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No description available.

Die Struktur der Plasmamembran ist von deren Lipid- und Proteinzusammensetzung abhängig und wird durch die Anbindung an das unterliegende Zytoskelett beeinflusst. Das Ziel der vorliegenden Arbeit war die Untersuchung eines neuen Modellsystems basierend auf po­ren­über­span­nen­den Membranen, welches sowohl die heterogene Lipidzusammensetzung als auch den Einfluss eines unterliegenden Netzwerks berücksichtigt. Lipidmembranen, zusammengesetzt aus der „raft“-ähnlichen Lipidmischung DOPC/Sphingo­myelin/Cho­les­terin (40:40:20), wurden auf porösen, hochgeordneten Siliziumsubstraten mit Po­ren­durch­messern von 0.8, 1.2 und 2.0 µm durch Spreiten und Fusion von Riesenvesikeln (giant unilamellar vesicles, GUVs) präpariert. Die mikroskopische Phasenseparation in koexistierenden flüssig-geordneten (liquid ordered, lo) und flüssig-ungeordneten (liquid disordered, ld) Domänen wurde stark durch das unterliegende poröse Substrat beeinflusst. Die Größe der lo-Domänen konnte durch die Porengröße des Siliziumsubstrats, die Temperatur und den Cholesteringehalt der Membran, welcher durch Zugabe von Methyl-β-Cyclodextrin moduliert wurde, kontrolliert werden. Die Bindung der Shiga Toxin B-Untereinheit (STxB) an po­ren­überspannende Membranen, dotiert mit 5 mol% des Rezeptorlipids Gb3, führte zu einem Anstieg des Anteils der lo-Phase. Außerdem wurde die Bildung von lo-Domänen in nicht-phasenseparierten Membranen, zusammengesetzt aus DOPC/Sphingomyelin/Cholesterin/Gb3 (65:10:20:5), durch die Shiga Toxin-Bindung induziert. Ein Anstieg des Anteils der lo-Phase konnte ebenfalls bei der Bindung der pentameren Cholera Toxin B-Untereinheit (CTxB) an po­ren­überspannende Membranen, dotiert mit 1 mol% des Rezeptorlipids GM1, beobachtet werden. Des Weiteren wurde der Einfluss der chemischen Struktur des Gb3-Moleküls auf die Shiga Toxin-Bindung und die Reorganisation von festkörperunterstützten Membranen (solid supported membranes, SSMs) untersucht. Die STxB-Bindung an α-hydroxyliertes Gb3 erhöhte signifikant den Anteil der lo-Phase, während eine cis-Doppelbindung zur Bildung einer weiteren lo-Phase führte, die vermutlich ungesättigte (Glyko-)Sphingolipide und Cholesterin enthält. Im Falles des ungesättigten Gb3 konnte außerdem eine Kondensation zu größeren Domänen nach der STxB-Bindung beobachtet werden. Die genaue Phasenzuordnung der eingesetzten Glykospingolipide vor der Proteinbindung ist bisher unbekannt. Daher wurde das Phasenverhalten eines fluoreszierenden Polyen-Ga­lac­to­ce­re­bro­sids untersucht, welches bevorzugt in der lo-Phase von GUVs angereichert war. Dieser neue, intrinsische Fluorophor vermag als Grundlage für weitere Studien zum Phasenverhalten von Glykosphingolipiden dienen.

