Wastewater-based epidemiology (WBE) has been a valuable public health surveillance tool used for monitoring the consumption of pharmaceuticals, illicit drugs, poliovirus, as well as exposure to chemical and other biological agents of public health concern (Orive et al. 2020; Huizer et al. 2021). During the global outbreak of the beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the use of WBE and associated wastewater surveillance methods proved to be a complementary monitoring tool in estimating real-time infection and coronavirus disease 2019 (COVID-19) burden in the community (Medema et al. 2020; Wu et al. 2020). SARS-CoV-2 genetic material and antimicrobial resistance (AR) genes/organisms can be shed through stool and remain detectable in wastewater both at the community-level (i.e., wastewater treatment plants) and at the facility level (e.g., primary schools, universities, and hospitals) (Wang et al. 2005; Acosta et al. 2021; Colosi et al. 2021; Harris-Lovett et al. 2021; Scott et al. 2021; Fielding-Miller et al. 2023). Wastewater surveillance in congregate settings, primarily university dormitories, has demonstrated its utility for the detection of SARS-CoV-2 (Gibas et al. 2021; Corchis-Scott et al. 2023). In the case of skilled nursing facilities (SNFs), residents were some of the most impacted individuals during the spread of SARS-CoV-2 (Abrams et al. 2020; Lau-Ng et al. 2020). SNF residents are a high-risk, high-acuity population typically having underlying, chronic medical conditions (D’Adamo et al. 2020), which likely contributed to the high mortality rates recorded during the early stages of the pandemic. As a result, several monitoring strategies, including testing of residents and healthcare workers coupled with facility-level wastewater surveillance, have been evaluated and used to inform guidelines for protecting these high-risk populations (Lee et al. 2021; Shrader et al. 2021; Tsoungui Obama et al. 2021). These enhanced surveillance strategies may also be extended to monitoring AR, an additional public health concern impacting SNFs. Determining success for detecting SARS-CoV-2 and antimicrobial-resistant threats at the SNF level and other long-term care facility equivalents has the potential for actionable results with public health impact.

Wastewater surveillance methods can provide trend data for specific targets in communities, university/college campuses, and prisons, representing hundreds to hundreds of thousands of individuals. Capturing the pathogen signal in a resident/patient population at an SNF also presents challenges due to the population being in the tens to hundreds and with only a limited portion of the population being mobile and capable of using the toilet and conducting hygienic practices (e.g., bathing or showering, sink use). Additionally, the erratic, low-flow effluent from the building contains debris and fresh residues from laundry, kitchen, etc. that can impact the detection of the target microorganism. While there are a few hospital wastewater surveillance studies published that demonstrate associations between SARS-CoV-2 detected in wastewater and clinical cases (Wang et al. 2005; Acosta et al. 2021; Colosi et al. 2021), there are only two peer-reviewed published studies currently available regarding SARS-CoV-2 detection in United States nursing homes (Gamage et al. 2024; Keck et al. 2024). In the work of Gamage et al. (2024), grab samples were collected and concentrated using centrifugation and ultrafiltration methods to demonstrate 60% sensitivity in detecting a SARS-CoV-2 infected resident. A study by Keck et al. study (2024) utilized 24 h composite samples and exclusion-based sample preparation to demonstrate a 48% sensitivity for identifying SARS-CoV-2 infections (Keck et al. 2024). An additional study conducted outside the United States applied an aluminum adsorption-precipitation concentration method (Davó et al. 2021). There is limited and inconsistent application of successful wastewater surveillance methods at the congregate-level to evaluate recovery for SARS-CoV-2 (CDC 2023a), and multiple studies in this setting are important to determine long-term feasibility.

In the few peer-reviewed papers regarding wastewater surveillance for AR genes, where five different isolation and detection approaches were taken, all the studies were conducted outside the United States (White et al. 2016; Buelow et al. 2018; Cahill et al. 2019; Buelow et al. 2020; Kagambèga et al. 2023). Implementing wastewater surveillance for AR organisms and genes in a SNF population or long-term care equivalent within the United States has yet to be thoroughly evaluated. A recent report by the National Academies of Sciences on wastewater-based disease surveillance highlighted the importance of facility-level testing as a more effective form of disease surveillance compared to community-wide surveillance, particularly when it comes to determining the spread of antibiotic resistance (National Academies of Sciences & Medicine 2024). Differing from SARS-CoV-2, where detection of the virus indicates an infection, detection of a bacterial pathogen and associated antimicrobial genes is complicated by colonized individuals contributing to this signal and even the natural environmental reservoir, not necessarily an active infection. If wastewater surveillance for AR is possible at SNFs in order to supplement infrequent point prevalence surveys, the gains in understanding would outweigh the challenges faced in attempting to reduce the estimated 35,000 deaths, 2.8 million infections, and $4.1–$5.1 billion spent in the United States per year with increased surveillance (CDC 2019). The AR focused targets for this project were the carbapenem genes bla_KPC, bla_NDM, bla_VIM, and bla_OXA-like, as these genes are responsible for a majority of resistant infections in facilities with high risk, high acuity populations and are recognized global AR threats.

