An extensive 10 year monitoring project was initiated in 1986 to examine the effects of a combination of BMPs on surface water quality within a watershed with complex land use. This research specifically examined bacteriological water quality and BMP impacts. Bimonthly grab samples were collected from four surface water monitoring stations, including the watershed outlet, and analyzed for fecal coliform, total coliform, and fecal streptococcus bacteria. Other data compiled from the watershed included hydrologic, meteorologic, geologic and land use data, also collected on a regular basis. Data were collected continuously throughout the project, and thus included both pre- and post-BMP monitoring data. BMP implementation included animal waste storage facilities, nutrient management plans, conservation tillage, alternative water sources for livestock, fences, vegetative filter strips, runoff diversions, and others. Statistical analysis of the monthly precipitation data indicated no significant difference in rainfall quantity between the pre-BMP and post-BMP monitoring periods. Monthly runoff totals increased 39% from the pre- to the post-BMP periods at the watershed outlet. Increases at all of the subwatershed outlets occurred as well (B, 40%; C, 38%; D, 16%). Statistical analysis did not show a significant difference in runoff between the two monitoring periods, except at station C, where post-BMP runoff was significantly greater than the values measured during the pre-BMP period. Overall reductions in the mean (geometric) levels of total coliform, fecal coliform and fecal streptococcus bacteria observed at the watershed outlet were 81%, 30% and 76%, respectively. Both parametric and nonparametric statistical analysis techniques were applied to the bacteriological data. Regression analysis of the fecal coliform data showed an increase during the pre-BMP period followed by a decrease post-BMP and a statistically significant difference between the two periods (p=0.004). No trends were evident. Only one of the four stations had a statistical difference between pre- and post-BMP fecal streptococcus data, however, a downward trend was present at every station. No statistically significant difference between the pre- and post-BMP total coliform bacteria was evident, although a downward trend was present at the watershed outlet. These findings indicate that the combination of BMPs implemented in the watershed were effective in reducing the loss of fecal bacteria to receiving streams via overland flow. / Master of Science

Sources of E. coli were determined from impaired waterways in the Appomattox River watershed (in the lower Piedmont and South-Central Virginia) for the development of bacterial Total Maximum Daily Loads (TMDLs). The Appomattox River watershed is primarily undeveloped with 70.8% of the land forested, 17.0% used for agriculture (mainly livestock production), and 7.7% classified as water, wetland or barren land. The remaining 4.5% is developed for residential, commercial, and industrial land uses (mainly within the city of Petersburg). Using Antibiotic Resistance Analysis, a known source library of 1,280 E. coli isolates (320 isolates per source) was constructed. Water samples were collected monthly for between eleven and fourteen months (11/02-12/03) from 40 locations throughout the Appomattox watershed and analyzed for fecal coliforms, E. coli, and resistance to 7 antibiotics of varying concentrations. A total of 486 water samples (9,907 isolates) were analyzed during the study. The objectives of this study were verify that each sampling site exceeded state bacterial count standards (using fecal coliform data), to compare the Discriminate Analysis and Logistic Regression statistical models for use in the classification of isolates, and finally to determine the source of contamination at each site. The fecal coliform and E.coli data was used to determine if each site exceeded state standards during the assessment period. Thirty-eight of the sites exceeded the fecal coliform standard at least 10% of the time, and thirty-three exceeded the E.coli standard at least 10% of the time. Discriminate Analysis (DA) is typically used to classify isolates, but the results obtained from the DA model were unrealistic based on the watershed land uses. By statistically analyzing the original 1,280 E.coli isolates six different ways, a more appropriate classification of isolates was determined. The six analyzing methods were Regular DA and Logistic Regression (LR); DA and LR where each isolate whose probability fell below 80% was deleted; DA and LR where each isolate whose probability fell below 80% was used to create an Unknown category. The Logistic Regression model with an Unknown category proved to be the most appropriate. By using the Logistic Regression model, with Unknown category, to classify isolates, twenty five of the forty sites were discovered to be contaminated predominately with Livestock and fourteen of the sites predominately by Wildlife. One site was equally divided between these two categories. Human and Pet contamination were not dominant at any of the forty sites. This comparison of the DA and LR statistical methods could change the analysis standard for Bacterial Source Tracking and suggests that the model required to classify isolates depends on the watershed characteristics. / Master of Science

