This thesis is missing a page between pages 60 and 70, and three pages between pages 70 and 81. Since the pages are not all numbered, the specific page numbers cannot be determined. Theses missing pages are not in the other copies of the thesis. -Digitization Centre / Replicate isogenic populations of E. coli were propagated and maintained for over 4000 generations in order to investigate the adaptation of E. coli to increased levels of the mutagen methanesulfonic acid ethyl ester (EMS). Control "C" cell lines were propagated through daily serial culture in the absense of any mutagenic treatment. EMS adapted cell lines "E" / "e" were propagated through daily serial culturing and treated daily with 25Jul of EMS following serial dilution. Mutation frequency and survival assays conducted in this investigation strongly suggest that prior long-term low dose exposure to EMS results in significantly higher levels of resistance to the lethal and mutagenic effects of larger challenge doses of EMS relative to long-term evolved control cell lines "C". In addition, both survival and inhibition disk assays suggest a cross adaptive response between EMS and MNNG, showing enhanced survival and reduced growth inhibition zones in cells adapted to EMS and challenged with MNNG. Preliminary competition experiments suggest relative fitness for the EMS adapted cell lines ( "E" / "e") compared to the "C" control cell lines in both the presence of EMS. Unexpectedly the fitness estimates also suggest a higher relative fitness for the "E" / "e" EMS adapted cell lines in the absence of EMS treatment, suggesting that the EMS specific adaptation may also result in improved fitness in novel environments. Despite the adaptive advantage for the "E" / "e" cell lines suggested by the fitness estimates, the results from the competition experiments are insignificant due to the high degree of variability among replicate fitness estimates. Attempts to induce the adaptive response repair pathway were not successful in either the control "C" or the EMS adapted "E" / "e" cell lines suggesting that enhanced resistance seen in the adapted "E" / "e" cell lines could likely be a result of enhanced activity of the constitutive transferase Ogt and the constitutive glycosylase Tag. The ada and the ogt genes encode the induced and the constitutively-active DNA methyl transeferases in E. coli. As such they appeared to be the most likely candidates for genetic changes responsible for the enhanced resistance to the lethal and mutagenic effects of large doses of alkylating agents in the long-term EMS adapted "E" / "e" cell line. However, the DNA sequences analyzed for the ogt and the ada genes for both the long-term evolved control E. coli cell line "C" and the long-term-evolved EMS adapted "E" / "e" cell line indicate no sequences differences between these two cell lines. Previous studies have primarily observed E. coli's ability to phenotypically acclimate over very short time intervals to EMS. This analysis has shown that long-term genetic adaptation to low doses of EMS results in enhanced resistance to both the lethal and mutagenic effects of larger challenge doses of EMS. / Thesis / Master of Science (MS)

Poultry litter can harbor pathogenic bacteria, including avian pathogenic Escherichia coli (APEC). Applying litter amendments is one strategy to improve bird health and potentially reduce pathogens. Biochar and PLT were applied as litter amendments in a live bird trial to study their effects on E. coli populations, APEC virulence genes (VAG), and antimicrobial resistance (AMR) genes. Samples were collected at days 0, 17, 29, and 41 to enumerate E. coli and store bacterial isolates for antimicrobial-resistance gene analysis. Data analysis showed litter amendments did not significantly affect overall E. coli populations. Grow-out time impacted E. coli populations, with reductions occurring over time. Litter treatment had no impact on the prevalence of VAG or AMR. Time showed VAGs were absent at d 0 while AMR genes were prevalent at d 0. This indicates chicks may have been the source of VAG, while AMR genes were prevalent in used litter.

Thesis (Ph.D.)--Baylor University, 2008. / Includes bibliographical references (p. 130-146).

Thesis--Göteborg University, 1997.

In contrast to the damage caused by far-UV, the damaging effects of UVA (320-400 nm) in living cells are not well understood. The damage caused by UVA irradiation is largely oxygen-dependent, suggesting UVA-mediated DNA damage involves reactive oxygen species produced through the action of an endogenous photosensitizer. Previous studies examining cellular responses to UVA irradiation in E. coli have been hindered by the fact that, at sublethal fluences, wild-type cells undergo a transient inhibition of cell growth termed a "growth delay". This effect is absent in nuvA⁻ strains, thereby facilitating the study of DNA repair factors required for the repair of UVA-mediated damage. Formamidopyrimidine (Fapy) glycosylase (encoded by fpg) and the UvrABC excinuclease are both capable of excising oxidatively damaged DNA bases. An fpg::kan mutation was placed into isogenic uvrA⁺ and uvrA⁻ strains of E. coli to evaluate the relative importance of these repair enzymes in the recovery from UVA-induced stress. In a nuvA⁻ background, the survival of fpg⁻ mutants exposed to UVA was significantly reduced relative to isogenic fpg⁺ control strains. This effect was enhanced in the absence of the UvrABC excinuclease, suggesting a role for both of these enzymes in repairing UVA-generated lesions. Survival of isogenic nuvA⁺ repair-deficient strains was significantly lower than nuvA⁻ strains, suggesting a role for the modified base 4-thiouridine in UVA-mediated lethality. An in vitro plasmid DNA irradiation assay in the presence and absence of 4-thiouridine was used to examine this possibility. When irradiated DNA was subsequently used to transform the fpg⁻ and uvrA⁻ mutant strains, no increase in DNA damage (as measured by a decrease in transformational efficiency) in the presence of 4-thiouridine was observed, suggesting that when present in solution this base does not play a photosensitizing role in UVA-mediated lethality. / Thesis / Master of Science (MSc)

