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Abstract: 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 &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Rapid isolation of Salmonella from food is essential for faster typing and source tracking in an outbreak. The objective of this study was to investigate a rapid isolation method that would augment the standard U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) method. Food samples with low microbial load, including egg salad and ice cream, moderately high-microbial-load tomatoes, and high-microbial-load ground beef were intentionally inoculated with 2 to 48 CFU of Salmonella enterica serotype Typhimurium. The samples were preenriched in buffered peptone water for 6 h, and then selectively concentrated by immunomagnetic separation and plated for isolation on xylose-lysine-desoxycholate agar: the 6IX method. Salmonella Typhimurium was presumptively identified from approximately 97% of the low-microbial-load and moderately high-microbial-load samples by the 6IX method 2 days before the BAM standard method for isolation of Salmonella. In 49% of the beef samples, Salmonella Typhimurium was presumptively identified 1 or 2 days earlier by the 6IX method. Given the inocula used, our data clearly indicated that for most of the food samples tested, with the exception of ground beef, Salmonella Typhimurium could be isolated two laboratory days earlier with the 6IX method compared with the BAM method. In conclusion, this 6IX method may expedite Salmonella isolation and, therefore, has the potential to accelerate strain tracking for epidemiological analysis in a foodborne outbreak. [ABSTRACT FROM AUTHOR]

The gram-negative bacterium Vibrio vulnificus is a natural inhabitant of estuarine waters and poses a significant health threat to humans who suffer from immune disorders, liver disease, or hemochromatosis (iron overload). V. vulnificus enters human hosts via wound infections or consumption of raw shellfish (primarily oysters), and infections frequently progress to septicemia and death in susceptible individuals. Prevalence in waters and shellfish is not correlated with fecal indicator organisms; therefore, species-specific detection and enumeration of V. vulnificus in the environment has become a priority for agencies that are responsible for shellfish safety. The many selective-differential media developed for isolation of Vibrio spp., and specifically for V. vulnificus detection, are reviewed here; however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms. Therefore, immunological and molecular protocols are needed for confirmation of the identity of the organism and are discussed in detail. Methods under development that hold promise for rapid, accurate, and sensitive detection and enumeration of the organism include multiplex and real-time PCR. Developing technologies that have proven useful for detection and investigation of other pathogens such as biosensors, spectroscopy and microarrays may provide the next generation of tools for investigation of the prevalence and ecology of V. vulnificus. [Copyright &y& Elsevier]

Huntington Beach, California, one of the most popular surfing spots in the world, is plagued by sporadic, elevated levels of fecal bacteria. To assist with pollution source identification, we analyzed antibiotic resistance patterns (ARPs) of enterococci from four known sources (bird feces, urban runoff, coastal marsh sediment and sewage effluent from local sanitation district) and one unknown source (seawater) using seven antibiotics at four concentrations each. Of 2491 enteroccoci tested, all were resistant to at least one antibiotic at some level. Discriminant analysis indicated that the average correct classification rates for bird feces and urban runoff sources were above 80%. Sewage effluent contained mixed fecal sources. Sixty-four percent of the sewage isolates classified with the sewage category, while the other 35% of isolates were assigned evenly across the other three categories. When enterococci isolated from the seawater were classified using the known ARP database, it was evident that bird feces were the source of surf zone contamination on some days while the coastal salt marsh and sewage plume may have impacted the surf zone water quality to various degrees during other times. [Copyright &y& Elsevier]

