<p><strong><br /></strong></p> / <p><strong>In Canada the incidence of yeast infections have increased over the past decade, which in turn has resulted in the increased mortality and morbidity rates among the immuno-compromised patients. Yeasts are ubiquitous in nature and constitute a healthy portion of human skin and gut flora. Factors such as the urban settings and food have been previous shown to influence the yeast flora people harbour. This makes us believe that to effectively tackle the rising yeast infections in Canada we need to not only conduct epidemiological yeast studies in clinical settings but should also understand the diversity and distribution of them in the urban environment. This thesis constitutes of an epidemiological fungemia study and an urban environmental yeast profiling study conducted in the city of Hamilton.</strong></p> <p><strong> </strong></p> <p><strong>In the first chapter of the thesis I discuss the results of the epidemiological candidemia study. We noticed that over the past decade the mean age of the population with candidemia in hospitals within Hamilton has increased by 10 years. DNA fingerprinting analysis suggested that 33% of the blood stream</strong><strong><em>Candida</em></strong><strong> </strong><strong>isolates from January 2005 to February 2009 belonged to 18 clusters, some of which were shared between wards and hospitals. we found that for each of the four species, strains isolated closer to each other temporally were overall genetically more similar to each other as well, which suggested that nosocomial sources likely caused repeated candidemia infections. The study is the first of its sort in Canada and the results of this chapter are expected to aid infection control practitioners in the Hamilton hospitals and make the stay of patients in hospitals safer.</strong></p> <p><strong> </strong></p> <p><strong>In the second chapter, we discuss the diversity and distribution of yeasts prevalent on trees in and around Hamilton. We identified a total of 88 environmental yeasts belonging to 20 species (based on ITS sequence data). The yeast populations were highly heterogeneous in both species and genotype composition. Among the 14 tree species sampled, yeasts were frequently found on cedar, cottonwood and basswood. Interestingly all the</strong><strong> <em>Candida parapsilosis</em> </strong><strong>strains were found from pine tree only. Some of the potential environmental factors shaping the distribution of yeast populations in Hamilton are discussed.</strong></p> / Master of Science (MSc)

Research regarding the distribution and structure of fungal communities in caves is lacking. The current study examines fungal communities in Kartchner Caverns, a mineralogically diverse cave located in the Whetstone Mountains, Arizona, USA. The first study examines culturable fungal diversity from speleothem surfaces. Twenty-one fungal genera represented by 43 genotypes and 53 distinct morphological taxonomic units (MTU) were recovered from 15 speleothems. Analysis of DGGE profiles indicated a significant effect of sampling site on community structure. The second study examined fungal diversity from speleothem and rock wall surfaces using the 454 FLX Titanium sequencing platform using the rDNA internal transcribed spacer 1 (ITS1) as a genetic marker. Fungal diversity was estimated and compared between speleothem and rock wall surfaces and its variation with distance from the natural entrance of the cave was quantified. Effects of environmental factors and nutrient concentrations in speleothem drip water at different sample sites on fungal diversity were also examined. Sequencing revealed 2219 fungal operational taxonomic units (OTUs) at 95% similarity. Speleothems supported a higher fungal richness and diversity than rock walls, but community membership and the taxonomic distribution of fungal OTUs did not differ significantly. OTU richness and diversity were negatively correlated with distance from the natural cave entrance. Community membership and taxonomic distribution of fungal OTUs differed significantly between the front and back of the cave. There was no observed effect of drip water nutrient concentration on fungal community structure. The third study examined fungal community structure from bat guano over the course of a year. There was no significant difference in fungal OTU richness, diversity, or community membership and taxonomic affiliations among sampling times. There were no significant differences in nutrient concentrations of guano samples among sampling times. Nutrient concentration did have a significant effect on community structure, especially the level of nitrogen and calcium.

