Unveiling the critical role of overlooked consumer protist-bacteria interactions in antibiotic resistance gene dissemination in urban sewage systems

Antibiotic resistance genes (ARGs) are emerging contaminants of significant concern due to their role in facilitating the spread of antibiotic resistance, especially high-risk ARGs, which are characterized by high human accessibility, gene mobility, pathogenicity, and clinical availability. Studies have shown that cross-domain interactions, such as those between consumer protists (consumers) and bacteria, can influence bacterial diversity, distribution, and function through top-down control. The consumers-bacteria interactions may also affect the occurrence and distribution of ARGs, yet this has been scarcely explored in field investigations. We conducted a city-scale investigation of ARGs, protists, and bacterial communities across each unit of the urban sewage system (USS), including 49 sewage pumping stations (SW), as well as influent (IF), activated sludge (AS), and effluent (EF) from seven wastewater treatment plants. Interestingly, consumers-bacteria interactions, as indicated by indices of bipartite relevance networks (i.e., connectedness and cohesion), increased from SW and IF to AS and EF. Structural equation modelling (SEM) revealed that consumers-bacteria interactions had a greater influence on the abundance of total ARGs and high-risk ARGs than seasonal or environmental factors. Notably, the total effects of consumers-bacteria interactions in SEM were significant (P < 0.05) and comparable in both IF and EF, even with the decrease in ARG abundance from IF to EF. This suggests a potential risk of ARG spread to the environment, facilitated by consumer protists in the EF. Additionally, the relevance network also demonstrated an increasing trend in the relationships between consumer protists and potential hosts of high-risk ARGs from raw sewage (SW and IF) to AS and EF. Overall, this study emphasizes the importance of integrating multitrophic microbial interactions to better understand and mitigate the dissemination of ARGs in sewage systems.