Antimicrobial resistance (AMR) is a worldwide public health concern. While it is evident that the use of antibiotics creates selection pressure for the evolution of antibiotic resistance genes, there are still considerable knowledge gaps relating to the status quo of antibiotic use, emergence of resistant pathogens in different livestock production systems and spread within human and animal communities. This thesis includes a survey of antibiotic use in the dairy sector within a specific area of Zambia and analysis of AMR and virulence factors in E. coli isolated from dairy cattle and diarrhoea human patients with the following objectives. 1. To investigate the usage of antibiotics in the dairy sector and the drivers for use. 2. To determine the prevalence and patterns of antimicrobial resistance in E. coli isolated from faecal samples of dairy cattle. 3. To use whole genome sequencing (WGS) to investigate the molecular epidemiology of resistance determinants in E. coli strains isolated from both dairy cattle and humans. 4. To assess the zoonotic potential of isolated E. coli focusing on Shiga toxin-producing E. coli (STEC) and relationship to STEC associated with clinical disease in the UK. In view of these objectives, the first part of the work was carried out in Zambia and involved a questionnaire, a field survey, isolation of E. coli from dairy cattle faecal samples and phenotypic testing for AMR. In addition, E. coli isolates were obtained from another study that was focused on human patients presenting with diarrhoea at the University Teaching Hospital in Lusaka. The second part involved whole genome sequencing and molecular analyses of E. coli for resistance and virulence genotypes at the Roslin Institute (UK). For the field study, a stratified random sample of 104 farms was studied, representing approximately 20% of all dairy farms in the region. On each farm, faecal samples were collected from a random sample of animals and a standardised questionnaire on the usage of antibiotics was completed. An E. coli isolate was obtained from 98.67% (371/376) of the sampled animals and tested for resistance against the six types of antibiotics (tetracycline, ampicillin, sulfamethoxazole/trimethoprim, cefpodoxime, gentamicin and ciprofloxacin). These E. coli were then analysed together with those from humans for genotypes in the laboratory and from Illumina short read whole genome sequences using bioinformatics tools. Tetracylines and penicillin were the commonly used antibiotics in dairy herds. This finding was in line with the resistance phenotypes detected in E. coli isolated from the dairy cattle. The most prevalent AMR was to tetracycline (10.61; 95%CI: 7.40-13.82), followed by ampicillin (6.02; 95%CI: 3.31-8.73), sulfamethoxazole/ trimethoprim (4.49; 95%CI: 2.42-6.56), cefpodoxime (1.91; 95%CI: 0.46-3.36), gentamicin (0.89; 95%CI: 0.06-1.84) and ciprofloxacin (0%). The risk analysis indicated that AMR was associated with livestock diseases (lumpy skin disease and foot rot), exotic breeds (Jersey and Friesian), location, farm size and certain management practices. Analysis of whole genome sequences showed that isolates from humans had both higher levels and a greater diversity of resistance alleles than the cattle isolates. Common genotypes in both populations were: tetA (16%), tetB (10%), tetC (2%) for cattle isolates with tetA (32%), tetB (22%) and tetD (1%) in human isolates. Other common genotypes were blaTEM (56%), sul1 (29%), sul2 (66%), strA4 (57%) and strB1 (64%) in isolates of human origin while blaTEM (15%), sul1 (3%), sul2 (17%), strA4 (13%) and strB1 (19%) were in the cattle isolates. Whilst the E. coli isolates from cattle encoded resistance to common antibiotics of limited significance to human clinical medicine, isolates from humans had additional extended spectrum beta-lactamases (blaOXA, blaCMY, blaNDM, and blaDHA, blaOKP and blaCTX-M) that encode for resistance to essential antibiotics such as third generation cephalosporins and carbapenems. This was an evidence that AMR is an ongoing public health subject in Zambia but the exclusivity of certain resistances in the human population points to limited or no exchange of genotypes between E. coli of human origin and those from cattle. AMR in humans was probably independently selected by the use of antibiotics of clinical importance such as cephalosporin and fluoroquinolones. The virulence analysis focused on STEC, 11% (41/371) of E. coli isolates from cattle contained Shiga toxin genes (stx) while none (0/73) of the human isolates were positive. Phylogenetic analysis showed a random distribution of bovine STEC, with no indication of clonal spread. Although 89% (16/18) of the STEC tested had a cytotoxic effect on Vero cells, indicative of Shiga toxin production, only three (O45, O111, O157) belonged to one of the seven serogroups (O26, O157, O111, O103, O121, O145 and O45) associated with life-threatening enterohaemorrhagic E. coli (EHEC) infections in humans. In line with this, only the O157 serotype encoded a type 3 secretion system. This shows that, while Stx-encoding strains are common in these dairy herds of Zambia, they are not strain backgrounds known to pose an immediate threat to human health as they lack colonisation factors that are found in typical human EHEC. However, we must remain vigilant as emergence of EHEC strains in these animals remains an ever-present threat.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

No description available.

