>Magister Scientiae - MSc / Despite the wide success of antimicrobial agents against waterborne pathogens, waterborne disease continues to pose a threat to both mankind and animals. A major concern is that certain bacteria have developed resistance to antimicrobial agents, as a result of their overuse. Silver (Ag) nanoparticles are widely used for antibacterial purposes such as medical dressings. However, they are highly toxic to human cells. Hence, there is a great interest in developing next generation antibacterial nanoparticles that are as effective as Ag nanoparticles for antibacterial functions, while having less toxicity to human cells. Several methods can be used to generate these antimicrobial nanoparticles, one of which is green nanotechnology. Green nanotechnology uses natural plants such as tea to synthesise nanoparticles rather than chemicals, thus reduce human and animal harm and improve sustainability of antibacterial agents. Silver-titanium nano-composites (Ag-TiO2 NCs) were synthesised with the hydrothermal method using a tea extract from Aspalathus linearis (Rooibos, RB), and distilled water in the presence of nitrogen. The resulting structures were characterised with high resolution transmission electron microscopy (HRTEM), energy-dispersive spectroscopy (EDS) analysis X-Ray Diffraction (XRD) and Thermogravimetric Analysis (TGA). The antibacterial characteristics of these new NCs were evaluated against 3 bacteria: Bacillus cereus, Cupriavidus metallidurans, and Escherichia coli. The optimum processing conditions to produce 6-nm spherical NPs included maintaining the temperature at 90 °C, the pH at 4.35, and using RB extract at a concentration of 2 mg/mL. The size of silver NPs was reduced in acidic conditions, agglomerated in neutral conditions, and highly reduced in alkaline conditions. Increasing the pH decreased the particle size and narrowed the particle size distribution. Gram-positive B. cereus showed slight resistance or tolerance to the Ag-TiO2 nanocomposite compared to the gram-negative bacteria E. coli and C. metallidurans. The treatment concentration required for total inhibition of E. coli and C. metallidurans growth was 100 mg/mL. Supported silver nanoparticles has shown to be a suitable way to obtain highly dispersed silver over higher surface area. This approach allowed Ag-TiO2 nanocomposite to be an efficient bactericide, with less silver amount employed.

Phytophthora pini Leonian, recently re-established from P. citricola I, is a pathogen with a wide range of forest and nursery hosts. It causes foliar infections in horticultural nurseries in Oregon, where recirculating irrigation systems are common. Increased use of recirculating irrigation systems may contribute to disease caused by waterborne plant pathogens. Simulated nursery chamber experiments were utilized to investigate the relationship between Phytophthora pini zoospore inoculum dose and disease on Rhododendron. Disease incidence in this system was unexpectedly low despite high inoculum levels tested, so further experiments under lab conditions were conducted to explore possible causes. Detached leaf assays were conducted to determine how inoculum dose, leaf wounding, and agitation of zoospore inoculum affected foliar infection of Rhododendron. Wounded and nonwounded leaves were dipped into suspensions of zoospores that were either untreated, mechanically agitated by vortexing to cause encystment, or pumped through an irrigation sprayer system. Disease severity (lesion area) and incidence (number of lesions per leaf area) were measured over seven days. At inoculum levels of ���10,000 propagules/mL, motile zoospores infected both wounded and nonwounded leaves. Vortexing or pumping resulted in zoospore encystment, and inoculation with these treatments caused disease almost exclusively on wounded leaves. No disease symptoms were observed following inoculation with any inocula at ��� 2,000 propagules/mL. Scanning electron microscopy of leaves inoculated with encysted propagules showed germinated cysts with hyphae growing over and around stomata without entering leaf tissue until reaching a wound site. Nonwounded leaves inoculated with motile spores showed stomata penetrated by hyphae. These findings indicate the importance of zoospore motility in reaching suitable infection sites, and demonstrate the impact of zoospore encystment on disease development. This has implications for disease management in nurseries where pruning wounds are common and the pumping of infested irrigation water may influence zoospore motility and infectivity. / Graduation date: 2013