Surface waters containing fecal bacteria present significant public health risks. Understanding the sources of and factors affecting the distribution of fecal-indicating bacteria is necessary to predict potential illnesses more effectively. This thesis presents two studies on the distribution of fecal bacteria in the James River through Richmond, Virginia. Chapter 1 describes nearly 11 years of water quality, climate, and hydrologic data that occurred with changes in Escherichia coli concentrations, concluding that Richmond contributes significant quantities of fecal bacteria to the James River, and that the distribution of these bacteria varies seasonally. Chapter 2 details the development of Polymerase Chain Reaction (PCR)-based methods to identify four common pathogenic taxa of fecal bacteria, and indicates that the factors controlling the distribution of these pathogens may be taxa-specific. Both studies, taken together, indicate that urbanization increases the presence of fecal bacteria and pathogens in this system, and that recreational contact with the river presents potential health risks.

Antibiotic resistance analysis (ARA) of Enterococci was used to determine sources of fecal contamination in three District of Columbia waterways: Rock Creek, the Anacostia River, and the Potomac River. These three waterways were identified as exceeding water quality standards set for fecal coliform levels and were designated by the District of Columbia to the Environmental Protection Agency's 303 (d) impaired waters list. A library profile of 1,806 enterococcus isolates from known sources was built based on antibiotic resistance patterns from thirty concentrations of nine antibiotics. These sources included human, cattle, chicken, horse, goat, sheep, deer, raccoon, muskrat, goose, seagull, coyote, duck, wild turkey, dog, and cat. Antibiotic profiles were characterized for 24 unknown enterococci isolates on each of 198 samples (38 samples from the Potomac River, 79 samples from the Anacostia River, and 81 samples from Rock Creek) collected periodically from July 2002 through April 2003. Two major storm events were also sampled during this period. These isolate profiles were compared to the known source library using logistic regression. Three dominant sources of fecal pollution were detected in the Potomac River: livestock (30%), human (29%), and wildlife (22%). Three dominant signatures were also detected in Rock Creek: horse (26%), human (26%), and wildlife (24%). Human was the only dominant source detected in the Anacostia River, averaging 43% over the sampling period. The results of this study indicate that human is a substantial contributor to the fecal contamination problems, especially in the Anacostia River, but there are significant agricultural and wildlife contributions as well. Significant and predictable seasonal variations were also detected, indicating the influence of precipitation on source distributions. The results of this study will aid the Metropolitan Washington D.C. Council of Governments in making important management decisions to help improve the water quality in and around the Washington D.C. area. Expanding the limits of ARA was also an integral part of this research. Three new and even controversial analytical techniques were run on the data collected from this project in an attempt to improve confidence and provide direction to the results of this study. The first was a comparison of the more commonly used statistical analysis model discriminate analysis (DA) with logistic regression (LR). No significant difference was found between the output of the two models for the known source libraries, therefore no suggestion could be made in favor of one model over the other. Another analytical test of the data was the introduction of a standard requiring isolates to meet a minimum of 80% similarity to the known source profiles where it was classified. With the 80% cutoff, between 41% and 44% of the isolates could not be classified to any source and were placed in an unknown category. Based on the remaining isolates, source distributions were recalculated and were not statistically different than those calculated with no restriction for isolate similarity for matching. The last major test of the data was the analysis of the library for representativeness via pulled sample cross validation and the exclusion of all duplicate patterns from the known source library. These analyses did not confirm the representativeness of the databases, but results were further analyzed based on the implications these analyses have on library based methods. / Master of Science

