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Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Amy Pruden reports financial support was provided by National Science Foundation. Amy Pruden reports financial support was provided by Centers for Disease Control and Prevention. Amy Pruden reports financial support was provided by US Bureau of Reclamation. Amy Pruden reports financial support was provided by Hampton Roads Sanitation District. Charles Bott is an an employee of Hampton Roads Sanitation District. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Surface water quality quantitative polymerase chain reaction (qPCR) technologies are expanding from a subject of research to routine environmental and public health laboratory testing. Readily available, reliable reference material is needed to interpret qPCR measurements, particularly across laboratories. Standard Reference Material® 2917 (NIST SRM® 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for estimation of total fecal pollution and identification of key fecal sources. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1008 instrument runs). Using a Bayesian approach, single-instrument run data are combined to generate assay-specific global calibration models allowing for characterization of within- and between-lab variability. Comparable data sets generated by two additional laboratories are used to assess new SRM 2917 data acceptance metrics. SRM 2917 allows for reproducible single-instrument run calibration models across laboratories, regardless of qPCR assay. In addition, global models offer multiple data acceptance metric options that future users can employ to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier Ltd.)

Tropical coastal waters are understudied, despite their ecological and economic importance. They also reflect projected climate change scenarios for other climate zones, e.g., increased rainfall and water temperatures. We conducted an exploratory microbial water quality study at a tropical beach influenced by sewage-contaminated rivers, and tested the hypothesis that fecal microorganisms (fecal coliforms, enterococci, Clostridium perfringens, somatic and male-specific coliphages, pepper mild mottle virus (PMMoV), Bacteroides HF183, norovirus genogroup I (NoVGI), Salmonella, Cryptosporidium and Giardia) would vary by season and tidal stage. Most microorganisms' concentrations were greater in the rainy season; however, NoVGI was only detected in the dry season and Cryptosporidium was the only pathogen most frequently detected in rainy season. Fecal indicator bacteria (FIB) levels exceeded recreational water quality criteria standards in >85% of river samples and in <50% of ocean samples, regardless of the FIB or regulatory criterion. Chronic sewage contamination was demonstrated by detection of HF183 and PMMoV in 100% of river samples, and in >89% of ocean samples. Giardia, Cryptosporidium, Salmonella, and NoVGI were frequently detected in rivers (39%, 39%, 26%, and 39% of samples, respectively), but infrequently in ocean water, particularly during the dry season. Multivariate analysis showed that C. perfringens, somatic coliphage, male-specific coliphage, and PMMoV were the subset of indicators that maximized the correlation with pathogens in the rivers. In the ocean, the best subset of indicators was enterococci, male-specific coliphage, and PMMoV. We also executed redudancy analyses on environmental parameters and microorganim concentrations, and found that rainfall best predicted microbial concentrations. The seasonal interplay of rainfall and pathogen prevalence undoubtedly influences beach users' health risks. Relationships are likely to be complex, with some risk factors increasing and others decreasing each season. Future use of multivariate approaches to better understand linkages among environmental conditions, microbial predictors (fecal indicators and MST markers), and pathogens will improve prediction of high-risk scenarios at recreational beaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Vancomycin resistant enterococci (VRE) cause 20,000 infections annually in the United States, most of which are nosocomial. Recent findings of VRE in sewage-contaminated surface waters demonstrate an alternate route of human exposure, and a possible setting for horizontal gene exchange facilitated by plasmids and other mobile genetic elements. Maintenance of antibiotic resistance genes and proteins may, however, present a fitness cost in the absence of selective pressure, particularly in habitats such as environmental waters that are not optimal for gut-associated bacteria. Nutrient levels, which are transiently elevated following sewage spills, may also affect survival. We tested the hypotheses that nutrients and/or plasmids conferring vancomycin resistance affect Enterococcus faecium survival in river water by measuring decay of strains that differed only by their plasmid, under natural and augmented nutrient conditions. In natural river water, decay rate (log 10 reduction) correlated directly with plasmid size; however, plasmid presence and size had no effect on decay rate when nutrients levels were augmented. Under natural nutrient levels, the vancomycin-resistant strain with the largest plasmid (200 kb) decayed significantly more rapidly than the plasmid-less, susceptible parent strain, in contrast to similar decay rates among strains under augmented nutrient conditions. This work is among the first to show that plasmids conferring antibiotic resistance affect fitness of Enterococcus species in secondary habitats such as surface water. The nutrient-dependent nature of the fitness cost suggests that conveyance of VRE to environmental waters in nutrient-rich sewage may prolong survival of these pathogens, providing greater opportunity for host infection and/or horizontal gene transfer., (Copyright © 2019. Published by Elsevier Ltd.)

