Interconnected reservoirs of multidrug-resistant bacteria and plasmids within hospital plumbing infrastructure

Hospital sinks and wastewater streams are hotspots for contamination and colonization with multidrug-resistant Gram-negative bacteria (MDR-GNB). While hospital water systems may facilitate MDR-GNB transmission during outbreaks, longitudinal population dynamics of environmental MDR-GNB and their long-term relationship with patient isolates remain unclear. Here we aimed to characterize the diversity and abundance of MDR-GNB within the hospital built environment and evaluate their relationship with clinical isolates. We performed nanopore sequencing on MDR-GNB cultured from sink drains (n=523) and wastewater (n=146) at a New York City hospital between March-August 2023. We assessed spatial and longitudinal genomic strain and plasmid dynamics and quantified relatedness to historical clinical isolates from the same hospital (n=602). Sink drains (54-73%) and wastewater (100%) demonstrated widespread, longitudinal colonization with MDR-GNB, driven primarily by carbapenem-resistant GNB including opportunistic organisms and Enterobacterales encoding blaKPC. While bacterial colonization differed across environmental niches, we identified both MDR-GNB strains and resistance plasmids that were shared between clinical and environmental sources, as well a distinct interplay between clinical and wastewater versus sink drain samples. This study supports the role of hospital plumbing as an important reservoir for clinically relevant MDR-GNB, but highlights differences across environmental sources, which may impact surveillance approaches.