Les Escherichia coli enterohémorragiques (EHEC) sont responsables de maladies graves comme la colitehémorragique (CH), le syndrome hémolytique et urémique (SHU) ou le purpura thrombotiquethrombocytopénique (PTT). Les infections humaines sont principalement dues à la consommationd’aliments contaminés, en particulier la viande d’origine bovine, le lait ou les légumes. Le principalréservoir naturel des EHEC est le tractus gastro-intestinal (TGI) des bovins. Les fèces de bovins sont, parconséquent, responsables de la contamination de plusieurs types d’aliments mais également de ladissémination environnementale de ces bactéries pathogènes. En relation avec cette problématique, lesdeux objectifs de ce projet étaient (i) de développer une nouvelle méthode d’immuno-capture pouraméliorer l’isolement des principaux sérogroupes de EHEC impliqués dans les infections humaines; et (ii)de développer un nouveau DFM (direct fed microbial) utilisable chez les bovins pour diminuer laprévalence des EHEC chez les animaux. Cette thèse a permis le développement d’une méthoded’immuno-capture basée sur l’utilisation d’une microplaque à 96 puits, coatée avec des anticorpsspécifiquement dirigés contre E. coli O157 ou d’autres sérogroupes. Cette méthode, appelée immunomicroplatecapture (IMC) est efficace et facile d’utilisation pour l’isolement de souches d’E. coli O157 ;O26 ; O103 et O111 dans les produits alimentaires. L’IMC pourrait être une alternative à l’utilisation desbilles immuno-magnétiques qui sont classiquement utilisées pour la détection des EHEC dans lesproduits alimentaires, mais qui représentent une technique longue et fastidieuse lors de l’analysesimultanée de nombreux échantillons. La seconde partie de cette thèse a permis de sélectionner 5souches de bactéries lactiques qui présentent une activité antagoniste vis-à-vis d’E.coli O157 et d’autressérogroupes in vitro. La résistance de ces souches aux conditions du tractus gastro-intestinal (conditionsacides, présence de sels biliaires, présence de jus de rumen) a été évaluée in vitro. Leur innocuité a étévérifiée chez des souris Balb-C par l’administration de chacune des souches incorporées à la nourriture(109 cfu/g). Enfin, la lyophilisation des souches n’a pas affecté leur activité antagoniste in vitro. Lesrésultats obtenus dans les différents tests in vitro suggèrent que les 5 souches pourraientpotentiellement être utilisées comme DFM chez les bovins pour diminuer la colonisation de leur TGI parles EHEC et par conséquent diminuer le risque de contamination des aliments et le risque dedissémination environnementale. / Enterohemorrhagic E. coli (EHEC) are responsible for important diseases such as hemorrhagic colitis,hemolytic and uremic syndrome or thrombotic thrombocytopenic purpura. Human infections occurprincipally by consumption of contaminated food particularly beef meat, milk or vegetables. The mainnaturally reservoir of EHEC is the gastro-intestinal tract of cattle. Cattle feces are therefore responsiblefor contamination of various types of food but also environment dissemination of the pathogenicbacteria. Related to this problematic, the two objectives of this project were (i) to develop a newimmuno-capture method to improve the isolation of the main serogroups of EHEC involved in humaninfections in food; and (ii) to develop a new direct fed microbial usable in cattle to decrease prevalenceof EHEC in animals. This thesis allowed the development of an immuno-capture method based on theuse of 96-well microplates coated with specific antibody directed against E. coli O157 and otherserogroups. This method, called immuno-microplate capture (IMC) was efficient and user-friendly forthe isolation of E. coli O157; O26; O103 and O111 in foods. This could be an alternative to the use ofimmuno-magnetic beads which are currently used for the detection of EHEC in foods, but are timeconsumingand labor intensive when large number of samples is analyzed simultaneously. The secondpart of this thesis allowed the selection of 5 lactic acid bacteria strains which presented highantagonistic activity against E. coli O157 and other serogroups in vitro. Resistance of these strains togastro-intestinal conditions (acidic conditions, presence of bile salts and rumen fluid) was evaluated invitro. The safety of the 5 strains was checked in Balb-C mice by administration of each strain mixed infeed at 109 cfu/g. Finally, freeze-drying did not affect the antagonistic activity of the 5 strains, suggesteda possible large scale use of these strains. According to the various results obtained in vitro, the 5 strainscould potentially be used as DFM in cattle to decrease colonization of their gastro-intestinal tract byEHEC and consequently decrease the risk of food and environment contaminations.

