Monitoring the microbial quality of wastewater in decentralized systems, including those serving small communities and individual buildings, is important for a variety of reasons, including wastewater surveillance and assessing the performance of decentralized wastewater treatment systems. However, temporal fluctuations in flow rates and contaminant loadings can cause high variations in sample concentrations. This requires the need to optimize methods to collect, process, and analyze samples in these settings. Composite samples have been recommended over grab samples, but there is still limited understanding about the influence of subsampling intervals on estimates of contaminant loadings. To address this gap in the literature, the effect of subsample collection frequency on virus concentrations measured in time-based composite samples from residential buildings at a university campus was assessed. For time-based composite samples, there was a relationship between subsampling frequency and the variance in measured concentrations, with variance increasing with decreasing subsampling frequency. The variability in the virus concentrations measured in composite samples with subsampling intervals of 1 to 3 h was similar to the variability in concentrations from composite samples with subsampling intervals of 15 min. The comparison of wastewater concentrations of SARS-CoV-2 with confirmed cases in the respective buildings showed higher positive predictive values and similar specificity but lower negative predictive values and lower sensitivity compared to other building-level wastewater surveillance studies. The inclusion or exclusion of grab samples did not significantly change the predictive metrics. Reducing the subsampling interval for composite samples can have meaningful implications for improving the energy efficiency and reducing the environmental impact, as well as the time and costs associated with maintaining and repairing sampling equipment.

Transboundary rivers worldwide face escalating wastewater pollution, highlighting the need for multinational monitoring and mitigation. The Tijuana River, shared by Mexico and the United States, is contaminated by untreated sewage, industrial waste, trash, and polluted runoff. In December of 2022, damaged wastewater infrastructure and wet weather shifted transboundary flow from intermittent to perennial. Average dry-weather transboundary flows increased from 3.18 × 10 4 m 3 /day during water years (WYs) 2021–2022 to 19.1 × 10 4 m 3 /day for WYs 2023–2024, a 6.3-fold increase. To evaluate the effects of the altered flow regime on pollutant export, over 280 water samples were analyzed for chemical constituents and fecal indicator bacteria. Chronic cross-border flows increased pollutant loading to the Pacific Ocean by an order of magnitude, with annual exports of ~ 1.06 million kg of total dissolved nitrogen (TDN) and 1.15 million kg of dissolved organic carbon (DOC). Chronic dry weather flows averaged 7.8 × 10 6 MPN/100mL for Escherichia coli , equivalent to untreated wastewater, and resulted in continuous beach closures since 2023. Under this continuous transboundary flow regime, dry-weather baseflow had significantly (p < 0.01) higher DOC, TDN, and tryptophan-like fluorescence than wet-weather, storm-driven runoff. Attenuation of chemical constituents, microbial source tracking markers, and enteric virus pathogens across the ~ 12 km from border to ocean was essentially absent, likely due to limited photodegradation in highly colored water. These findings establish a baseline for evaluating infrastructure investments, reducing public health and ecosystem risks, and supporting sustained binational monitoring and data sharing.

Coastal waters impacted by fecal contamination pose health risks to recreational users through direct water contact; however, risk to users, workers and surrounding communities from potential airborne exposure remain poorly understood. This study quantified two human-associated sewage markers (HF183 and pepper mild mottle virus (PMMoV)) and two enteric viruses (norovirus GII (NoVGII) and human adenovirus (HAdV)) in paired water and air samples collected in San Diego County, USA, from the Tijuana River, a binational river flowing into the US from Mexico that is polluted with untreated and treated wastewater, a community site on the beach (Imperial Beach), and a northern reference site (La Jolla Shores). A total of 23 paired water and air samples were collected during the dry season, with 10 water samples collected during the wet season. All microbial targets were consistently detected at high concentrations in the Tijuana River water, while concentrations in Imperial Beach water were significantly lower. In contrast, airborne concentrations were comparable between the two sites, with PMMoV detected in all air samples. Log-transformed ratios of the concentrations in air and water samples indicate enrichment in the air samples from Imperial Beach compared to the Tijuana River. PMMoV showed the most consistent detection and the strongest relationship with airborne HAdV and NoV levels, suggesting PMMoV as a conservative indicator of aerosolized viruses of human fecal origin in such settings. These findings indicate that coastal surf zones may serve as important interfaces for aerosolization of wastewater-associated microorganisms and suggest that inhalation could represent a previously underrecognized exposure pathway in contaminated coastal environments.