Although infectious disease risk from recreational exposure to waterborne pathogens has been an active area of research for decades, beach sand is a relatively unexplored habitat for the persistence of pathogens and fecal indicator bacteria (FIB). Beach sand, biofilms, and water all present unique advantages and challenges to pathogen introduction, growth, and persistence. These dynamics are further complicated by continuous exchange between sand and water habitats. Models of FIB and pathogen fate and transport at beaches can help predict the risk of infectious disease from beach use, but knowledge gaps with respect to decay and growth rates of pathogens in beach habitats impede robust modeling. Climatic variability adds further complexity to predictive modeling because extreme weather events, warming water, and sea level change may increase human exposure to waterborne pathogens and alter relationships between FIB and pathogens. In addition, population growth and urbanization will exacerbate contamination events and increase the potential for human exposure. The cumulative effects of anthropogenic changes will alter microbial population dynamics in beach habitats and the assumptions and relationships used in quantitative microbial risk assessment (QMRA) and process-based models. Here, we review our current understanding of microbial populations and transport dynamics across the sand-water continuum at beaches, how these dynamics can be modeled, and how global change factors (e.g., climate and land use) should be integrated into more accurate beachscape-based models., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

CrAssphage are recently-discovered DNA bacteriophages that are prevalent and abundant in human feces and sewage. We assessed the performance characteristics of a crAssphage quantitative PCR (qPCR) assay for quantifying sewage impacts in stormwater and surface water in subtropical Tampa, Florida. The mean concentrations of crAssphage in untreated sewage ranged from 9.08 to 9.98 log 10 gene copies/L. Specificity was 0.927 against 83 non-human fecal reference samples and the sensitivity was 1.0. Cross-reactivity was observed in DNA extracted from soiled poultry litter but the concentrations were substantially lower than untreated sewage. The presence of the crAssphage marker was monitored in water samples from storm drain outfalls during dry and wet weather conditions in Tampa, Florida. In dry weather conditions, 41.6% of storm drain outfalls samples were positive for the crAssphage marker and the concentrations ranged from 3.60 to 4.65 log 10 gene copies/L of water. After a significant rain event, 66.6% of stormwater outlet samples were positive for the crAssphage marker and the concentration ranged from 3.62 to 4.91 log 10 gene copies/L of water. The presence of the most commonly used Bacteroides HF183 marker in storm drain outfalls was also tested along with the crAssphage. Thirteen samples (55%) were either positive (i.e., both markers were present) or negative (i.e., both markers were absent) for both the markers. Due to the observed cross-reactivity of this marker with DNA extracted from poultry litter samples, it is recommended that this marker should be used in conjunction with additional markers such as HF183. Our data indicate that the crAssphage marker is highly sensitive to sewage, is adequately specific, and will be a valuable addition to the MST toolbox., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Indicator bacteria, which are conventionally used to evaluate recreational water quality, can originate from various non-human enteric and extra-enteric sources, hence they may not be indicative of human health risk nor do they provide information on the sources of contamination. In this study we utilized traditional (enterococci and Escherichia coli) and alternative (Clostridium perfringens) indicator bacteria, F + -specific coliphage, molecular markers for microorganisms associated with human sewage (human-associated Bacteroides and polyomaviruses), and microbial community analysis tools (16S rRNA gene fragment amplicon sequencing), to identify and evaluate human sewage-related impact in the Manoa watershed in Honolulu, Hawaii. Elevated concentrations of enterococci (geometric mean ranging from 1604 to 2575 CFU 100 mL -1 ) and C. perfringens (45-77 CFU 100 mL -1 ) indicated impairment of the urbanized section of the stream, while indicator bacteria concentrations decreased downstream in the tidally influenced Ala Wai Canal. The threshold values triggering water quality violation notifications in Hawaii were exceeded in 33.3-75.0% of samples collected at sites in the urbanized section of Manoa Stream, but were not exceeded in any of the samples collected at an upstream