This thesis assessed the prevalence, patterns of antibiotic resistance, and molecular characteristics of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli (EC) and Klebsiella pneumoniae (KPN) isolated from Canadian hospitals. Bacterial isolates were collected as part of the CANWARD national surveillance study. The prevalence of ESBL-EC [2007: 3.4%, 2012: 7.6%], AmpC-EC [2007: 0.7%, 2012: 2.2%], and ESBL-KPN [2007: 1.5%, 2012: 3.6%] increased between 2007 and 2012. Antimicrobials demonstrating the greatest activity against isolates in this study were colistin, amikacin, ertapenem, and meropenem, while 78.8%, 34.9%, and 66.7% of ESBL-EC, AmpC-EC, and ESBL-KPN, respectively, were multidrug resistant. Isolates were generally unrelated by PFGE; however, ST-131 was identified among 56.9% and 31.7% of ESBL-EC and AmpC-EC, respectively. CTX-M-15 was the dominant genotype in ESBL-EC (66.5%) and ESBL-KPN (48.1%), while the dominant genotype in AmpC-EC was CMY-2 (53.2%). Carbapenemase production was identified in 0.03% of EC and 0.05% of KPN, all of which produced KPC-3.

Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity on glycoproteins. The scarce supply of sialic acid hinders its advancement in basic research, diagnostic development and therapeutic production. In this work, the recombinant E. coli BRL04 (pBRL89) producing sialic acid was studied by some batch and fed batch runs of high cell density cultivation using a 3-L fermentor. Some cultivation conditions including carbon source, induction time, dissolved oxygen were optimized and different feeding strategies were compared to enhance sialic acid production. The results may be helpful to the further scale-up of sialic acid production and the production of other recombinant proteins by high cell density cultivation of E. coli.

The spread of resistance to the cephalosporins in the Enterobacteriaceae and more specifically within E. coli is a continuing cause of public health concern, with such resistance increasingly seen in community- and nosocomial-acquired infections. Extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase (AmpC) enzymes cause most cephalosporin resistance in E. coli by hydrolysis of the antimicrobial and continue to jeopardize patient outcome. The purpose of this thesis was to determine the prevalence of ESBL-producing Enterobacteriaceae and to molecularly characterize ESBL and AmpC producers found to be associated with the increasing cephalosporin resistance among E. coli within Canadian hospitals from 2005 to 2009. Isolates were collected as part of the Canadian Intensive Care Unit and Canadian Ward surveillance studies. ESBL and AmpC producers were molecularly characterized for resistance genes, virulence factors and phylogenetic groups. All strains were typed using PFGE and ESBL-producing E. coli were further typed by MLST. Plasmids bearing the ESBL and AmpC genes were characterized by BglII RFLP analysis and a multiplex PCR for replicon typing. ESBL-producing E. coli and K. pneumoniae and AmpC-producing E. coli were found to be firmly established in Canadian hospitals; whereas, ESBL-producing K. oxytoca and P. mirabilis have yet to emerge. Increasing resistance to several unrelated antimicrobials leading to multi-drug resistance among these pathogens is concerning. The successful dissemination of ESBL-producing E. coli in Canada occurs through a diversity of different mechanisms and does not correspond to a single ESBL determinant, or a single clone, or a single plasmid but rather through the combination of clonal spread of virulent strains and the acquisition of diverse ESBL-bearing plasmids. However, the predominance of CTX-M-15-producing E. coli in this study was mainly due to the virulent ST131 clone and the diverse IncFII plasmids bearing the blaCTX-M-15 gene. Whereas, horizontal transfer of genetically similar IncI1, IncA/C and IncK/B plasmids bearing blaCMY-2 and the clonal spread of virulent strains, including ST131 with ampC promoter/attenuator mutations, appears to be playing a role in the spread of AmpC-producing E. coli isolates in Canadian hospitals. The increasing prevalence of these multi-drug resistant pathogens in Canadian hospitals demonstrates the need for increased surveillance and understanding of these emerging pathogens. The continued surveillance will help guide proper infection control procedures and identify optimal treatment of these clinically important pathogens in Canadian hospitals.

