Wastewater-based surveillance (WBS) entails monitoring targets reflective of human behavior, such as illicit drugs (1) and alcohol (2). WBS has been successfully implemented as an early warning system for polio (3) and was rapidly adopted throughout the world for SARS-CoV-2 monitoring. Now, many WBS programs, including the US National Wastewater Surveillance System (4), are looking to expand to other public health targets, including antimicrobial resistance (AMR) (5).
AMR limits the effectiveness of antibiotics towards bacterial pathogens and is an urgent global health threat (6). Excessive use and misuse of antibiotics can contribute to AMR by exerting selection pressures that result in elevated levels of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the human gut, excreta, and sewage (7). There have been concerted efforts both nationally and internationally to improve antibiotic stewardship, including reducing and optimizing use of antibiotics and disinfectants whenever possible (8). However, it is challenging to directly measure the benefits of such efforts. WBS could be a powerful means to address this data gap (9). Examination of WBS data collected during the COVID-19 pandemic provides a dynamic scenario to relate antimicrobial use data to AMR measures in sewage. Concerns raised during this period include decreased implementation of antibiotic stewardship programs (10), over prescription and misuse of antibiotics for COVID-19 patients (11), and greater use of disinfectants due to increased hygiene vigilance (12). If antibiotic resistant infections in patients increased, then increased release of ARB and ARGs into the sewage would be expected (12–15).
At the onset of the pandemic, there was uncertainty regarding appropriate COVID-19 treatment. Antibiotics were often prescribed to prevent secondary bacterial infections that might arise from respiratory distress or other co-morbidities. For US hospitals in February-March 2020, > 70% of COVID-19 patients were administered at least one antibiotic, with 81% of prescriptions commencing immediately upon admission (16). Such practices occurred even though coinfection rates were lower (~ 3–16%) than expected (17–19). Areas with high COVID-19 burdens especially struggled to maintain antibiotic stewardship. For example, US hospital data (n = 716 hospitals) indicated that, on average during March–October 2020, there were greater numbers of antibiotic days of therapy (DOT) per 1000 patient days than the years preceding the pandemic for cefepime (a beta-lactam), azithromycin (a macrolide), and doxycycline (a tetracycline) (16). Despite increases in inpatient antibiotic prescription rates during the early stages of the pandemic, outpatient prescription rates appear to have drastically fallen in 2020. Between 2011–2018 outpatient prescriptions in the US decreased by 10%; while from 2019–2020 there was a 25% decline, primarily attributed to decreased outpatient visits and healthcare seeking behavior (8). Between 2019 and 2020 the rates of outpatient oral antibiotic prescriptions decreased. These rates increased in 2022, returning to levels observed prior to the pandemic (20–22). Fewer respiratory illness diagnoses in young adults during the pandemic (23), may have contributed to changes in antibiotic prescriptions, for example, from 2010–2018 influenza associated antibiotic prescribing (e.g., misuse) was highest in a cohort study for adults aged 18–19 (compared with adults aged 18–59) (24).
Increased hygiene vigilance was initially thought to be a logical intervention to mitigate COVID-19. The US Centers for Disease Control (CDC) recommended daily surface disinfection (25) using one of the EPA List N of disinfectants (26). Global disinfectant and sanitizer demand increased during the pandemic and remained higher than pre-pandemic levels through March 2021 (27). Quaternary ammonium compounds (QACs) are the most common active ingredients in these EPA-approved products. QAC biocides are frequently used in solvents, cleaning products (soap and cleaning wipes), personal care products, and biomedical materials (28–30). The presence of a positively-charged quaternary amine group and one or two long alkyl chains within the QAC structure (28) enables QACs to sorb to negatively charged solids such as sludges, sediments, and soils. Correspondingly, QACs are widely found in the environment (28). During the pandemic, wet disinfecting wipes, often containing QACs (31), were flushed at such quantities that blockages were reported in many sewer systems (32, 33).
Concerns about the unanticipated negative consequences of disinfectant overuse with respect to AMR were raised early during the COVID-19 pandemic. Chen and colleagues (27) noted that the lack of diversity in disinfectant-types could potentially promote bacterial resistance since bacteria were repeatedly exposed to the same biocidal agent. Disinfectants can promote antibiotic resistance by imposing a selective advantage for ARG carrying bacteria and thus increase the tendency for horizontal gene transfer (12). In particular, some Gram-negative bacteria, such as Pseudomonas aeruginosa, exhibit natural resistance to QACs due to their intrinsic carriage of efflux pumps. Other bacteria acquire QAC resistance by either increasing expression of existing efflux pumps, acquiring efflux pumps that remove QACs, reducing membrane permeability, or increasing membrane stability (34). Importantly, the QAC resistance gene, qacE, is embedded in the class 1 integron intI1, which plays a critical role in the dissemination of multi-antibiotic resistance (35). Prior research suggests an increase in co-resistance (different resistance determinants on the same genetic element) and cross-resistance (one genetic determinant responsible for antibiotic and QAC resistance (36)) between QACs and a range of clinically-important antibiotics and disinfectants. Some efflux pumps mediate cross-resistance due to their ability to export both QACs and other antimicrobial agents. For example, qacH, detected in clinical isolates on an integron on a conjugative plasmid, also encodes resistance to aminoglycosides, chloramphenicol, tetracycline, trimethoprim, beta-lactams, and sulfonamide (34, 37).
The objective of this study was to assess the potential for WBS to track AMR trends by comparing metagenomes and other relevant data pertaining to two small WWTPs serving neighboring, but distinct, communities. One WWTP (6.5 MGD) primarily served a university town of ~ 52,500 (university WWTP) and the other WWTP (3 MGD) served a community of ~ 21,500 (community WWTP). The two WWTPs were monitored during the COVID-19 pandemic to assess ARG profiles as they related to data available on diagnoses of COVID-19 and other illnesses, clinical antibiotic use, disinfectant/QAC use, measured antibiotics/antimicrobials in the sewage, and differences in populations served. Shotgun metagenomic sequencing was applied to comprehensively profile ARGs, mobile genetic elements (MGEs), and QAC resistance genes in the sewage. The findings shed light on how antibiotic/disinfectant stewardship practices can be reflected in sewage AMR measurements and more broadly on how the COVID-19 pandemic may have influenced community level antibiotic resistance carriage.