Impact of Wastewater Effluent on Surface Water Resistomes in a Small Lower-Income Community with Deteriorated Sewage Infrastructure

Abstract

Antimicrobials are known for their lifesaving benefits, but their widespread use has increased selective pressure favoring the development of resistant bacteria and antibiotic resistance genes (ARGs). Consequently, this has led to a global antimicrobial resistance (AMR) crisis associated with an estimated 4.71 million deaths globally in 2021. Due to inputs of antimicrobials and antibiotics excreted and flushed down the drain, nutrient rich conditions, and high active microbial density, wastewater treatment plants (WWTPs) are hypothetical hotspots for the evolution and dissemination of AMR, but also function as a crucial checkpoint for restricting discharge of resistant bacteria, ARGs, and other pollutants into the environment. In this study, we evaluated the levels of ARGs and antibiotic-resistant bacteria in wastewater influent, wastewater effluent, and surface water samples collected upstream and downstream of a deteriorating WWTP serving a small, lower-income, community in a rural region of Virginia. Samples were collected monthly over a 12-month period. Multiple AMR indicators were assessed by bacterial culture, droplet digital polymerase chain reaction (ddPCR), and DNA-sequencing. A significant difference was observed in the relative abundance of total ARGs between upstream and effluent samples (p =1.9x10^(-5) ), whereas no significant differences were observed between effluent and downstream (p=0.16 or upstream and downstream (p= 0.21). Across all sampling dates, the WWTP achieved a 56% reduction in relative abundance of total ARGs. Analysis of unique ARGs by sampling site revealed that, despite an overall reduction in distinct ARG types` across the treatment process, the downstream site still harbored substantially more unique ARGs (n=402) compared to the upstream site (n=106). Furthermore, most of these downstream-specific ARGs (n=272) were also detected in the effluent. Positive trends between precipitation and ARG abundance in effluent and downstream river samples were observed, though none were statistically significant. This suggests that while precipitation may play a role in ARG composition, its influence varies across different water matrices and other environmental factors. ddPCR enumeration of blaCTXm, an ARG that encodes resistance to a broad spectrum of beta lactam antibiotics and is of key clinical concern, displayed the same trend as the total ARG relative abundance. Specifically, blaCTXm, was significantly higher downstream than upstream of the WWTP. Culture-based enumeration of cefotaxime-resistant Escherichia coli confirmed occasional elevated levels in the WWTP effluent discharged to the river, though there were also upstream sources. Whole genome sequencing of resistant Escherichia coli and carbapenem-resistant bacteria provided a means to assess the range of strains encountered and can be compared with putative sources and clinical data in future studies. These findings provide valuable insights into the role of a deteriorating WWTP in the dissemination of AMR within rural, low-income community. This study underscores the importance of maintaining the integrity of wastewater infrastructure for protecting water quality and public health.