The mannose-binding lectin from snowdrop (Galanthus nivalis agglutinin; GNA) was produced in Escherichia coli and purified as a functional protein after denturation/renaturation. Incorporation of the four extra C-terminal residues recently revealed from X-ray crystallographic data demonstrated that these residues increase binding to the glycoprotein carboxypeptidase Y. However, no differences in activities were observed in haemagglutination assays when compared to native GNA and toxicity towards rice brown planthopper (Nilaparvata lugens', BPH) in artificial diet bioassays was unaltered. Site-directed mutagenesis of the carbohydrate-binding site of GNA provided evidence of a direct correlation between the binding potential of GNA to BPH gut glycoprotein 'receptors' and the toxicity levels of GNA towards BPH nymphs. Functional recombinant plant lectins GNA and PHA (Phaseolus vulgaris agglutinin) were expressed in Pichia pastoris using native signal peptides or the Saccharomyces a-factor prepro-sequence to direct secretion. The a-factor prepro-sequence was inefficiently processed unless Glu-Ala repeats were added at the C-terminal end. In the latter case, removal of the Glu-Ala repeats was itself inefficient leading to recombinant lectins with heterogenous N-termini. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed and fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs in Pichia. A fusion protein containing both GNA and GFP (GNA-GFP) was expressed in Pichia pastoris. Simultaneous dual activities (i.e. carbohydrate binding and fluorescence) of recombinant GNA-GFP were demonstrated. Partial cleavage in the linker region resulted in co-purification of GNA which increased the binding activity of the fusion protein. Selective binding of GNA-GFP to haemocytes in the haemolymph of Lacanobia oleracea was observed, both in vitro and when the protein was fed to insects in diet.

This multi-phase project was designed (1) to evaluate existing post-harvest process controls and intervention strategies used to reduce Escherichia coli O157:H7, (2) to evaluate the impacts of cattle source and environmental factors on Salmonella prevalence in bovine lymph nodes, and (3) to evaluate sanitary conditions of feedyards in South Texas. The ultimate goal of this project was to identify and implement measures that reduce E. coli O157:H7 in beef harvest facilities, and Salmonella prevalence in feedyards. To evaluate process control of E. coli O157:H7 throughout the beef harvest process, samples were collected from harvest floor processing areas at two commercial beef slaughter establishments, and enumerated for aerobic plate counts, E. coli/coliform, and Enterobacteriaceae. To survey existing Salmonella prevalence, bovine lymph nodes (n = 307) were collected from beef carcasses at a commercial beef processing plant. Lymph nodes were extracted from cattle sourced from seven feedyards. Salmonella prevalence in lymph nodes was found to be 0% in cattle sourced from only one of the seven yards. Lymph nodes from cattle sourced from the other feedyards yielded positive samples, with varying prevalence. Of the remaining six feedyards, one feedyard yielded 88.2% prevalence of Salmonella in bovine lymph nodes, which was significantly higher than all other feedyards (42.9, 40.0, 40.0, 24.0, and 4.0%). The prevalence of Salmonella in the feedlot environment was compared among three feedyards; one yard had 65.0% environmental prevalence of Salmonella, which was statistically higher than the other feedyards surveyed. Of the two remaining yards, one had 0% prevalence of Salmonella in fecal and soil samples, which was also the feedyard with 0% prevalence of Salmonella in lymph nodes. Findings include (1) the significance of effective sanitary dressing procedures and intervention strategies in a beef harvest environment, (2) that there is clear feedyard-to-feedyard variation with relation to Salmonella prevalence in bovine lymph nodes, and (3) that differences in environmental factors existed among feedyards although the reasons remain unclear.

