A model study has been conducted to understand the influence of hydrodynamic features, environmental conditions as well as bather shedding and animal fecal sources on the fate and transport of indicator microbe enterococci at a subtropical marine beach in South Florida. The model being used is based on an existing finite element hydrodynamic and transport model CAFE3D to which a first order microbe deactivation function due to solar radiation is added. The decay coefficient is assumed to be linearly proportional to the solar insolation value, while the constant coefficient linking the two is determined to be 0.368[m2/MJ] using local experimental data. This value corresponds to decay coefficients in the range of 0 to 1 hr-1 and is of similar magnitude to values found in the literature. The hydrodynamic model produces water current velocities, which are used in the transport model to simulate water enterococci concentration in space and time. Previous measurements have suggested that enterococci originate from the beach shoreline area. In this case the simulated velocity fields and Lagrangian particle trajectories indicate that the small velocity (generally less than 0.05m/s) and weak dilution at the water?s edge may cause enterococci accumulation and elevated concentration. Among human and animal sources, the impacts on maximum enterococci concentration in descending order is a dog fecal event, human bather shedding and seagull fecal events. A single dog fecal event at the middle part of the beach is found to cause enterococci far field concentrations to reach hundreds of CFU/100ml, which exceeds the EPA water quality standard (104 CFU/100ml). These high concentrations, however, only impact a small area. Concentration due to tens of bathers shedding enterococci during a course of 4 hours reaches 0.01 CFU/100ml, while the concentration due to thousands of bather shedding during a Memorial Day weekend reaches about 1 CFU/100ml. Concentration due to tens of seagull fecal events reaches only 0.0001 CFU/100ml. Thus, only the presence of dogs could explain the high enterococci observations infrequently found in monitoring data. The limited spatial extent compared to beach survey data of the enterococci plume from a dog fecal event suggests that other microbial sources are present, most likely microbes released from the beach sand.

Bioretention cells (BRCs) are low-impact development stormwater management structures that integrate water quantity and quality management. Although BRCs have a predicted design life of about 25 years, most current research focuses on performance of cells less than two years old. This project evaluated the effectiveness of a BRC installed in 2007 to treat a 0.16-ha parking lot in Blacksburg, VA. After installation, this BRC was monitored for five months to determine initial flow reduction and total suspended solids, and nutrient removal. By monitoring for the same parameters, changes in cell performance since installation were quantified. ISCO automated stormwater samplers collected inflow and outflow composite samples from the cell, which were then analyzed for fecal indicator bacteria (total coliforms, E. coli, and enterococci), total suspended solids (TSS), total nitrogen (TN), and total phosphorus (TP). To determine if denitrification is occurring within the BRC, media samples taken throughout the cell were analyzed using qPCR. The bioretention media was also sampled to quantify changes in media nutrient content and particle size over the past seven years. Results indicate the bioretention media has not accumulated nitrogen and phosphorus since installation, and that the BRC remains effective at reducing flow volume and peak flow rates, as well as TSS, TN, TP, total coliforms, E. coli, and enterococci loads. Bacterial analysis of the media show most of the denitrifiers are present in the top layers of the bioretention media, despite an internal water storage layer and the bottom of the cell designed specifically for denitrification. / Master of Science

The contamination of inland surface water by point and nonpoint sources is a widespread human health concern. To address this problem, a thorough understanding of the sources and persistence of this pollution is necessary. Using fecal indicator bacteria (FIB) as a surrogate for enteric pathogens, the Environmental Protection Agency (EPA) and state agencies have developed surface water quality standards. If a segment does not meet these standards, a Total Maximum Daily Load (TMDL) must be developed for the watershed to identify sources of bacterial pollution. Currently, FIB pollution is the leading pollution type addressed by TMDLs in the country. One source of FIB identified in TMDLs is colonies of birds roosting under bridges. It has been proposed that the birds’ feces can augment the FIB concentrations downstream of bridges. In this year-long study of Bull Creek in Austin, Texas, it was determined that the concentrations of Escherichia coli and fecal coliform downstream of a bridge were significantly greater when migratory cliff swallows were nesting under the bridge. The downstream concentrations of both FIB exceeded contact recreation standards. Data also suggest that FIB from the feces could be swept into the stream by runoff from storm events. No enterococci loading was observed