Verocytotoxin-producing Escherichia coli (VTEC) are important human pathogens that may cause diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome (HUS). Worldwide, non-sorbitol-fermenting (NSF) VTEC O157:H7 is the most common serogroup associated with HUS but several non-O157:H7 serogroups have emerged as causes of this disease. This research investigated the pathogenic potential of two non-O157:H7 serogroups: O26 and sorbitol-fermenting (SF) O157:NM. While VTEC O26 have emerged as a significant cause of HUS in continental Europe, human infections associated with this pathogen are uncommon in Scotland and generally only result in simple diarrhoea. The study characterised E. coli O26 isolates recovered from human infections in Europe and Scotland and isolates collected from Scottish cattle with the objectives to identify factors which may allow strains to cause more serious clinical disease and to investigate the potential of bovine VTEC O26 in Scotland to cause human infection. MLST analysis of housekeeping genes found little genetic variation in the genomic ‘backbone’ among the vast majority of E. coli O26 isolates. The gene for verocytotoxin 2 (vtx2) alone was carried by VTEC O26 isolates recovered from patients in continental Europe but was found in no Scottish human isolate, where the majority of isolates did not harbour a vtx gene. It was demonstrated that among the European VTEC O26 human isolates, 67% carried a specific allele within the promoter region for LEE1 and 87% harboured the tccP2 gene. In contrast, no Scottish VTEC O26 human isolate carried this allele or the tccP2 gene. The impact these genotypic characteristics have on the pathogenic potential of a strain remains uncertain. There were no clear differences in verocytotoxin titres, levels of LEEencoded protein secretion or levels of adherence to Caco-2 cells between VTEC O26 isolates recovered from human infections of varying severity. However, levels of LEE-encoded protein secretion from cattle isolates were generally higher than those from many of the human isolates. The differences in pathogenic potential between isolates are likely to be due to horizontally acquired DNA, including vtx2 carriage and the O-island-phage-associated effector protein repertoire. Further work is required to determine if the differences identified may also impact on shedding levels from cattle and therefore the likelihood of transmission to humans. Since 1988, SF VTEC O157:NM strains have emerged and have been associated with a higher incidence of progression to HUS than NSF VTEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF VTEC O157:NM. While no evidence of toxin or toxin expression differences between the two VTEC O157 groups was found, the SF VTEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. The capacity of SF VTEC O157:NM strains to express curli at 37C may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn this could lead to increased toxin exposure and an increased likelihood of progression to HUS.

The EspF protein is translocated into host cells by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF sequences differ between EPEC and EHEC serotypes in terms of the number of SH3-binding polyproline rich repeats and specific residues in these regions as well as residues in the amino domain involved in cellular localization. In this study we have compared the capacity of different espF alleles to inhibit: (i) bacterial phagocytosis by macrophages; (ii) translocation through an M-cell co-culture system; (iii) uptake by and translocation through cultured bovine epithelial cells. The espFO157 allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast, espFO157 was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonisation site of EHEC O157 in cattle and a site containing M-like cells. As functional differences could not be simply assigned to variation in established interactions of EspF with Sorting Nexin 9 and N-WASP, yeast-2-hybrid screening was used to identify additional host proteins that may interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was identified from this screen. Mad2L2 was then demonstrated to interact with EspF variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays. While Mad2L2 has been shown to be targeted by the non homologous Shigella effector protein IpaB to limit epithelial cell turnover, we presume that EspF interactions with this protein may indicate a similar function to promote EPEC and EHEC colonization. The final section of work addressed whether bacterial interactions can actually induce M-cell differentiation on follicle-associated epithelium. The work focused on bovine rectal primary cell cultures interacting with Salmonella enterica serovar Typhimurium. The type III secreted protein, SopB, was required for Salmonella to: III (i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii) transform a subset of epithelial cells to a cell type that phenotypically and functionally resembles specialized antigen sampling M cells; (iii) induce RANKL and downstream RelB dependent NFkB signaling. The work suggests that Salmonella may induce this cellular transformation to promote its invasion and colonization of intestinal mucosa.

