Urban green roofs host intrinsic resistomes shaped by management but not dominated by pathogenic resistance

Urban green roofs are increasingly introduced to enhance urban biodiversity and ecosystem services, yet their role in shaping antimicrobial resistance in cities remains unclear. Using long-read metagenomic sequencing, we characterized antimicrobial resistance genes (ARGs) across an experimental extensive green roof system with plots under four different management regimes specifically designed to test the influence of vegetation and organic amendments, as green waste, which although widely used to improve substrate quality, has been flagged as a potential ARG source.

We detected 62 ARGs across the four management regimes, which were dominated by target-modification and mixed mechanisms conferring resistance to naturally occurring antibiotics such as bacitracin (bacA) and rifamycin (arr, rox, rph), rather than efflux-based multidrug resistance, which is typically co-selected by anthropogenic pollutants. The ARGs were mainly chromosomally encoded, with only two ARGs located on plasmids, and associated with non-pathogenic environmental taxa. The management regime had a significant effect on ARG richness, ARG composition and plasmid abundance, but not on average genome size-normalized ARG abundance. We also detected aph3-II and tlmA as enriched in the unamended samples, which were carried by oligotrophic bacteria, pointing towards microbial competition in a nutrient-limited environment.

Overall, our findings indicate that green roof management supports a substrate resistome driven by ecological constraints rather than clinical threats. However, further research is required to evaluate potential risks and support the safe integration of green roofs within a One Health framework.