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Surveillance of wastewater from healthcare facilities has the potential to identify the emergence of multidrug-resistant organisms (MDROs) of public health importance. Specifically, wastewater surveillance can provide sentinel surveillance of novel MDROs (e.g., emergence ofCandida auris) in healthcare facilities and could help direct targeted prevention efforts and monitor longitudinal effects. Several knowledge gaps need to be addressed before wastewater surveillance can be used routinely for MDRO surveillance, including determining optimal approaches to sampling, processing, and testing wastewater for MDROs. To this end, we evaluated multiple methods for wastewater collection (passive, composite, and grab), concentration (nanoparticles, filtration, and centrifugation), and PCR quantification (real-time quantitative PCR vs. digital PCR) forC. aurisand 5 carbapenemase genes (bla_KPC, bla_NDM, bla_VIM, bla_IMP, andbla_OXA-48-like) twice weekly for 6 months at a long-term acute care hospital in Chicago, IL. We also tested the effects of different transport and sample storage conditions on PCR quantification. All genes were detected in facility wastewater, withbla_KPCbeing the most consistently abundant. Experiments were done in triplicate with gene copy, variance, and number of detections between triplicates used to determine method efficacy. We found that passive samples processed immediately using a combination of centrifugation followed by bead-beating and dPCR provided the most reliable results for detecting MDROs. We also present the trade-offs of different approaches and use culture and metagenomics to elucidate clinical relevance.

This study establishes a practical approach for wastewater surveillance as a potential tool for public health monitoring of MDRO burden in healthcare facilities.