Degraded surface water quality is a growing public health concern. While indicator organisms are frequently used as a surrogate measure of pathogen contamination, poor correlation is often observed between indicators and pathogens. Because of adverse health effects associated with poor water quality, an assessment of the factors influencing the fate and transport of fecal pollution is necessary to identify sources and effectively design and implement Best Management Practices (BMPs) to protect and restore surface water quality. Sinking Creek is listed on the State of Tennessee’s 303D list as impaired due to pathogen contamination. The need to address the listing of this and other water bodies on the 303D list through the Total Maximum Daily Load (TMDL) process has resulted in increased research to find methods that effectively and universally identify sources of fecal pollution. The main objective of this research is to better understand how microbial, chemical, and physical factors influence pathogen fate and transport in Sinking Creek. This increased understanding can be used to improve source identification and remediation. To accomplish this objective, physical, chemical, and microbial water quality parameters were measured and the data were analyzed using multivariate statistical methods to identify those parameters influencing pathogen fate and transport. Physical, chemical, and microbial water and soil properties were also characterized along Sinking Creek to determine their influences on the introduction of fecal pollution to surface water. Results indicate that the 30-day geometric mean of fecal indicator organisms is not representative of true watershed dynamics and that their presence does not correlate with the presence of bacterial, protozoan, or viral pathogens in Sinking Creek. The use of multivariate statistical analyses coupled with a targeted water quality-monitoring program has demonstrated that nonpoint sources of fecal pollution vary spatially and temporally and are related to land use patterns. It is suggested that this data analysis approach can be used to effectively identify nonpoint sources of fecal pollution in surface water.

The development and refinement of FA has been adequately investigated with major emphasis on pathogenic microorganisms. The development of this technique has reduced both the time and number of biochemical tests necessary to identify a diversity of organisms. The organisms included are the protozoans, as described by Goldman (1953 and 1957) and by Ingram (1961), viruses, as reported by Liu (1955a) and Burgdorfer and Lackman (1960a), pathogenic bacteria which have been investigated by Moody, Goldman, and Thomason (1956), Moody and Winter (1959), Deason, Falcone, and Harris (1957) and Thomason, Cherry, and Moody (1957). Various fungi have been studied with FA by Kaufman and Brandt (1964), Kaufman and Kaplan (1961 and 1963) and Gordon (1958). Therefore, due to the success of the fluorescent antibody technique in many areas of microbiology in previous investigations, the logical assumption was that immunofluorescence might be incorporated into an efficient system in which a specific organism associated with fecal pollution, such as S. faecalis, could be rapidly identified. Based on this assumption, the feasibility of fluorescent antibody techniques, using S. faecalis was investigated as a means of rapid determination of bacterial pollution in water. Although much progress has been achieved in the study of cytochemical reactions by immunofluorescence, no attention has been focused on the application of this method as a determinative tool by which water contamination, due to the presence of the enterococci, could be demonstrated. Specifically, the purpose of the research reported in this dissertation was to devise an applicable, valid, and rapid method that could be employed in the detection and identification of S. faecalis.

Antibiotic resistance analysis (ARA) was used to determine sources of fecal pollution in the Blackwater River in South-central Virginia. The Department of Environmental Quality designated six segments as impaired due to high fecal coliform concentrations with non-point source (NPS) agriculture the suspected source of impairment. The Blackwater River watershed encompasses 72,000 ha of dairy, beef, and intensive production agriculture, abundant wildlife populations and many homes with onsite septic systems. A library of antibiotic resistance profiles based on 30 concentrations of 9 antibiotics was developed for 1,451 enterococci isolates from human, cattle, chicken, horse, goat, sheep, deer, raccoon, muskrat, goose, duck, coyote, and wild turkey fecal samples. Each isolate was classified as human, wildlife or livestock. Correct classification rates were 82.3% for human, 86.2% for livestock and 87.4% for wildlife isolates when profiles were analyzed with discriminant analysis. Profiles were also determined for 48 isolates from 128 stream samples collected periodically from August 1999 thru April 2001 and compared to the known sources using discriminate analysis. A human signature was found at each site at least once during the year, ranging from 0.0% to 85.0% of the sample isolates. The livestock signature varied from 2.3% to 100% over sites and months, and the wildlife signature varied from 0.0% to 79.5%. The results indicate that both humans and wildlife contribute to fecal pollution in addition to the suspected source, livestock, and reducing fecal pollution will require consideration of all three sources. The results from this research are being used to develop a total maximum daily load (TMDL) project allocations for fecal coliforms in the Blackwater River. Isolates identified by ARA were also profiled using the Biolog metabolic identification system. A library of metabolic profiles was constructed from known source isolates. Stream isolates were identified by Biolog and the metabolic profile was compared to the Biolog library. Of ten stream isolates identified by ARA as human, the Biolog library identified one as human, four as livestock, and five as wildlife. Of ten isolates identified by ARA as livestock, the Biolog library identified seven as livestock and three as wildlife. Of ten isolates identified by ARA as wildlife, one was identified as human, three as livestock and six as wildlife. The overall correct classification of Blackwater isolates in the Biolog library was 14 of 30 isolates, or 47%. Although the Biolog library was constructed with some isolates from the Blackwater basin, there may not be enough isolates in the Biolog library to adequately represent the variability shown by the Blackwater isolates, resulting in lower than expected correct classifications. In spite of these results, Biolog remains promising as one of several tools with potential as a bacterial source tracking method. / Master of Science

