Thesis (Ph.D.)--University of Western Sydney, Hawkesbury, 2004. / A thesis presented to the University of Western Sydney, Hawkesbury, Centre for Advanced Food Research, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

Escherichia coli (E. coli) is a widespread species that includes a broad variety of strains, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to virulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. The aim of this study was to evaluate the prevalence and antibiogram profiles of E. coli pathotypes previously isolated from Kat River and Fort Beaufort abstraction water. A total of 171 E. coli isolates showed at least one pathogenic determinant among the isolated 278 E. coli. The other 107 isolates were negative for the tested virulence genes. All 278 presumptive isolates tested positive for the UidA gene, and were therefore classified as non-categorized pathogenic E. coli. The 171 pathogenic isolates had at least one characteristic gene of pathogenic E. coli and were identified and classified as enteropathogenic E. coli (6%), enterotoxigenicE. coli (131), uropathogenic E. coli (6), neonatal meningitis E. coli (14), diffusely adherent E. coli (1) and enterohaemrrhagic E. coli (1). Interestingly, no virulence genes were detected for the enteroinvasive E. coli and the enteroaggregative E. coli. The antibiotic resistance profiles for all isolates that were identified as E. coli showed 100% resistance to penicillin G, 98% resistance to ampicillin, 38% resistance to trimethoprim-sulphamethoxazole and 8% resistance to streptomycin. Multiple antibacterial resistance (MAR) was also observed, where 44% of the isolates were resistant to three antibiotics and 8% resistant to four antibiotics. The results of this study showed the Kat River and Fort Beaufort abstraction water are reservoirs of pathogenic strains of E. coli which harbour antibiotic resistance determinants that can cause serious health risks to the people in the surrounding communities.

Mucin was isolated for incorporation in bacteriological media by reduction and proteolysis of mucous gel from porcine small intestine. Mucin prepared in this study contained (by weight) 37.2% protein, 58.7% carbohydrate (4.6% fucose, 9.4% mannose, 10.7% sialic acid, 13.5% galactose, and 20.5% hexosamine), and 3.7% ester sulfate. Fractionation of mucin on Sepharose CL-4B yielded one peak which eluted at the void volume. However, sodium dodecyl sulfate gel electrophoretic patterns contained 9 polypeptide bands of which 6 stained with periodic acid—Schiff reagent. Proline, serine, and threonine residues accounted for 26% (by weight) of the total protein in the preparation. Half-cysteine residues made up another 1%. Intrinsic viscosity of mucin prepared by reduction and proteolysis was 135 ml/g. Mucin was incorporated into a minimal bacteriological medium as the sole-source of carbon and nitrogen. Enterotoxigenic and non-enterotoxigenic Escherichia coli grew equally well in mucin medium at levels comparable to growth in 3 mM glucose medium. Growth did not appear to be limited by availability of metabolizable substrates. Spent mucin medium supported growth in comparable numbers. E. coli P-155 produced heat-stable and heat-labile enterotoxins during growth in both mucin medium and fresh mucosal scrapings medium. E. coli utilized total hexose and protein in mucin medium at comparable levels (6 to 10%). Increases in reducing end groups (0.28 μ moles/ml) and free amino sugar end groups (0.04 μmoles/ml) during growth were detected. E. coli used approximately 15% of the total carbohydrate in mucin medium including 36% of the galactose, 15% of the fucose, and 27% of the mannose. Utilization of mucin by QL ggli produced minor changes in gel filtration patterns on Sepharose CL-4B. Twelve strains of E. coli were examined for glycosidase activity during growth on mucin. All twelve produced a cell-bound and an extracellular α-fucosidase although the majority of activity was cell-bound. Although α-fucosidase was a constitutive enzyme of E. coli P-155, maximum activity was observed during exponential growth in mucin medium. Eleven strains produced cell-bound α-galactosidase. No extracellular activity of this enzyme was detected. Maximum levels of induced α-galactosidase activity were obtained in late exponential to early stationary growth of E. coli. E. coli ATCC 23723, a mutant of E. coli K12 lacking the galactoside permease gene, did not produce α-galactosidase activity during growth on mucin. No α-mannosidase activity was detected using nitrophenylmannoside as substrate. Porcine small intestinal mucin was a positive chemoattractant for E. coli in capillary assays. Optimal chemotactic response by E. coli P-155 in capillary experiments was obtained at a mucin concentration of 1 mg dry wt/ml at a pH of 7.0. Spent mucin was still a positive chemoattractant for E. coli P-155 and 123 despite losing 15% of the total mucin carbohydrate. / Ph. D.

Colibacillosis is considered one of the more costly diseases encountered in the production of market turkeys. It is responsible for a significant amount of mortality in birds between the ages of 6-12 weeks. Research conducted over the past 5 years has shown that within the Shenandoah Valley production area, multiple primary infectious agents are responsible for the predisposition of turkeys to colibacillosis. These agents were first identified as potential contributors through field case studies. They include hemorrhagic enteritis (HE) virus, Newcastle disease virus, and Bordetella avium. Further retrospective serologic studies affirmed the role of these primary agents and uncovered the potential involvement of Mycoplasma meleagridis. Trials were conducted to determine the reproducibility of some multiple agent interactions under laboratory conditions. It was found that Newcastle disease virus or B. avium infection followed by HE virus and Escherichia coli challenge produced clinical colibacillosis. It is believed that hemorrhagic enteritis virus is the pivotal agent in this process of predisposition. Almost all turkeys are vaccinated for hemorrhagic enteritis in the field. The virulent strains of the virus are known to be immunosuppressive. It is suspected that the vaccine strains are mildly so. Infection with HE vaccine virus was shown to cause an increase in CT8+ cells in peripheral blood. These cells are believed to be suppressor T-cells and may account for the reputed immunosuppressive effects of the virus. Thus, interactions of multiple infectious agents including Newcastle disease virus, B. avium, M. meleagridis, and HE virus appear to be involved in the predisposition of turkeys to secondary E. coli infections. / Ph. D.