This paper describes a pilot application of wastewater surveillance at an SNF in the Atlanta-metro area during the fall of 2021, with a primary focus on evaluating three methods for concentrating wastewater samples followed by DNA/RNA extraction and detection/quantification of genetic material from SARS-CoV-2, and endogenous and exogenous virus surrogates. Two of the three methods, electronegative membrane filtration (enMF) and polyethylene glycol precipitation (PEG), have traditionally been used for concentrating viruses from a variety of environmental matrices, including wastewater (La Rosa et al. 2020; Wu et al. 2020). The third, Nanotrap^® magnetic virus particles (NP), is a more recent technological development for capturing viral particles in wastewater and environmental matrices and was included to evaluate how it compares to the more traditional methods. Additionally, enriched wastewater samples were assessed for the presence of carbapenem resistance genes and AR Gram-negative Enterobacterales using selective agars and PCR assays typically used for clinical isolates. These findings will help to further inform potential approaches and considerations for wastewater surveillance, particularly at SNFs.

This study was approved by the CDC COVID-19 Response Laboratory Task Force and met the CDC safety policies and recommendations in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (Meechan & Potts 2020). This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.^§

The importance of WBE as a public health monitoring tool was highlighted during the height of the COVID-19 pandemic (Gamage et al. 2024). SNFs and other long-term care facilities were heavily impacted by the pandemic, highlighting the potential role that wastewater surveillance, coupled with clinical testing, could play in detecting transmission and informing infection control measures. The wastewater surveillance study by Gamage et al. (2024) highlighted the utility of WBE over eight distinct facilities and demonstrated how in some cases wastewater surveillance served as an early indicator of asymptomatic or presymptomatic SARS-CoV-2 residents; however, they noted that it was not reliable enough to replace other established practices (Gamage et al. 2024). Keck et al. (2024) suggest that ‘such testing (wastewater) could provide an early warning to trigger enhanced clinical testing or infection prevention activities’ (Keck et al. 2024). To that end, protocols should be developed carefully as factors such as sample type, frequency of collection, concentration methods, and type of analytical detection (e.g., RT-PCR vs ddPCR) can impact the reproducibility of wastewater surveillance results (Pecson et al. 2021). Establishing clear standard operating procedures that incorporate quality control parameters along with optimal method sensitivity is also important (Pecson et al. 2021).

Additionally, the characteristics of SNF wastewater effluents and their associated water quality parameters (e.g., pH, temperature, BOD, and TSS) should be considered, as they may have an impact on the detection of microbial targets. In this study, these parameters did not appear to greatly impact the recovery of endogenous controls in the wastewater likely due to the short residence time (i.e., minutes) from the point of flush to the point of sampling. Taking the factors of variable front-end concentration methods, macro-pollutants, and small populations into consideration, this study successfully demonstrated the detection of SARS-CoV-2, endogenous controls, and antimicrobial-resistant genes/organisms at the SNF level.

A review by Achak et al. (2021) highlighted how hospital wastewater can be a source of both micro- and macro-pollutants, including pathogenic microorganisms which can be a source of disease transmission (Achak et al. 2021). The physicochemical properties of facility wastewater effluents could provide insight into the disease burden at a given facility; therefore, there is value in measuring water quality parameters. In this study, for example, measured values for conductivity, BOD, TSS, total coliforms, and E. coli fell within the ranges previously reported for hospital and urban wastewaters (Verlicchi et al. 2012). It should be noted that any cleaning or disinfection interventions facility personnel undertake could impact the recovery of target microorganisms. It was also noted that recovery of E. coli was impacted in at least two of our samples, and despite discussions with facility administrators and personnel, no external facility factors could be attributed to these occurrences including the water quality parameters measured. Future use of facility-level data can be helpful in understanding how facility signals align with community-level contributions.

One of the primary aims of this study was to determine which wastewater concentration method would provide the best recovery with minimal processing time. The concentration methods evaluated included traditional methods (e.g., enMF and PEG precipitation) and a more novel approach utilizing magnetic NPs. The recovery of both endogenous and exogenous viral targets using all three methods, combined with ddPCR for detection, suggested that each of the methods was a viable option for the wastewater surveillance approach in this study. When it came to detecting the exogenous control BRSV, we noted no statistical difference between enMF, PEG, and NP. However, the percent recovery range for enMF was greater than those observed for PEG and NP. Unlike crAssphage and PMMoV, BRSV is spiked into the wastewater samples and has a relatively short residence time in the wastewater matrix before concentration. It is possible that this difference in residence time, compared to the endogenous controls, may have resulted in no clear distinction between the three methods tested. Notably, the single positive SARS-CoV-2 sample in this study was recovered by enMF, but not from the other two concentration methods, suggesting that enMF may provide a bit more flexibility in its ability to capture a target organism, particularly when its presence in the wastewater is highly variable or limited. Unfortunately, with only a single positive SARS-CoV-2 sample and several samples that were detectable but not quantifiable (DNQ), the data was insufficient to conduct a proper analysis of the sensitivity and specificity of these methods. The results of this study do not necessarily point to enMF performing the best; however, taking into account that it was the only method that yielded a sample with detectable and quantifiable genetic material from SARS-CoV-2, and it provided a relatively quick and efficient workflow, made it stand out among the methods tested. Notably, rapid turnaround times and streamlined workflows were also observed for the NP method. Thus, for future wastewater surveillance studies, these concentration methods may be considered.