Orientador: Luiz Augusto do Amaral / Banca: Laudicéia Giacometti Lopes / Banca: Maria da Glória Buzinaro / Resumo: A qualidade da água in natura é de grande importância nos sistemas de tratamento de águas superficiais, pois uma vez que haja falhas no tratamento pode haver o risco de produzir água contaminada tornando-se prejudicial à saúde dos consumidores. O objetivo do presente trabalho foi conhecer a dinâmica temporal da poluição fecal, durante 24 horas, na água do manancial de abastecimento público da cidade de Jaboticabal, SP. Para isso foram colhidas amostras da água do manancial, com intervalos de 2 horas, durante 24 horas e foram determinados os Números Mais Prováveis de coliformes totais, Escherichia coli e Clostrídios sulfito redutores e as concentrações de amônia nos períodos de chuvas e estiagem. Concluiu-se que em alguns eventos a água do Córrego Rico não poderia ser utilizada para o abastecimento da população por tratamento convencional, pois com as incidentes chuvas, principalmente no período vespertino, o córrego apresenta contaminação acima do limite para sua classe. Assim podendo apresentar risco à saude da população. Os resultados obtidos podem nortear ações visando promover a qualidade da água fornecida à população no sentido de prevenir as enfermidades de veiculação hídrica. / Abstract: Considering the quality of the water "in natura" has a great importance in the systems of superficial water treatment, once time that is fails it can be had a risk to produce contaminated water being harmful to the consumers health, the present work was elaborated with the objective to know the secular dynamics of pollution fecal, during 24 hours, in the source water of public supplying of the city of Jaboticabal/SP. Samples of source water will be harvested, with intervals of 2 hours, during 24 hours and will be determined the Most Probable Numbers of total coliformes, Escherichia coli and Clostrídios sulfite reducing and the ammonia concentrations in the periods of rains and dries. Conclude that at times the water of the Córrego Rico can not be used for the supply of the population by conventional treatment, because the incidents rain, mainly during the evening, the river shows contamination above the limit for its class. May present risk to the health of the population. The gotten results will be able to guide actions aiming promote the best quality of the supplied water to the population, preventing the hydric diseases propagation. / Mestre

Monitoring viruses circulating in the human population and the environment is critical for protecting public and ecosystem health. The goal of this dissertation was to incorporate a viral metagenomic approach into virus surveillance efforts (both clinical and water quality control programs) to enhance traditional virus detection methods. Clinical surveillance programs are designed to identify and monitor etiological agents that cause disease. However, the ability to identify viruses may be compromised when novel or unsuspected viruses are causing infection since traditional virus detection methods target specific known pathogens. Here we describe the successful application of viral metagenomics in a clinical setting using samples from symptomatic patients collected through the Enterovirus Surveillance (EVS) program in the Netherlands (Appendix A). Despite extensive PCR-based testing, the viruses in a small percentage of these samples (n = 7) remained unidentified for more than 10 years after collection. Viral metagenomics allowed the identification of viruses in all seven samples within a week using minimal sequencing, thus rapidly filling the diagnostic gap. The unexplained samples contained BK polyomavirus, Herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) which dominated the unexplained samples (n = 4). This study demonstrated that metagenomic analyses can be added as a routine tool to investigate unidentified viruses in clinical samples in a public-health setting. In addition, metagenomic data gathered for SAFV was used to complete four genotype 3 SAFV (SAFV-3) genomes through primer walking, doubling the number of SAFV-3 full genomic sequences in public databases. In addition to monitoring viruses in symptomatic patients, it is also important to monitor viruses in wastewater (raw and treated) to protect the environment from biological contamination and prevent further spread of pathogens. To gain a comprehensive understanding of viruses that endure wastewater treatment, viral metagenomics was used to survey the total DNA and RNA viral community in reclaimed water (the reusable end-product of wastewater treatment) (Appendix B). Phages (viruses that infect bacteria) dominated the DNA viral community while eukaryotic viruses similar to known plant and insect viruses dominated RNA metagenomic libraries suggesting that highly stable viruses may be disseminated through this alternative water supply. A plant virus, the Pepper mild mottle virus (PMMoV), was identified as a potential indicator of wastewater contamination based on metagenomic data and quantitative PCR assays (Appendix C). The metagenomic analysis also revealed a wealth of novel single-stranded DNA (ssDNA) viruses in reclaimed water. Further investigation of sequences with low-level similarities to known ssDNA viruses led to the completion of ten novel ssDNA genomes from reclaimed water and marine environments (Appendix D). Unique genome architectures and phylogenetic analysis suggest that these ssDNA viruses belong to new viral genera and/or families. To further explore the ecology of the novel ssDNA viruses, a strategy was developed to take metagenomic analysis to the next level by combining expression analysis and immunotechnology (Appendix E). This dissertation made a significant contribution to current microbiological data regarding wastewater by uncovering viruses that endure the wastewater treatment and identifying a new viral bioindicator.