Master of Science / Food Science Institute / Randall K. Phebus / The USDA declared seven STEC serotypes to be adulterants in raw, non-intact beef products due to their severe health implications. STEC contamination of carcasses is most likely to occur during hide removal. This study evaluated the efficiency of a mixed STEC-7 inoculum to attach to raw beef carcasses (predominantly lean muscle and adipose tissue), and compared the efficacy of 4.5% lactic acid (LA) to a water (W) spray to reduce STEC populations. Four carcass contamination scenarios, representing potential points whereby STEC could come into contact with raw beef surfaces during slaughter operations, were evaluated: (A) pre-rigor surface STEC inoculated (ca. 7 log cfu/cm[superscript]2), 30-min ambient temperature attachment, spray with LA or W; (B) pre-rigor inoculated, 24-h chilled attachment, spray; (C) tissue chilled 24 h, inoculated, 30-min attachment, spray; and (D) tissue chilled 24 h, rewarmed to 30°C, inoculated, 30-min attachment, spray. Predominantly lean muscle and adipose tissue were collected from four fed cattle immediately after harvest and assigned to the four scenarios for STEC inoculation, followed by a post-inoculation water (control) or LA spray. Tissue excision samples were collected pre- and post-treatment and analyzed to enumerate STEC-7 populations. Data were collected in a completely randomized design and analyzed using a mixed-model ANOVA. Pairwise comparisons of treatment means were made at α = 0.05 with p-values adjusted using Tukey-Kramer. Initial STEC attachment levels to predominantly lean muscle and adipose tissues were not significantly different across all scenarios. Scenarios C and D showed greater STEC attachment compared to scenarios B and A. The LA spray reduced STEC levels more effectively than water across all scenarios. A significant treatment by tissue type interaction was observed for STEC reductions. A greater STEC reduction was observed for adipose tissue than for predominantly lean muscle when lactic acid spray was applied. A significant treatment by scenario interaction was observed for STEC reductions. Scenarios A and B presented greater log reductions (1.77 ± 0.27 and 1.85 ± 0.25 log CFU/cm[superscript]2, respectively) than scenario C (1.04 ± 0.10 Log CFU/cm[superscript]2). LA spray presented the same level of effectiveness when applied to pre-rigor warm tissues and chilled tissues for reducing STEC. Greater post-LA spray reductions were observed when STECs were inoculated onto pre-rigor meat surfaces and submitted to a 24 h chill cycle, suggesting that cold storage temperatures (~2 °C) may stress or injure the STEC cells prior to subsequent antimicrobial spray applications to chilled surfaces. For laboratory studies, consideration must be given to when inocula are applied to tissue surfaces to accurately determine and/or compare the effectiveness of antimicrobial treatments. These findings provide insight to beef processors and researchers regarding inoculation protocols for comparative validation studies, and potential impacts on microbiological results from application of antimicrobial interventions at different points during raw beef processing.

L’introduction commence avec la description de toxines d’origines bactérienne et végétale, en particulier la toxine Shiga ainsi que les toxines de la même famille (chapitre 9.1.2). Les petites molécules inhibitrices de ces toxines sont ensuite résumées dans le chapitre 9.1.3, en particulier le composé Retro-2. L’efficacité de ces toxines à atteindre leurs cibles reposant sur le trafic intracellulaire, un aperçu général de l’endocytose et du trafic endosomal sont présentés (chapitre 9.2). Puis, l’entrée de la voie rétrograde est décrite (chapitre 9.2.5), avec un intérêt particulier porté sur la clathrine, le rétromère et GPP130, une protéine qui circule de manière continue entre le Golgi, la membrane plasmique et les endosomes. Les protéines SNARE, en particulier la syntaxine-5 et le syntaxine-16, sont ensuite introduites (chapitre 9.2.6). Après une brève section sur les micro-ARNs de la famille 199 (chapitre 9.3), l’introduction se termine avec la description des techniques clés utilisées au cours de mon travail, tels que la chimie click bio-orthogonale, la synchronisation du trafic antérograde par rétention grâce à des hameçons spécifiques (RUSH), et la ligation par proximité basé sur des anticorps (chapitre 9.4).Ci-inclus, mon article en cours de soumission ouvre la partie résultats (chapitre 10.1), dans laquelle je présente l’intérêt de la chimie click bio-orthogonale pour identifier les cibles cellulaires