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

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, some of these targets may persist in pipe biofilms and detach into wastewater, complicating accurate WS interpretation. To investigate biofilm contributions to wastewater pathogen and ARG signals, paired sink-drain biofilm, branch-drain-plumbing biofilm (sewer biofilm), and wastewater were collected from five hospital sites over a four-month period and analyzed using 16S rRNA gene amplicon sequencing and probe-capture metagenomics. Overall, sewer biofilm bacterial communities were as diverse as wastewater. Across sites, a mean of 9% (0.9 to 23.3%) of wastewater bacterial communities could be attributed to sewer biofilm communities. Many clinically relevant pathogens were consistently detected both in sewer biofilm and wastewater, including environmentally persistent and/or biofilm-associated taxa (e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae). While many ARGs overlapped between wastewater and biofilms (e.g., tetA, sul1, blaCTX-M, vanA), others were significantly enriched in sewer biofilms (e.g., qacL, van-operon and OXA genes). Together, these findings confirm that wastewater pathogen and resistome profiles integrate inputs from both human shedding and pipe-resident communities and therefore need to be considered when selecting WS targets and interpreting signal.