Introduction

Wastewater-based epidemiology (WBE) relies on the assumption that antibiotics and antimicrobial resistance (AMR) markers remain stable during sewer transport, allowing wastewater measurements to reflect community-level antibiotic consumption and resistance trends. However, evidence suggests that in-sewer processes, largely linked to biofilm activity, can cause antibiotic degradation and may affect the distribution of antimicrobial resistance genes (ARGs). This creates uncertainty as to whether wastewater signals represent population-level trends or whether they are influenced by in-sewer changes. To address this, we investigated the stability and behaviour of 11 antibiotics and 54 ARGs in laboratory-scale sewer pipes (operated for > 12 months prior to sampling), using influent wastewater from the North-East of Scotland. Clarithromycin and its associated resistance gene ermB_1 were examined in detail.

Methods

Four pipes (clay and plastic, with and without biofilms) were operated in triplicate. Wastewater was sampled over 24 hours without disturbing biofilms. Antibiotics were quantified using direct injection UPLC-MS/MS, while ARG behaviour was assessed using SmartChip high-throughput qPCR.

Results

Biofilm pipes showed greater antibiotic degradation than biofilm-free controls, with clarithromycin degradation reaching 59–65% over 24 hours compared to 20–23% in controls, confirming a biofilm-driven effect on degradation. This suggests that estimation of community antibiotic usage using WBE may require corrections for in-sewer losses. Despite this, the abundance of ermB_1 did not increase in the wastewater. Instead, biofilm systems exhibited equal or lower ermB_1 levels than biofilm-free pipes, with plastic biofilm pipes showing both the highest antibiotic degradation and the lowest ARG signal. This indicates that tracing ARG abundance using WBE may be better suited to assessing long-term trends rather than short-term changes.

Conclusions

These findings demonstrate that sewer biofilms influence antibiotic stability without necessarily promoting increased ARG abundance in wastewater. This supports more robust interpretation of WBE data across multiple antibiotics and resistance genes, improving confidence in wastewater-based AMR surveillance.