Mapping antimicrobial resistance landscape at a city scale sewage network

Wastewater is a valuable source for monitoring contaminants of biotic or abiotic origin. Antimicrobial resistance (AMR) has emerged as a public health threat that consists of the ability of microorganisms to resist the effects of antimicrobial compounds, rendering them very difficult or impossible to eradicate in case of infection. Considering the dissemination of antimicrobial resistance genes (ARGs) to a wide number of ecosystems, there is a need for the identification of hotspots that concentrate antimicrobial resistance determinants. A comprehensive investigation conducted at a city-scale in Sabadell (Barcelona, Spain) has integrated both phenotypic and genotypic methodologies, including metagenomics and culture-based techniques coupled with whole-genome sequencing (WGS), to monitor ARG presence in seven different spots of the sewage system. Metagenomics approach identified 262 ARG variants across analyzed sampling sites, grouped into 15 resistance categories. The most prevalent ARGs were macrolides-lincosamides-class B streptogramins (MLS B ) (35.1 %) and beta-lactams (28.7 %), including carbapenems (5.9 %) and cephalosporins (5.3 %). MLS B resistance featured dominant msr(E) and mph(E) genes, the most abundant ARGs in our study. ARGs conferring resistance to beta-lactam were dominated by blaOXA-464 , blaOXA-491 , and blaNPS . Key genes for carbapenem ( blaOXA-372, blaKPC-2 ) and cephalosporin ( blaOXA-10, blaOXA-1 ) resistance were identified. The hospital sector exhibited the highest relative abundance of ARGs, dominated by beta-lactams, MLS B , and aminoglycosides. Wastewater treatment plant (WWTP) entrance points and residential areas displayed similar ARG profiles, while WWTP effluent and industrial zones had the lowest ARG levels. WWTP significantly reduced ARG presence (93.3 %). The characterization of antibiotic-resistant bacterial isolates found that most abundant ARGs were predominantly plasmid-borne, favoring ARG spread across bacterial genera. This finding confirmed the significant role of plasmids in ARG dissemination, increasing both diversity and prevalence within waterborne bacterial communities. City-scale surveillance programs can play a pivotal role in guiding effective measures to reduce the dissemination of AMR and mitigate their environmental impact. • Hospital effluent presented the highest amount and diversity of ARGs and ARB. • Plasmids play a key role in ARG dissemination in wastewater. • blaOXA-1 , blaKPC-2 , blaTEM-1, msr(E) and erm(F) were proposed as AMR tracking makers. • On-site wastewater treatments can contribute to AMR mitigation strategies.