Intra‐City Assessment of Antibiotic Resistance across the Entire Sewer System of a Small Urban Area

ABSTRACT Emerging and spreading of antibiotic resistance continues to pose significant global health challenges, highlighting the need for rapid surveillance to reduce the dissemination of antibiotic resistant bacteria. In this context, the present intra‐city case study aimed to propose a monitoring approach for investigating the distribution of selected antibiotic resistance genes (ARGs) throughout a whole town's sewer system. The study focused on the quantification of azithromycin and ofloxacin and selected associated resistance genes, including erm B and qnr S, as well as class 1 integrons, using the integrase gene ( intI 1) as a proxy for the presence of ARGs. Quantifications were carried out using qPCR for genetic targets and ultra‐high‐performance liquid chromatography coupled with Orbitrap mass spectrometry for antibiotics. Samples were collected in March and July 2023 to assess seasonal variations in micropollutant presence. While concentrations of the antibiotics azithromycin and ofloxacin remained relatively stable (849.7 µg/L in March vs . 775.8 µg/L in July), significant temporal differences were observed in gene abundances. ARGs were more abundant in March, except i ntI 1, which was significantly higher in July than in March. Spatial variation was also detected, with erm B differing significantly between sampling sites on certain dates. These findings, particularly those related to erm B dinamycs, suggest that the proposed intra‐city monitoring approach is a valuable complement to conventional surveillance systems, offering a targeted and practical tool for tracking selected ARG dynamics and guiding public health interventions.