Fecal indicator bacteria (FIB) have historically been used to assess public health risks associated with contact water recreation, and microbial source tracking (MST) has been introduced to distinguish human-associated fecal pollution from natural sources of animal feces. While MST can identify human-associated fecal pollution, without interpretation it cannot differentiate between small amounts of fresh fecal contamination and large amounts of decayed pollution, which can present vastly different health risks. This study examined the ratio of HF183 and pepper mild mottle virus (PMMoV) as an indicator of the age of fecal contamination. The correlation was also assessed between these two individual markers, the HF183:PMMoV ratio, and two enteric viruses, norovirus genogroup II (NoV GII) and human adenovirus (HAdV). A total of 364 samples were collected under varying weather conditions over two years at impacted and reference surface water sites in southern California, including estuaries and adjacent ocean locations. The mean concentrations of MST markers and enteric viruses were higher during the rainy season compared to the dry season, especially at the impacted sites. NoV GII showed seasonality with higher detections and concentrations during the rainy season, while HAdV did not show such seasonal trends. The HF183:PMMoV ratio demonstrated a stronger correlation with enteric viruses than either marker alone, but when seasonal and weather effects were factored out, the HF183:PMMoV ratio and PMMoV had a stronger relationship with the concentration of enteric viruses. The results of this study indicate that the HF183:PMMoV ratio is a more reliable indicator of human fecal pathogens than the two individual markers by themselves. Synopsis The HF183:PMMoV ratio is a reliable and robust indicator for recreational water quality.

Two photo-sequencing batch reactors (PSBR) fed with real wastewater were evaluated to understand the elimination and particle association of fecal indicator bacteria (FIB) and coliphages.

The direct and indirect potable reuse of wastewater has recently gained significant attention in water scarce regions globally. Some advanced treatment facilities utilize ozone and biological activated carbon (BAC) filtration to remove persistent low molecular weight compounds, but the role of microorganisms in these systems has not been well studied. Previous studies of microbial communities in potable reuse systems have been limited to culture-based methods, targeted molecular methods, or 16S rRNA sequencing. The goal of this study was to use shotgun sequencing to characterize the phylogenetic and functional diversity at each point within a potable reuse treatment train in southern California, which consisted of primary clarification, activated sludge with nitrification and partial denitrification, tertiary filtration, ozonation, BAC filtration, membrane filtration, reverse osmosis, and UV/advanced oxidation. Samples were collected from the untreated wastewater and from the effluent of each treatment process. The data revealed significant shifts in phylogenetic and functional diversity at each point in the treatment train. The biofilm of the BAC filter was enriched with genes capable of degrading aromatic compounds, possessed mostly by Bradyrhizobia. The extracted final effluent sample only contained 0.3 ng/mu L of microbial DNA-this was insufficient for shotgun sequencing, which requires at least 1 ng/mu L.

Enterococci, widely used as a fecal indicator in coastal recreational waters, can be quantified using different methods, including the culture-based Enterolert method and the newer, more rapid droplet digital polymerase chain reaction (ddPCR) method. Previous studies on the correlation between culture-based and PCR-based methods have shown mixed results. To address this knowledge gap, data from a study conducted at three beaches in the city of Coronado, California during the summer of 2023 were analyzed to compare ddPCR-based concentrations of Enterococcus with Enterolert-based concentrations of enterococci in samples collected daily for approximately two months. The concentrations using both methods were consistent with the lognormal distribution. A linear regression between ddPCR and Enterolert concentrations was statistically significant with an R2 value of 0.41, but the slope was significantly lower than slopes reported in previous studies. The index of agreement between the two methods was 0.25, but it increased to 0.47 when the ddPCR concentrations were scaled to Enterolert equivalents using the regression. When comparing beach action values based on Enterolert and ddPCR, the false positive rate associated with the use of ddPCR was 56.3 %. Overall, the results of this study showed a discordance between the Enterolert and ddPCR methods at Coronado beaches during the summer of 2023, indicating that the relationship may be influenced by spatial and temporal factors.

Challenges from globalization, population growth, and climate change require science, technology, and engineering (STEM) professionals to have global competency. However, the impact of international experiences on STEM students' development of these abilities has not been well studied. We assessed the effects of international research experiences in Latin America and the Caribbean (LAC) and Europe on the development of global competency for STEM graduate students from the United States. Research placements in LAC were generally field-based, involving interactions with community members, while placements in Europe were mostly lab-based. Surveys and interviews with participants before and after their trips revealed increases in intercultural abilities for students from all groups. Students who traveled to LAC had higher intercultural abilities before the trip but experienced smaller gains than their counterparts who traveled to Europe. Despite the value in community-based activities for students outside of university settings, more effort is needed to eliminate students' barriers to understanding communication styles in their host communities.