site located in a forested area. Correlation between indicator bacteria concentrations and rainfall amounts was weak to moderate but significant (E. coli R = 0.251, P = 0.009; enterococci R = 0.369, P < 0.001; C. perfringens R = 0.343, P < 0.001), while concentrations of human fecal-associated molecular markers were not significantly correlated with rainfall (human-associated Bacteroides, R = 0.131, P = 0.256; human-associated polyomaviruses, R = 0.213, P = 0.464). Presence of human sewage was confirmed by detection of human-associated Bacteroides and human polyomavirus in the urbanized section of Manoa Stream (83.3-100% and 41.7-66.7% positive samples respectively). It was further confirmed by microbial community analyses which suggested that an average 2.4-3.4% of the total bacterial population in this section was associated with sewage. Microbial community profiles were significantly influenced by rainfall (R 2  = 0.4390, P < 0.001), pH (R 2  = 0.3077, P = 0.006), salinity (R 2  = 0.2614, P = 0.038), and conductivity (R 2  = 0.2676, P = 0.031). Although microbial diversity fluctuated throughout the watershed, it was lower in the impaired section. Leaking sewer systems and illegal cross-connections are implicated in the impairment of the watershed, hence both the sewer and the storm water lines should be routinely inspected. Collectively, our data suggest that information derived from the analysis of microbial communities complements current marker-based microbial source tracking techniques and environmental monitoring programs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Understanding the decomposition of microorganisms associated with different human fecal pollution types is necessary for proper implementation of many water quality management practices, as well as predicting associated public health risks. Here, the decomposition of select cultivated and molecular indicators of fecal pollution originating from fresh human feces, septage, and primary effluent sewage in a subtropical marine environment was assessed over a six day period with an emphasis on the influence of ambient sunlight and indigenous microbiota. Ambient water mixed with each fecal pollution type was placed in dialysis bags and incubated in situ in a submersible aquatic mesocosm. Genetic and cultivated fecal indicators including fecal indicator bacteria (enterococci, E. coli, and Bacteroidales), coliphage (somatic and F+), Bacteroides fragilis phage (GB-124), and human-associated genetic indicators (HF183/BacR287 and HumM2) were measured in each sample. Simple linear regression assessing treatment trends in each pollution type over time showed significant decay (p ≤ 0.05) in most treatments for feces and sewage (27/28 and 32/40, respectively), compared to septage (6/26). A two-way analysis of variance of log 10 reduction values for sewage and feces experiments indicated that treatments differentially impact survival of cultivated bacteria, cultivated phage, and genetic indicators. Findings suggest that sunlight is critical for phage decay, and indigenous microbiota play a lesser role. For bacterial cultivated and genetic indicators, the influence of indigenous microbiota varied by pollution type. This study offers new insights on the decomposition of common human fecal pollution types in a subtropical marine environment with important implications for water quality management applications., (Published by Elsevier Ltd.)

Chicken feces commonly contain human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a previously developed SYBR green qPCR assay (LA35) to a novel TaqMan qPCR assay (CL) for the environmental detection of poultry-associated fecal pollution. We tested both assays against chicken litter (n = 40), chicken fecal samples (n = 186), non-chicken fecal sources (n = 484), and environmental water samples (n = 323). Most chicken litter samples (i.e., ≥ 98%) were positive for both assays with relatively high signal intensities, whereas only 23% and 12% of poultry fecal samples (n = 186) were positive with the LA35 and the CL assays, respectively. Data using fecal samples from non-target animal species showed that the assays are highly host-associated (≥ 95%). Bayesian statistical models showed that the two assays are associated with relatively low probability of false-positive and false-negative signals in water samples. The CL marker had a lower prevalence than the LA35 assay when tested against environmental water samples (i.e., 21% vs. 31% positive signals). However, by combining the results from the two assays the detection levels increased to 41%, suggesting that using multiple assays can improve the detection of chicken-fecal pollution in environmental waters., (Copyright © 2013. Published by Elsevier Ltd.)