Call number: LD2668 .T4 1985 G733 / Master of Science

"February 2003." Addendum and corrigenda inserted at back Includes bibliographical references (leaves 249-272) Aims to identify and characterise potential virulence-associated factors from the locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21 strain 98NK2 which was responsible for an outbreak of haemolytic uremic syndrome. Particular attention was focused on putative virulence genes encoded on the megaplasmid of this strain.

"February 2003." / Addendum and corrigenda inserted at back / Includes bibliographical references (leaves 249-272) / xi, 272 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to identify and characterise potential virulence-associated factors from the locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21 strain 98NK2 which was responsible for an outbreak of haemolytic uremic syndrome. Particular attention was focused on putative virulence genes encoded on the megaplasmid of this strain. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, 2003

Escherichia coli diarrhoea is the most important source of mortality in piglets. The most frequently isolated strain in enterotoxigenic E. coli diarrhoea is F4ab/ac. Recent studies in South Africa reported non-fimbrial strains such as PAA and EAST-1 to be prevalent. The objective of the study was to determine whether there are breed differences among pigs with respect to E. coli adhesion phenotypes and correlate them to polymorphisms at selected candidate genes in the South African population. A total of 225 pigs aged 3-12 weeks of the imported (Large White, Landrace and Duroc), local and crossbreds, were sampled from the Eastern Cape and Limpopo provinces of South Africa and genotyped for PCR-RFLP polymorphisms at four candidate genes associated with E. coli F4ab/ac resistance/susceptibility. These genes were Mucin 4 (MUC4), Mucin 13, (MUC13), Mucin 20 (MUC20) and Transferrin Receptor (TFRC). The TFRC and MUC13 genes were less polymorphic, the C allele was close to fixation and the homozygous CC genotype was the most frequent in all three pig populations. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for the TFRC locus. The g.8227G>C polymorphism in MUC4 segregated in all three breeds and the marker was moderately polymorphic. There was a significant difference (P <0.05) in genotypic distribution amongst breeds for MUC4.The g.191C>T polymorphism in MUC20 segregated in the local and crossbred pigs and was close to fixation in the imported pigs. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for MUC20, which was moderately polymorphic. There was a reduction in heterozygosity in both the TFRC and MUC13 loci, although MUC4 and MUC20 genes had higher heterozygosity levels. The MUC4 gene had a negative FIS value, indicating outbreeding at this locus. The MUC20, MUC13 and TFRC genes had a positive FIS value, indicating inbreeding at these loci. Overall, the studied population was outbred. Imported pigs in TFRC and MUC20 deviated from Hardy-Weinberg equilibrium (HWE). All breeds were in HWE at the MUC4 and MUC13 genes. There was no linkage disequilibrium observed amongst the analysed loci. iv A total of 109 piglets of three breeds (Large White, indigenous and crossbred) aged 3-5 weeks, were investigated for the susceptibility to E. coli F4, PAA strains and EAST-1 toxin. Adhesion tests were conducted on pig intestinal cells, which were viewed under a phase contrast microscope. Three phenotypes were identified as, adhesive, weakly adhesive and non-adhesive. There was a significant association (P <0.05) between breed and level of adherence of the F4 and PAA strains. Highest frequencies of adhesion phenotypes were observed in the indigenous pigs for both F4 and PAA E. coli strains. Large White pigs had the lowest frequency of non-adhesion in F4 and PAA E. coli strains. The F4 strain had a higher (P <0.05) level of adherence compared to PAA and EAST-1 in Large White pigs. Age of pigs had a significant effect on the level of E. coli adherence in indigenous and crossbred pigs (P <0.05). Adhesion of F4 and EAST-1 was higher in weaned indigenous and crossbred pigs, respectively, than in suckling piglets. There was no significant difference between F4 adhesion and the genotypes at all four candidate genes genotypes. The study showed that both imported and local pig populations carry receptors and are susceptible to F4, PAA and EAST-1 E. coli infections. Indigenous pigs were less susceptible than Large White to E. coli infection. Although polymorphic and segregating in the populations, the MUC4 g.8227G>C and MUC20 g.191C>T mutations were not associated with the adhesion phenotypes and cannot be used in the selection of susceptible animals. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.

Rotavirus is responsible for the death of many children annually, and current vaccines have lower efficiency in developing countries. A reverse translated consensus gene sequence of the rotavirus VP6 cloned into a pET-28a(+) plasmid was used to transform BL21 and KRX Escherichia coli cells. Optimal expression of soluble protein was induced in KRX cells by adding 0.05% L-rhamnose and 0.0001 M IPTG, with an incubation temperature of 25ºC for 6 h. VP6 was purified by combining anion exchange chromatography followed by affinity chromatography. Far-UV circular dichroism and intrinsic fluorescence were used as probes to assess the native structure of VP6 and structural in the presence of a denaturant, high sodium chloride concentrations and varying temperatures. The 0.2 M sodium chloride had an impact on the VP6’s tertiary structure and also influenced the proteins conformational changes as detected during thermal unfolding to 90ºC. Although treatment with 3 M urea showed tertiary structural changes no secondary structural loss occurred due to the presence of a denaturant. / Life Sciences / M. Sc. (Life Sciences)

Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.