in conjunction with the swallows. This study also investigated the affects of sediment resuspension on surface water FIB concentrations and the persistence of FIB in shady, inland streams. The resuspension of sediments with attached FIB could also increase the FIB concentration in the water column and increase its persistence. The results of reactor-based experiments demonstrated that the concentration of E. coli in water from an effluent-dominated stream increased by a factor of 3 when riverine sediments were resuspended and exceeded single sample standards for contact recreation, suggesting sediments as a reservoir of E. coli. Additionally, concentrations of E. coli decreased by approximately 90% and 70% over 2 days in reactors containing stream water and sediment-laden stream water, respectively. / text

Fecal indicator bacteria (FIB) such as Escherichia coli and enterococci are used to assess microbiological water quality in recreational waters worldwide. FIB are used with the assumption that their presence correlates with that of fecal-associated pathogens in recreational waters. In aquatic habitats, several factors can interfere with the predictive relationship between FIB and pathogens including extended survival of FIB in secondary habitats such as sediment, vegetation and sand. Furthermore, many biotic (e.g. predation from bacterivorous protozoa and competition from indigenous bacteria) and abiotic factors (e.g. temperature, salinity, ultraviolet (UV) light irradiation, and nutrient availability) can influence the fate of FIB and pathogens associated with gastrointestinal tracts of animals (enteric pathogens) in secondary habitats. The relative importance of these factors is not well characterized, thus limiting our knowledge on the efficacy of FIB as indicators of fecal contamination and microbial pathogens in water. The studies presented in this dissertation investigated the influence of biotic (predation from bacterivorous protozoa and competition from indigenous bacteria) and abiotic factors (e.g. nutrient availability) on the survival of FIB (E. coli and Enterococcus faecalis) and pathogens (E. coli O157 and Salmonella enterica) in aquatic habitats. Water and sediment samples were collected from a fresh water river source (Hillsborough River, Tampa, FL) and used to prepare a series of outdoor mesocosm experiments. In each experiment, biota treatments were varied to include various combinations of predation and competition, both or neither. Manipulation of biota treatments involved disinfection of water and baking of sediments to remove indigenous microbiota, or addition of cycloheximide or kanamycin to diminish the effect of predation from natural protozoa or competition from indigenous bacteria respectively. Bacterial levels in all experiments were monitored over a five day period. In the mesocosms investigating the effect of predation and competition on FIB (E. coli and Ent. faecalis) and a pathogen (E. coli O157:H7), predation had a detrimental effect on the survival of the FIB and pathogen in the water column but only influenced the survival of the FIB in the sediment. Unlike predation, competition from indigenous bacteria influenced the survival of E. coli but not Ent. faecalis in both water and sediment. The second set of mesocosms investigated the effect of predation on two motile and non-motile enteric bacteria types (E. coli O157 and S. enterica), each with a motile and non-motile counterpart. An allochthonous predation source (Tetrahymena pyriformis) was added into the mesocosms to supply a consistent level of predation. Motility had a significant positive effect on the survival of S. enterica in the water and sediment but had negative significant effect for E. coli O157 in sediment only. Motility also played a more important role in the sediment compared to predation while predation played a more important role in the water column for both bacteria types. The third study compared the relative effects of predation, competition and nutrients on the survival of E. coli. Natural waters (not amended with nutrients) served as a baseline condition to which organic nutrients were added in two increments. Significant interactions among predation, nutrients and competition (all possible combinations) were observed. Interactions between predation and nutrients as well as competition and predation also accounted for the greatest effects (10% and 8% respectively). The interaction between predation and competition was particularly pronounced at the highest nutrient level. These studies reveal that predation, competition and nutrients are all important factors in the survival of FIB and enteric bacteria in water and sediment, and provide new observations on the relative magnitude of these effects. I show that survival characteristics of FIB and enteric bacteria in secondary habitats can vary depending on bacteria type (FIB or pathogen), location (water or sediment), prey characteristics (motile or non-motile) and specific environmental stressor present (predation, competition or nutrients). The findings of this dissertation provide new insights on the ecology of FIB and enteric bacteria in secondary habitats and underscore the importance of biotic and abiotic factors as determinants of the fate of FIB and enteric bacteria in secondary habitats.

