Valerie J. Harwood

Exposures to both human and animal feces pose human health risks, particularly for young children in low- and middle-income country (LMIC) settings where domestic animals are common, water and sanitation infrastructure is often limited, and enteropathogen transmission is high. Microbial source tracking (MST) markers specific to feces from humans and particular animal types can be used to identify the provenance of microbial contamination, yet most MST studies explore few household environmental sample types, limiting the understanding of how marker utility varies by matrix. We validated qPCR assays for six MST markers and quantified their prevalence in 585 samples from 59 households spanning an urban–rural gradient in northwestern Ecuador. We used GenBac3 to test for general fecal contamination and HF183, Rum2Bac, Pig2Bac, DG37, and GFD to test for human, ruminant, swine, dog, and avian contamination, respectively. Approximately 10 sample types were collected per household, including the following: rinses of child and adult hands, swabs of floors and surfaces, soil, domestic and drinking water, and food. GenBac3 and HF183 were detected in 77.82% and 15.36% of samples, respectively. Animal-associated markers were detected less frequently, in 0.5%–4.1% of samples. However, when present, animal marker concentrations were comparable to HF183. Host-associated markers were most often detected in adult and child hand rinse and floor samples, and GenBac3 concentrations were highest in hand rinse samples. HF183 detection on adult caregiver hands was associated with increased odds of HF183 detection on children’s hands and floors. Together, these findings identify hands and floors as reservoirs of fecal contamination and highlight the need for integrated interventions that address both human and animal sources to address household exposures to reduce exposures to enteric pathogens. IMPORTANCE: Understanding the sources and pathways of detectable household environmental fecal contamination is critical for identifying how exposures occur and for developing targeted interventions to reduce risk of enteric infection. By linking contamination on caregiver hands to that on children’s hands and floors, we highlight a likely route for pathogen transfer in the home. The inclusion of multiple host-associated markers across a wide range of sample types reveals patterns that narrower studies may miss, offering new insights into the complex ecology of fecal contamination. These findings can inform sampling strategies, guide risk assessments, and support the design of interventions aimed at reducing child exposure to enteric pathogens in similar high-risk settings.

Antibiotic-resistant bacteria (ARB) such as Escherichia coli and Enterococcus released into surface waters have strong potential to impact human health. We assessed the prevalence of antibiotic-resistant bacteria (ARB) and multidrug-resistant (MDR) bacteria in undisinfected wastewater effluent in a tropical estuary that receives the discharge from a major wastewater treatment plant (WWTP) in Costa Rica. Methods: We quantified culturable ampicillin-resistant (ampR) and (MDR) E. coli and Enterococcus in wastewater influent from hospital and residential sources, effluent, and estuarine receiving waters of the secondary-treated effluent of a WWTP. AmpR isolates confirmed to species or genus were tested for resistance against six additional antibiotic classes. Results: The proportion of ampR E. coli (18%) was significantly greater than that of ampR enterococci (4%) but neither differed among sites. AmpR E. coli concentrations were significantly different by site (estuary, 3.9 log₁₀ CFU/100 mL vs. untreated residential wastewater, 6.4 log₁₀ CFU/100 mL), but ampR enterococci concentrations were consistent among sites. MDR E. coli and Enterococcus were most prevalent in hospital wastewater (57% and 45% of ampR isolates, respectively), but were found at all sites. MDR E. coli and Enterococcus isolates resistant to five antibiotics were isolated from the estuary, and gentamicin-resistant Enterococcus were isolated only from effluent. Conclusions: Undisinfected effluent is a source of ARB and MDR opportunistic pathogens in the tropical estuary and has the potential to impact the health of beachgoers. Our findings highlight the importance of rigorous treatment of wastewater effluent, including disinfection, as a contribution to efforts to achieve effective stewardship of antibiotics.

Exposures to both human and animal feces pose human health risks, particularly for young children in low- and middle-income country (LMIC) settings where domestic animals are common, water and sanitation infrastructure is often limited, and enteropathogen transmission is high. Microbial source tracking (MST) markers specific to feces from humans and particular animal types can be used to identify the provenance of microbial contamination, yet most MST studies explore few household environmental sample types, limiting understanding of how marker utility varies by matrix. We validated qPCR assays for six MST markers and quantified their prevalence in 585 samples from 59 households spanning an urban–rural gradient in northwestern Ecuador. We used GenBac3 to test for general fecal contamination, and HF183, Rum2Bac, Pig2Bac, DG37, and GFD to test for human, ruminant, swine, dog, and avian contamination, respectively. Approximately 10 sample types were collected per household, including: rinses of child and adult hands, swabs of floors and surfaces, soil, domestic and drinking water, and food. GenBac3 and HF183 were detected in 77.82% and 15.36% of samples, respectively. Animal-associated markers were detected less frequently, in 0.5–4.1% of samples. However, when present, animal marker concentrations were comparable to HF183. Host-associated markers were most often detected in adult and child hand rinse and floor samples, and GenBac3 concentrations were highest in hand rinses. HF183 detection on adult caregiver hands was associated with increased odds of HF183 detection on children’s hands and floors. Importance: Understanding the sources and pathways of detectable household environmental fecal contamination is critical for identifying how exposures occur and for developing targeted interventions to reduce risk of enteric infection By linking contamination on caregiver hands to that on children’s hands and floors, we highlight a likely route for pathogen transfer in the home. The inclusion of multiple host-associated markers across a wide range of sample types reveals patterns that narrower studies may miss, offering new insights into the complex ecology of fecal contamination. These findings can inform sampling strategies, guide risk assessments, and support the design of interventions aimed at reducing child exposure to enteric pathogens in similar high-risk settings.