In surface waters with fecal pollution, the age of fecal pollution has a direct connection to human health risk. Current microbial source tracking methods distinguish human-associated fecal markers from animal-associated biomarkers. However, in waters containing human-associated fecal pollution, current source tracking methods do not effectively distinguish small amounts of fresh (new) fecal pollution from large amounts of aged (old) fecal pollution. We introduced a concept for a model that considers environmental factors such as sunlight and temperature to estimate human fecal pollution age based on the ratio of two human-associated fecal biomarkers with different levels of persistence in the environment: HF183 and pepper mild mottle virus (PMMoV). We assessed the HF183/PMMoV ratio using (RT-)qPCR in 98 wastewater samples from two university residence halls in southern California, finding that HF183 was 15 times higher than PMMoV on average. This ratio decreased under sunlight and at higher temperatures due to differing decay rates of the two biomarkers. We propose that monitoring the concentration of HF183 and PMMoV, as well as the HF183/PMMoV ratio, can help to distinguish between a small amount of fresh contamination and a large amount of aged contamination.

Microbial water quality is an integral to water security and is directly linked to human health, food safety, and ecosystem services. However, specifically pathogen data and even faecal indicator data (e.g., E. coli), are sparse and scattered, and their availability in different water bodies (e.g., groundwater) and in different socio-economic contexts (e.g., low- and middle-income countries) are inequitable. There is an urgent need to assess and collate microbial data across the world to evaluate the global state of ambient water quality, water treatment, and health risk, as time is running out to meet Sustainable Development Goal (SDG) 6 by 2030. The overall goal of this paper is to illustrate the need and advocate for building a robust and useful microbial water quality database and consortium worldwide that will help achieve SDG 6. We summarize available data and existing databases on microbial water quality, discuss methods for producing new data on microbial water quality, and identify models and analytical tools that utilize microbial data to support decision making. This review identified global datasets (7 databases), and regional datasets for Africa (3 databases), Australia/New Zealand (6 databases), Asia (3 databases), Europe (7 databases), North America (12 databases) and South America (1 database). Data are missing for low- and middle-income countries. Increased laboratory capacity (due to COVID-19 pandemic) and molecular tools can identify potential pollution sources and monitor directly for pathogens. Models and analytical tools can support microbial water quality assessment by making geospatial and temporal inferences where data are lacking. A genomics, information technology (IT), and data revolution is upon us and presents unprecedented opportunities to develop software and devices for real-time logging, automated analysis, standardization, and modelling of microbial data to strengthen knowledge of global water quality. These opportunities should be leveraged for achieving SDG 6 around the world.

The safe management of fecal sludge from the 3.4 billion people worldwide that use onsite sanitation systems can greatly reduce the global infectious disease burden. However, there is limited knowledge about the role of design, operational, and environmental factors on pathogen survival in pit latrines, urine diverting desiccation toilets, and other types of onsite toilets. We conducted a systematic literature review and meta-analysis to characterize pathogen reduction rates in fecal sludge, feces, and human excreta with respect to pH, temperature, moisture content, and the use of additives for desiccation, alkalinization, or disinfection. A meta-analysis of 1,382 data points extracted from 243 experiments described in 26 articles revealed significant differences be-tween the decay rates and T99 values of pathogens and indicators from different microbial groups. The overall median T99 values were 4.8 days, 29 days, >341 days, and 429 days for bacteria, viruses, protozoan (oo)cysts, and Ascaris eggs, respectively. As expected, higher pH values, higher temperatures, and the application of lime all significantly predicted greater pathogen reduction rates but the use of lime by itself was more effective for bacteria and viruses than for Ascaris eggs, unless urea was also added. In multiple lab-scale experiments, the application of urea with enough lime or ash to reach a pH of 10 - 12 and a sustained concentration of 2,000 - 6,000 mg/L of non-protonated NH3-N reduced Ascaris eggs more rapidly than without urea. In general, the storage of fecal sludge for 6 months adequately controls hazards from viruses and bacteria, but much longer storage times or alkaline treatment with urea and low moisture or heat is needed to control hazards from pro-tozoa and helminths. More research is needed to demonstrate the efficacy of lime, ash, and urea in the field. More studies of protozoan pathogens are also needed, as very few qualifying experiments were found for this group.