This study presents the spatial and temporal variability of Salmonella enterica in urban and rural streams in a model watershed (Grand River watershed, Ontario, Canada), and examines the antimicrobial resistance (AMR) and genetic diversity of various serotypes. Using a swab collection method and various media types, Salmonella were detected in 78.4% of samples between November 2003 and July 2005. A diverse range of Salmonella serotypes (n=38) were isolated from water. Predominant serotypes and phagetypes (PT), including S. Typhimurium PT 104 and S. Heidelberg PT 19, and the proportion of isolates demonstrating AMR (33%), was similar to those for humans and farm animals locally and across Canada, a trend not commonly reported. There was a greater diversity of serotypes and AMR profiles in isolates from the urban stream compared to the rural/agricultural streams. Plasmid-borne resistance was observed in 28.6% of AMR isolates, with two different plasmids responsible for resistance; the TEM-1 plasmid (8.1Kb plasmids carrying blaTEM-1, responsible for ampicillin resistance) and CMY-2 plasmid (95.5Kb plasmids carrying blaCMY-2, responsible for 3rd generation cephalosporin resistance). CMY-2 plasmids were only found in the urban stream and did not create a biological burden under non-selective conditions, indicating the long-term permanence of these plasmids. Seasonal differences in the overall diversity of serotypes and predominance of serotypes of human health significance (S. Typhimurium and S. Heidelberg) were observed. The lower occurrence of S. Typhimurium and S. Heidelberg in February and March was not the result of lower survival of these serotypes at low temperatures. Peaks in occurrence of S. Typhimurium and S. Heidelberg in the summer and spring, respectively, were pronounced in the rural/agricultural streams, as opposed to the urban stream. Pulsed-field gel electrophoresis and plasmid-typing revealed diversity within multiple drug resistant S. Typhimurium PT 104 isolates, indicating genetic differences among tributaries. The ubiquitous nature of Salmonella in water and the predominance of serotypes/phagetypes of human or veterinary health significance suggest that environmental exposure through consumption or contact with contaminated water is plausible. These streams may act as a vehicle for the dissemination of these organisms and their resistance genes between different hosts or environments.

Three experiments were conducted to characterize the concentration of microorganisms in biosolids, the plume of aerosols created during land application of biosolids and the occupational risk of infection due to pathogens aerosolized during land application of biosolids in the United States. In all, more than three-hundred air samples were collected immediately downwind of biosolids applications throughout the United States using liquid impingers, and more than one-hundred air samples were collected downwind of microbially seeded, land applied water, which served as a conservative model system of aerosol generation. The novel model system made it possible to calculate the flux of microorganisms through a virtual plane defined by air samplers in vertical and horizontal arrays, located immediately downwind of a passing spray applicator. The rate of aerosolization during land application of biosolids near Tucson, Arizona, was calculated to be less than 33 plaque forming units (PFU) of coliphage and 10 colony forming units (CFU) of coliform bacteria per meter traveled by the spray applicator. Rates of aerosolization from the model system were shown to be much greater. To assess the risk to occupational health from bioaerosols generated during land application of biosolids, coliform bacteria, coliphages, and heterotrophic plate count (HPC) bacteria were enumerated from air and biosolids at 10 land application sites throughout the nation. The method of land application strongly influenced aerosolization, while relative humidity, temperature and wind speed showed limited correlation to concentrations of fecal indicator microorganisms in air. Occupational risks of infection and illness from aerosolized Salmonella and enteroviruses were calculated for a variety of land application scenarios. Realistic exposure scenarios carried occupational risks of Salmonella infection ranging from of 0.0001% to 0.013% per year. The corresponding occupational risk of infection from enteroviruses, using coxsackievirus A-21 as a model, ranged from 0.78% to 2.1% per year, depending on the type of activity performed by the worker. In addition, samples of biosolids from the Southwestern United States were characterized to provide up-to-date information about pathogens in biosolids for environmental regulators, biosolids producers, researchers, and public health agencies.

Climate change is expected to cause changes in precipitation and runoff patterns, likely increasing the risk of waterborne infectious disease in some areas. In this context, the research objectives were to describe links between weather, water quality, and infectious gastrointestinal illnesses (IGI) in Nunatsiavut, Canada, which necessarily involved evaluating the quality and usefulness of data captured by the local health registry system. For this evaluation, IGI was used as a reference syndrome. Community-based meteorological stations captured weather data; trained local personnel conducted water quality testing. Clinic records provided IGI-related data (2005-2008). This study is the first to systematically gather and describe baseline empirical data on weather, water quality, and health in Nunatsiavut. It showed the necessity of improving Inuit health data quality and monitoring environmental health variables consistently and systematically across all Arctic regions. These data are critical to inform adaptation strategies for managing impacts of climate change on health.

