The use of the larvacidal delta-endotoxin of the sporeforming bacterium Bacillus thuringiensis var. israelensis has been examined as a promising means to control insects that carry diseases such as malaria. An ultimate goal of this project was to genetically engineer both E. coli and the cyanobacterium Synechococcus PCC 7942 to express high levels of this delta-endotoxin and to construct the recombinant to carry a gene which would allow for monitoring of recombinants in the field. Previous research performed by a member of our laboratory involved cloning the gene fragment encoding the delta-endotoxin into a hybrid plasmid yielding recombinant E. coli clones which were toxic to mosquito larvae. Unfortunately, upon further examination of these recombinants using agarose gel electrophoresis and mosquitocidal assays, the clones were found to be unstable and lost their toxin encoding genes readily. Furthermore, cloning of the stabilizing parB locus into one of the recombinant plasmids did not enhance segregational stability as had been shown with some plasmids in E. coli. In another approach oligonucleotide primers were constructed which flanked the 130 kDa toxin gene but excluded a transposon-likesequence postulated to contribute to instability. These primers were used in the polymerase chain reaction in order to amplify this smaller DNA fragment for cloning experiments. Only a small quantity of primers were made and amplification of the DNA was not achieved prior to depletion of the primers. Future work will involve synthesizing new primers to be used for amplification and cloning of the B.t.i. toxin gene.In order to construct a traceable recombinant, the luciferase reporter gene (Luc) had been previously cloned into a hybrid plasmid that was capable of transforming both E. coli and the cyanobacterium Synechococcus PCC 7942. The new construction was then transformed into E. coli, to yield a pool of uncharacterized recombinants. In the present work, I determined that the luciferase enzyme was being expressed in the E. coli recombinants in the presence of the substrate luciferin. Initially, bioluminescence of these E. coli clones was detected by using OG-1 film which fogs in the presence of light. In order to quantify expression of the clones, lysates of the E. coli recombinants were also examined using a luminometer. Comparisons of bioluminescence were made between lysates with the parent E. coli plasmid harboring the luciferase gene and recombinants in which the Luc gene was placed downstream of the powerful rightward lambda promoter. Luminometer readings indicated that luciferase expression was enhanced six fold (from 2.0 X 10-6 to 3.0 X 10-5 by units/cell) in the recombinant plasmid. Plasmid DNA was isolated from the two luciferase expressing E. coli clones. Recombinants were obtained as determined by agarose gel electrophoresis examination of the plasmid DNA. This recombinant DNA was used to transform Synechococcus PCC 7942. However, because enzyme releasing methods were unsuccessful for the more rigid Synechococcus PCC 7942, the level of expression of the Luc gene could not be determined by either method mentioned above. Apparently, the methods used either failed to lyse the cells or they were too harsh and inactivated the enzyme. Future endeavors will involve the use of a French press to more gently lyse the cells so that the level of expression can be determined. / Department of Biology

Environmental pathogens, such as Escherichia coli and Streptococcus uberis, are currently the major cause of mastitis within dairy herds. This leads to severe financial losses, lower production rates and deterioration of the general health of the herd. E. coli mastitis is becoming a major threat to high milk-producing dairy herds. This is because of its increasing resistance to antibiotics, rendering antibiotic treatment regimes against E. coli infections mostly ineffective. The aim of this study was to develop a method to select mastitis causing E. coli isolates for the formulation of effective herd specific vaccines. Two methods, namely a genotyping method (Random Amplification of Polymorphic DNA; RAPD) and an antibiogram based method, were used. A dairy farm milking approximately 1000 Holstein cows in the Darling area, Western Cape Province, was selected for this study. The study was conducted over a period of 48 months and mastitis samples were analysed for mastitis pathogens. Antibiogram testing (disk diffusion method) and an in-house developed RAPD analysis method were used to analyse the E. coli isolates. A total of 921 milk samples were analysed from which 181 E. coli isolates were recovered. The number of all other common mastitis pathogens combined was 99 isolates (Streptococcus uberis 18, Streptococcus dysgalactiae 46, Streptococcus agalactiae 1, Staphylococcus epidermidis 21, Arcanobacterium pyogenes 13). All E. coli isolates, except for one, were resistant to at least three antibiotics. Antibiotic variance profiles were also highly erratic. The RAPD analysis revealed high levels of polymorphisms and clear epidemiological trends were observed over time. No similarities in the variance profiles between the antibiotic variance data and phylogenetic data were observed. Formalin inactivated autogenous vaccines were produced containing E. coli isolated from the herd. The vaccines were formulated using the RAPD or antibiogram data of the E. coli isolates. A total of 5 vaccines were formulated using RAPD data (Rvaccines) and one vaccine was formulated using antibiotic variance data (A-vaccine). The RAPD formulated vaccines were more effective than the antibiotic variance formulated vaccine. After each R-vaccination, the number of E. coli mastitis cases declined within the herd. The A-vaccinations seemed to have had no effect, which lead to a rise in E. coli mastitis cases. RAPD analysis on new emerging isolates was able to detect genetic variation from vaccine strains, which in turn facilitated the formulation of new updated vaccines with higher effectiveness than the previous vaccine. Mastitis data prior to and after the vaccination period revealed significant higher incidences of mastitis in the herd than during the vaccination period. This study demonstrated that sufficient sampling practices coupled with a reliable genotyping method, resulted in the formulation of updatable vaccines which were highly effective in controlling E. coli mastitis within the herd. / Thesis (M Environmental Sciences)--North-West University, Potchefstroom Campus, 2012

A method for transformation of Geobacillus debilis by conjugation was developed using a recombinant plasmid, pNW33N-pxyl-bs2-mob, derived from pNW33N. The plasmid includes the mob region of RP4 for mobilization, is mobilized from E. coli S17-1 to G. debilis, and can stably propagate in G. debilis trans-conjugants grown at 50 oC and 55 oC, in the presence of thiamphenicol. Successful conjugation was depended on the cell density and viability of G. debilis when harvested for conjugation, as well as the metabolic activity of E. coli S17-1 used for conjugation. Substantial reduction in size of the plasmid DNA was observed when G. debilis transconjugants were cultured at 60 oC in the presence of thiamphenicol, and uniform rearrangement of the plasmid DNA was observed after culturing G. debilis transconjugants in the presence of spectinomycin, even at 50 oC.

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.