Integrating digital PCR and metagenomics to quantify potential soilborne bacterial pathogens in urban ecosystem

Understanding the environmental occurrence patterns of soilborne pathogens is essential for public health, yet a comprehensive and accurate assessment remains challenging. This study presents an innovative technical framework integrating metagenomic pathogen screening with quantitative validation using chip-based digital PCR (dPCR) targeting the overall bacteria community as well as three dominant species-Ralstonia pickettii, Saccharomonospora viridis, and Gordonia terrae. This approach enabled a comprehensive quantification of potential human-, plant-, and zoonotic pathogens and elucidation of their environmental drivers across urban soil habitats in Beijing. Farmland and hospital greenspaces exhibited higher potential pathogen richness (15.55 ± 5.87 and 10.70 ± 4.52) and abundance (22,475.52 ± 15,559.92 and 26,217.62 ± 19,299.90 copies g⁻¹ soil) compared with forests and campus greenspaces. The composition of potential pathogens varied among habitats, with farmlands containing the highest number of unique species, and four taxa were detected across all habitats, showing strong adaptive capacity. Pathogen diversity was positively correlated with total and available phosphorus and with total bacterial α- and β-diversity, while negatively associated with soil organic carbon, reflecting limited pathogen inputs in carbon-rich forest soils and the key role of phosphorus in pathogen enrichment. Climatic and soil physicochemical factors indirectly influenced pathogen diversity by modulating bacterial communities, whereas human activities directly increased pathogen abundance. Molecular ecological network analysis demonstrated that 81% of the associations between pathogenic and non-pathogenic taxa were significantly negative, suggesting competitive exclusion as a key regulatory mechanism. Collectively, these findings provide a precise monitoring framework and new insights into cross-species interactions, contributing to improved risk assessment and One Health strategies for the prevention of soilborne diseases.