Environmental conditions influence fecal indicator bacteria (FIB) levels, which are routinely used to characterize recreational water quality. This study examined 15 years of environmental and FIB data at Puntarenas and Jacó beach, Costa Rica. FIB relationships with sea level, wave height, precipitation, direct normal irradiance (DNI), wind, and turbidity were analyzed. Pearson's correlations identified lags between 24 and 96 h among environmental parameters and FIB. Multiple linear regression models composed of environmental parameters explained 24% and 27% of fecal coliforms and enterococci variability in Jacó, respectively. Puntarenas's models explained 17–26% of fecal coliforms and 12–18% enterococci variability. Precipitation, sea level anomalies, and wave height most frequently explained FIB variability. Hypothesis testing often identified significant differences in precipitation, wave height, daily sea level anomalies, and maximum sea level 24 h prior between days with and without FIB threshold exceedance. Unexpected FIB interactions with DNI, sea level, and turbidity highlight the importance of future investigations. Unlabelled Image • Environmental parameters explained 12 to 27% of FIB geometric mean variability. • Precipitation, sea level anomalies, and wave height most frequently explained FIB variability. • Higher precipitation was associated with exceedance of FIB thresholds. • Greater wave height was associated with exceedance of FIB thresholds. • Greater maximum sea level 24 h prior was associated with exceedance of FIB thresholds. [ABSTRACT FROM AUTHOR]

• Metagenomic and culture methods revealed fate of ARG/ARB in water reuse systems. • Activated sludge removed the majority of ARB/ARGs compared to tertiary treatments. • Ozonation resulted in enrichment of multidrug ARGs. • Non-potable reuse distribution systems are vulnerable to ARG/ARB regrowth. • Resistome risk is reduced by chlorination and lowest in the recharged aquifer. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Abstract: Propidium monoazide (PMA) was used to differentiate live from membrane-compromised bacteria in PCR methods. We have adapted this technique for use on membrane-filtered water samples and determined its efficacy using qPCR. Independent labs at three institutions replicated these findings. [Copyright &y& Elsevier]

Escherichia coli (E. coli) is frequently used in assessment and regulation of recreational water quality, but it is a general fecal indicator that provides no information about fecal contamination source. Sewage-associated microorganisms and related marker genes have proven useful for microbial source tracking (MST) applications that link fecal contamination to host sources, but many MST marker genes are carried in taxa not used in regulatory contexts. A more direct connection with regulatory concerns, including human health risk and total maximum daily load (TMDL) assessments, could be accomplished with tools such as the human-associated marker genes of E. coli. We evaluated the performance of E. coli H8, H12, H14, and H24 marker genes for detection of domestic sewage at the E. coli isolate level in Florida. E. coli isolates (n = 1, 380) from reference fecal and wastewater samples were first tested by binary PCR for the presence of each H marker gene. H8 and H12 were >90% specific and sensitive for domestic sewage, while H14 and H24 were ≤86% specific. Therefore, quantitative PCR (qPCR) assays were used to quantify H8 and H12 marker genes at the sample level. Specificity values for the H8 and H12 qPCR assays were 96 and 93%, respectively, while both marker genes showed 100% sensitivity. H12 concentrations were tenfold lower in wastewater than H8 (~6–7 log 10 gene copies (GC)/100 mL). H8 concentrations in wastewater and contaminated environmental water samples were correlated with the sewage-associated Bacteroides HF183 marker gene. This study suggests that E. coli H genes, and H8 in particular, can be useful for sewage contamination tracking and TMDL development in subtropical waters. Unlabelled Image • Performance characteristics of E. coli H gene assays were tested by analysing fecal samples. • H8 and H12 were >90% specific and sensitive, and were developed into qPCR assays. • H8 qPCR was superior for field study based on performance and concentration in sewage. • H8 correlated with the human-associated Bacteroides HF183 in contaminated samples. • Direct connection with E. coli makes H8 qPCR a superior new tool for TMDL assessment. [ABSTRACT FROM AUTHOR]