Transport of fecal bacteria and nutrients from point and nonpoint sources to surface water bodies is of significant concern in Virginia and the United States. In Virginia, 4,320 river miles are impaired for one or more beneficial use and 72% of the streams are impaired due to pathogen indicators (VDEQ, 2002). Land applications of manure from confined animal systems and by direct deposit by grazing animals are both major sources of fecal bacteria and nutrients in runoff. Therefore, an understanding of the overland transport mechanisms for fecal bacteria and nutrients is very important for the development of best management practices to reduce loading of pathogens and nutrients to surface water bodies. The objectives of this study were to quantify the release and transport potential of three fecal bacterial indicators: E. coli, Enterococcus, and fecal coliforms; and nitrogen and phosphorus from land applied manure during runoff events. Another objective was to identify the Enterococcus species present in dairy manure and determine which species have the highest potential to be transported by runoff. Release plots were established to study the in-field bacteria and nutrient release. The bacteria and nutrients released from the plots are available to be transported to the edge of the field in runoff. Four manure treatments (turkey litter, liquid dairy manure, cowpies, and none or control) and three land type treatments: pasture with a history of poultry litter application (Turkey Farm), pasture with a history of liquid dairy manure application (Dairy Farm), and pasture with no prior manure application (Tech Research Farm) were studied. During a short but intense rainfall event, the highest bacterial release was measured under the cowpie treatment (E. coli concentrations ranging from 37,000 to >300,000 and FC concentrations ranging from 65,000 to >300,000). Pasturelands with a history of previous manure applications did not release higher bacteria concentrations compared with pasturelands which had never received manure applications. Pasturelands with a history of land application of liquid dairy manure and turkey litter had 143% and 94% higher TSS concentrations available to be transported off the field during overland flow events because of the build up of organic material on the soil surface. TP concentrations released from the cowpie, liquid dairy, and turkey litter treatments were 3.12 mg/L, 3.00 mg/L, and 1.76 mg/L, respectively. Transport plots were developed to measure the concentrations of fecal bacteria and nutrients present in overland flow at the edge of the field. The bacteria flow-weighted concentrations were highest in runoff samples from the plots treated with cowpies (200,000 CFU/100 mL of E. coli and 234,000 CFU/100 mL of FC). The turkey litter had the highest concentration of dissolved phosphorus in runoff from pasturelands (1.22 mg/L), but the cowpie treatment had the highest concentrations of sediment bound phosphorus in runoff (0.73 mg/L). All three treatments investigated in this study contributed to phosphorus loading in surface waters and could potentially increase the risk of eutrophication. Total nitrogen concentrations from the transport plots exceeded the threshold for likely eutrophication problems for all treatments and the total nitrogen concentrations from plots treated with cowpies exceeded the threshold for severe eutrophication problems. The Biolog System, a method of bacterial source tracking, was used to identify the different species of Enterococcus present both in the cowpie source manure and in the runoff collected from the transport plots treated with cowpies. The source manure is dominated by the Enterococcus mundtii (55%), Enterococcus gallinarum (20%), Enterococcus faecium (10%), and Enterococcus faecalis (10%). Enterococcus faecalis had the highest percentage of isolates present in runoff with a total of 37%, followed by Enterococcus mundtii which was present in 21% of the runoff events and Enterococcus gallinarum and Enterococcus faecium (11%). Improvements in understanding the bacterial release and overland processes will enhance modeling of bacteria and nutrient transport, and provide a basis for a more realistic evaluation of the impacts of management practices implementation. The data from this study will serve as a baseline to model the release and transport of fecal bacteria and nutrients from agricultural watersheds to surface waters. / Master of Science

El objetivo principal del estudio fue establecer el nivel de resistencia a antimicrobianos en un total de 222 cepas comensales de E. coli de origen fecal, en Perú. Las frecuencias de resistencia encontrados, frente los antimicrobianos evaluados, fueron: ampicilina (62,6%), cotrimoxazol (48,6%), tetraciclina (43,0%) y cloranfenicol (15,8%). Destacan los elevados niveles de resistencia a quinolonas: 32% al ácido nalidíxico (NAL) y 12% a ciprofloxacino (CIP). Estos elevados niveles hacia las quinolonas en cepas comensales aisladas en niños de esta franja de edad, realzan el uso extendido y el impacto de consumo de este tipo de antimicrobianos en la comunidad, mostrando el riesgo potencial de su pérdida de utilidad en el área. / The main aim of this study was to establish the resistance levels to antimicrobial agents, in 222 non-pathogenic E. Coli strains of fecal origin in Peru. The proportion of resistance found to the evaluated antimicrobials was ampicillin (62.6%), cotrimoxazole (48,6%), tetracycline (43,0%) and chloramphenicol (15,8%). We emphasize the high resistance levels found for quinolones: 32% for nalidixic acid (NAL) and 12% for ciprofloxacin (CIP). These high levels of quinoloneresistance in non-pathogenic strains isolated from children in this age group highlight the extensive use and the impact of the intake of this kind of antimicrobials in the community, showing the potential risk of the loss of their utility in the area.

