The secondary metabolites of citrus such as limonoids and flavonoids constitute an important part of human diet. The present work was undertaken to elucidate the effect of citrus limonoids and flavonoids on the bacterial cell-cell signaling in Vibrio harveyi, Escherichia coli O157:H7 and Salmonella Typhimurium LT2. The first experiment was focused on purification of limonoids from grapefruit and sour orange seeds. The limonoids were extracted using organic solvents and purified by chromatographic techniques. A total of ten limonoids (7 aglycones and 3 glucosides) were purified. Currently, simultaneous measurement of aglycones and glucosides of limonoids is not available. To address this limitation, an analytical method using high performance liquid chromatography was developed with the capability of measuring both aglycones and glucosides in a single run. Furthermore, its applicability in the fruit and juice samples was demonstrated. The third study investigated the V. harveyi cell-cell signaling inhibitory potential of purified limonoids. Isolimonic acid, ichangin, obacunone and nomilin were showed potent inhibitory activity. Furthermore, isolimonic acid and ichangin inhibit the signal transduction pathway by up-regulating the response regulator luxO. Isolimonic acid was also found to be a potent inhibitor of Escherichia coli O157:H7 cell-cell signaling in the fourth study. The results demonstrated that isolimonic acid inhibits the autoinducer/epinephrine mediated cell-cell signaling, biofilm and virulence in QseBC and QseA dependent fashion. Further investigations using limonin analogues, in the fifth study, demonstrated that the analogue limonin-7-methoxime inhibited the E. coli biofilm in type 1 pili and antigen 43 dependent-fashion, by preventing the binding of the adhesins to plastic surfaces. Another limonoid, obacunone was demonstrated to attenuate the Salmonella virulence by repressing Salmonella Pathogenicity Island 1 (SPI-1) in EnvZ/OmpR dependent mecahnism. The seventh study showed that naringenin, among the flavonoids, was the most potent inhibitor of V. harveyi and E. coli O157:H7 cell-cell signaling. Furthermore, naringenin was found to repress the (SPI-1) in PstS-HilD dependent fashion in the eighth study. In conclusion, the current project identified several limonoids and flavonoids with cell-cell signaling inhibitory property in three bacterial species.

Shiga toxin-producing <i>E. coli</i> (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE).<p> It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non-O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific.<p> To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty-mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by <i>Mannheimia haemolytica</i>. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included.<p> In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects.<p> Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®.

Shiga toxin-producing <i>E. coli</i> (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE).<p> It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non-O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific.<p> To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty-mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by <i>Mannheimia haemolytica</i>. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included.<p> In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects.<p> Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®.

This study was conducted to determine the survival of Escherichia coli O157:H7 and Salmonella Typhimurium in marinade that was used to vacuum tumble beef inside skirts and beef tri-tip roasts. The depth of penetration of each microorganism into the individual meat products, and the survival of these microorganisms in the products as well as marinade stored over time were evaluated. Two commercial marinades were used, Reo TAMU Fajita Marinade and Legg's Cajun Style Marinade. Eighteen beef inside skirts and 18 tri-tips were used during this study. Both inside skirts and tri-tips were vacuum tumbled for a total of 1 h. Samples of products were tested immediately following tumbling (day 0), or were vacuum packaged and stored in the cooler (approximately 2 degrees C) to be tested 7 and 14 days following tumbling. Samples of the spent marinade were taken and tested initially following tumbling (day 0), and were also stored in a cooler and tested 3 and 7 days after the marinade was used. The results of the study showed that with both marinades S. Typhimurium and E. coli O157:H7 penetrated throughout the skirt meat. After having been stored for 7 days following tumbling, the log value of both S. Typhimurium and E. coli O157:H7 decreased in the meat. After 14 days of storage following tumbling, the log value of both S. Typhimurium and E. coli O157:H7 continued to decrease; however, both pathogens were still detectable. The penetration of the pathogens in the tri-tip roast varied depending on the thickness of the roast. The thicker roasts had undetectable levels of both pathogens in the geometric center; however, the thinner tri-tip roasts had detectable levels at the geometric center. The spent marinade tested on day 0, 3, and 7 showed that the microorganisms were able to survive in the marinade at refrigerated temperatures. The results of this study demonstrated that pathogens may penetrate into the interior of beef skirts and tri-tips during vacuum tumbling with contaminated marinade, and that pathogens survive during refrigerated storage of spent marinade. Industry should consider these data when evaluating potential food safety risks associated with the production of vacuum tumbling beef products.