Nitrification is a biological oxidation of NH3 to NO2- and then to NO3-. Understanding how the nitrifier community responds to agricultural management is essential because the community composition is complex and functional distinction of subgroups occurs. Better managing nitrifiers could benefit the environment by increasing nitrogen (N) fertilizer use efficiency, decreasing NO3- leaching, and reducing NO and N2O emissions. This study examined how long-term N fertilization and tillage influenced nitrifier density, ratios, nitrification rates, and the community structure of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and nitrite-oxidizing bacteria (NOB). The study site was a long-term (>40 years) continuous maize (Zea mays L.) experiment with three N fertilization rates (0, 168, and 336 kg ha-1) and either no-tillage (NT) or plow tillage (PT). Most Probable Number method was used to estimate the density of AOB and NOB; the shaken slurry method was used to measure potential nitrification rates; PCR-denaturing gradient gel electrophoresis (DGGE) was used to analyze nitrifier communities. Tillage, fertilization, and their interaction all significantly influenced the AOB and NOB densities, the ratio of AOB to NOB, and potential nitrification rate. Nitrifier densities and potential nitrification rates increased with increased N fertilization; NOB density increased faster than AOB density with fertilization. The influence of tillage on nitrification was different for different fertilization rates. The trends for nitrifier density and potential nitrification rate were not consistent. Nitrifier community structure was influenced by sample season, N fertilization rates, tillage, and their interaction. Different nitrifier groups had different responses to the treatments. The AOB became more diverse with increasing N input; tillage rather than N fertilizer played a dominant role affecting the AOA community; two NOB genera had different responses to N fertilization rates: Nitrobacter diversity increased with more N applied; Nitrospira was the opposite. Unique bands/members were discovered in different treatments, manifesting environmental selection. Long-term field trials were useful in better understanding how soil management influenced the relationship between nitrifier densities, nitrification rates, and community structure, which may facilitate new approaches to optimize nitrification and provide new clues to discover which environmental factors most influence the nitrifier community in agroecosystems.

Saltwater intrusion due to global change is expected to have a detrimental effect on the biogeochemistry of tidal freshwater wetlands. Of particular concern is that fact that salinization can alter the role of these ecosystems in the global carbon cycling by causing shifts in microbial metabolism that alter greenhouse gas emissions and increase carbon mineralization rates. However, our understanding of how wetland microbial community dynamics will respond to saltwater intrusion is limited. To address this knowledge gap and increase our understanding of how microbial communities in tidal freshwater wetlands change over time (1, 3, 12, and 49 weeks) under elevated salinity conditions, an in situ soil transplant was conducted. Throughout the 49 weeks of saltwater exposure, salinity had no effect on soil quality (organic matter content and C:N ratio). In contrast, the concentration of porewater ion species (SO4-2, NO3-, and NH4+) considerably increased. The activity of hydrolytic enzymes, (ß-1,4-glucosidase and 1,4-ß-cellobiohydrolase) gradually decreased with prolonged exposure to saline conditions; by the final sampling event (49 weeks), activity was reduced by ~70% in comparison to the freshwater controls. Short term exposure to salinity (3 and 12 weeks) had a greater effect on phenol oxidase, decreasing activity by 10-20%. Saltwater exposure had an immediate (1 week) effect on potential rates of carbon mineralization; overall, carbon dioxide production doubled and methane production decreased by ~20-fold. These changes in gas production were correlated to increased salinity and to changes in the abundance of methanogens and sulfate reducing bacteria, suggesting a shift in the terminal step in organic matter degradation from methanogenesis to sulfate reduction. Principal component analysis revealed distinct changes in soil environmental conditions and carbon metabolism within weeks, but the response of the microbial community was slower (months to a year). Taken together, results from this study indicate that the response of tidal freshwater wetlands to salinization is driven by complex interactions of microbial related processes and environmental changes that are dependent on the duration of exposure. Assessing the impact of environmental perturbation on ecosystem function may be better achieved by complementary analysis of both microbial community structure and function.