No description available.

When developing a Watershed Protection Plan (WPP) or a Total Maximum Daily Load (TMDL), it is often difficult to accurately assess the pollutant load for a watershed because not enough water quality monitoring data are available. According to the Texas Commission on Environmental Quality (TCEQ), there are 274 bacteria impairments in Texas water bodies out of 386 impaired water bodies. Bacteria water quality data are often more sparse than other types of water quality data, which hinders the development of WPPs or TMDLs. The Spatially Explicit Load Enrichment Calculation Tool (SELECT) was used to develop watershed protection plans for four rural watersheds in Texas that are impaired due to E. coli bacteria. SELECT is an automated Geographical Information System (GIS) tool that can assess pathogen loads in watersheds using spatial factors such as land use, population density, and soil type. WPPs were developed for four rural Texas watersheds: Buck Creek, Lampasas River, five sub watersheds of the Little Brazos River, and Geronimo Creek. A spatial watershed model was developed to simulate bacteria concentrations in streams resulting from non point sources using SELECT combined with a simple rainfall-runoff model and applied to the Geronimo Creek watershed. The watershed model applies a rainfall-driven loading function to the potential E. coli loads calculated by the output of SELECT. The simulated runoff volumes and E. coli concentrations from the model were compared to actual monthly E. coli data collected at two sampling sites near the outlet of a subwatershed. The results show how SELECT methodology was applied to each watershed and adapted based on stakeholder concerns and data availability. The highest potential contributors were identified and areas of concern were highlighted to more effectively apply best management practices (BMPs). The runoff volumes were predicted with very good agreement (E = 0.95, RSR = 0.21 to 0.22) for both sampling sites. The predicted E. coli concentrations did not agree with measured concentrations for both sites using eight different methods. The results indicate that the model does not include significant factors contributing to the transport of E. coli bacteria but can be modified to include these factors.

There are approximately 10^14 bacteria belonging to nearly 1000 different species in the human gastrointestinal (GI) tract that co-exist with host cells. Within the GI tract, signaling molecules secreted by both eukaryotic and prokaryotic cells are abundant. Recent studies have shown that both bacteria and human cells recognize and respond to the signals from each other, presumably to gain a competitive advantage. The cross-recognition of signals is known as Inter-kingdom (IK) signaling and this phenomenon is considered to be important in the onset of infections in the GI tract. Of the eukaryotic signaling molecules present in the GI tract, the neuroendocrine hormone norepinephrine (NE) is considered to be important in the context of infections as NE is produced at very high concentration in the intestine under post traumatic stress, is known to increase bacterial virulence and infection, and has also been shown to be a potent chemoattractant for GI tract pathogens such as enterohemorrhagic E. coli (EHEC). The focus of this study is on elucidating the mechanisms underlying the recognition and chemotaxis of bacteria towards NE. While chemotaxis has been typically investigated in the context of bacteria moving towards a metabolizable source (e.g., amino acids), chemotaxis is potentially important in the onset of infections in the human GI tract. In this study we use a microfluidic plug assay to investigate the receptor and mechanism utilized by a model bacterium Escherichia coli in its chemotactic response to NE. A series mutant of E. coli RP437 strains of knockouts for four MCP-encoding genes was used in this study. The results from the microfluidic plug assay were then confirmed quantitatively by capillary assay. We have shown that Tsr receptor is necessary for chemotaxis of NE for E. coli RP437, and attraction of E. coli towards NE may require an additional receptor. Results from the priming experiments suggest that exposure to NE may result in the de novo expression of co-receptor(s) that are crucial to chemotaxis towards NE. The requirement for high cell density also suggests the possibility that NE per se may not be an attractant for E. coli, but could be a precursor that is modified into a chemoattractant by cells. These results are expected to further our understanding of bacterial chemotaxis and its role in bacterial colonization and infection of the human GI tract.

