Grantee Research Project Results

Final Report: Understanding the role of wastewater treatment for mitigating antimicrobial resistance: leveraging historical trajectories, current day mass balances, and clinical relevance

EPA Grant Number: R840827
Title: Understanding the role of wastewater treatment for mitigating antimicrobial resistance: leveraging historical trajectories, current day mass balances, and clinical relevance
Investigators:
Institution:
EPA Project Officer:
Project Period: September 1, 2024 through April 23, 2025
Project Amount: $2,038,572
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:

The overarching project goal is to provide more complete estimates of the impact of wastewater treatment processes on the removal and/or selection of antimicrobial resistance (AMR). This research will provide an evaluation of the trajectory of change over a decade in antimicrobial resistance gene (ARG) concentration and load entering and being discharged from the municipal wastewater treatment process, the impacts of various wastewater treatment processes on ARG and antimicrobial resistant bacteria (ARB) removal or selection and subsequent loading to the environment, and an understanding of the fate and transport of resistance genes and bacteria in treated effluent discharge. Emphasis will be placed on quantifying ARG and ARB environmental loading and the clinical relevance of the resistance genes and bacteria that are ultimately discharged from the wastewater treatment process. In this way, the current risks from wastewater outflows may be put into perspective with other industrial and agricultural sectors that influence antimicrobial resistance dissemination from one environment to another.

Summary/Accomplishments (Outputs/Outcomes):

For this project, our findings were confined to preliminary methods development and procedural quality control and the collection of initial samples from a subset of proposed wastewater treatment plants and receiving waters. New sample collection was initiated in September 2024 on the Root River, which only recently received treated wastewater effluent. Four sampling trips were conducted to collect water for ARG quantification and associated water quality parameters. Including these new samples, we conducted 22 sampling trips for the project and collected approximately 120 samples upstream and downstream of the newly implemented treated wastewater discharge. These samples are archived and ready for ARG-related analyses.

We also initiated a preliminary sampling of wastewater treatment plants. Samples were collected on two occasions from four wastewater treatment plants as a pilot for our sampling and DNA extraction procedures. Sampling was conducted according to the plans outlined in the QAPP. Based on previous research among our groups, two primary DNA extraction methods were tested for filtered and pelleted water and sludge samples. Various combinations of
sample preparation methods were tested between the two base DNA extraction setups. We found the MPBio FastDNA Spin Kit yielded a combination of highest DNA yield and purity across sample types and was chosen as the primary method for all samples in this project.

Using preliminary wastewater treatment plant samples, we quantified the presence of potentially clinically-relevant and antibiotic-resistant bacteria by focusing on Aeromonas spp., Escherichia coli and Enterococcus spp.. Our preliminary data showed that among E. coli isolates, 5.10% (110 CFU/mL) had CTX-resistance in the influent to the treatment plant (a daily loading of 3.71 x 1013) and this resistance percentage was reduced to 0.99% (1 CFU/ml) after treatment but prior to chlorination. After the final disinfection stage using chlorine, the percentage of CTX-resistance in E. coli dropped further to 0.75% or 0.004 CFU/mL. This results in an average of load of 1.35 x 109 CTX-resistant E.coli discharged daily to natural waters. Overall, this shows the wastewater treatment train reduced CTX-resistant E. coli populations by 99.9964% of the incoming population.

The wastewater treatment train had a similar effect on Gram-positive vancomycin-resistant Enterococci. We found that 1.53% (33 CFU/mL) of the influent Enterococci were vancomycin-resistant (1.11 x 1013 CFU per day). The percentage of vancomycin-resistant Enterococci increased through the treatment train, rising to 5.06% of all Enterococci in the pre-chlorinated effluent, but the concentration decreased. In this preliminary dataset, we were not able to detect vancomycin-resistant Enterococci in the final post-chlorinated effluent (detection limit at 1 per 500 mL). For Aeromonas spp., 0.8% of the culturable Aeromonas spp. had CTX-resistance. Unlike the other genera, the percent of resistant Aeromonas increased through the treatment train, increasing to 5.9% in the pre-chlorinated water and to 8.6% in the post-chlorinated effluent. Additional screening of these isolates is needed to confirm their affiliation as Aeromonas spp. as there have been documented cases of non-Aeromonas growing on the selective media used here. As part of our research to determine the clinical context for antimicrobial resistance in wastewater isolates, we also obtained >100 isolates from local clinics. These bacteria were isolated from human infections and had resistance profiles of leading antimicrobial resistance threats. The genomes from three vancomycin-resistant Enterococci isolates were sequenced, assembled, and annotated. Two strains harbored plasmids with vancomycin resistance gene cassettes. Ultimately these data provide comparative genomes and gene targets to identify and quantify in wastewater samples so that the extent of connection between resistant bacteria from the clinic and those found in the wastewater treatment system can be quantified.

Supplemental Keywords:

antibiotics, ARGs, ARBs, wastewater-based epidemiology, activated sludge, anaerobic digestion, disinfection, ESBLs, E. coli, Enterobacterales, Acinetobacter