Because of the potential health hazards associated with the use of sludge for agricultural purposes, Dudley et al (1980) published a scheme for the routine analysis of bacteria in municipal sewage sludge. In this study, the Dudley et al scheme (1980) was modified by updating some of the procedures. Aerobically digested sludge generated by the Bowling Green Wastewater Treatment Plant, Bowling Green, Kentucky, was analyzed using the modified scheme. Sludge samples were collected once every two months over a one-year period from October 1989 to August 1990. Egg yolk-free tryptose sulfite cycloserine agar in conjunction with the revewrse CAMP test was used to assay for Clostridium perfringens. This procedure improved the one proposed by Dudley et al. (1980) by achieving a higher confirmation rate, reducing testing time, allowing for easier interpretation of results, and increasing accuracy. Selective and differential media by Rippey and Cabelli (1979) were added to the scheme to isolate Aeromonas, Aeroomonas hydrophila and Aeromonas caviae were successfully isolated wand were identified using the system by Cunliffe and Adcock (1989) for speciating aeromonads. Baird-Parker medium was compared to mannitol salt agar for effectiveness in isolating Staphylococcus from sludge. Statistical analysis showed Baird-Parker medium to be significantly more effective than mannitol salt agar. However, neither agar reduced background flora to acceptable levels. Staphylococcus isolates were subject to species identification by the API Staph Ident system (Analytab Products, Plainview, New York). Staphylococcus xylosus, Staphylococcus haemolyticus, and Staphylococcus epidermidis were found to be present in the sludge. A procedure by Ottolenghi and Hamparian (1987) was employed to isolate Salmonella in sludge. No salmonellae were isolated over the one year period. Over the year-long study, bacterial numbers, with the exception of Clostridium perfringens and the total aerobic count, fluctuated with variations in the aerobic digester temperature. Numbers decreased as temperature increased. Clostridium perfringens counts were the most consistent throughout the year and exceeded fecal coliform and fecal streptococci counts in five of the six samplings.

Pseudomonas syringae produz núcleos de gelo biológicos de grande eficiência. Bioaerossóis destas células tem potencial de participar na glaciação de nuvens, podendo influenciar a precipitação. Foram estudadas como as condições as quais P. syringae está sujeita em suspensão na atmosfera afetam sua sobrevivência e sua atividade de nucleação de gelo. Duas cepas foram testadas, e ambas apresentaram baixa tolerância ao UV-C e ao UV-B, mas exibiram uma maior resistência quando expostas a um espectro semelhante ao encontrado no ambiente. A atividade de congelamento de uma das cepas (pv. syringae) não foi afetada pelo UV, enquanto que para a outra (pv. garcae) houve uma redução moderada. Em resposta à dessecação, pv. garcae foi substancialmente mais resistente que pv. syringae. Isto também afetou a nucleação de gelo das cepas. Em ensaios adicionais, estas bactérias foram expostas em um voo de balão estratosférico, e a uma simulação em laboratório das condições no topo da troposfera. Nestes dois experimentos, sobreviventes protegidos do UV foram recuperados. / Pseudomonas syringae produces biological ice nuclei of great efficiency. Bioaerosols of these cells have the potential to take part in cloud glaciation, possibly influencing the precipitation. It was studied how the conditions to which P. syringae is subjected while in suspension in the atmosphere affect its survival and its ice nucleation activity. Two strains were tested, and both showed a low tolerance to UV-C and UV-B, but exhibited a higher resistance when exposed to a spectrum similar to the one found in the environment. The freezing activity of one of the strains (pv. syringae) was not affected by the UV, while that for the other (pv. garcae) there was a moderate reduction. In response to desiccation, pv. garcae was substantially more resistant than pv. syringae. This also affected the ice nucleation by the strains. In additional assays, these bacteria were exposed in a stratospheric balloon flight, and to a laboratory simulation of the conditions at the top of the troposphere. After these two experiments, survivors protected from the UV were recovered.

