To investigate temporal trends of ARGs, four wastewater samples per month from Bath were chosen to investigate selected ARGs using digital PCR (dPCR). Of these samples, two were selected to further investigate using DNA sequencing (one wastewater sample from November 2018 and one sample from March 2019). Influent wastewater samples (100 mL) were filtered through Nalgene™ Sterile Analytical Filter Units (Thermo Scientific, UK) containing 0.2 μm cellulose filter papers. DNA was then extracted from the membrane directly from the filter paper using FastDNA SPIN Kit for Soil (MP Bio, UK). The amount of extracted DNA was determined using a Qubit 4 Fluorometer (Thermo Scientific, UK). DNA was kept at -20 °C before further analysis.