In the nal study of this thesis four di erent genetic and epidemiological source attribution methodologies were applied to the same dataset in a comparative modelling framework. iv The studies in this thesis show that epidemiological studies combined with molecular tools and modeling can provide valuable risk-based tools to inform the surveillance and control of zoonotic pathogens. Methods from these studies may be readily applied to the control of other (food borne) zoonoses and provide new opportunities for epidemiological investigations and source attribution modelling of major pathogens.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 6 uppsatser.

No description available.

This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel. In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.

Campylobacter est la principale cause de gastro-entérite bactérienne d’origine alimentaire à travers le monde. Chez les consommateurs, les campylobactérioses d’origine alimentaire sont en grande majorité dues au contact et à la consommation de produits de volaille, le poulet à griller étant particulièrement mis en cause. La contamination de la carcasse se fait souvent lors de l’abattage des oiseaux. Bien que plusieurs données concernant la distribution de Campylobacter à l’abattoir soient disponibles dans certains pays, aucune étude récente visant à décrire la présence et la distribution de ce pathogène, tout au long de la chaîne d’abattage, n'a été réalisée au niveau des établissements d'abattage canadiens. Notre hypothèse était que l’on pouvait identifier des étapes clés d’intervention pour contrôler la contamination par Campylobacter sur les carcasses de volaille à l’abattoir en étudiant la présence de ce pathogène sur les produits de viande et dans l’environnement de production. Il y avait deux objectifs principaux dans cette étude. Premièrement, nous voulions décrire la distribution de Campylobacter lors des différentes étapes de production dans deux établissements de transformation québécois. Deuxièmement, nous voulions déterminer si les moyens de gestion du risque mis en place au moment de l’étude étaient suffisants pour prévenir la contamination du produit de viande destiné au consommateur. Pour répondre à ces objectifs, un oiseau par lot d’élevage a été échantillonné par rinçât de carcasse pour chaque étape suivante de la transformation (n=4) : avant l'abattage, tout juste après la saignée, au transfert entre les chaînes d'abattage et d'éviscération, après l'éviscération et après l’étape du refroidissement dans deux abattoirs québécois. Cette procédure fut répétée pour un total de 379 échantillons de rinçâts de carcasses de poulets de chair qui ont été collectés à l’occasion de multiples visites à l’abattoir et ce, de février à juillet 2017. Un total de 217 échantillons environnementaux pouvant être impliqués dans la contamination croisée des oiseaux ont aussi été récupéré pendant les diverses visites. Les échantillons ont été dilués dans de l'eau peptonée et une identification de Campylobacter par PCR a été faite à l’aide d’amorces spécifiques au gène codant pour l’ARN ribosomal 16S. Les résultats obtenus pour la période étudiée indiquent que la positivité des carcasses de poulets de chair à Campylobacter est significativement plus élevée pour les échantillons effectués l’été comparé au printemps. En revanche, la présence de la bactérie dans l’environnement des abattoirs étudiés apparaît plus élevée durant l’hiver. Puisque la présence de la bactérie sur les carcasses de poulets a diminué tout au long de la chaîne de production, qu’aucune carcasse positive n’a été retrouvée après un refroidissement à l’air et que la positivité des carcasses suite au refroidissement à l’eau était aussi très basse, nos résultats suggèrent, malgré certains enjeux associés à la sensibilité de la méthode d’identification des échantillons positifs, que les mesures actuelles de gestion du risque sont efficaces pour contrôler Campylobacter dans les deux abattoirs québécois suivis. / Campylobacter is responsible of the highest number of bacterial gastroenteritis worldwide. Most diseases in humans attributed to meat can be associated to consumption of or contact with poultry derived products; broiler chicken being involved in the majority of cases. The contamination of carcasses often occurs during the slaughter process. While many studies from various countries reported the distribution of Campylobacter among various critical steps of the slaughter process, none has been published, to our knowledge, in Canada regarding the presence and distribution of this bacterium within the abattoir. Our hypothesis was that it would be possible to identify key steps to control the contamination of carcasses by this bacterium by studying the presence and distribution of Campylobacter on carcasses and within the environment during the slaughter process. This study had two objectives. The first objective was to describe the distribution of Campylobacter within two selected slaughterhouses in Quebec in order to understand which processing step(s) play(s) a critical role in carcasses contamination. In the second objective, we aimed to verify if actual management procedures applied in these abattoirs were efficient in preventing consumer’s exposition. To meet these goals, four birds by production lot, one at each of the following steps (after bleeding, at transfer between killing and evisceration chain, after evisceration and after the cooling process) were sampled for a total of 379 birds from February 2017 to July 2017 in two slaughterhouses located in the province of Quebec. Furthermore, 217 environmental samples were collected during these visits in various sites possibly in contact with birds. Samples were suspended in peptone water and submitted to a PCR assay, using a specific 16S ribosomal probe, for detection of Campylobacter. Overall, for the year of the study, we observed a significantly higher number of positive carcasses in summer compared to spring, while the environmental samples were more often positive in winter compared with summer. Furthermore, our results indicated that the number of positive carcasses decreased over the various processing steps, being either negative (air chilling) or low (water chilling) after the cooling process. Although we experienced some issues associated with the sensitivity of the procedure we used in this study to recover Campylobacter, taken together, these results suggest that the actual management procedures of Campylobacter in studied slaughterhouses are efficient.

Unsafe sanitation systems poses a risk for pathogen transmission, wherefore it is important to both inactivate pathogens present in human excreta and conduct safe sanitation systems from use to end-use. The Peepoo toilet, using ammonia sanitisation, have been suggested as a low-cost sanitation solution and is implemented in schools in Kibera, an urban slum in Kenya. This master thesis aim to study the inactivation efficiency of ammonia sanitisation when treating human excreta with urea, and to quantify the risks of exposure to microbial hazards from the Peepoo sanitation system using faecal indicator bacteria. Excreta was collected from four schools in Kibera. After adding urea to mimic the inactivation of the Peepoo in the laboratory, the inactivation rate was correlated to temperature and free ammonia concentration for Campylobacter spp., Escherichia coli and Enterococcus spp.. Campylobacter spp. and E. coli both had a high inactivation rate even at low temperature and low addition of urea. Inactivation rate of Enterococcus spp. was lower and close to zero when 1.87 % urea was added for 15 °C. For Enterococcus spp. a lag-phase was observed, which was not affected by temperature but by concentration of free ammonia. For investigated bacteria, inactivation rate increased with increased temperature and free ammonia concentration. Along the Peepoo management chain, several hazardous events were identified such ascontamination during usage, handling and transportation. Sampling showed a higher contamination of Enterococcus spp. than of E. coli. Enterococcus spp. was used as a faecal indicator for Ascaris and E. coli was used as an indicator of E. coli O157:H7 in a quantitative microbial risk assessment (QMRA). Through the QMRA, the risk of infection of Ascaris and E. coli O157:H7 for one exposure event was simulated based on a Exponential and a Beta-Poisson dose-response model respectively. The risk of infection of Ascaris was around 12 % regardless of where exposure occurs, if Ascaris eggs were present. For risk for infection with E. coli O157, the simulated risks were below 10 % at almost all exposure points, with most of the high risk exposure points located in the schools. There are risks of pathogen transmission in the Peepoo management chain that should be further investigated. Ammonia sanitisation permits a high degree of microbial inactivation but to secure a safe end-product it is recommended to be kept in room temperature (24.05±0.62 °C) or higher.

No description available.