Realizamos o isolamento de leveduras do complexo Cryptococcus a partir da cloaca e do inglúvio de papagaios do gênero Amazona aestiva. Para a realização das coletas, as aves foram anestesiadas, e em seguida foi realizado lavado do inglúvio e coleta de material da cloaca. As amostras coletadas foram inoculadas em ágar Sabouraud dextrose com cloranfenicol, de onde foram isoladas colônias leveduriformes. Por meio de análises macro e micromorfológicas, os isolados condizentes com as características do gênero Cryptococcus foram submetidos à provas bioquímicas, testes de suscetibilidade aos antifúngicos e pesquisa de exoenzimas. Todos os isolados foram provenientes da cloaca. Dos isolamentos, 90% das cepas corresponderam à espécie C. albidus, e 10% à espécie C. laurentii; 80% foram produtores de fosfolipase e 100% de proteinase. Estes resultados sugerem que não só o ambiente, como também as aves podem ser carreadoras de Cryptococcus albidus. / We performed the isolation of yeasts of Cryptococcus complex from the cloaca and the crop of parrots of the genus Amazona aestiva. To carry out the sampling, the birds were anesthetized to perform a lavage of the crop and the collection of material from the cloaca. The samples were inoculated on Sabouraud dextrose agar with chloramphenicol, which were isolated from yeast colonies. Through macro and micromorphological analysis, isolates consistent with the characteristics of the genus Cryptococcus were subjected to biochemical tests, antifungal susceptibility testing and research exoenzymes. All isolates were from the cloaca. Of the isolates, 90% of the strains corresponded to the species C. albidus, and 10% of the species C. laurentii; 80% of the isolates were producing phospholipase and 100% were producing proteinase. These results suggest that not only environmental but also birds can be Cryptococcus albidus carrier. These results suggest that there is not only an environmental source but also birds can be Cryptococcus albidus carriers

Cubatão, o maior pólo industrial da américa latina também já foi uma das cidades mais poluídas do mundo. Os 30 anos de intensa atividade industrial vêm pressionando o meio ambiente com substâncias tóxicas e afetando gravemente a saúde da população. Dentre as substâncias contaminantes mais importantes da região estão os derivados de petróleo como o benzeno, tolueno, etilbenzeno e xilenos. Conhecidos como BTEX, eles são produzidos e utilizados em larga escala e a contaminação ocorre frequentemente através de vazamentos. Nos solos, devido à sua solubilidade em água, essas substâncias podem se espalhar por longas distâncias a partir do ponto afetado contaminando locais distantes. Já foi comprovada a capacidade de micro-organismos de sobreviver e até utilizar BTEX como fonte de carbono. Os micro-organismos adaptados catabolizam os contaminantes transformando-os em substâncias menos tóxicas e até mesmo eliminando-os do ambiente, capacidade de grande interesse econômico e ambiental. Nessa linha, nossa proposta visa o estudo das comunidades microbianas de solos afetados e não afetados por BTEX. Para isso foi utilizada a metagenômica como abordagem de estudo identificando-se diferenças qualitativas e quantitativas nas estruturas microbianas de três diferentes locais do município de Cubatão, sendo um deles afetado diretamente por BTEX. Pelo método utilizado e aqui desenvolvido, foi possível identificar um panorama metabólico geral identificando-se genes relevantes e o potencial de degradação de hidrocarbonetos aromáticos de micro-organismos conhecidos e desconhecidos, revelando melhor o potencial metabólico dos solos identificados. Os resultados apresentados podem contribuir para um melhor entendimento da dinâmica in situ de uma comunidade microbiana afetada por BTEX assim como melhorar o conhecimento sobre a comunidade microbiana de um local altamente impactado como Cubatão. / Cubatão is the largest industrial site in Latin America and was in the past one of the most polluted cities in the world. 30 years of intense industrial activity has pushed environmental limits with toxic substances and has severely affected the inhabitants\' health. Among the contaminants found in the region, the petroleum derivatives benzene, toluene, ethylbenzene and xylenes are the most important. Known collectively as BTEX, they are produced and used at a large scale and contamination frequently occurs. Because it is highly soluble in water, when in soil BTEX can spread long distances from the original contamination site, thus affecting large areas. Some microorganisms are known to live in contaminated environments and use contaminants such as BTEX as a unique carbon source for energy production. They catabolize contaminants into less dangerous products or even eliminate them from environment, a feature which has great commercial and environmental interest. We therefore compared the microbial communities in soils which were affected and un-affected by BTEX contamination. To this end, we used a metagenomics approach and developed a comparison method to identify microorganisms and degradation potential of soils studied. We found qualitative and quantitative differences in microbial structures from three different sites in Cubatão County, one of which is contaminated with BTEX. We constructed a metabolic overview identifying important genes, degradation potential and microorganisms related to BTEX degradation. The results presented here could contribute to understanding the in situ dynamics of a BTEX affected microbial community as well as improving our knowledge of the microbial community of Cubatão, a highly environmentally impacted place.

