Abstract
BACKGROUND: Bacterial antiphage defense systems play essential roles in microbial ecology, yet their dynamics within urban wastewater systems (UWS) remain poorly characterized. RESULTS: In this study, we performed comprehensive metagenomic and plasmidome analyses on 78 wastewater samples collected during two seasons and four sampling points across UWS from three European countries. We observed a significant reduction in the abundance, diversity, and mobility potential of defense systems during biological treatment. However, these reductions were not directly correlated with changes in microbial abundance. Defense systems were significantly enriched on plasmids, particularly conjugative plasmids, where their gene density was approximately twice as high as on chromosomes and remained relatively stable across compartments. In contrast to chromosomal defense systems, plasmid-borne systems exhibited more frequent co-localization with a wide range of mobile genetic elements (MGEs)-associated genes, thereby facilitating multilayered dissemination networks. Furthermore, we detected a strong correlation between phage abundance and host defense system profiles, indicating ongoing phage-host co-evolutionary dynamics in these environments. CONCLUSIONS: In summary, our results demonstrate that UWS reduce the abundance and diversity of bacterial defense system genes. However, plasmid-associated defense systems can persist through shared mobile genetic reservoirs. These findings underscore the critical role of plasmids in bacterial immunity and provide new insights into defense system dynamics within urban wastewater environments.