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-17Bitstream added on 2014-06-13T19:55:41Z : No. of bitstreams: 1 scholten_c_me_jabo.pdf: 337005 bytes, checksum: 42ae9fd40b2c65ddf42df554a9389874 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A qualidade da água in natura é de grande importância nos sistemas de tratamento de águas superficiais, pois uma vez que haja falhas no tratamento pode haver o risco de produzir água contaminada tornando-se prejudicial à saúde dos consumidores. O objetivo do presente trabalho foi conhecer a dinâmica temporal da poluição fecal, durante 24 horas, na água do manancial de abastecimento público da cidade de Jaboticabal, SP. Para isso foram colhidas amostras da água do manancial, com intervalos de 2 horas, durante 24 horas e foram determinados os Números Mais Prováveis de coliformes totais, Escherichia coli e Clostrídios sulfito redutores e as concentrações de amônia nos períodos de chuvas e estiagem. Concluiu-se que em alguns eventos a água do Córrego Rico não poderia ser utilizada para o abastecimento da população por tratamento convencional, pois com as incidentes chuvas, principalmente no período vespertino, o córrego apresenta contaminação acima do limite para sua classe. Assim podendo apresentar risco à saude da população. Os resultados obtidos podem nortear ações visando promover a qualidade da água fornecida à população no sentido de prevenir as enfermidades de veiculação hídrica. / Considering the quality of the water “in natura” has a great importance in the systems of superficial water treatment, once time that is fails it can be had a risk to produce contaminated water being harmful to the consumers health, the present work was elaborated with the objective to know the secular dynamics of pollution fecal, during 24 hours, in the source water of public supplying of the city of Jaboticabal/SP. Samples of source water will be harvested, with intervals of 2 hours, during 24 hours and will be determined the Most Probable Numbers of total coliformes, Escherichia coli and Clostrídios sulfite reducing and the ammonia concentrations in the periods of rains and dries. Conclude that at times the water of the Córrego Rico can not be used for the supply of the population by conventional treatment, because the incidents rain, mainly during the evening, the river shows contamination above the limit for its class. May present risk to the health of the population. The gotten results will be able to guide actions aiming promote the best quality of the supplied water to the population, preventing the hydric diseases propagation.

