Principal Investigator: LaPara, Timothy Institution: University of Minnesota-Twin Cities Proposal No: CBET-0967176

Antibiotics have profoundly impacted medical practice, as these drugs can successfully eradicate most bacterial infections. While the medical community now recognizes the growing problem of antibiotic resistance, effective solutions are currently lacking and there is an urgent need to find new approaches to help solve the problem of broadly disseminated antibiotic resistant bacteria. This research proposal focuses on the importance of municipal wastewater and municipal wastewater treatment on the proliferation of antibiotic resistance. Even though municipal wastewater has been clearly and repeatedly shown to contain antibiotic resistant bacteria, the scientific community has generally failed to recognize municipal wastewater as a principal reservoir of antibiotic resistance. There are two primary routes by which antibiotic resistant bacteria can leave a municipal wastewater treatment facility: (1) with the treated effluent, and (2) with the residual wastewater solids. Of these two routes, residual wastewater solids are much more likely to be important for the spread of resistance ? and thus the primary focus of this proposal. The proposed research will provide seminal knowledge, allowing design engineers to select technologies that will optimize pathogen inactivation, the elimination of antibiotic resistant bacteria, the reduction of antibiotic resistance genes, and the stabilization of wastewater solids.

The proposed research will test two hypotheses concerning the ecology of antibiotic resistance: (1) municipal wastewater treatment facilities can be intentionally designed to inactivate antibiotic resistant bacteria and to eliminate the genetic determinants that encode resistance, and (2) municipal wastewater treatment facilities are a source of antibiotic resistant bacteria because the conditions intentionally designed by environmental engineers to treat municipal wastewater are simultaneously (albeit unintentionally) conducive for horizontal gene transfer. The first hypothesis will be tested by quantifying the reductions of genes encoding antibiotic resistance in anaerobic digestors and other unit operations used to treat residual wastewater solids (Objective 1) and by determining the fate of genes encoding antibiotic resistance when treated wastewater solids are applied to agricultural soils (Objective 2). The second hypothesis will be tested by elucidating the rates of horizontal gene transfer and characterizing the genes that are horizontally transferred within the aeration tanks and anaerobic digestors of several municipal wastewater treatment facilities (Objective 3).

The proposed project will have broad impacts that extend beyond the scientific research. The most pertinent of these broader impacts will focus on developing future scientists and engineers whose professional efforts will benefit society. The proposed project will directly support the education of a doctoral student at the University of Minnesota. In addition, the principal investigators will leverage the proposed project to augment the educational experience of two formal lecture/laboratory courses offered by the University of Minnesota. Similarly, the principal investigators will leverage the proposed project to mentor undergraduate students that will be financially supported the Research Experience for Undergraduates (REU) program at the University of Minnesota. These REU programs have been particularly successful at recruiting students from underrepresented groups to the University of Minnesota, thus likely providing another broader impact as defined by the National Science Foundation. Finally, the results of the proposed research will be broadly disseminated to the peer-reviewed technical literature, various agencies who operate municipal wastewater treatment facilities throughout the state of Minnesota, and to Minnesota State legislators.

Project Report

Bacterial resistance to antibiotic chemotherapy is a growing problem. Recent publications from the Centers for Disease Control and Prevention suggest that antibiotic resistance leads to tens of thousands of preventable deaths each year in addition to $20-$40 billion in extra medical costs. Although the problem of antibiotic resistance has historically been viewed as a strictly medical problem, emerging research suggests that the environment plays a key role in the spread of antibiotic resistant bacteria and antibiotic resistance genes (ARGs). Specifically, untreated municipal wastewater is believed to be a very important reservoir of ARGs. The goal of this study was to investigate the current and potential role of applying municipal wastewater treatment processes to mitigate the spread of antibiotic resistance. The first phase of this research was to investigate the importance of treated municipal wastewater on the levels of ARGs in surface waters. To achieve this goal, ARGs were tracked in the St. Louis River, Duluth-Superior Harbor, Lake Superior, and the treated wastewater discharged by the Western Lake Superior Sanitary District. This result showed that ARGs were highest in the wastewater discharge, detectable within the harbor, but very low in the St. Louis River and in Lake Superior. This research demonstrated that the wastewater treatment discharge was the primary source of ARGs in Duluth-Superior Harbor. A similar study was performed on the Upper Mississippi River basin, examining the quantities of ARGs from 14 different wastewater treatment discharges and 12 different locations in the Mississippi River. In this study, ARGs were again very high in the treated wastewater, but low throughout a 500+ mile stretch of the Mississippi River; mathematical modeling suggests ARG levels in the Mississippi River remain unaffected because the flow rate of the river is much higher than the combined flow rates of all of the wastewater discharges. The second phase of the research was to investigate the fate of ARGs in several technologies used to treat municipal wastewater solids (a.k.a. sewage sludge) -- these experiments were performed in laboratory-scale systems that simulated full-scale operations. In general, the technologies were capable of removing ARGs from wastewater solids, although the decay rates varied substantially. The half-lives for most ARGs treated by aerobic digestion, air drying and conventional anaerobic digestion were typically 1-2 weeks; in contrast, the half-lives for most ARGs treated by thermophilic anaerobic digestion were typically less than one day. The third phase of the research was to investigate the fate of ARGs in soils following the application of treated wastewater solids. In these experiments, wastewater solids were obtained from full-scale treatment processes or treated by various laboratory-scale treatment processes. Using wastewater solids from a full-scale treatment process, the half-lives of ARGs were typically 1-2 months. Substantial differences, however, were observed such that more aggressive treatment processes (lime stabilization, pasteurization, thermophilic anaerobic digestion) resulted in scenarios in which ARGs decayed more rapidly and to a greater extent than conventionally treated wastewater solids. This research demonstrated that untreated municipal wastewater is a signifincant and important reservoir of antibiotic resistance. Current wastewater treatment technology is somewhat effective at mitigating the spread of ARGs, but treatment efficiency could be improved substantially by incorporating more aggressive technologies for the treatment of wastewater solids. This research also had substantial broader impacts, including supporting (partially or fully) two PhD students and one master's student. The research was integrated with teaching; one of the research publications from this project was performed as part of a graduate level course at the University of Minnesota.

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University of Minnesota Twin Cities
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