Grantee Research Project Results

Final Report: Quantifying Wastewater Sources of Antibiotic Resistance to Aquatic and Soil Environments and Associated Human Health Risks

EPA Grant Number: R840828
Title: Quantifying Wastewater Sources of Antibiotic Resistance to Aquatic and Soil Environments and Associated Human Health Risks
Investigators:
Institution:
EPA Project Officer:
Project Period: August 1, 2024 through April 24, 2025
Project Amount: $2,374,575
RFA: National Priorities: Evaluation of Antimicrobial Resistance in Wastewater and Sewage Sludge Treatment and Its Impact on the Environment Request for Applications (RFA) (2023) RFA Text |  Recipients Lists
Research Category: Human Health , Water , Water Treatment

Objective:

Antimicrobial resistance (AMR) refers to the ability of microbes to survive treatment with antimicrobials, such as antibiotics, resulting in deadly infections that threaten human health globally. Sewage contains antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) that are excreted and washed down the drain or toilet in wastewater, some of which may persist, be amplified, or attenuated through wastewater treatment.

The goal of this research was to support the development of comprehensive strategies to mitigate the spread of AMR and potential impact to human health. The objectives of this project were to quantify outputs of ARB/ARGs from wastewater treatment plants (WWTPs) into the environment and to characterize their amplification or attenuation in aquatic and soil environments and potential impact to human health.

The investigative team responded to EPA-G2023-ORD-F1: National Priorities and proposed a comprehensive study of ARB/ARGs in WWTP effluent and biosolids, with the aim of quantifying and modeling their fate in aquatic and soil environments. The main objective was to characterize exposure potentials and create a comprehensive risk assessment modeling framework that was tailored to the unique aspects of AMR and could support evaluation of mitigation options. Such mitigation options could include:

• Improving or adjusting the design of the WWTP process.
• Altering the manner in which WWTP effluents are disinfected.
• Optimizing biosolids treatment processes prior to land application.
• Improving advisories for recreational water use to account for the risk of acquiring an antibiotic-resistant infection.

The research team built a network of representative sampling of rivers and streams across the United States.  The team was led by The Water Research Foundation, Virginia Tech, ASU, West Virginia University, and the University of South Florida, with subawards to Emory University, University at Buffalo, East Tennessee State University, and InterAmerican University of Puerto Rico. A partnership was established with over 20 water utilities and public health departments across the United States to provide access to sampling locations and data sharing to support the research goals. Additionally, the team collectively secured 33% in non-federal cost share to support this research with individual funding and in-kind services, a proportion exceeding the required 25%.

This proposal was selected for funding through the EPA merit review process, which included an internal review of relevancy and responsiveness to the request for proposals, peer-review by an external panel of experts in the field, and verification of the quality of the past performance of our team members. The five specific objectives of this research were as follows:

• Objective 1. Determine ARB/ARG mass loading ranges in WWTP effluents and biosolids as a function of WWTP characteristics.
• Objective 2. Estimate the degree to which WWTP effluent and biosolid-borne ARB/ARGs attenuate or amplify in the environment and identify controlling factors.
• Objective 3. Evaluate evidence of WWTPs as a source of AMR infections in humans, relative to other sources, across a range of U.S. communities that reflect a spectrum of wastewater management scenarios.
• Objective 4. Develop a human health risk assessment for WWTP effluent and biosolid sources of ARB/ARGs based on the knowledge gaps addressed through Objectives 1–3.
• Objective 5. Engage with stakeholders and translate the research into actionable solutions.

The original proposed project was designed and funded as a three-year project, commencing August 1, 2024. However, the research team received a stop-work order on April 24, 2025, about 9 months into the project, and therefore were not able to fully realize the goals that were originally proposed. Nonetheless, the team made noteworthy progress, which is detailed here in this final report

Summary/Accomplishments (Outputs/Outcomes):

Objective 1. Determine ARB/ARG mass loading ranges in WWTP effluents and biosolids as a function of WWTP characteristics.

• The team compiled in-house and publicly available data across multiple databases covering over 100 WWTPs around the world to provide a resource for investigating ARB and ARG occurrence patterns associated with wastewater treatment and discharge. Although the team could not complete the compilation due to the termination, this will still be a valuable resource, contributing to national and global efforts to understand typical baseline occurrence patterns and levels of ARB and ARGs in rivers and streams, and the extent to which WWTP discharge alters or augments them.
• A preliminary analysis was conducted using this database to map occurrences of ARB and ARGs across the United States and find hot spots where these AMR markers are elevated.