In this study we investigated the composition of the sediment from Sergipe River Estuary, located in the metropolitan area of Aracaju, in two periods: the winter of 2010 and the summer of 2011. In the recent years, this river has been undergoing a process of degradation due to release of sewage, without proper treatment, and the process of industrialization in its basin. We performed the sediments characterization from the analysis of total organic carbon (TOC), total organic matter (TOM), total nitrogen (TN) and the amount of silt, clay and sand. The lipid compounds, such as sterols and linear alcohols, were identified and quantified in extracts of surface sediments in the Sergipe River Estuary by gas chromatography coupled to mass spectrometry (GC-MS). The autochthonous and allochthonous contributions were characterized in the study area through the determination of sterols and alcohols in sediments. The autochthonous contribution was determined using the amount and distribution of the linear alcohols of short chain (<C20), which characterize the presence of aquatic organisms such as algae and bacteria. The allochthonous contribution was evidenced by the presence of linear long chain alcohols (> C20) and the identification of sterols of 29 carbon atoms. The C30 alcohol was the compound which presented the higher concentration within all samples, with values ranging from 0.62 to 2.46 μg.g-1 in the winter and 0.37 to 21.41 μg.g-1 in the summer. The most prominent sterol concentration was observed for β-sitosterol (C29), in concentrations ranging from 1.15 to 15.69 μg.g-1 in the winter and from 1.23 to 8.09 μg.g-1 in the summer. These compounds are frequently found in large quantities in vascular plants. Therefore, suggesting a strong contribution of terrigenous materials to the estuary. Contamination by sewage in the Sergipe River estuarine system was assessed by the presence of coprostanol (fecal biomarker) in the sediments and the different sterols ratios and indexes. This contamination was attributed to the possible discharge of untreated sewage into the Sergipe river basin. / Neste trabalho foi feita a análise do sedimento do Estuário do Rio Sergipe, situado na região metropolitana de Aracaju, em dois períodos: no inverno de 2010 e no verão de 2011. Nos últimos anos, esse Rio vem passando por um processo de degradação devido ao lançamento de esgotos domésticos, sem tratamento apropriado, em suas águas e pela concentração industrial nessa Bacia. Foi realizada a caracterização dos sedimentos a parir da análise de teores de carbono orgânico total (COT), matéria orgânica total (MOT), nitrogênio total (NT) e teores de silte, argila e areia. Os compostos lipídicos, esteróis e álcoois lineares, foram identificados e quantificados nos extratos de sedimentos superficiais no Estuário do Rio Sergipe através da cromatografia em fase gasosa acoplado a espectrômetro de massas (CG-EM). As contribuições autóctone e alóctone foram caracterizadas na área de estudo através da determinação dos esteróis e álcoois nos sedimentos. A contribuição autóctone foi determinada a partir dos álcoois lineares de cadeia curta (<C20) que caracteriza a presença de organismos aquáticos como algas e bactérias. A contribuição alóctone foi evidenciada pela presença dos álcoois lineares de cadeia longa (>C20) e pela identificação de esteróis com 29 átomos de carbono na estrutura. O álcool identificado em maior concentração em todas as amostras foi o C30, em concentrações que variaram de 0,62 a 2,46 μg.g-1 no inverno e de 0,37 a 21,41 μg.g-1 no verão. O esterol de concentração mais proeminente foi o β-sitosterol (C29) em concentrações que variaram de 1,15 a 15,69 μg.g-1 no inverno e de 1,23 a 8,09 μg.g-1 no verão. Esses compostos são frequentemente encontrados em grandes quantidades em plantas superiores, sugerindo assim uma forte contribuição de material terrígeno no estuário. A contaminação por esgoto no estuário do Rio Sergipe foi avaliada pela presença do coprostanol (biomarcador de origem fecal) nos sedimentos analisados e pelas razões de diferentes esteróis. Esta contaminação foi atribuída ao possível lançamento de esgoto não tratado na Bacia do Rio Sergipe.

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The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners in detergents) were used in the summers of 2004 and 2005 to determine the origins of fecal pollution at beaches with a past history of, or the potential for, high enterococci counts and posted advisories. At Hilton and Anderson beaches, ARA and fluorometry in the summer of 2004 detected substantial human-origin pollution in locations producing consistently high counts of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of these mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE). Results at other beaches indicated that birds and/or wildlife were largely responsible for elevated enterococci levels during 2004 and 2005. The application of fluorometry proved difficult in opens waters due to high levels of dilution, but showed potential for use in storm drains. An additional study developed and tested a new library-based MST approach based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer region, and subsequent digestion using the restriction endonuclease MboI. Initial results from small known-source libraries yielded high average rates of correct classification (ARCC). However, an increase in the library size was accompanied by a reduction in the ARCC of the library and the method was deemed unsuccessful, and unsuitable for field application. A final study focused on the potential for classification bias with disproportionate source category sizes using discriminant analysis (DA), logistic regression (LR), and k-nearest neighbor (K-NN) statistical classification algorithms. Findings indicated that DA was the most robust algorithm for use with source category imbalance when measuring both correct and incorrect classification rates. Conversely k-NN was identified as the most sensitive algorithm to imbalances with the greatest levels of distortion obtained from the highest k values. Conclusions of this project include: 1) application of a validation set, as well as a minimum detectable percentage to known-source libraries aids in accurately assessing the classification power of the library and reducing the false positive identification of contributing fecal sources; 2) the validation of MST results using multiple methods is recommended for field applications; 3) fluorometry displayed potential for detecting optical brighteners as indicators of sewage leaks in storm drains; 4) the digestion of the 16S-23S rDNA intergenic spacer region of Enterococcus spp. using MboI does not provided suitable discriminatory power for use as an MST method; and 5) DA was the least, and k-NN the most, sensitive algorithm to imbalances in the size of source categories in a known-source library. / Ph. D.