Microbial contamination of drinking water poses an important health risk which causes severe illnesses and epidemics. In order to improve surface and drinking water quality, the understanding of fecal pathogen contamination processes including their prevention and control needs to be enhanced. The watershed model soil water assessment tool (SWAT) is commonly used to simulate the complex hydrological, meteorological, erosion, land management and pollution processes within river basins. In recent years, it has been increasingly applied to simulate microbial contamination transport at the watershed scale. SWAT is used in this study to simulate Escherichia coli (E.coli) and fecal coliform densities for the agriculturally dominated Payne River Basin in Ontario, Canada. Unprecedented extensive monitoring data that consist of 30 years of daily hydrological data and 5 years of bi-weekly nutrient data have been used to calibrate and validate the presented model here. The calibration and validation of the streamflow and nutrients indicate that the model represent these processes well. The model performs well for periods of lower E. coli and fecal coliform loadings. On the other hand, frequency and magnitude of higher microbial loads are not always accurately represented by the model.

Microbial contamination of drinking water poses an important health risk which causes severe illnesses and epidemics. In order to improve surface and drinking water quality, the understanding of fecal pathogen contamination processes including their prevention and control needs to be enhanced. The watershed model soil water assessment tool (SWAT) is commonly used to simulate the complex hydrological, meteorological, erosion, land management and pollution processes within river basins. In recent years, it has been increasingly applied to simulate microbial contamination transport at the watershed scale. SWAT is used in this study to simulate Escherichia coli (E.coli) and fecal coliform densities for the agriculturally dominated Payne River Basin in Ontario, Canada. Unprecedented extensive monitoring data that consist of 30 years of daily hydrological data and 5 years of bi-weekly nutrient data have been used to calibrate and validate the presented model here. The calibration and validation of the streamflow and nutrients indicate that the model represent these processes well. The model performs well for periods of lower E. coli and fecal coliform loadings. On the other hand, frequency and magnitude of higher microbial loads are not always accurately represented by the model.

Microbial contamination of drinking water poses an important health risk which causes severe illnesses and epidemics. In order to improve surface and drinking water quality, the understanding of fecal pathogen contamination processes including their prevention and control needs to be enhanced. The watershed model soil water assessment tool (SWAT) is commonly used to simulate the complex hydrological, meteorological, erosion, land management and pollution processes within river basins. In recent years, it has been increasingly applied to simulate microbial contamination transport at the watershed scale. SWAT is used in this study to simulate Escherichia coli (E.coli) and fecal coliform densities for the agriculturally dominated Payne River Basin in Ontario, Canada. Unprecedented extensive monitoring data that consist of 30 years of daily hydrological data and 5 years of bi-weekly nutrient data have been used to calibrate and validate the presented model here. The calibration and validation of the streamflow and nutrients indicate that the model represent these processes well. The model performs well for periods of lower E. coli and fecal coliform loadings. On the other hand, frequency and magnitude of higher microbial loads are not always accurately represented by the model.

AIG, CFL, SM, JR participaron en la concepción y diseño del estudio; AIG, CFL, MM, TJO y JR en el aporte de pacientes o material de estudio; CFL, TJO y JR en la obtención del financiamiento; TJO, SM, MJP, JR, y LJdV participaron el análisis e interpretación de los datos. Todos los autores participaron de la recolección de resultados, revisión crítica del manuscrito, aprobación de su versión final. / The main aim of this study was to establish the resistance levels to antimicrobial agents, in 222 non-pathogenic E. Coli strains of fecal origin in Peru. The proportion of resistance found to the evaluated antimicrobials was ampicillin (62.6%), cotrimoxazole (48,6%), tetracycline (43,0%) and chloramphenicol (15,8%). We emphasize the high resistance levels found for quinolones: 32% for nalidixic acid (NAL) and 12% for ciprofloxacin (CIP). These high levels of quinoloneresistance in non-pathogenic strains isolated from children in this age group highlight the extensive use and the impact of the intake of this kind of antimicrobials in the community, showing the potential risk of the loss of their utility in the area. / Este trabajo fue parcialmente financiado por Agència Catalana de Cooperació al Desenvolupament proyecto U2006 (LJdV), Centre de Cooperació per al Desenvolupament - Universitat Politècnica de Catalunya (LJdV), Agencia Española de Cooperación Internacional al Desarrollo proyectos numero A/4892/06 (LJdV), D/019499/08 y D/024648/09 (JR), Fogarty International Center, National Institute of Health, USA, proyecto 1K01TW007405 (TJO) Sanofi Pasteur y fondos de investigación del Dr. Lanata, Instituto de Investigación Nutricional, Lima, Perú. La investigación de JR es financiado por el proyecto CP05/0130 del FIS (Fondo de Investigaciones Sanitarias, España). / Revisión por pares.