Wastewater surveillance of AR bacteria and genes was of particular interest to this study to understand their presence in the SNF population, particularly during clinical presentations of SARS-CoV-2 early in the pandemic. One of the goals was to pilot selective agars, typically used for clinical screening, to screen for multi-drug-resistant organisms in wastewater, as well as evaluate front-end approaches from filtered or enriched samples directly to PCR from SNF wastewater. Various clinically and environmentally relevant bacterial isolates, exhibiting carbapenem-resistant and extended-spectrum β-lactamase-producing phenotypes, were successfully isolated and confirmed using MALDI-TOF. The TSB enrichment after Colilert-18 was not beneficial in increasing carbapenem-resistant or ESBL-producing phenotype detection as compared to direct plating.

The molecular detection of the bla_KPC gene from filtered wastewater samples via PCR was successful, whereas E. coli-positive Colilert-18 samples did not yield any carbapenem resistance gene detection. It should be noted that the filters represented 10 ml of wastewater, went through a more robust extraction process, and did not bias growth. Although molecular detection of resistance genes was achieved from filtered wastewater samples, a limitation of this study is the lack of resistance mechanism confirmatory PCR for isolates exhibiting AR phenotypes. Future studies should include this confirmatory PCR testing to determine if the resistance mechanism is present in the isolates and also correlate it to available facility-level clinical data. This study relied on both culture- and non-culture-based methods that were compatible with the facility sampling schedule, sample storage capacities, and laboratory workflow. An examination of the Antimicrobial Resistance and Patient Safety Portal data of the percent AR from healthcare-associated infection types (i.e., catheter-associated urinary tract infections, central line-associated bloodstream infections, surgical site infections) in Georgia in 2021 indicated that 7.8 and 6.6% were associated with carbapenem-resistant Klebsiella and Enterobacter, respectively, whereas only 0.8% were associated with carbapenem-resistant E. coli (CDC 2023b). Although recovered from an environmental source and not directly from patients, in this study a greater percentage of the isolates that exhibited a carbapenem-resistant phenotype were in the Klebsiella and Enterobacter genera and a much lower percentage was observed for E. coli. While the AR data presented here and the application of wastewater surveillance at healthcare facilities is nascent, evaluating existing specimen approaches for wastewater samples is a feasible starting place. Expanding these approaches to complement existing patient care data and infection control practices could bridge knowledge gaps and challenges raised by public health agencies (CDC 2022).

Similarly, this study also provided some insight into the relationship between real-time, patient case data and the ability of wastewater surveillance to capture corroborating data on the levels of viral circulation in specific populations. Although linking sequencing data from wastewater samples to positive clinical samples was beyond the scope of this study and limited by the facility’s capacity at the time, it is interesting to note that during the same period (September–November 2021), data provided by reporting facilities to both Centers for Medicare & Medicaid Services and the Georgia Department of Public Health (GADPH) indicated reductions in both COVID-19 cases and hospitalizations among nursing home residents (CMMS 2021). For example, according to the GADPH, rolling 7- and 14-day percent positive thresholds decreased by nearly 50% from month to month (September–November 2021), indicating a reduction in SARS-CoV-2 infections during this period. During our study period, only one of eighteen composite wastewater samples had detectable levels of SARS-CoV-2 that could be quantified by ddPCR. Sequencing data confirmed the presence of SARS-CoV-2 but could not confidently assign the variant, although the circulating variant at the time in Georgia was delta. In several instances there were samples that were DNQ, suggesting that additional factors such as waste discharge from the facility and variable shedding rates, including from healthcare workers and visitors, could impact the levels of virus that can be detected and quantified. Furthermore, at the time, the SARS-CoV-2 ddPCR kit used for detection had been granted Emergency Use Authorization for the qualitative detection of nucleic acids from SARS-CoV-2, from nasal swab specimens, and may have been expanded beyond its scope. Based on our findings and the reporting of other groups, wastewater-based surveillance would be sensitive enough to detect both increases and decreases associated with pathogen transmission among the SNF population (Fernandez-Cassi et al. 2021; D’Aoust et al. 2021).

Overall, this study successfully demonstrated the detection of SARS-CoV-2, endogenous controls, and antimicrobial-resistance genes/organisms at the SNF level. Future efforts using the confirmed methodological workflow from this study will be expanded to include additional SNFs to establish best practices and longitudinal measurements needed to correlate SARS-CoV-2 in wastewater with COVID-19 resident infections. Wastewater surveillance provides unique insight into the spread of existing and emerging disease-causing pathogens in a given population and can help to inform infection control strategies in real-time and long-term public health policies. While the COVID-19 pandemic presented unique challenges to the existing public health framework, it also provided an opportunity to evaluate the potential strengths and weaknesses of wastewater surveillance applications at SNFs.