de Retro-2. Je décris un des candidats potentiels, Sec16A, et illustre comment grâce à la technique de RUSH, perturber la fonction de Sec16A conduit à la relocalisation partielle de la syntaxin-5 au niveau du reticulum endoplasmique via l’inhibition du transport antérograde de la syntaxine-5. La seconde partie de l’article décrit comment la relocalisation de la syntaxine-5 induit l’inhibition du trafic de la toxine Shiga des endosomes au TGN. Je présente une nouvelle interaction entre la syntaxine-5 et la protéine TGN GPP130, qui ont déjà été caractérisées en relation avec le trafic de la toxine Shiga. Mon travail connecte à la fois les facteurs de trafic avec le trafic rétrograde au niveau de l’interface endosome-TGN. De manière frappante, cette interaction est très probablement basée sur une fonction non-SNARE de la syntaxine-5 car le domaine de fixation sur GPP130 est structurellement non lié à toute fonction SNARE.En collaboration avec Juan Francisco Aranda et Carlos Fernandez aux Etats-Unis, nous avons placés des micro-ARNs dans un contexte de régulation endogène du trafic rétrograde de la toxine Shiga (chapitre 11.2). Une discussion plus approfondie sera apportée dans le chapitre 12.Enfin, une vue d’ensemble des projets en cours est apportée dans la section des perspectives (chapitre 12), dans laquelle les collaborations plus approfondies sont mises en lumière.Mots clés : transport rétrograde, toxine Shiga, toxine de la famille Shiga, STxB, syntaxin-5, Sec16A, GPP130, Retro-2, Retro-2.1, chimie click sans cuivre, identification des cibles de petites molécules, spétrométrie de masse, function non-SNARE, inhibition du trafic antérograde, miARN, miR199, rétromère, VPS26 / The introduction of my PhD manuscript starts with describing plant and bacterial toxins (chapter 9.1), in particular Shiga toxin and Shiga-like toxins (SLTs) (chapter 9.1.2). Small molecule inhibitors of these toxins are summarized afterwards in chapter 9.1.3, notably the Retro-2 compound. Since these toxins rely on intracellular trafficking to reach their molecular targets, a general overview of endocytosis and endosomal trafficking is provided (chapter 9.2). Next, the retrograde route entry is presented (chapter 9.2.5), with focus on clathrin, the retromer and GPP130, a protein that constantly cycles between Golgi, plasma membrane, and endosomes. SNARE proteins, particularly syntaxin-5 and syntaxin-16, are then introduced (chapter 9.2.6). After a brief section of the micro RNA family 199 (chapter 9.3), the introduction finishes with the description of some salient techniques that were used in my work, such as - bio-orthogonal Click-Chemistry, anterograde trafficking synchronization with the retention using selective hooks (RUSH) assay, and the antibody-based proximity ligation assay (chapter 10.6.1, 0, 10.11.1).Herein, my submitted publication opens the results part (chapter 11.1), in which I present the utility of biorthogonal click chemistry for the search of the cellular targets of Retro-2, a small molecule inhibitor that was previously shown to protect cells and animals against Shiga toxin and ricin. I describe that Sec16A is a likely cellular target candidate, and illustrate using the RUSH approach how interfering with Sec16A functions leads to the partial relocalization of syntaxin-5 to the endoplasmic reticulum (ER) by slowing-down its anterograde transport. The second part of the paper describes how syntaxin-5 relocalization causes the inhibition of Shiga toxin trafficking from endosomes to the TGN. I present a novel interaction between syntaxin-5 and the Golgi protein GPP130, which both have been already described in relation to Shiga toxin trafficking. My work connects both trafficking factors in retrograde trafficking at the endosomes-TGN interface. Strikingly, I demonstrate that this interaction is most probably based on a non-SNARE function of syntaxin-5.In collaboration with Juan Francisco Aranda and Carlos Fernandez in the US, we put micro RNAs into an endogenous regulation context of Shiga toxin retrograde trafficking (chapter 11.2). An extended discussion will be given in chapter 12.Last, a general outlook of ongoing projects is given in the perspectives section (chapter 13), in which further collaborations are highlighted.Keywords: Retrograde transport, Shiga toxin, Shiga-like toxin (SLT), STxB, syntaxin-5, Sec16A, GPP130, Retro-2, Retro-2.1, azide-functionalized Retro-2, copper-free click chemistry, small molecule target identification, mass spectrometry, non-SNARE function, anterograde trafficking inhibition, miRNA, miR199, retromer, VPS26

No description available.