京都大学 / 新制・課程博士 / 博士(理学) / 甲第24184号 / 理博第4875号 / 新制||理||1698(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 半谷 吾郎, 准教授 Huffman Michael Alan, 教授 今井 啓雄 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

<p> Large high density populations, up to 3,000/m^2 of the bivalve Macoma balthica inhabit the muddy intertidal areas in the Bay of Fundy, Nova Scotia. The fecal pellets produced by Macoma b. are resistant to breakdown. Off Spencer's Point, the pellets are transported via small intertidal channels away from the shore onto a sand bar, at low tide. At high tide the channels act as traps for fecal pellets. The pellets breakdown over the sand bar at high tide, but a large proportion of the pellet remains in suspension as a mucous bound mud agglomerate. In an area of 2000 Macoma b./m^2, the rate of fecal pellet production is approximately 1 Kg dry wt/m^2/yr. </p> <p> The rate of pseudofecal production increases with water turbidity. The rate of sediment reprocessing is approximately 9 Kg dry wt. of sediment/m^2/yr., or a layer of wet sediment approximately 3.3 cm. deep. </p> <p> The Macoma b. in Cobequid Bay are largely deposit feeding. In areas of high population density and coarser sediment, this depletes the surface of sediment mud and fine sand. The influx of sediment necessary to sustain such a population is, in part, pseudofecal material from Macoma b. populations higher on the mud flats. </p> / Thesis / Bachelor of Arts (BA)

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Sources of fecal pollution were evaluated as a function of sampling frequency with stream samples from Mill Creek, Montgomery County, VA. Samples were collected monthly for one year, plus weekly for four consecutive weeks during seasonal high flows (March), and seasonal low flows (September-October), plus daily for seven consecutive days within the weekly schedules. Thirty stream samples were collected from each of two sites (60 total) in Mill Creek, and 48 isolates of E. coli per sample (total of 2,880 stream isolates) were classified by source using antibiotic resistance analysis (ARA) and comparing the resulting patterns against a known-source E. coli library (1,158 isolates). The same process was performed with enterococci isolates against an enterococci library (1,182 isolates). The average rate of correct classification (ARCC) for the E. coli library with a three-way split (human, livestock, and wildlife) was 89.0%, and the ARCC of the species-specific E. coli library (cattle, deer, goose, human, misc. wildlife) was 88.9%. The ARCC of the enterococci library with a three-way split was 85.3%, and the ARCC of the species-specific enterococci library was 88.1%. The results did not justify the need for daily or weekly sampling, but indicated that monthly was adequate (quarterly and every-other-month were not). There was a seasonal effect as the human signature was highest during high flow while the livestock signature dominated during low flow. The results also indicated that sampling should be done over a period of time that includes both seasonal wettest and driest periods (at least 8 months). / Ph. D.

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.

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

Anthropozoonotic diseases, defined as infectious diseases caused by pathogens transmitted from humans to wildlife, pose a significant health threat to wildlife populations. Many of these pathogens are also able to move from wildlife reservoirs to humans, termed zoonotic diseases, creating the possibility for bi-directional transmission between humans and wildlife. Recent studies show that a significant proportion of emerging infectious diseases in humans originate in wildlife reservoirs and that the frequency of emergence is increasing, yet the specific transmission pathways still remain speculative in most cases. Human fecal waste is persistent across human-modified landscapes and has been identified as a potential source of disease exposure for wildlife populations living near humans. As part of a long-term study of banded mongoose (Mungos mungo) that live in close association with humans and human fecal waste I used Escherichia coli and banded mongoose (Mungos mungo) for evaluating exchange of fecal waste-borne microorganisms at the human-wildlife interface. Antibiotic resistance was found in 57.5% ° 10.3% (n=87) of mongoose fecal samples and 37.2% ° 5.9% of isolates (n=253). Multidrug resistance was detected in 13.8% ° 4.2% of isolates (n=253). Mongoose and human fecal waste isolates consistently clustered together in phylogenetic analyses and statistical analysis of genetic variation showed no significant differences (p=0.18) between E. coli from human and mongoose populations. These results suggest that human fecal waste contamination is an important mechanism for the transmission of pathogens to both humans and animals, including the spread of antibiotic resistance in the environment, an emerging global health threat. / Master of Science

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