Safe drinking water is a basic human right. This study mainly focused on the physicochemical and microbiological drinking water quality of selected rural, peri-urban and urban communities and schools in the North West Province, South Africa. Parameters measured to determine the physico-chemical quality of drinking water were temperature, pH, total dissolved solids (TDS), electric conductivity, carbonate hardness, total hardness, NO2 -, NO3 - and chlorine. These parameters indicated hard water in the informal settlement (Sonderwater) as well as in the rural area (Ganyesa). Nitrate content were troubling for both areas, and total dissolved solids were higher than the standard in the water from Ganyesa. For microbiological quality of the water, heterotrophic plate count (HPC) bacteria, total coliforms, faecal coliforms, faecal streptococci, and staphylococci were enumerated on appropriate selective media using standard procedures. In the water from Sonderwater, faecal indicator bacteria were isolated, but none were found in the water from Ganyesa. Heterotrophic plate count bacteria and total coliforms were detected at levels above the standard in water samples from both areas. Staphylococci and faecal streptococci were present in low numbers in the water from both sites. Faecal coliforms isolated from Sonderwater showed multiple antibiotic resistances to beta-lactams. Identification of faecal coliforms from Sonderwater by API 20E strips and sequencing showed that they were Aeromonas spp. and Enterobacter spp.. Bacteria in the water from Sonderwater were tested for the potential to form biofilms. Scanning electron microscopy revealed multi-species biofilms developing in the water container after 5 days of storage. Water was sampled from four areas outside of Potchefstroom to determine a settlement gradient in water quality. Areas ranged from a formal area, through an established informal area and a newly established informal area to the newest established informal area. The water from these areas was classified as hard according to physico-chemical parameters measured, and TDS for the water from all areas were above the standard for domestic use. The established informal area had high numbers of total coliforms present in the water. Staphylococci and HPC bacteria were detected in levels higher than the standard for domestic use in all water samples. No faecal coliforms were found in the water from any of the areas. There was no visible gradient in the water quality between the areas. The water samples collected from rural, peri-urban and urban schools were also analysed in terms of physico-chemical and microbiological parameters. Water from all schools was classified as hard water. Only one school (peri-urban) had a pH above the standard. One rural school and one peri-urban school had TDS and electrical conductivity levels above the standard for domestic use. All rural and peri-urban schools had alarmingly high levels of nitrates present in the water. These schools receive groundwater as drinking water. Total coliform bacteria were present at high levels in all water samples from the schools. Rural and peri-urban schools presented levels of staphylococci and HPC bacteria higher than the standard for domestic use. Streptococci were present in water from some of the rural and peri-urban schools and one urban school. Faecal coliform/faecal streptococci ratios for rural schools indicated faecal pollution potentially of human origin, and in other schools faecal pollution from both human and animal origin. Before the vacation, faecal coliform bacteria were detected in water from all rural schools, two peri-urban schools and one urban school. After the vacation, faecal coliforms were only detected in water from two rural schools and one peri-urban school. Faecal coliforms identified and characterized showed multiple antibiotic resistances to beta-lactams, oxy-tetracycline and trimethoprim. Identification by API 20E strips and sequencing confirmed that faecal coliforms from schools were Escherichia coli and Klebsiella spp. It was concluded that water from Sonderwater were of poor quality and water from Ganyesa were acceptable with only the nitrates a troubling factor. There was no settlement gradient observed in terms of water quality between areas. Water from rural schools were generally of unacceptable quality in terms of both physico-chemical and microbiological parameters. The water quality of these schools was also very poor when compared to urban schools. Periurban schools had water quality better than rural schools, but poorer than urban schools. Surveys of water quality are recommended for all areas sampled, and education on the sanitary quality of water and related health implications is advisable for residents of informal and rural areas. / Thesis: B.Sc. Microbiology and Biochemistry School of Environmental Science and Development Faculty of Natural Sciences North-West University: Potchefstroom campus 2006.