The rural population of Grampian was reported to be two times more likely to suffer from <i>E. coli</i> O157 infection than the urban population. <i>E. coli</i> O157 was isolated from minced beef and lamb sampled in butcher shops and supermarkets in rural and urban areas, environmental samples (sheep and cattle faeces on farms) and tap water from private water supplies in the countryside using enrichment of sample, followed by immunomagnetic separation and culture on CT-SMAC agar. Clinical <i>E. coli</i> O157 isolates were recovered from patients in parallel. All <i>E. coli</i> O157 were genotyped by multilocus variable number of tandem repeats analysis (MLVA). Results from the meat survey (Nov04-Aug06) revealed a very low incidence of <i>E. coli </i>O157 in minced meat i.e. 0.75 % (4/530). The winter <i>E. coli</i> O157 sheep study (Jan-Mar05) revealed a group prevalence of 42.8 % and an individual animal prevalence of 5.8 %, consistent with a previous summer study (40 % and 6.5 % respectively) and the cattle farm prevalence was 22 % (Oct06-Jun07), consistent with previous prevalence studies in Scotland. According to MLVA data analysis, it appears that the rural community becomes ill through ingestion of <i>E. coli</i> O157 originating mostly from cattle, from which, a small proportion are infected by drinking water contaminated by cattle. Four human cases of infection living in urban areas shared the same MLVA types as rural human cases emphasising the fact that both populations can become ill from the same source i.e. city dwellers visiting the countryside or by a food widely distributed. The rest of the urban cases MLVA types were significantly different from cattle and sheep types and another source of infection was proposed i.e. travel outwith Grampian or ingestion of food produced in other countries.

A Prolactina (PRL) é um dos hormônios mais versáteis em termos de ação biológica. Sua ação mais conhecida está relacionada com o estímulo da lactação e regulação do crescimento e da diferenciação da glândula mamária; também apresenta importante aplicação diagnóstica. Somando os crescentes estudos sobre suas possíveis aplicações terapêuticas, fica cada vez mais notória a necessidade da obtenção desse hormônio puro, biologicamente ativo e na sua forma autêntica.O objetivo fundamental desse projeto foi a produção de hPRL em escala laboratorial a partir de bactérias (E.coli) modificadas geneticamente, utilizando um sistema de expressão baseado no promotor Lambda () PL, o mesmo utilizado com sucesso em nosso laboratório na expressão do hGH. Descrevemos nesse trabalho um processo de cultivo em biorreator, onde não foi utilizado o repressor cIts, uma proteína termo-sensível que usualmente é utilizada para inibir o funcionamento do promotor PL durante crescimento a 30ºC. O processo de cultivo apresenta basicamente três etapas: na primeira etapa o crescimento é realizado sem adição contínua de nutrientes (cultivo em batch), na segunda etapa ocorre adição contínua de nutrientes e carboidrato (cultivo em fed-batch) e na última etapa é realizada a ativação, caracterizada pelo aumento da temperatura mantendo-se a adição de nutrientes e carboidrato. Esse processo de fermentação rápido e flexível, com duração média de 20 horas, permitiu obter uma biomassa final correspondente à densidade óptica de aproximadamente 30 A600nm (unidades ópticas de absorbância em 600nm) e com uma expressão da ordem de 1g de hPRL mL-1 A600 -1, as mais altas já relatadas para secreção de prolactina no espaço periplásmico. A hPRL monomérica foi purificada e caracterizada por métodos físico-químicos e biológicos, os quais confirmaram a sua atividade biológica e imunológica, o seu correto processamento e uma massa molecular relativa (Mr) de 22.906. / Prolactin (PRL) is one of the most versatile hormones in terms of biological action. His best known action is related to the stimulation of lactation and regulation of growth and differentiation of the mammary gland; it also has wide important diagnostic applications. Considering all the increasing studies on its potential therapeutic applications, the need for obtaining this hormone in its pure, biologically active and authentic form becomes clearer and clearer. The fundamental objective of this project was the production of hPRL on the laboratory scale, from genetically modified bacteria (E.coli), using an expression system based on Lambda () PL promoter, the same successfully used in our laboratory for the expression of hGH. We set up a cultivation process in bioreactor, where the repressor (cIts), a thermo-sensitive protein that is usually used to inhibit the PL promoter during the growth phase (30°C). The cultivation process presents basically three stages: the first step in was not used the growth is carried out without the continuous addition of nutrients (batch cultivation), the second step in which a continuous addition of nutrients and carbohydrate occurs (fed-batch cultivation) and a final step when activation is carried out. The latter is characterized by an increased temperature, still maintaining the addition of nutrients and carbohydrate. This fast and flexible process of fermentation, with the average duration of 20 hours, led to a final biomass of approximately 30 A600nm (units of optical absorbance at 600nm), with the expression of about 1g of hPRL mL-1A600 -1, the highest ever reported for the secretion of prolactin in the periplasmic space. Monomeric hPRL was purified and characterized by physical-chemical methods and biological assays, which confirmed its biological and immunological activity, correct processing and a relative molecular mass (Mr) of 22,906.

Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics. / October 2007