Increased antimicrobial and multidrug resistance downstream of wastewater treatment plants in an urbanizing watershed

Authors: MUKHERJEE, MAITREYEE - University Of Southern Mississippi; LAIRD, EDWARD - Texas A&M University; GENTRY, TERRY - Texas A&M University; Brooks, John; KARTHIKEYAN, RAGHUPATHY - Texas A&M University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2021
Publication Date: 5/24/2021
Citation: Mukherjee, M., Laird, E., Gentry, T.J., Brooks, J.P., Karthikeyan, R. 2021. Increased antimicrobial and multidrug resistance downstream of wastewater treatment plants in an urbanizing watershed. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2021.657353.
DOI: https://doi.org/10.3389/fmicb.2021.657353

Interpretive Summary: Overall, water quality in sub urban waterways, such as rivers or tributaries, can be compromised by wastewater discharge. While the discharge is cleaned to an approved standard, those standards do not include treatment for antibiotic resistant bacteria. This constitutes one facet of a much larger global health concern. In the current study, we collected surface water samples from locations in waterways upstream of wastewater treatment plants in an urbanizing watershed in Bryan-College Station, Texas. Escherichia coli (E. coli) was collected from these waters along with general bacteria and were tested against antibiotics to determine the antibiotic effect against the bacteria. Additionally, DNA was collected from these waters and tested for antibiotic resistance DNA. Overall, numbers were greater in downstream water sources, which was expected. Likewise, antibiotic resistant general bacteria and the majority of tested antibiotic resistance DNA were also greater in downstream waters. Some DNA genes associated with antibiotic resistance were also located at higher numbers downstream of the wastewater treatment plants. This suggests that wastewater treatment plants can significantly increase the level of antibiotic resistant bacteria in surface water.

Technical Abstract: Development and spread of antimicrobial resistance (AMR) and multidrug resistance (MDR) through propagation of antibiotic resistant genes (ARG) in various environments is a global emerging public health concern. The role of wastewater treatment plants (WWTPs) as hot spots for the dissemination of AMR and MDR has been widely pointed out by the scientific community. In this study, we collected surface water samples from sites upstream and downstream of two WWTP discharge points in an urbanizing watershed in the Bryan-College Station (BCS), Texas, over a period of nine months. E. coli isolates were tested for resistance to ampicillin, tetracycline, sulfamethoxazole, ciprofloxacin, cephalothin, cefoperazone, gentamycin, and imipenem using the Kirby-Bauer disc diffusion method. Heterotrophic Plate Counts (HPCs) were enumerated on R2A amended with ampicillin, ciprofloxacin, tetracycline, and sulfamethoxazole. In addition, quantitative real-time polymerase chain reaction (qPCR) method was used to measure eight ARG – tetA, tetW, aacA, ampC, mecA, ermA, blaTEM, and intI in the surface water collected at each time point. Significant associations (p < 0.05) were observed between the locations of sampling sites relative to WWTP discharge points and the rate of E. coli isolate resistance to tetracycline, ampicillin, cefoperazone, ciprofloxacin, and sulfamethoxazole together with an increased rate of isolate MDR. The abundance of antibiotic-resistant heterotrophs was significantly greater (p < 0.05) downstream of WWTPs for all tested antibiotics and the concentrations of all ARG were substantially higher in the downstream sites compared to the upstream sites, particularly in the site immediately downstream of the WWTP effluent discharges (except mecA). In addition, the Class I integron (intI) genes were detected in high amounts at all sites and all sampling points, and were about 20 times higher in the downstream sites (2.5 × 107 copies/ 100 mL surface water) compared to the upstream sites (1.2 × 106 copies/ 100 mL surface water). Results suggest that the treated WWTP effluent discharges into surface waters are potentially significant contributors to the spread and persistence of AMR in the surrounding watershed. In addition to detecting increased ARG in the downstream sites by qPCR, findings from this study also report an increase in viable AMR (HPC) and MDR (E. coli) in these sites. This data will benefit establishment of improved environmental regulations and practices to help manage AMR/ MDR and ARG discharges into the environment.