Abstract Fecal contamination of recreational waters with cattle manure can pose a risk to public health due to the potential presence of various zoonotic pathogens. Fecal indicator bacteria (FIB) have a long history of use in the assessment of recreational water quality, but FIB quantification provides no information about pollution sources. Microbial source tracking (MST) markers have been developed in response to a need to identify pollution sources, yet factors that influence their decay in ambient waters are often poorly understood. We investigated the influence of water type (freshwater versus marine) and select environmental parameters (indigenous microbiota, ambient sunlight) on the decay of FIB and MST markers originating from cattle manure. Experiments were conducted in situ using a submersible aquatic mesocosm containing dialysis bags filled with a mixture of cattle manure and ambient water. Culturable FIB (E. coli , enterococci) were enumerated by membrane filtration and general fecal indicator bacteria (GenBac3, Entero1a, EC23S857) and MST markers (Rum2Bac, CowM2, CowM3) were estimated by qPCR. Water type was the most significant factor influencing decay (three-way ANOVA, p : 0.006 to <0.001), although the magnitude of the effect differed among microbial targets and over time. The presence of indigenous microbiota and exposure to sunlight were significantly correlated (three-way ANOVA, p : 0.044 to <0.001) with decay of enterococci and CowM2, while E. coli , EC23S857, Rum2Bac, and CowM3 (three-way ANOVA, p : 0.044 < 0.001) were significantly impacted by sunlight or indigenous microbiota. Results indicate extended persistence of both cultivated FIB and genetic markers in marine and freshwater water types. Findings suggest that multiple environmental stressors are important determinants of FIB and MST marker persistence, but their magnitude can vary across indicators. Selective exclusion of natural aquatic microbiota and/or sunlight typically resulted in extended survival, but the effect was minor and limited to select microbial targets. Graphical abstract Unlabelled Image Highlights • In situ decay comparison of eight cattle manure derived water quality indicators. • Extended persistence of all indicators of bovine fecal pollution was noted. • In general, decay was most influenced by water type (freshwater versus marine). • Sunlight and microbiota effects were minimal and varied across different indicators. [ABSTRACT FROM AUTHOR]

Microbial source tracking (MST) methods have provided the means to identify sewage contamination in recreational waters, but the risk associated with elevated levels of MST targets such as sewage-associated Bacteroides HF183 and other markers is uncertain. Quantitative microbial risk assessment (QMRA) modeling allows interpretation of MST data in the context of the risk of gastrointestinal (GI) illness caused by exposure to pathogens. In this study, five sewage-associated, quantitative PCR (qPCR) MST markers [ Bacteroides HF183 (HF183), Methanobrevibacter smithii nifH ( nifH ), human adenovirus (HAdV), human polyomavirus (HPyV) and pepper mild mottle virus (PMMoV)] were evaluated to determine at what concentration these nucleic acid markers reflected a significant health risk from exposure to fresh untreated or secondary treated sewage in beach water. The QMRA models were evaluated for a target probability of illness of 36 GI illnesses/1000 swimming events (i.e., risk benchmark 0.036) for the reference pathogens norovirus (NoV) and human adenovirus 40/41 (HAdV 40/41). Sewage markers at several dilutions exceeded the risk benchmark for reference pathogens NoV and HAdV 40/41. HF183 concentrations 3.22 × 10 3 (for both NoV and HAdV 40/41) gene copies (GC)/100 mL of water contaminated with fresh untreated sewage represented risk >0.036. Similarly, HF183 concentrations 3.66 × 10 3 (for NoV and HAdV 40/41) GC/100 mL of water contaminated with secondary treated sewage represented risk >0.036. HAdV concentration as low as 4.11 × 10 1 GC/100 mL of water represented risk >0.036 when water was contaminated with secondary treated sewage. Results of this study provide a valuable context for water quality managers to evaluate human health risks associated with contamination from fresh sewage. The approach described here may also be useful in the future for evaluating health risks from contamination with aged or treated sewage or feces from other animal sources as more data are made available. [ABSTRACT FROM AUTHOR]