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human and ecosystem health can be damaged by fecal contamination of recreational waters. Microbial source tracking (MST) can be used to specifically detect domestic sewage containing human waste, thereby informing both risk assessment and remediation strategies. Previously, an inter-laboratory collaboration developed standardized PCR methods for a bacterial, an archaeal, and a viral indicator of human sewage. Here we present results for two subsequent years of field testing in fresh and salt water by five laboratories across the U.S. Gulf Coast (two in Florida and one each in Mississippi, Louisiana and Texas) using common standard operating procedures (SOPs) developed previously. Culturable enterococci were enumerated by membrane filtration, and PCR was used to detect three MST markers targeting domestic sewage: human-associated Bacteroides (HF183), Methanobrevibacter smithii and human polyomaviruses BK and JC (HPyVs). Detection of sewage markers in surface waters was significantly associated with higher enterococci levels and with exceedance of the recreational water quality standard in four or three regions, respectively. Sewage markers were frequently co-detected in single samples, e.g., M. smithii and HF183 were co-detected in 81% of Louisiana samples, and HPyVs and M. smithii were co-detected in over 40% of southwest Florida and Mississippi samples. This study demonstrates the robustness and inter-laboratory transferability of these three markers for the detection of pollution from domestic sewage in the waters impacting the Gulf of Mexico over a coastal range of over 1000 miles., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We investigated the potential for a variety of environmental reservoirs to harbor or contribute fecal indicator bacteria (FIB), DNA markers of human fecal contamination, and human pathogens to a freshwater lake. We hypothesized that submerged aquatic vegetation (SAV), sediments, and stormwater act as reservoirs and/or provide inputs of FIB and human pathogens to this inland water. Analysis included microbial source tracking (MST) markers of sewage contamination (Enterococcus faecium esp gene, human-associated Bacteroides HF183, and human polyomaviruses), pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses), and FIB (fecal coliforms, Escherichia coli, and enterococci). Bayesian analysis was used to assess relationships among microbial and physicochemical variables. FIB in the water were correlated with concentrations in SAV and sediment. Furthermore, the correlation of antecedent rainfall and major rain events with FIB concentrations and detection of human markers and pathogens points toward multiple reservoirs for microbial contaminants in this system. Although pathogens and human-source markers were detected in 55% and 21% of samples, respectively, markers rarely coincided with pathogen detection. Bayesian analysis revealed that low concentrations (<45 CFU × 100 ml(-1)) of fecal coliforms were associated with 93% probability that pathogens would not be detected; furthermore the Bayes net model showed associations between elevated temperature and rainfall with fecal coliform and enterococci concentrations, but not E. coli. These data indicate that many under-studied matrices (e.g. SAV, sediment, stormwater) are important reservoirs for FIB and potentially human pathogens and demonstrate the usefulness of Bayes net analysis for water quality assessment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Recent evidence of extended survival of fecal indicator bacteria in sediments and submerged aquatic vegetation (SAV) has raised concerns about using indicator bacteria to reliably detect fecal contamination. We monitored enterococci densities and population structure in water, sediment and SAV simultaneously at sites across a subtropical watershed (Tampa Bay, FL, USA) over one year to determine the extent to which each matrix serves as a potential reservoir of enterococci. SAV harbored significantly higher mean densities of enterococci than sediments, which harbored higher densities than water. Mean enterococci densities were also greater at sites located further upstream in the watershed. The population structure assessed by BOX-PCR genotyping was relatively dissimilar in each sample, although some similarities among samples suggested grouping by location. Strain diversity ranged from very high to negligible, with lowest overall diversity in lake samples taken during the summer. Several strains were highly abundant and cosmopolitan (found across sites, seasons, and matrices) and were identified by 16S rRNA gene sequencing as the Enterococcus species casseliflavus, faecalis, faecium, hirae, and mundtii. The proportional dominance of certain strains suggests the existence of persistent and possibly naturalized indicator bacteria populations that are not directly related to pollution events., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10(-5)-10(-6) dilution). LODs for HPyVs and M. smithii assays were similar to each other (10(-3)-10(-4)), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts' workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2.

Bacteria such as fecal coliforms are used as indicators of fecal pollution in natural waters. These bacteria are found in the feces of most wild and domestic animals and thus provide no information as to the source of fecal contamination, yet identification of indicator bacteria sources allows improved risk assessment, remediation, and total daily maximum load (TDML) assessment of environmental waters. This bacterial source tracking study was initiated in order to identify the dominant source(s) of fecal contamination in the urban watershed of Stevenson Creek in Clearwater, Florida. Five sites that represent areas where routine monitoring has previously shown high levels of fecal coliforms were sampled over 7 months. Fecal coliforms were enumerated by membrane filtration, and antibiotic resistance analysis was used to "fingerprint" a subset of randomly selected isolates and statistically match them to fingerprints of fecal coliforms from known sources (the library). A field test of the classification accuracy of the library was carried out by isolating fecal coliforms from the soil and waters surrounding a failing onsite wastewater treatment and disposal system (OSTDS). The vast majority of the isolates were classified into the human category. The major sources of fecal pollution in Stevenson Creek over the course of the study were wild animal, human, and, to a lesser extent, dog. Overall, wild animal feces were identified as the dominant source when fecal coliform levels were high, but when fecal coliform levels were low, the dominant source was identified as human. The results of this study demonstrate that the sources of fecal indicator bacteria within one urban watershed can vary substantially over temporal and spatial distances.