Current understanding of potential toxicity of engineered nanomaterials to aquatic microorganisms is limited for risk assessment and management. Here we evaluate if the MetPLATE™ test can be used as an effective and rapid screening tool to test for potential aquatic toxicity of various metal-based nanoparticles (NPs). The MetPLATE bioassay is a heavy metal sensitive test based on β-galactosidase activity in Escherichia coli. Five different types of metal-based NPs were screened for toxicity: (1) citrate coated nAg (Citrate-nanosilver), (2) polyvinylpyrrolidone coated nAg (PVP-nAg), (3) uncoated nZnO, (4) uncoated nTiO2 and (5) 1-Octadecylamine coated CdSe Quantum Dots (CdSe QDs); and compared with their corresponding ionic salt toxicity. Citrate-nAg was further fractionated into clean Citrate-nAg, unclean Citrate-nAg and permeate using a tangential flow filtration (TFF) system to eliminate residual ions and impurities from the stock Citrate-nAg suspension and also to differentiate between ionic- versus nano-specific toxicity. Our results showed that nAg, nZnO and CdSe QDs were less toxic than their corresponding ionic salts tested, while nano- or ionic form of TiO2 was not toxic as high as 2.5 g L− 1 to the MetPLATE™ bacteria. Although coating-dependent toxicity was noticeable between two types of Ag NPs evaluated, particle size and surface charge were not adequate to explain the observed toxicity; hence, the toxicity appeared to be material-specific. Overall, the toxicity followed the trend: CdCl2 > AgNO3 > PVP-nAg > unclean Citrate-nAg > clean Citrate-nAg > ZnSO4 > nZnO > CdSe QDs > nTiO2/TiO2. These results indicate that an evaluation of β-galactosidase inhibition in MetPLATE™ E. coli can be an important consideration for rapid screening of metal-based NP toxicity, and should facilitate ecological risk assessment of these emerging contaminants.

The increasing number of polluted watersheds and water bodies with total maximum daily loads (TMDLs) has resulted in increased research to find methods that effectively and universally identify fecal pollution sources. A fundamental requirement to identify such methods is understanding the microbial and chemical processes that influence fate and transport of fecal indicators from various sources to receiving streams. Using the Watauga River watershed in northeast Tennessee as a model to better understand these processes, multivariate statistical analyses were conducted on data collected from four creeks that have or are expected to have pathogen TMDLs. The application of canonical correlation and discriminant analyses revealed spatial and temporal variability in the microbial and chemical parameters influencing water quality, suggesting that these creeks differ in terms of the nature and extent of fecal pollution. The identification of creeks within a watershed that have similar sources of fecal pollution using this data analysis approach could change prioritization of best management practices selection and placement. Furthermore, this suggests that TMDL development may require multiyear and multisite data using a targeted sampling approach instead of a 30-d geometric mean in large, complex watersheds. This technique may facilitate the choice between watershed TMDLs and single segment or stream TMDLs.