A survey was developed and sent out to each sector of the beef industry (slaughter, non-intact processing and grinding) by using the FSIS Meat, Poultry and Egg Product Inspection Directory. Survey questions were specific to processes and interventions being applied, and the use and familiarity with Industry Best Practices documents for beef processing. Returned completed surveys. A total of 469 beef processing operations responded and of survey respondents, 119 establishments were called and asked additional questions. Critical Control Points (CCPs) and testing for E. coli O157:H7 were common discussion point during phone calls. Plant visits were made to confirm the answers that were provided in the written survey. Plants that further processed beef were found to need to reassess their HACCP plan based on their response to the question, "Is E. coli O157:H7 a reasonably likely to occur food safety hazard?" E. coli O157:H7 is considered an adulterant in the products that they produced if they answered yes to this question. Based on survey responses, slaughter establishments were using available technologies to reduce or eliminate possible microbiological contamination. Further process operations, especially those plants that produced intact steaks and roasts, marinated/enhanced steaks and roasts, and plants that produced needle/blade tenderized steaks and roasts, used documentation such as supplier purchasing specifications instead of using processes to control, reduce, or eliminated microbiological food safety hazards. Industry Best Practices were being utilized most frequently by slaughter and ground beef operations. Plants that further process beef still need to implement the use of the Industry Best Practices specific to them. Plants used testing for E. coli O157:H7 throughout the beef industry regardless of plant size or type.

Increase in consumption of fresh cut produce over the past decade has resulted in a rise in incidents of food borne outbreaks due to pathogens. Conventional techniques of sanitizing washes may not be effective since the organic matter released from the fresh produce use up the free chlorine thus reducing the sanitizing potential of wash water just when it is needed most and a heat treatment step to kill pathogens cannot be applied if the purpose is to consume fresh produce. Electron beam (e-beam) irradiation was used to treat cut cantaloupe, cut roma tomatoes, baby spinach, romaine lettuce which were surface inoculated with a cocktail of Salmonella and E. coli O157:H7. Results showed that irradiation reduced Salmonella and E. coli O157:H7 significantly with increasing doses at 0.2, 0.4, 0.6, 0.8, and 1.0 kGy. The D10-value for Salmonella on irradiated cut cantaloupe, cut roma tomatoes, baby spinach, and romaine lettuce was found to be 0.71 kGy, 0.64 kGy, 0.19 kGy, and 0.23 kGy respectively. The D10-value for E. coli O157:H7 on the produce listed above was found to be 0.73 kGy, 0.54 kGy, 0.18 kGy, and 0.20 kGy respectively. Low dose e-beam irradiation was found to be an excellent tool for ensuring the reduction of spoilage organisms and extending shelf life in cut cantaloupe, cut roma tomatoes, baby spinach, romaine lettuce, strawberries, and green onion. The produce were tested for 12 days of storage for aerobic plate count, yeast and mold, lactic bacteria, color, texture, and respiration rate as a function of irradiation doses 0, 1, 3, and 5 kGy. Aerobic plate counts, yeast counts, and lactic acid bacteria were reduced appreciably at all doses tested on all commodities. Molds did not grow on any samples including control for cut cantaloupe, cut tomatoes, and green onion but for the other commodities, mold was reduced at the same rate as yeasts and vegetative bacteria. Lactic acid bacteria were reduced at all doses while the reduction was highest with 5 kGy in all commodities. When irradiated with 5 kGy, during storage, strawberries, spinach, and green onion displayed wet, soggy and mushy appearance, romaine lettuce leaves were wilted, had a translucent midrib and brown pigmentation. E-beam irradiation increased respiration rate for all samples on day 0 compared to non-irradiated control irrespective of the commodity type and the effect was dose dependent. Firmness reduced appreciably for cut roma tomatoes, baby spinach, strawberries, romaine lettuce, and green onion with increasing doses. Cut cantaloupe was low in firmness but the effect was not dose dependent. Irradiation at low doses is a promising tool to reduce pathogens and enhance keeping quality of cut cantaloupe, cut tomatoes, baby spinach, romaine lettuce, strawberries, and green onion. Irradiation is to be implemented as part of an overall HACCP plan and is not meant to replace existing control measures.