The risk of waterborne infections acquired from the consumption of contaminated water is related to changes in source water fecal contamination, which is often influenced by land-use and hydro-meteorological conditions in the surrounding watershed. The impact of land-use composition on surface water contamination was explored in order to determine the risk of surface water contamination associated with land-use change. Highest contamination was observed in watersheds characterized by more than 12.5% agricultural and more than 1.6% urban land (mean fecal coliform (FC) concentration of these 5 sites = 135 CFU 100ml-1 while the British Columbia (BC) raw water quality guideline = 100 CFU 100ml-1). Contamination increased exponentially, and violated BC raw water quality guidelines with greater frequency, in relation to greater agricultural land in the upstream watershed. Additional factors, such as sewage treatment plants, low dilution in smaller streams, and higher temperatures were also associated with greater contamination. These results indicate the high level of risk posed by agricultural and urban development and the need for source water protection. Fecal contamination levels in source water are also influenced by rainfall and snowmelt-induced surface runoff that transport diffuse fecal contaminants into surface water. Seasonal levels of fecal contamination in surface water was related to the watershed hydro-climatic regime for around half of the watersheds examined. Watersheds with snowmelt-dominant (SD) runoff regimes showed stronger evidence of hydro-meteorological variability driving seasonal contamination levels than those with rainfall and snowmelt-influenced (RSI) and rainfall-dominant (RD) runoff regimes, and thus are more prone to experiencing changes to seasonal variability resulting from climate change. Projected increases in mean annual temperatures of between 1.70C and 4.00C towards the end of the 21st century will alter existing runoff regimes within watersheds. For SD watersheds that remain below freezing and continue to accumulate snowpack during the cold season, transport of fecal contamination will likely occur earlier in the year with greater intensity. Fecal coliform transport in summer is likely to decrease, especially in SD watersheds in which fecal contamination is driven by summer rainfall events. Snowmelt-dominant watersheds transitioning toward a RD runoff regime will experience less contamination during spring but increased contamination during late fall and winter. The extent to which these changes in runoff regime will influence surface water fecal contamination will vary among watersheds. Further investigation is required to identify factors that enhance or mitigate the association of surface water fecal contamination with rainfall and snowmelt-induced runoff in order to identify specific site vulnerability to changing seasonal contamination levels. Total precipitation within BC is projected to increase by 20-30% towards the end of the 21st century. The association of annual FC variability with snowmelt and rainfall variability was examined in order to assess the capacity of such increases to raise the level of surface water fecal contamination. Greater total annual and seasonal rainfall and/or river discharge increased surface water fecal contamination for 58% (11/19) of the sites examined. Hydro-meteorological variability influenced FC concentration during winter, the season of greatest precipitation, and spring, the season of greatest snowmelt, but not during summer or fall. Reduced contamination levels during the El Niño event in 2002/03 were associated with a mean reduction in river discharge during spring and summer. These associations suggest that the risk of increased surface water fecal contamination in response to higher precipitation is likely to be greatest in winter for RD watersheds and spring for SD watersheds, although the magnitude of impact will vary among sites. Climate change and land-use activities within watersheds have the capacity to alter the timing and amount of surface water fecal contamination. These factors are likely to act synergistically by increasing the presence and transport of fecal contaminants within watersheds. Such relationships should be carefully considered to aid the assessment and mitigation of the risk of source water contamination associated with land-use and climate change. / Graduate

This study presents the spatial and temporal variability of Salmonella enterica in urban and rural streams in a model watershed (Grand River watershed, Ontario, Canada), and examines the antimicrobial resistance (AMR) and genetic diversity of various serotypes. Using a swab collection method and various media types, Salmonella were detected in 78.4% of samples between November 2003 and July 2005. A diverse range of Salmonella serotypes (n=38) were isolated from water. Predominant serotypes and phagetypes (PT), including S. Typhimurium PT 104 and S. Heidelberg PT 19, and the proportion of isolates demonstrating AMR (33%), was similar to those for humans and farm animals locally and across Canada, a trend not commonly reported. There was a greater diversity of serotypes and AMR profiles in isolates from the urban stream compared to the rural/agricultural streams. Plasmid-borne resistance was observed in 28.6% of AMR isolates, with two different plasmids responsible for resistance; the TEM-1 plasmid (8.1Kb plasmids carrying blaTEM-1, responsible for ampicillin resistance) and CMY-2 plasmid (95.5Kb plasmids carrying blaCMY-2, responsible for 3rd generation cephalosporin resistance). CMY-2 plasmids were only found in the urban stream and did not create a biological burden under non-selective conditions, indicating the long-term permanence of these plasmids. Seasonal differences in the overall diversity of serotypes and predominance of serotypes of human health significance (S. Typhimurium and S. Heidelberg) were observed. The lower occurrence of S. Typhimurium and S. Heidelberg in February and March was not the result of lower survival of these serotypes at low temperatures. Peaks in occurrence of S. Typhimurium and S. Heidelberg in the summer and spring, respectively, were pronounced in the rural/agricultural streams, as opposed to the urban stream. Pulsed-field gel electrophoresis and plasmid-typing revealed diversity within multiple drug resistant S. Typhimurium PT 104 isolates, indicating genetic differences among tributaries. The ubiquitous nature of Salmonella in water and the predominance of serotypes/phagetypes of human or veterinary health significance suggest that environmental exposure through consumption or contact with contaminated water is plausible. These streams may act as a vehicle for the dissemination of these organisms and their resistance genes between different hosts or environments.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 120-131). / Microfiche. / xiii, 131 leaves, bound ill. 29 cm

Thesis (Ph. D.)--University of Texas at El Paso, 2009. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (M.Sc.)(Microbiology)--University of Pretoria, 2005. / Includes summary. Title from opening screen (viewed March 20, 2006). Includes bibliographical references.