Sewage contamination of environmental waters is increasingly assessed by measuring DNA from sewage-associated microorganisms in microbial source tracking (MST) approaches. However, DNA can persist through wastewater treatment and reach surface waters when treated sewage/recycled water is discharged, which may falsely indicate pollution from untreated sewage. Recycled water discharged from an advanced wastewater treatment (AWT) facility into a Florida stream elevated the sewage-associated HF183 marker 1,000-fold, with a minimal increase in cultured Escherichia coli . The persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities. E. coli (EC23S857) and sewage-associated markers HF183, H8, and viral crAssphage CPQ_056 were always detected in untreated sewage (6.5–8.7 log ₁₀ GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4–5 log ₁₀ through CWT, but CPQ_056 was ~100-fold more persistent. In AWT facilities, the log ₁₀ reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated ( P ≤ 0.0136; tau ≥ 0.44); however, CPQ_056 was not correlated with any marker, suggesting varying drivers of decay. Concentrations of cultured E. coli carrying the H8 marker (EcH8) in untreated sewage were 5.24–6.02 log ₁₀ CFU/100 mL, while no E. coli was isolated from recycled water. HF183 and culturable EcH8 were also correlated in contaminated surface waters (odds ratio β ₁ = 1.701). Culturable EcH8 has a strong potential to differentiate positive MST marker signals arising from treated (e.g., recycled water) and untreated sewage discharged into environmental waters. IMPORTANCE: Genes in sewage-associated microorganisms are widely accepted indicators of sewage pollution in environmental waters. However, DNA persists through wastewater treatment and can reach surface waters when recycled water is discharged, potentially causing false-positive indications of sewage contamination. Previous studies have found that bacterial and viral sewage-associated genes persist through wastewater treatment; however, these studies did not compare different facilities or identify a solution to distinguish sewage from recycled water. In this study, we demonstrated the persistence of bacterial marker genes and the greater persistence of a viral marker gene (CPQ_056 of crAssphage) through varying wastewater treatment facilities. We also aim to provide a tool to confirm sewage contamination in surface waters with recycled water inputs. This work showed that the level of wastewater treatment affects the removal of microorganisms, particularly viruses, and expands our ability to identify sewage in surface waters.

Escherichia coli is a promising subject for globally coordinated surveillance of antimicrobial resistance (AMR) in water environments due to its clinical relevance and widespread use as an indicator of fecal contamination. Cefotaxime-resistant E. coli was recently evaluated favorably for this purpose by the World Health Organization TriCycle Protocol, which specifies tryptone bile x-glucuronide (TBX) medium and incubation at 35°C. We assessed comparability with the U.S. Environmental Protection Agency-approved method for E. coli quantification, which uses membrane-thermotolerant E. coli (mTEC) agar and incubation at 44.5°C, in terms of recovery of E. coli and cefotaxime-resistant E. coli from wastewater influent and surface waters. Total E. coli concentrations in wastewater influent were 10 ⁶ –10 ⁸ CFU/100 mL, while cefotaxime-resistant E. coli were ~100-fold lower. Total E. coli in surface waters were ~10 ² CFU/100 mL, and cefotaxime-resistant isolates were near the limit of detection (0.4 CFU/100 mL). Total and putative cefotaxime-resistant E. coli concentrations did not differ significantly between media or by incubation method; however, colonies isolated on mTEC were more frequently confirmed to species (97.1%) compared to those from TBX (92.5%). Incubation in a water bath at 44.5°C significantly decreased non-specific background growth and improved confirmation frequency on both media (97.4%) compared to incubation at 35°C (92.3%). This study helps to advance globally coordinated AMR in water environments and suggests that the TriCycle Protocol is adaptable to other standard methods that may be required in different locales, while also offering a means to improve specificity by decreasing the frequency of false-positive identification of cefotaxime-resistant E. coli by modifying incubation conditions. IMPORTANCE: As antibiotic-resistant bacteria in water environments are increasingly recognized as contributors to the global antibiotic resistance crisis, the need for a monitoring subject that captures antibiotic resistance trends on a global scale increases. The World Health Organization TriCycle Protocol proposes the use of cefotaxime-resistant Escherichia coli isolated on tryptone bile x-glucuronide agar. The U.S. Environmental Protection Agency (USEPA) criteria for safe recreational waters also use E. coli as an indicator but specify the use of mTEC agar at a higher incubation temperature (44.5°C vs 35°C). We assessed the comparability of these methods for isolating total and cefotaxime-resistant E. coli , finding overall good agreement and performance, but significantly higher specificity toward E. coli selection with the use of the USEPA incubation protocol and mTEC agar. This study is the first to directly compare these methods and provides evidence that the methods may be used interchangeably for global surveillance of antibiotic resistance in the environment.