Stormwater contamination can threaten the health of aquatic ecosystems and human exposed to runoff via nutrient and pathogen influxes. In this study, the concentrations of 11 bacterial pathogens and 47 antibiotic resistance genes (ARGs) were determined by using high-throughput microfluidic qPCR (MFQPCR) in several storm drain outfalls (SDOs) during dry and wet weather in Tampa Bay, Florida, USA. Data generated in this study were also compared with the levels of fecal indicator bacteria (FIB) and sewage-associated molecular markers (i.e., Bacteroides HF183 and crAssphage markers) in same SDOs collected in a recent study ( Ahmed et al., 2018 ). Concentration of FIB, sewage-associated markers, bacterial pathogens and many ARGs in water samples were relatively high and SDOs may be potentially hotspots for microbial contamination in Tampa Bay. Mean concentrations of culturable E. coli and Enterococcus spp. were tenfold higher in wet compared to dry weather. The majority of microbiological contaminants followed this trend. E. coli eaeA , encoding the virulence factor intimin, was correlated with levels of 20 ARGs, and was more frequently detected in wet weather than dry weather samples. The bla KPC gene associated with carbapenem resistant Enterobacteriaceae and the beta-lactam resistant gene ( bla NPS ) were only detected in wet weather samples. Frequency of integron genes Intl2 and Intl3 detection increased by 42% in wet weather samples. Culturable E. coli and Enterococcus spp. significantly correlated with 19 of 47 (40%) ARG tested. Sewage-associated markers crAssphage and HF183 significantly correlated ( p  < 0.05) with the following ARGs: intl1 , sul1 , tet (M), ampC , mexB , and tet (W). The presence of sewage-associated marker genes along with ARGs associated with sewage suggested that aging sewage infrastructure contributed to contaminant loading in the Bay. Further research should focus on collecting spatial and temporal data on the microbiological contaminants especially viruses in SDOs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Millions of tons of fecal-contaminated poultry litter are applied to U.S. agricultural fields annually. Precipitation and irrigation facilitate transport of fecal-derived pathogens and fecal indicator bacteria (FIB) to groundwater. The goal of this study was to compare transport of pathogens, FIB, and a microbial source tracking marker gene for poultry litter (LA35) in a simulated soil-to-groundwater system. Nine laboratory soil columns containing four different soil types were used to evaluate microbial transport to groundwater via infiltration. Quantitative polymerase chain reaction was used to monitor Salmonella enterica Typhimurium, Escherichia coli , Enterococcus spp., Brevibacterium sp. LA35 and Bacteroidales leached from soil columns inoculated with poultry litter. S . enterica was correlated with LA35 poultry litter marker gene and FIB concentrations in column soils containing organic matter, but not in acid washed sands. In contrast, S . enterica was found to correlate with LA35 and FIB in the leachate from columns containing sand, but not with leachate from organic soil columns. The majority of recovered DNA was found in leachate of predominately sandy soil columns, and in the soil of loamy columns. At least 90% of the DNA retained in soils for each microbial target was found in the top 3 cm of the column. These studies suggest that poultry litter associated pathogens and FIB are rapidly released from litter, but are influenced by complex attenuation mechanisms during infiltration, including soil type. This study advances our understanding of the potential for subsurface transport of poultry litter associated pathogens and FIB, and support the use of the LA35 marker gene for evaluating poultry litter impacts on groundwater. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• SRM 2917 performance is evaluated across 16 labs with 12 qPCR assays. • Calibration model linearity (R 2) was ≥ 0.992 regardless of lab or assay. • Acceptable amplification efficiencies observed in 99.5% of single run models. • Global model within-lab variability was ≤ between-lab for each assay. • Novel data acceptance metrics are proposed for SRM 2917. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: The debate over the suitability of molecular biological methods for the enumeration of regulatory microbial parameters (e.g. Faecal Indicator Organisms [FIOs]) in bathing waters versus the use of traditional culture-based methods is of current interest to regulators and the science community. Culture-based methods require a 24–48hour turn-around time from receipt at the laboratory to reporting, whilst quantitative molecular tools provide a more rapid assay (approximately 2–3h). Traditional culturing methods are therefore often viewed as slow and ‘out-dated’, although they still deliver an internationally ‘accepted’ evidence-base. In contrast, molecular tools have the potential for rapid analysis and their operational utility and associated limitations and uncertainties should be assessed in light of their use for regulatory monitoring. Here we report on the recommendations from a series of international workshops, chaired by a UK Working Group (WG) comprised of scientists, regulators, policy makers and other stakeholders, which explored and interrogated both molecular (principally quantitative polymerase chain reaction [qPCR]) and culture-based tools for FIO monitoring under the European Bathing Water Directive. Through detailed analysis of policy implications, regulatory barriers, stakeholder engagement, and the needs of the end-user, the WG identified a series of key concerns that require critical appraisal before a potential shift from culture-based approaches to the employment of molecular biological methods for bathing water regulation could be justified. [Copyright &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

