The role of wastewater treatment and water reclamation on antibiotic resistance in water and soil environments in the United States and Mexico
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The role of wastewater treatment and water reclamation on antibiotic resistance in water and soil environments in the United States and Mexico

  • Author(s): Whitener, Victoria Ann
  • Advisor(s): Jay, Jennifer
  • et al.
Abstract

While improvements in medical management of antibiotics has resulted in decreased of some antibiotic resistant infections and associated deaths in past years, some other antibiotic resistant infection rates are increasing. In order to slow the rate of spread of antibiotic resistant threats, scientists are now looking at the environment as a source and means of transport of resistance. Antibiotic resistant bacteria or genes are quantified in three different environmental compartments in this dissertation – water, animals, and soil. The effect of biological treatment in conventional municipal wastewater treatment plants on antibiotic resistance gene (ARG) levels is crucial to an understanding of environmental antibiotic resistance (AR). This is the first study to compare ARG abundances through two full-scale biological treatment processes in parallel, and one of few to address trickling filters (TFs). In this work, we studied the comparative effects of TFs and activated sludge (AS) bioreactors on the absolute and relative removal of ARGs. We quantified eight target genes via qPCR: tetA, tetW, sul1, sul2, blaSHV, vanA, intI1, and the 16S rRNA gene in the primary effluent (PE) (common to both treatment trains), TF effluent, and AS effluent. We evaluated the similarity of ARG removal to COD and TSS removals as potential indicators, but found that ARGs are more readily removed by TF or AS treatment than either COD or TSS. Absolute ARG abundances decreased by up to 1.7 and 2.8 logs after TF and AS treatments, respectively, and the difference between the two treatments’ removal rates ranged as high as 2.8. The changes in relative abundances of tetA and tetW decreased respectively up to 5.6�10−3 and 9.7�10−3 gene copies per 16S rRNA. Potential for conjugative HGT was evaluated via the linear association between ARG and intI1 relative abundances. The relative abundance of sul1 positively correlated (r ≥ 0.74, p ≤ 4.0�10−4) to intI1 in all sampling points, while sul2 did not (p > 0.05), corresponding to what is known about plasmid location. These findings are particularly relevant where secondary effluent is the final effluent to the environment. Our results show that while absolute abundances of all target genes decreased in secondary treatment, TF and AS bioreactors affect ARGs differently. The use of antibiotics for therapeutic and especially non-therapeutic purposes in livestock farms promotes the development of antibiotic resistance in previously susceptible bacteria through selective pressure. In this work, we examined E. coli isolates using the standard Kirby-Bauer disk diffusion susceptibility protocol and the CLSI standards. Companies selling retail chicken products in Los Angeles, California were grouped into three production groupings – Conventional, No Antibiotics, and Humane Family Owned. Humane Family Owned is not a federally regulated category in the United States, but shows the reader that the chicken is incubated, hatched, raised, slaughtered, and packaged by one party, ensuring that the use of antibiotics in the entire production of the chicken is known and understood. We then examined the antibiotic resistance of the E. coli isolates (n = 325) by exposing them to seven common antibiotics, and resistance was seen to two of the antibiotics, ampicillin and erythromycin. As has been shown previously, it was found that for both ampicillin and erythromycin, there was no significant difference (p > 0.05) between Conventional and USDA (United States Department of Agriculture)-certified No Antibiotics chicken. Unique to this work, we additionally found that Humane Family Owned chicken had fewer (p ≤ 0.05) antibiotic-resistant E. coli isolates than both of the previous. Although not considered directly clinically relevant, we chose to test erythromycin because of its ecological significance to the environmental antibiotic resistome, which is not generally done. To our knowledge, Humane Family Owned consumer chicken has not previously been studied for its antibiotic resistance. This work contributes to a better understanding of a potential strategy of chicken production for the overall benefit of human health, giving evidentiary support to the One Health approach implemented by the World Health Organization. While increased use of reclaimed water is a solution to water-scarce regions, it is necessary to carefully consider the long-term effects of applying reclaimed water to agricultural soil. In the Maneadero Valley, Mexico, tertiary effluent from El Naranjo Wastewater Treatment Plant is used for the irrigation of flowers and livestock crops. In the past, groundwater was used, but over extraction of the Maneadero aquifer resulted in severe seawater intrusion to the region. In this work, we evaluate antibiotic resistance genes (ARGs) through qPCR as well as the viability method, PMA-qPCR (propidium monoazide). Genes were evaluated in the reclaimed water and at different farms where reclaimed water has been used for either 6, 4, 2, 0 (never) years. The investigation aims to better understand any potential public health impact of the 1) the use of the reclaimed water, 2) the effect of the distribution system, and 3) the effect of reclaimed water holding tanks. Additionally, the effect that the use of reclaimed water has on the soil environment with increasing times is evaluated. Because the use of the reclaimed water is a solution to the water scarcity of the area, we use the PMA-qPCR assay to better characterize any public health effect that the ARGs could create. While it is widely acknowledged that shifts in diet could play a large role in mitigating climate change with important health co-benefits, knowledge on how to accomplish these shifts is lacking. Our previous study showed a statistically significant reduction in the dietary carbon footprint of students who had completed a college course on the connections between food and the environment compared to a control group enrolled in an unrelated course. An extension of the previous study, this research evaluates the sustainability of female and male diets in both the intervention and control groups from baseline to follow up with respect to the following planetary boundaries: greenhouse gases, land use, water use, nitrogen loss, and phosphorus use. Student dietary footprints are compared to a per capita limit allowable for food according to the planetary boundaries concept. In addition, a 50-point Healthy Eating Index was calculated at baseline and follow up for all students. Female students enrolled in the intervention course reported diets with statistically significant reductions in their footprints from baseline to follow up for greenhouse gases (p=0.011), land use (p=0.012), and phosphorus (p=0.045), and the female diets were statistically different from the control groups for those three boundaries. For water use, female diets increased in footprint from baseline to follow up due to an increase in vegetable intake. Males enrolled in the intervention showed similar trends (reductions in footprints for greenhouse gases, land use, and phosphorus use and an increase in blue water use), but differences were not statistically significant, partially due to the smaller number of male respondents. For all of the planetary boundaries except blue water use, the student dietary footprints were well above the per capita boundary for food-related sources.

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