Objective 2. Estimate the degree to which WWTP effluent and biosolid-borne ARB/ARGs attenuate or amplify in the environment and identify controlling factors.

• The team developed standard operating procedures (SOPs) for sampling WWTP effluent and affected upstream and downstream aquatic samples. These protocols will be made publicly available to encourage consistent standards for aquatic AMR testing, enabling data comparison across systems and studies.
• A protocol for monitoring antibiotic-resistant Escherichia coli in aquatic environments was updated and was tested across multiple aquatic sites. The team also demonstrated the protocol’s efficacy for soil testing. This protocol adapts the World Health Organization Tricycle Protocol to conform with EPA standard methods for E. coli.
• An SOP was developed for monitoring antibiotic-resistant Enterococcus spp. in aquatic environments and tested across multiple aquatic sites. This protocol adapts existing EPA standard methods for testing AMR Enterococcus spp.
• An SOP was developed for monitoring antibiotic-resistant Pseudomonas aeruginosa in aquatic environments and tested across multiple wastewater and surface water samples. This SOP is particularly noteworthy given that current methods for P. aeruginosa enumeration from water samples are lacking, and there are no existing standard methods.
• A pilot data was gathered on the occurrence of AMR E. coli, Enterococcus spp. and P. aeruginosa at multiple sites across five states (FL, VA, WV, TN, GA), including WWTP effluent and upstream and downstream samples.
• A refined protocol for metagenomic profiling of WWTP effluents and affected aquatic environments was completed. The collected DNA samples from the pilot sampling were stored for future analysis, if resources are available.
• The team estimated attenuation rates of ARB/ARGs when biosolids are applied to soil, derived from a preliminary mesocosm study.

Objective 3. Evaluate evidence of WWTPs as a source of AMR infections in humans, relative to other sources, across a range of U.S. communities that reflect a spectrum of wastewater management scenarios.

• This objective entailed sequencing the genomic DNA of AMR bacterial isolates from Objective 2 and comparing it with clinical isolates. It was not possible to complete the objective due to premature termination of funding.

Objective 4. Develop a human health risk assessment for WWTP effluent and biosolid sources of ARB/ARGs based on the knowledge gaps addressed through Objectives 1–3.

• This objective was designed to identify scenarios where AMR spread is most likely and will inform estimates of rates of amplification/attenuation of ARB/ARGs in affected soil and aquatic environments to improve exposure estimates. The team made progress on a model for dose-response of Extended-spectrum beta-lactamases-ESBL E. coli, developed a semi-quantitative relative ranking system, and began factoring horizontal gene transfer into models. Due to the termination, progress on this objective was limited. An AMR-tailored human health risk assessment framework is urgently needed to inform and prioritize regulatory and management options for mitigating wastewater sources of AMR.

Objective 5. Engage with stakeholders and translate the research into actionable solutions.

• The investigative team consisted of individuals from over 10 universities, institutions, and consulting organizations, bringing together distinct expertise to execute this important, multi-faceted project. The team engaged with a range of academics, water utilities, environmental agencies, public health departments, hospitals, and other stakeholders to develop the sampling plan and to execute the pilot sampling. A multi-layer program and project advisory committee of independent experts and stakeholders were established to evaluate research progress. Collectively, the research team hopes that there are future avenues for this team and partnerships to collaborate so that efforts can continue to provide a forum for translating the research into mitigation approaches that results in sustainable solutions to help stem the spread of AMR.
• The team made limited progress on Objective 5 due to the premature termination of funding.
• In summary, while the research team was not able to fully achieve the originally proposed goals due to the termination, the team produced several deliverables that will be of value towards supporting national and global efforts to stem the spread of AMR and maintain the efficacy of antimicrobials to fight deadly diseases for future generations. These can be built upon in future national and global research efforts, supporting harmonized and comparable data collection.

Supplemental Keywords:

Water Reuse, Risk Assessment, Bacteria, Treatment 

Relevant Websites:

YouTube: How to Sample Wastewater Effluent, Surface Water, and Sediment for Microbiological Analysis Exit

YouTube: How to Filter Water for Metagenomic Analysis Exit

YouTube: How to Filter Water for Culture Analysis Exit