Biofilm Contributions of Bacterial Pathogens and Antimicrobial Resistance Genes to Wastewater Surveillance Signal at the Hospital Scale

Abstract Wastewater surveillance (WS) has been widely adopted as a cost-effective and population-representative infectious disease monitoring tool and is increasingly being applied to bacterial and antimicrobial resistance gene (ARG) targets. However, there is potential for some targets to persist in pipe biofilms and detach into wastewater, complicating WS interpretation. To investigate shared pathogen and ARG signals, paired sink-drain biofilm, branch-drain (secondary horizontal pipes carrying wastewater from multiple fixtures) biofilm, and wastewater were collected from five hospital sites over four months and analyzed using 16S rRNA gene amplicon sequencing and probe-capture metagenomics. Branch-drain biofilm bacterial communities were as diverse as wastewater, and across sites, a median of 8.5% (0–24.4%) of wastewater bacterial communities were attributed to branch-drain biofilm. Many clinically relevant pathogens were consistently detected in both branch-drain biofilm and wastewater, including taxa previously reported as environmentally persistent and/or biofilm-associated(e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae). While many ARGs overlapped between wastewater and biofilms (e.g., tetA, sul1, blaCTX-M), others were significantly enriched in branch-drain biofilms (e.g., qacL, van-operon and OXA genes). Together, these findings support that wastewater pathogen and resistome profiles may integrate inputs from both human shedding and pipe-resident communities, requiring further investigation and consideration when selecting WS targets and interpreting signal.