Three water, sanitation and hygiene (WASH) support tools were applied to Kampala city, Uganda, to evaluate areas with the highest health hazard due to poor wastewater and faecal sludge management and to develop interventions to improve sanitation and reduce exposure. The Pathogen Flow and Mapping Tool (PFMT) assessed how different sanitation management interventions influence pathogen emissions to surface water using rotavirus as the indicator pathogen, while the HyCRISTAL health hazard tool evaluated how flooding and drainage infrastructure influence the presence of human excreta in the environment. The SaniPath tool identified common high-risk pathways of exposure to faecal contamination in food, open drains and floodwater. An overlap in high health hazard hotspot areas was identified by the PFMT and the HyCRISTAL tools. Across the city, the most important hazard sources were the indiscriminate disposal of faecal waste into open stormwater drains from onsite sanitation technologies, open defecation and the insufficient treatment of wastewater. The SaniPath tool identified drain water, floodwater, street food and uncooked produce as the dominant faecal exposure pathways for selected parishes in the city, demonstrating the presence of excreta in the environment. Together, the tools provide collective evidence guiding household, community, and city-wide sanitation, hygiene and infrastructure management interventions from a richer assessment than when a single tool is applied. For areas with high spatial risks, those practising open defecation, and for low-lying areas, these interventions include the provision of watertight pit latrines or septic tanks that are safely managed and regularly emptied. Faecal sludge should be emptied before flood events, direct connections of latrines to open storm drains should be prevented, and the safe handling of food and water promoted. The tools enhance decision making for local authorities, and the assessments can be replicated in other cities.

Effective wastewater surveillance of SARS-CoV-2 RNA requires the rigorous characterization of the limit of detection resulting from the entire sampling process - the process limit of detection (PLOD). Yet to date, no studies have gone beyond quantifying the assay limit of detection (ALOD) for RT-qPCR or RT-dPCR assays. While the ALOD is the lowest number of gene copies (GC) associated with a 95% probability of detection in a single PCR reaction, the PLOD represents the sensitivity of the method after considering the efficiency of all processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays) to determine the number of GC in the wastewater sample matrix with a specific probability of detection. The primary objective of this study was to estimate the PLOD resulting from the combination of primary concentration and extraction with six SARS-CoV-2 assays: five RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab), and E_Sarbeco RT-qPCR, and one RT-dPCR assay (US CDC N1 RT-dPCR) using two models (exponential survival and cumulative Gaussian). An adsorption extraction (AE) concentration method (i.e., virus adsorption on membrane and the RNA extraction from the membrane) was used to concentrate gamma-irradiated SARS-CoV-2 seeded into 36 wastewater samples. Overall, the US CDC N1 RT-dPCR and RT-qPCR assays had the lowest ALODs (< 10 GC/reaction) and PLODs (<3,954 GC/50 mL; 95% probability of detection) regardless of the seeding level and model used. Nevertheless, consistent amplification and detection rates decreased when seeding levels were < 2.32 x 103 GC/50 mL even for US CDC N1 RT-qPCR and RT-dPCR assays. Consequently, when SARS-CoV-2 RNA concentrations are expected to be low, it may be necessary to improve the positive detection rates of wastewater surveillance by analyzing additional field and RT-PCR replicates. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater.

Water quality benchmarks for fecal indicator bacteria (FIB) are often exceeded in many urban streams in southern California. Possible sources of elevated stream FIB concentrations within urban areas include sanitary sewer exfiltration, sanitary sewer overflows (SSOs), illegal discharges, and human or animal fecal material on the ground surface. Teasing apart the different sources remains a challenge, especially when untreated wastewater and runoff from open defecation sites both contain human fecal material. To distinguish the various sources of microbial contamination in an urban stream, temporal trends in biological and chemical markers of anthropogenic contamination were evaluated in the San Diego River and its tributaries during storm events in two consecutive hydrologic years. Temporal trends in FIB, HF183, pepper mild mottle virus (PMMoV), caffeine, sucralose, chloride, bromide, specific ultraviolet absorbance, and fluorescence index indicated that untreated wastewater flushed from the vadose zone was the main source of microbial pollution to the San Diego River, while open defecation near homeless encampments in the river margins was not a major source. We demonstrated that the combined use of caffeine/sucralose ratios and HF183 and PMMoV holds promise for identifying sewage inputs to surface waters. These findings highlight the need for maintenance and repair of aging sewer infrastructure.

