Sewer Monitoring for Antimicrobial Resistance Genes and Organisms at Healthcare Facilities

Caryn Tyler
13 minutes ago
3 min read

Sewer monitoring for antimicrobial resistance genes and organisms at healthcare facilities

Rachel Poretsky, Dolores Sanchez Gonzalez, Adam Horton, Michael Schoeny, Chi-Yu Lin, Modou Lamin Jarju, Michael Secreto, Cecilia Chau, Ellen Gough, Erin Newcomer, Adit Chaudhary, Lisa Duffner, Nidhi Undevia, Angela Coulliette-Salmond, Amanda K. Lyons, Florence Whitehill, Mary K. Hayden, Stefan J. Green, Michael Y. Lin

medRxiv 2025.03.16.25324079; doi: https://doi.org/10.1101/2025.03.16.25324079

This article is a preprint and has not been peer-reviewed [what does this mean?]. It reports new medical research that has yet to be evaluated and so should not be used to guide clinical practice.

Abstract

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 of Candida 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) for C. auris and 5 carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-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, with blaKPC being 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.

Background

Surveillance of wastewater from healthcare facilities has the potential to identify the emergence of multidrug-resistant organisms (MDROs) of public health importance [1]. Optimal approaches to sampling, processing, and testing wastewater need to be established before wastewater surveillance (WWS) can be used routinely for MDRO surveillance. Although genes of interest can be detected using similar molecular approaches in both wastewater samples and patient samples, there are multiple interrelated and unique challenges to WWS. First, wastewater is a complex medium, containing a mixture of organic matter, particles, microorganisms, and flow variability. Second, laboratory methods for WWS have not yet been standardized, leading to poor understanding of measurement uncertainties and the effects of wastewater composition on detection [2–4]. Finally, target abundance can hinder detection; previous metagenomics-based studies in municipal and hospital wastewater have shown that many antimicrobial resistance (AR) genes are present at low relative abundances [5–7].

Here, we present an evaluation of approaches to address these knowledge gaps, focusing on healthcare associated pathogens: Candida auris and carbapenemase-producing organisms (CPOs, represented by five target genes blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like). We collected wastewater from a region of the United States where C. auris and CPOs are endemic [8–11]. This study employed a longitudinal design at a single long-term acute care hospital over a period of 6 months. We compared three sample collection methods (passive, composite, and grab), three sample processing methods (magnetic nanoparticle [NP] concentration, InnovaPrep Concentrating Pipette [ICP] filtration, and centrifugation followed by bead-beating), and two different detection methods (quantitative real-time PCR [qPCR] and digital PCR [dPCR]) with MDRO diagnostic assays that have been developed and validated for use in clinical samples; therefore, additional evaluation is needed to optimize performance in wastewater samples. We report the findings of a series of seminal experiments that establish an effective protocol for surveillance of C. auris and CPOs in healthcare facility wastewater, helping to define the use of WWS as an important tool for monitoring MDRO burden in healthcare facilities.