The growing crisis of antibiotic resistance is a major threat to ecosystems and human health. Infections caused by known and emerging antibiotic resistant pathogens are on the rise globally, with approximately 700,000 deaths per year caused by antibiotic resistant bacteria (1). In the United States, infections from antibiotic resistant bacteria cause more than 2 million illnesses and 23,000 deaths (2). Antibiotic resistant bacteria and antibiotic resistance genes are released into aquatic ecosystems through hospital waste, residential sewer lines and animal agricultural waste streams. Animal agriculture accounts for approximately 70% of antibiotic use in the United States (3). In agricultural ecosystems, runoff, land-applied fertilizer and waste lagoons can all contribute to the spread of antibiotic resistance. In urban ecosystems, sewage spills and other wastewater inputs contribute to the spread of antibiotic resistance. Environmental matrices, such as soil and water, can provide habitat, serving as reservoirs to potentially promote the spread of resistance. Research addressing antibiotic resistance primarily focuses on monitoring clinical occurrence and nosocomial infections (acquired in hospitals),but the natural environment also plays a role in the spread of antibiotic resistance. The consequences to aquatic ecosystems are not often studied and not well understood. Antibiotic resistance genes can transfer between bacteria through transduction, transformation and conjugation, potentially persisting in non-pathogenic environmental bacteria. Environmental reservoirs of antibiotics, antibiotic resistant bacteria and antibiotic resistance genes should be considered and integrated into frameworks to improve monitoring, regulation and management of urban and rural watersheds. The research presented in this doctoral dissertation includes field and laboratory studies designed to assess the prevalence and persistence of antibiotic resistant bacteria and antibiotic resistance genes in aquatic environments, with a focus on vancomycin-resistant enterococci, which are considered a major threat in the United States and top priority pathogens according to the Centers for Disease Control (2). The vanA gene associated with high-level resistance is located on mobile plasmids and associated with clinical infections, predominantly in the species Enterococcus faecium. E. faecium can cause bacteremia, endocarditis, pelvic infections and more (4). When vancomycin, often the last line of treatment for these infections, is no longer effective, the health burdens increase both financially and physically and infections can be fatal. Chapter 1 summarizes background and review of antibiotic resistance in the environment, including a co-authored review of culture-based methods to detect antibiotic resistant bacteria and antibiotic resistance genes in in the environment (previously published in the Journal of Environmental Quality (5). In Chapter 2, a field study was performed to investigate the occurrence and persistence of vancomycin-resistant enterococci and vanA in a sewage spill in Pinellas County, Florida, previously published in the journal Applied and Environmental Microbiology (6). In Chapter 3, antibiotic resistance genes were quantified to study their persistence in poultry litter microcosms (manuscript in prep). In Chapter 4, microcosms were used to assess how nutrients and plasmid-associated vancomycin resistance affect survival among E. faecium strains (in process of submitting for publication at Applied and Environmental Microbiology). Antibiotic resistance is a public health crisis and the results of the studies presented here contribute data towards a better understanding of environmental reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. The research has broad implications for public health, environmental policy and ecosystem management.

[pt] A matéria orgânica (MO) sedimentar de origem natural na Ria de Aveiro e no estuário Mondego foi caracterizada usando a composição elementar (C e N) e lipídios biomarcadores (esteróis, n-álcoois e triterpenoides). A contribuição por esgotos foi avaliada através de coprostanol e outros esteróis fecais. Amostras de sedimento superficial foram coletadas em 22 estações ao longo das regiões. Os lipídios biomarcadores foram extraídos, purificação com sílica-gel e analisados por cromatografia em fase gasosa acoplada a espectrometria de massas. O carbono orgânico total (9,94 e 43,00 mg g(-1)) teve uma correlação com o nitrogênio total (r(2) = 0,86). A razão C/N molar (14,34 mais ou menos 1,75) foi um valor típico de MO continental. Existe uma contribuição maior de MO terrestre nas amostras, como mostra as evidencias: a predominância de n-álcoois de cadeia longa (maior ou igual à C22), maior abundancia no somatório de sitosterol, estigmasterol e campesterol sobre outros fitoesterois, como o colesterol e o brassicasterol; e a alta concentração de triterpenoides de plantas superiores, como Beta-amirina e a Alfa amirina. A concentração de coprostanol (0,05 a 16,6 microgramas g(-1)) indicou a presença de esgotos em sete estações localizadas no largo do Laranjo (S4, S5, S6, S7, S10), porto de Aveiro (S14) e no canal de Ílhavo (S16). Os aportes mais significativos foram no largo do Laranjo (S8 e S9) e no porto do Mondego (S19). / [en] The sedimentary organic matter (OM) of natural origin in the Ria de Aveiro and the estuary of Mondego River was characterized using elemental composition (C and N) and lipid biomarkers (sterols, alcohols and triterpenoids). In addition, the contribution by sewage was evaluated by coprostanol and other fecal sterols. Surface sediment samples were collected at 22 stations along the two regions. The lipid biomarkers was extracted, separated with silica-gel and analyzed by gas chromatography coupled to mass spectrometry. Concentrations of total organic carbon (9.94 and 43.00 mg g(-1)) was highly correlated with total nitrogen (r(2) = 0.86). The C/N molar ratio: 14,34 plus-minus 1,75 are typical values of OM of continental origin. There is a major contribution of terrestrially OM to the sediments, as suggested by proxies, including: predominance of long-chain (greater-than or equal to C22) alcohols, higher abundance of sitosterol, estigmasterol and campesterol over other phytosterols, like cholesterol and brassicasterol, and elevated concentrations of triterpenoids from higher plants, as Beta-amyrin and Alpha-amyrin. The concentrations of coprostanol (0.05 to 16.6 micrograms g(-1)) indicated the presence of sewage in seven stations located in the Largo do Laranjo (S4, S5, S6, S7, S10), Port of Aveiro (S14) and in Ílhavo Channel (S16). The more significant contribution of sewage was identified in the Largo do Laranjo (S8 and S9) and the Port of Mondego (S19).