• Rainfall was the best predictor of microbial variables when river and ocean data were separated. • Sewage indicators HF183 and PMMoV were detected in >88% of samples. • Norovirus was detected only during dry season; Cryptosporidium was more prevalent in rainy season. • Multiple indicators, particularly viruses, correlated with pathogens; single indicators did not. • Multivariate analysis identified relationships of environmental and indicator variables with pathogens. 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. Image, graphical abstract [ABSTRACT FROM AUTHOR]

The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic acids in a variety of sample types. These methods share dependence on primers and probes with quantitative PCR, but they differ in the specific enzymes and instruments employed, and are frequently conducted in a binary, rather than quantitative format. IAMs typically rely on simpler instruments than PCR analyses due to the maintenance of a single temperature throughout the amplification reaction, which could facilitate deployment of IAMs in a variety of environmental and field settings. This review summarizes the mechanisms of the most common IAM methods and their use in studies of pathogens, harmful algae and fecal indicators in environmental waters, feces, wastewater, reclaimed water, and tissues of aquatic animals. Performance metrics of sensitivity, specificity and limit of detection are highlighted, and the potential for use in monitoring and regulatory contexts is discussed. • Isothermal amplification methods (IAMs) are an alternative to qPCR • IAMs are less expensive than qPCR, but quantification is less developed • Additional comparisons of IAMs and PCR performance metrics are needed • IAMs offer a promising field-deployable screening tool for microorganisms [ABSTRACT FROM AUTHOR]

The use of microbial source tracking (MST) marker genes has grown in recent years due to the need to attribute point and non-point fecal contamination to specific sources. Quantitative microbial risk assessment (QMRA) is a modeling approach used to estimate health risks from exposure to feces-contaminated water and associated pathogens. A combination of these approaches [quantitative MST (qMST) and QMRA] can provide additional pathogen-related information for prioritizing and addressing health risks, compared to reliance on conventional fecal indicator bacteria (FIB). To inform expansion of this approach, a review of published qMST-QMRA studies was conducted to summarize the state of the science and to identify research needs. The reviewed studies primarily aimed to identify what levels of MST marker genes in hypothetical recreational waterbodies would exceed the United States Environmental Protection Agency (USEPA) risk benchmarks for primary contact recreators. The QMRA models calculated relationships between MST marker gene(s) and reference pathogens based on published data in the literature. The development of a robust, accurate relationship was identified as an urgent research gap for qMST-QMRA. This metric requires additional knowledge to quantify the relationship between MST marker genes and the degree of variability in decay of pathogens as a dynamic function of environmental conditions and combinations of fecal sources at multiple spatial and temporal scales. Improved characterization of host shedding rates of host-associated microorganisms (i.e., MST marker genes), as well as fate and transport of these microorganisms and their nucleic acids, would facilitate expansion of this approach to other exposure pathways. Incorporation of information regarding the recovery efficiency, and host-specificity of MST marker genes into QMRA model parameters, and the sensitivity analysis, would greatly improve risk management and site-specific water monitoring criteria. • qMST and QMRA can provide information for prioritizing health risks. • Accurate relationship was identified as an urgent research gap for qMST-QMRA. • Recovery efficiency, host-specificity and sensitivity analysis need to be considered for QMRA model. [ABSTRACT FROM AUTHOR]