Despite their beneficial effects, concerns have been raised about the role of antimicrobial growth promoters (AGP) in the emergence of antibiotic resistant bacteria. This study evaluated the effects of approved AGP on the emergence of antibiotic resistance in commensal E. coli and foodborne pathogen Salmonella. A survey of antibiotic resistance levels in commercial broiler chicken farms in the Fraser Valley (B.C.) and an experimental feeding trial were conducted from May 2004 to February 2005 and May to November 2005, respectively. The latter examined the effects of ten AGP formulations (bambermycin, penicillin, salinomycin, bacitracin, combination of salinomycin and bacitracin, chlortetracycline, virginiamycin 11ppm, virginiamycin 22ppm, monensin and narasin) on bird performance as well. Multiple antibiotic resistant commensal E. coli and Salmonella carrying virulence genes were found at commercial broiler chicken farms and therefore may serve as reservoirs for these genes. There was no significant difference between feed formulations on the phenotypic or genotypic characteristics of the isolates, except for tetracycline resistance gene tet(B). In the experimental feeding trial, broiler chickens were fed a diet including or excluding AGP. Birds were sampled prior to and weekly during feeding of the control and the AGPP containing diets. Although not detected on day 0, E. coli increased after day 7 to more than 9.9 log10 CFU/g in ceca. Multi-drug resistant E. coli were isolated from birds fed the ten AGP containing diets as well as the control diet. Except for penicillin, none of the AGP containing diets significantly improved bird performance compared to the control diet (P>0.05). Good management practices can significantly improve broiler chickens performance and decrease the mortality rate.

The entrainment of microbes from agricultural land into overland flow during rainfall events is recognised as an important source of pathogenic microbes to surface water bodies and yet this transport process is poorly understood. In this study, a method has been developed to separate bacteria into the forms in which they have been postulated to exist in overland flow. Then Escherichia coli was used as a model organism to investigate the transported state of bacteria eroded from cowpats and their subsequent transport in overland flow. Simulated rainfall experiments were used to generate runoff direct from cowpats. Concentrations of E. coli in the runoff direct from cowpats were found to be directly proportional to the concentration in the cowpat, regardless of the age of the cowpat. It was also observed that E. coli were predominantly eroded from cowpats as individual cells. The interactions between E. coli and soil particles in overland flow were then examined in a small laboratory scale model system and showed that E. coli attached to large (>45 [mu]m) soil particles were transported significantly less than unattached cells. However, in the runoff from the model system, E. coli were found to be attached mainly to clay particles that were similar in size to the bacterial cells. Furthermore, the transport of E. coli through the model system appeared to follow the transport of a conservative chemical tracer implying that (a) the cells were being transported as a solute with the bulk of the water flow, and (b) that E. coli attached to small clay particles were as mobile in the overland flow as unattached cells. These observations imply that E. coli predominantly interact with small clay particles that are also being carried along in the overland flow. The transport of E. coli at a larger scale was then investigated using 5-metre long, 1-metre wide buffer strips operated under saturation excess conditions. In buffer strips using intact soils and existing pasture cover, E. coli removal was very poor (26 % removal) at the low flow rate of 2 L min⁻� with no removal observed at the higher flow rates of 6 and 20 L min⁻�. E. coli removal rates were increased to 41 % removal at 2 L min⁻� by cultivating the soils, with the removal rate again decreasing with increasing flow rate. E. coli in the overland flow from the buffer strips did not form into large flocs or attach to large soil particles, but were transported in small neutrally buoyant particles that remain entrained in the overland flow. Under saturation excess runoff conditions, E. coli in overland flow were not effectively removed by buffer strips as the small particles are transported either over the soil surface or, through large pores in the soil. This Thesis has shown that E. coli is transported in overland flow in small particle sizes that are difficult to trap or remove from overland flow thereby explaining the high fluxes of faecal bacteria observed in overland flow from agricultural land.