A l'heure actuelle, les Escherichia coli producteurs de Shiga-toxines (STEC) sont considérés comme des pathogènes émergents importants en santé publique. Cependant, il n'existe aujourd'hui aucune réglementation officielle stipulant les procédures à suivre pour l'échantillonnage et la recherche des STEC dans les denrées alimentaires. <br />Ce travail a pour objectif d'étudier les différents protocoles utilisés pour la recherche des STEC, de manière à pouvoir proposer aux industriels des protocoles optimisés leur permettant une réelle maîtrise du « danger STEC » dans leur filière. Dans ce but, la cinétique de croissance de diverses souches de STEC a, dans différentes conditions d'enrichissement, été suivie simultanément à celle de la flore annexe de la matrice, puis modélisée.<br />Notre étude souligne qu'un enrichissement trop court, comme les 6 heures d'incubation dans le cas de l'IMS, peut conduire à l'obtention de résultats faussement négatifs. Il s'avère néanmoins inutile, dans certaines conditions, de prolonger l'étape d'enrichissement car une interaction de type compétition simple avec la flore annexe arrête la croissance des STEC. Cet arrêt est plus ou moins rapide selon la matrice analysée et sa densité en flore annexe naturelle (de 4 à 7 h pour les fèces et de 10 à 12 h pour le steak haché dans nos expérimentations). Dans le lait, des interactions plus complexes entraînent un arrêt de la croissance des STEC avant celui de la flore naturelle (8,5 à 11 h dans nos expérimentations).<br />L'utilisation d'agents sélectifs a pour but de freiner la croissance de la flore annexe, ce qui peut avoir pour impact de prolonger la croissance des STEC. L'ajout de sels biliaires dans le milieu d'enrichissement a un effet positif dans le cas de l'enrichissement d'échantillons de fèces de bovins et de lait cru mais n'a pas d'effet significatif pour la matrice « steak haché ». En revanche, l'addition de novobiocine dans le milieu peut inhiber certaines souches de STEC non-O157:H7 et ralentir la croissance de E. coli O157:H7. L'usage de cet antibiotique, potentiellement responsable de résultats faussement négatifs, devrait être abandonné.<br />Par ailleurs, cette étude a permis d'optimiser le protocole de recherche de E. coli O157:H7 dans le steak haché (ISO 16140) en validant, d'une part, l'analyse d'une plus grosse masse d'échantillon dans un même volume de milieu (ratio plus élevé) et en réduisant, d'autre part, le temps d'analyse grâce à l'utilisation d'une température d'incubation plus élevée de 41,5°C.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Protection of potable water supplies demands a better understanding of the factors controlling migration of disease causing bacteria in subsurface environments. In this study, the migration behaviour of the waterborne pathogenic microorganisms Escherichia coli O157:H7 and Yersinia enterocolitica was investigated in water saturated granular systems. Both facultative bacteria were grown under aerobic and anaerobic conditions and further acclimatized to a microaerophilic or fully aerated environment for 21 h. Experiments were conducted using laboratory-scale packed columns over controlled extreme dissolved oxygen (DO) concentrations. The observed differences in the transport potential of these pathogens were found to depend strongly on the antecedent growth conditions under the tested environmental settings as well with the environmental DO in certain conditions. Further microbial characterization using cell titrations and FTIR spectroscopy gave a greater insight on the source of the surface charge that was found to dominate the attachment phenomena in sand packed columns. Techniques also revealed a probable role of other cell surface macromolecules (LPS) that could account for non-DLVO behaviour. The results illustrate the importance of considering physicochemical conditions relevant to the natural subsurface environment when designing laboratory transport experiments as evidenced by variations in microbe migration as a function of the DO under growth and acclimation. / Keywords: bacterial adhesion, bacterial transport, DLVO, physicochemical characterization, dissolved oxygen, porous media.