Awareness of the need for surveillance of antimicrobial resistance (AMR) in water environments is growing, but there is uncertainty regarding appropriate monitoring targets. Adapting culture-based fecal indicator monitoring to include antibiotics in the media provides a potentially low-tech and accessible option, while quantitative polymerase chain reaction (qPCR) targeting key genes of interest provides a broad, quantitative measure across the microbial community. The purpose of this study was to compare findings obtained from the culture of cefotaxime-resistant (cefR) Escherichia coli with two qPCR methods for quantification of antibiotic resistance genes across wastewater, recycled water, and surface waters. The culture method was a modification of US EPA Method 1603 for E. coli, in which cefotaxime is included in the medium to capture cefR strains, while qPCR methods quantified sul1 and intI1. A common standard operating procedure for each target was applied to samples collected by six water utilities across the United States and processed by two laboratories. The methods performed consistently, and all three measures reflected the same overarching trends across water types. The qPCR detection of sul1 yielded the widest dynamic range of measurement as an AMR indicator (7-log versus 3.5-log for cefR E. coli), while intI1 was the most frequently detected target (99% versus 96.5% and 50.8% for sul1 and cefR E. coli, respectively). All methods produced comparable measurements between labs (p < 0.05, Kruskal–Wallis). Further study is needed to consider how relevant each measure is to capturing hot spots for the evolution and dissemination of AMR in the environment and as indicators of AMR-associated human health risk.

This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates.

Shotgun metagenomic sequencing of the collective genomic information carried across microbial communities is emerging as a powerful approach for monitoring antibiotic resistance in environmental matrices. Metagenomics is advantageous in that known and putative antibiotic resistance genes (ARGs) (i.e., the resistome) can be screened simultaneously without a priori selection of targets. Additionally, as new ARGs are discovered and catalogued, stored sequencing data can be reanalyzed to assess the prevalence of emerging genes or pathogens. However, best practices for metagenomic data generation and processing are needed to support comparability across space and time. To support reproducible downstream analysis, guidance is first needed with respect to sampling design, sample preservation and storage, DNA extraction, library preparation, sequencing depth, and experimental controls. Here we conducted a systematic review to assess current practices for the application of metagenomics for AR profiling of wastewater, recycled water, and surface water and to offer recommendations to support comparability in the collection, production, and analysis of resulting data. Based on integrated analysis of findings and data reported across 95 articles identified, a field to benchtop metagenomic workflow is discussed for optimizing the representativeness and comparability of generated data. Through the reanalysis of 1474 publicly-available metagenomes, appropriate sequencing depths per environment and uniform normalization strategies are provided. Further, there is opportunity to harness the quantitative capacity of metagenomics more overtly through inclusion of sequencing controls. The recommendations will amplify the overall value of the metagenomic data generated to support within and between study comparisons, now and in the future. Graphical Abstract

Purpose of Review: Mounting evidence indicates that habitats such as wastewater and environmental waters are pathways for the spread of antibiotic-resistant bacteria (ARB) and mobile antibiotic resistance genes (ARGs). We identified antibiotic-resistant members of the genera Acinetobacter, Aeromonas, and Pseudomonas as key opportunistic pathogens that grow or persist in built (e.g., wastewater) or natural aquatic environments. Effective methods for monitoring these ARB in the environment are needed to understand their influence on dissemination of ARB and ARGs, but standard methods have not been developed. This systematic review considers peer-reviewed papers where the ARB above were cultured from wastewater or surface water, focusing on the accuracy of current methodologies. Recent Findings: Recent studies suggest that many clinically important ARGs were originally acquired from environmental microorganisms. Acinetobacter, Aeromonas, and Pseudomonas species are of interest because their ability to persist and grow in the environment provides opportunities to engage in horizontal gene transfer with other environmental bacteria. Pathogenic strains of these organisms resistant to multiple, clinically relevant drug classes have been identified as an urgent threat. However, culture methods for these bacteria were generally developed for clinical samples and are not well-vetted for environmental samples. Summary: The search criteria yielded 60 peer-reviewed articles over the past 20 years, which reported a wide variety of methods for isolation, confirmation, and antibiotic resistance assays. Based on a systematic comparison of the reported methods, we suggest a path forward for standardizing methodologies for monitoring antibiotic resistant strains of these bacteria in water environments.