Persistence of sewage-associated genetic markers in advanced and conventional treated recycled water: implications for microbial source tracking in surface waters.

Sewage contamination of environmental waters is increasingly assessed by measuring DNA from sewage-associated microorganisms in microbial source tracking (MST) approaches. However, DNA can persist through wastewater treatment and reach surface waters when treated sewage/recycled water is discharged, which may falsely indicate pollution from untreated sewage. Recycled water discharged from an advanced wastewater treatment (AWT) facility into a Florida stream elevated the sewage-associated HF183 marker 1,000-fold, with a minimal increase in cultured Escherichia coli. The persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities. E. coli (EC23S857) and sewage-associated markers HF183, H8, and viral crAssphage CPQ_056 were always detected in untreated sewage (6.5-8.7 log10 GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4-5 log10 through CWT, but CPQ_056 was ~100-fold more persistent. In AWT facilities, the log10 reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated (P ≤ 0.0136; tau ≥ 0.44); however, CPQ_056 was not correlated with any marker, suggesting varying drivers of decay. Concentrations of cultured E. coli carrying the H8 marker (EcH8) in untreated sewage were 5.24-6.02 log10 CFU/100 mL, while no E. coli was isolated from recycled water. HF183 and culturable EcH8 were also correlated in contaminated surface waters (odds ratio ß1 = 1.701). Culturable EcH8 has a strong potential to differentiate positive MST marker signals arising from treated (e.g., recycled water) and untreated sewage discharged into environmental waters. IMPORTANCE: Genes in sewage-associated microorganisms are widely accepted indicators of sewage pollution in environmental waters. However, DNA persists through wastewater treatment and can reach surface waters when recycled water is discharged, potentially causing false-positive indications of sewage contamination. Previous studies have found that bacterial and viral sewage-associated genes persist through wastewater treatment; however, these studies did not compare different facilities or identify a solution to distinguish sewage from recycled water. In this study, we demonstrated the persistence of bacterial marker genes and the greater persistence of a viral marker gene (CPQ_056 of crAssphage) through varying wastewater treatment facilities. We also aim to provide a tool to confirm sewage contamination in surface waters with recycled water inputs. This work showed that the level of wastewater treatment affects the removal of microorganisms, particularly viruses, and expands our ability to identify sewage in surface waters.

Interlaboratory performance and quantitative PCR data acceptance metrics for NIST 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.

An assessment of three methods for extracting bacterial DNA from beach sand.

AIMS: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. METHODS AND RESULTS: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20-80 gene copies g-1 , recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. CONCLUSIONS: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. SIGNIFICANCE AND IMPACT OF STUDY: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches.

Ubiquity and functional uniformity in CO2 concentrating mechanisms in multiple phyla of Bacteria is suggested by a diversity and prevalence of genes encoding candidate dissolved inorganic carbon transporters.

Autotrophic microorganisms catalyze the entry of dissolved inorganic carbon (DIC; = CO2 + HCO3- + CO32-) into the biological component of the global carbon cycle, despite dramatic differences in DIC abundance and composition in their sometimes extreme environments. "Cyanobacteria" are known to have CO2 concentrating mechanisms (CCMs) to facilitate growth under low CO2 conditions. These CCMs consist of carboxysomes, containing enzymes ribulose 1,5-bisphosphate oxygenase and carbonic anhydrase, partnered to DIC transporters. CCMs and their DIC transporters have been studied in a handful of other prokaryotes, but it was not known how common CCMs were beyond "Cyanobacteria". Since it had previously been noted that genes encoding potential transporters were found neighboring carboxysome loci, α-carboxysome loci were gathered from bacterial genomes, and potential transporter genes neighboring these loci are described here. Members of transporter families whose members all transport DIC (CHC, MDT and Sbt) were common in these neighborhoods, as were members of the SulP transporter family, many of which transport DIC. 109 of 115 taxa with carboxysome loci have some form of DIC transporter encoded in their genomes, suggesting that CCMs consisting of carboxysomes and DIC transporters are widespread not only among "Cyanobacteria", but also among members of "Proteobacteria" and "Actinobacteria".

Quantitative microbial risk assessment of microbial source tracking markers in recreational water contaminated with fresh untreated and secondary treated sewage.

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 × 103 (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 × 103 (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 × 101 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.

Precipitation influences pathogenic bacteria and antibiotic resistance gene abundance in storm drain outfalls in coastal sub-tropical waters.

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 blaKPC gene associated with carbapenem resistant Enterobacteriaceae and the beta-lactam resistant gene (blaNPS) 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.

Application of SourceTracker for Accurate Identification of Fecal Pollution in Recreational Freshwater: A Double-Blinded Study.

The efficacy of SourceTracker software to attribute contamination from a variety of fecal sources spiked into ambient freshwater samples was investigated. Double-blinded samples spiked with ≤5 different sources (0.025-10% vol/vol) were evaluated against fecal taxon libraries characterized by next-generation amplicon sequencing. Three libraries, including an initial library (17 nonlocal sources), a blinded source library (5 local sources), and a composite library (local and nonlocal sources), were used with SourceTracker. SourceTracker's predictions of fecal compositions in samples were made, in part, based on distributions of taxa within abundant genera identified as discriminatory by discriminant analyses but also using a large percentage of low abundance taxa. The initial library showed poor ability to characterize blinded samples, but, using local sources, SourceTracker showed 91% accuracy (31/34) at identifying the presence of source contamination, with two false positives for sewage and one for horse. Furthermore, sink predictions of source contamination were positively correlated (Spearman's ρ ≥ 0.88, P < 0.001) with spiked source volumes. Using the composite library did not significantly affect sink predictions ( P > 0.79) compared to those made using the local sources alone. Results of this study indicate that geographically associated fecal samples are required for SourceTracker to assign host sources accurately.

Evaluation of the novel crAssphage marker for sewage pollution tracking in storm drain outfalls in Tampa, Florida.

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 log10 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 log10 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 log10 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.