Safe drinking water is a basic human right. This study mainly focused on the physicochemical and microbiological drinking water quality of selected rural, peri-urban and urban communities and schools in the North West Province, South Africa. Parameters measured to determine the physico-chemical quality of drinking water were temperature, pH, total dissolved solids (TDS), electric conductivity, carbonate hardness, total hardness, NO2 -, NO3 - and chlorine. These parameters indicated hard water in the informal settlement (Sonderwater) as well as in the rural area (Ganyesa). Nitrate content were troubling for both areas, and total dissolved solids were higher than the standard in the water from Ganyesa. For microbiological quality of the water, heterotrophic plate count (HPC) bacteria, total coliforms, faecal coliforms, faecal streptococci, and staphylococci were enumerated on appropriate selective media using standard procedures. In the water from Sonderwater, faecal indicator bacteria were isolated, but none were found in the water from Ganyesa. Heterotrophic plate count bacteria and total coliforms were detected at levels above the standard in water samples from both areas. Staphylococci and faecal streptococci were present in low numbers in the water from both sites. Faecal coliforms isolated from Sonderwater showed multiple antibiotic resistances to beta-lactams. Identification of faecal coliforms from Sonderwater by API 20E strips and sequencing showed that they were Aeromonas spp. and Enterobacter spp.. Bacteria in the water from Sonderwater were tested for the potential to form biofilms. Scanning electron microscopy revealed multi-species biofilms developing in the water container after 5 days of storage. Water was sampled from four areas outside of Potchefstroom to determine a settlement gradient in water quality. Areas ranged from a formal area, through an established informal area and a newly established informal area to the newest established informal area. The water from these areas was classified as hard according to physico-chemical parameters measured, and TDS for the water from all areas were above the standard for domestic use. The established informal area had high numbers of total coliforms present in the water. Staphylococci and HPC bacteria were detected in levels higher than the standard for domestic use in all water samples. No faecal coliforms were found in the water from any of the areas. There was no visible gradient in the water quality between the areas. The water samples collected from rural, peri-urban and urban schools were also analysed in terms of physico-chemical and microbiological parameters. Water from all schools was classified as hard water. Only one school (peri-urban) had a pH above the standard. One rural school and one peri-urban school had TDS and electrical conductivity levels above the standard for domestic use. All rural and peri-urban schools had alarmingly high levels of nitrates present in the water. These schools receive groundwater as drinking water. Total coliform bacteria were present at high levels in all water samples from the schools. Rural and peri-urban schools presented levels of staphylococci and HPC bacteria higher than the standard for domestic use. Streptococci were present in water from some of the rural and peri-urban schools and one urban school. Faecal coliform/faecal streptococci ratios for rural schools indicated faecal pollution potentially of human origin, and in other schools faecal pollution from both human and animal origin. Before the vacation, faecal coliform bacteria were detected in water from all rural schools, two peri-urban schools and one urban school. After the vacation, faecal coliforms were only detected in water from two rural schools and one peri-urban school. Faecal coliforms identified and characterized showed multiple antibiotic resistances to beta-lactams, oxy-tetracycline and trimethoprim. Identification by API 20E strips and sequencing confirmed that faecal coliforms from schools were Escherichia coli and Klebsiella spp. It was concluded that water from Sonderwater were of poor quality and water from Ganyesa were acceptable with only the nitrates a troubling factor. There was no settlement gradient observed in terms of water quality between areas. Water from rural schools were generally of unacceptable quality in terms of both physico-chemical and microbiological parameters. The water quality of these schools was also very poor when compared to urban schools. Periurban schools had water quality better than rural schools, but poorer than urban schools. Surveys of water quality are recommended for all areas sampled, and education on the sanitary quality of water and related health implications is advisable for residents of informal and rural areas. / Thesis: B.Sc. Microbiology and Biochemistry School of Environmental Science and Development Faculty of Natural Sciences North-West University: Potchefstroom campus 2006.

