Time-series sewage metagenomics can separate the seasonal, human-derived and environmental microbial communities, holding promise for source-attributed surveillance

Sewage metagenomics has risen to prominence in urban population surveillance of pathogens and antimicrobial resistance (AMR). Unknown species with similarity to known genomes cause database bias in reference-based metagenomics. To improve surveillance, we designed this study to recover sewage genomes and develop a quantification and correlation workflow for these genomes and AMR over time. We used longitudinal sewage sampling in seven treatment plants from five major European cities to explore the utility of catch-all sequencing of these population-level samples. Using metagenomic assembly methods, we recovered 2,332 metagenome-assembled genomes (MAGs) from prokaryotic species, 1,334 of which were previously undescribed. These genomes account for ~69% of sequenced DNA and provide insight into sewage microbial dynamics. Rotterdam (Netherlands) and Copenhagen (Denmark) showed strong seasonal microbial community shifts, while Bologna, Rome, (Italy) and Budapest (Hungary) had occasional blooms of Pseudomonas-dominated communities, accounting for up to ~95% of sample DNA. Seasonal shifts and blooms present challenges for effective sewage surveillance. We find that bacteria of known shared origin, like human gut microbiota, form communities, suggesting the potential for source-attributing novel species and their ARGs through network community analysis. This could significantly improve AMR tracking in urban environments.