Free chlorine and thermal treatment are used in water disinfection, sludge treatment, and food sterilization processes to inactivate pathogens. Previous research has shown that RNA viruses and bacteriophages can evolve to develop increased resistance to chlorination and thermal disinfection. However, DNA bacteriophages are also commonly used as indicators and surrogates for enteric viruses, but knowledge about their adaptation to disinfection processes is more limited. We repeatedly subjected T7 coliphage to small doses of free chlorine or to heat treatment at 60 degrees C for a long enough time to cause an inactivation of 90-99.9%, then cultivated the surviving phage and assessed the resistance to these treatments to understand if increased resistance emerged. After 10 repeated exposures to free chlorine, T7 coliphage did not develop increased resistance compared with the unexposed strain. However, T7 did develop an increased resistance to thermal treatment at 60 degrees C after four consecutive exposures. The heat-adapted population of T7 had a significantly lower decay rate than the original strain, but its breakpoint temperature (a measure of its thermal stability) did not change. Results from this study provide important implications for scientific and practitioner communities, specifically that not all bacteriophages are equal indicators, proxies, or surrogates for human pathogens in all treatment processes, and caution is warranted when choosing an appropriate indicator. Further research is needed to better understand the influence of virus genome type, mutation rate, and capsid reactivity on virus evolvability to chlorine and heat treatment processes.

The COVID-19 pandemic and the detection of SARS-CoV-2 RNA in sewage has expanded global interest in wastewater surveillance. However, many underserved communities throughout the world lack improved sanitation and use informal combined sanitary and storm sewer systems. Sewage is transported via open channels, ditches, and rivers, where it mixes with surface water and/or stormwater. There is a need to develop better methods for the surveillance of pathogens such as SARS-CoV-2 RNA in this context. We developed a simplified surveillance system and monitored flow rates and concentrations of SARS-CoV-2 RNA in the Tijuana River at two locations downstream of the United States-Mexico border in California, United States. SARS-CoV-2 RNA was detected in the upstream location on six out of eight occasions, two of which were at concentrations as high as those reported in untreated wastewater from California sanitary sewer systems. The virus was not detected in any of the eight samples collected at the downstream (estuarine) sampling location, despite the consistent detection of PMMoV RNA. Synchrony was observed between the number of cases reported in Tijuana and the SARS-CoV-2 RNA concentrations measured with the CDC N1 assay when the latter were normalized by the reported flow rates in the river.

Water and sanitation (wastewater) infrastructure in the United States is aging and deteriorating, with massive underinvestment over the past several decades. For many years, lack of attention to water and sanitation infrastructure has combined with racial segregation and discrimination to produce uneven access to water and wastewater services resulting in growing threats to human and environmental health. In many metropolitan areas in the U.S., those that often suffer disproportionately are residents of low-income, minority communities located in urban disadvantaged unincorporated areas on the margins of major cities. Through the process of underbounding (the selective expansion of city boundaries to exclude certain neighborhoods often based on racial demographics or economics), residents of these communities are disallowed municipal citizenship and live without piped water, sewage lines, and adequate drainage or flood control. This Perspective identifies the range of water and sanitation challenges faced by residents in these communities. We argue that future investment in water and sanitation should prioritize these communities and that interventions need to be culturally context sensitive. As such, approaches to address these problems must not only be technical but also social and give attention to the unique geographic and political setting of local infrastructures.

Sewer overflows and exfiltration can potentially contaminate water bodies with pathogens from wastewater. Microbial source tracking (MST) methods such as the detection of the HF183 gene target of Bacteroides have been proposed to monitor human fecal pollution inputs to surface waters; however, the persistence of HF183 and other MST markers in water flushed from soils after contamination events is not well understood. In this study, the persistence and decay of two culture-based fecal indicators, Escherichia coli and enterococci, and two molecular MST markers, HF183 and pepper mild mottle virus (PMMoV), were evaluated in riverbank soils spiked with untreated sewage, which were left idle for 1, 14, 28, 60, and 121 days under dark conditions and then flushed with synthetic rainwater. All four microbial indicators were still detected in flush water 4 months after the soil was contaminated. PMMoV persisted much longer and had a slower decay rate than the other microbial indicators, and E. coli degraded most rapidly. In consecutive flushing experiments with fresh (1 day) sewage-spiked soils, HF183, E. coli , and enterococci were all detected after 20 consecutive flushes with rainwater, but PMMoV was not detected after the fifth flush. Our findings indicate that water (e.g., stormwater interflow) flushing through riverbank soils that have previously been contaminated by sewer overflows or sewer exfiltration can potentially be a source of microbial pollution to surface waters, even for several months after the contamination occurs. Results from this study also demonstrate the benefits of using multiple human-associated fecal indicators to distinguish pollution from different microbial groups in water bodies. Graphical abstract