Foodborne outbreaks have continued to be associated with produce contamination originating from on-farm sources, such as soil or agricultural water. Additionally, the heterogeneity of the pre-harvest environment complicates the development of universal strategies for managing produce safety risks. Understanding the ecology and epidemiology of foodborne pathogens and fecal indicator bacteria (FIB) by growing regions, sample types, scale of analysis, and detection method is essential for developing targeted mitigation strategies. This dissertation utilized quantitative research methods and statistical modeling to examine the impact of sampling method, spatial, temporal, meteorological, and physicochemical factors on pathogen prevalence and FIB levels. Key findings highlight that the drivers of prevalence differ between pathogens and were influenced by sample type, scale, and region.. The variations in associations emphasize that risk varies by space and time. Therefore, results support regional and scale-dependent food safety standards and guidance documents for controlling hazards to minimize risk. Additionally, the method used for pathogen detection influences prevalence highlighting the need for standard methods since methodological differences confound comparisons across studies. Furthermore, since agricultural water quality is an important food safety priority, this dissertation aimed to determine the efficacy of chemical antimicrobial sanitizers against Salmonella in pre-harvest agricultural water. Results demonstrated that certain sanitizer treatments and conditions can significantly reduce Salmonella populations in preharvest agricultural water sources and thus may serve as a risk reduction option when used correctly. / Doctor of Philosophy / Fresh fruits and vegetables are continually implicated in foodborne outbreaks. Additionally, the source of the pathogen that causes illness in these outbreaks is often due to contact with contaminated soil or water on the farm. Since the environment is extremely diverse, the risk of foodborne pathogens is not uniform across a farm and between farms. Therefore, the development of a one-size-fits-all plan to reduce the risk of foodborne pathogens from contaminating produce on a farm is difficult. Understanding the incidence and distribution of foodborne pathogens and fecal bacteria and how these microorganisms interact with the environment is important to develop strategies to manage risk. Additionally, understanding how the prevalence of bacteria varies by state, medium (water vs soil), and farm is needed to develop targeted mitigation plans. This dissertation utilized laboratory and field-based experiments to understand how space, time, weather, and physical properties impact the occurrence of foodborne pathogens and fecal bacteria. The primary results show factors that impact prevalence are different between pathogens (Salmonella vs Listeria vs E. coli). Furthermore, the occurrence differed by sampling method (molecular vs culture), sample type (water vs soil), scale (within a farm vs between multiple farms), and region emphasizing that the risk from foodborne pathogens varies over space and time. Overall, this dissertation's results suggest that both regional and scale-specific guidelines are needed to reduce foodborne pathogen risks in the farm environment. Lastly, since the quality of the water used in growing fresh produce is an important food safety priority, the effectiveness of chemical antimicrobial sanitizers against Salmonella in agricultural water was evaluated. Results demonstrated that certain sanitizer treatments and conditions (sanitizer concentrations, water temperatures) can significantly reduce Salmonella populations in pre-harvest water sources and may serve as a risk reduction option when sanitizers are used correctly.

The reduced capacity of antibiotics to treat infections is one of the greatest health concerns that society faces. There is substantial evidence that links this reduced capacity with the widespread use of antibiotics in livestock production. Livestock can act as reservoirs of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria, which can pass resistance on in the livestock's manure. It is important to understand the fate of antibiotic resistance genes and resistant bacteria in the environment after land-application of manure-based amendments. The goal of this field-scale study was to identify the effects of soil amendments (inorganic fertilizer, compost, or raw manure) and crop cover (lettuce or radish) on sediment transfer, fecal indicator bacteria (FIB), and release of ARGs in runoff over six storm events. Two FIB (Escherichia coli and enterococci) and two ARGs (sulI and ermB) were quantified in runoff from each of the constructed plots throughout the growing season. FIB and ARGs were recovered from all plots, including control plots indicating a background level within the soil. Additionally, only the effects of variability among individual storms had an impact on the concentration of FIB in runoff. Vegetative cover and storm variability affected sediment release. A trend of higher sul1 and ermB in runoff from compost and raw manure-amended plots for at least 2 months after planting crops was observed. Only one of these ARGs (ermB) is associated with the class of drugs given to the dairy cows used for the manure and compost, indicating inherent carriage of some ARGs independent of the type of antibiotic administered, and such genes can persist in the environment. These results suggest that there is a risk of ARGs being carried into areas downgradient from agricultural plots that have been amended with compost or manure. / MS

Bacterial water pollution is a significant problem because it is associated with reduction in the ‘quality’ of water systems with a potential impact on human health. Faecal indicator bacteria (FIB) are usually used to monitor the quality of water, and to indicate the presence of pathogens in water bodies. However, enumeration alone does not enable identification of the precise origin of these pathogens. This study aimed to monitor the quality of bathing water and associated fresh water in and out of the ‘bathing season’ in the UK, and to evaluate the use of microbial source tracking (MST) such as the host-specific based polymerase chain reaction (PCR) and quantitative PCR (qPCR) to recognize human and other animal sources of faecal pollution. The culture-dependent EU method of estimating FIB in water and sediment samples was performed on beach in the South Sands, Kingsbridge estuary, Devon, UK- a previously ‘problematic’ site. FIB were present at significant levels in the sediments, especially mud, as well as fresh water from the stream and pond flowing onto South Sands beach. However, the quality of bathing water was deemed to be ‘good’ and met with the EU bathing water directive 2006. Using MST it was possible to successfully classify the nature of the source from which the bacteria came. PCR was applied to detect the Bacteroides species 16S rRNA genetic markers from human sewage and animal faeces. All water and sediment samples displayed positive results with a general Bacteroides marker indicating the presence of Bacteroides species. Host-specific PCR showed the human Bacteroides genetic marker only in the sediment of the stream. However, limitations in the ‘types’ of probes available and in the persistence of these markers were identified. Thus, novel dog-specific Bacteroides conventional PCR and qPCR primer sets were developed to amplify a section of the 16S rRNA gene unique to the Bacteroides genetic marker from domestic dog faeces, and these were successfully used to quantify those markers in water samples at a ‘dog permitted’ and ‘dog banned’ beach (Bigbury-on-Sea, Devon, UK). Generic, human and dog Bacteroides PCR primer sets were also used to evaluate the persistence of Bacteroides genetic markers in controlled microcosms of water and sediment at differing salinities (< 0.5 and 34 psu) and temperature (10 and 17 ºC). The rates of decline were found did not differ significantly over 14 and 16 days for the water and sediment microcosms, respectively. Beach sediments which were studied in this project may act as a reservoir for adhesive FIB, and this was confirmed using fluorescence in situ hybridisation (FISH). The similarity in the persistence of these Bacteroides 16S rRNA genetic markers in environmental water and sediment suggests that viable but non-culturable (VBNC) Bacteroides spp. do not persist in the natural environment for long. Therefore, 16S rRNA genetic markers can be of value as additional faecal indicators of bathing water pollution and in source tracking. Thus, in this study MST methods were successfully used and in future applications, dog-specific primer sets can be added to the suite of host-specific Bacteroides genetic markers available to identify the source(s) of problem bacteria found on failing beaches.

<p>The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used.</p><p>The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum).</p><p>Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces.</p><p>In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.</p>