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Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci--an important consideration in a public health context. Control assay and delta-delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta-delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms., (Published by Elsevier B.V.)

Urban runoff can carry a variety of pollutants into recreational beaches, often including bacterial pathogens and indicators of fecal contamination. To develop complete recreational criteria and risk assessments, it is necessary to understand conditions under which human contamination could be present at beaches solely impacted by urban runoff. Accurately estimating risk requires understanding sources, concentrations, and transport mechanisms of microbial contaminants in these environments. By applying microbial source tracking methods and empirical modeling, we assessed the presence and level of human contamination at urban runoff impacted recreational beaches. We also identified environmental parameters and pollution sources that can influence the concentration and transport of culturable and molecular fecal indicator bacteria (FIB) in systems impacted solely by urban runoff. Water samples and physico-chemical parameters were collected from shoreline locations from three South Carolina (SC) beaches (five locations per beach) and two Florida (FL) beaches (three locations per beach). Each SC beach was directly impacted by swashes or tidal creeks receiving stormwater runoff from the urbanized area and therefore were designated as swash drain associated (SDA) beaches, while FL beaches were designated as non-swash drain associated (NSDA). Sampling in swash drains (SD; three sites per SD) directly impacting each SC beach was also conducted. Results indicate that although culturable (enterococci) and real-time quantitative polymerase chain reaction (qPCR) (EC23S857, Entero1, and GenBac3) FIB concentrations were, on average, higher at SD locations, SDA beaches did not have consistently higher molecular FIB signals compared to NSDA beaches. Both human-associated markers (HF183 and HumM2) were concomitantly found only at SDA beaches. Bacteroidales species-specific qPCR markers (BsteriF1 and BuniF2) identified differences in the Bacteroidales community, depending on beach type. The marker for general Bacteroidales was most abundant at SD locations and exhibited a high correlation with both culturable and other molecular markers. Combining molecular information with predictive modeling allowed us to identify both alongshore movement of currents and SD outflow as significant influences on the concentration of molecular and culturable indicators in the bathing zone. Data also suggests that combining methodologies is a useful and cost effective approach to help understand transport dynamics of fecal contamination and identify potential sources of contamination at marine beaches., (Published by Elsevier Ltd.)

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r(2), 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Enterococci target sequence density estimates from analyses of diluted river water DNA extracts by EPA Methods 1611 and 1609 and estimates with lower detection limits from undiluted DNA extracts by Method 1609 were indistinguishable. These methods should be equally suitable for comparison with U.S. EPA 2012 Recreational Water Quality Criteria values., (Published by Elsevier B.V.)

Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler(®) II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (ΔCT and ΔΔCT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log(10) unit. Platform had the smallest effect, generally exerting <0.1 log(10) unit difference in final results. Quantification model led to small differences (0.04-0.2 log(10) unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log(10) unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for uncertainty, the high degree of precision attainable by the MPN approach and the need to account for the differences in target sequence recoveries from different calibrator sample cell sources when they are used in the comparative Ct method., (Published by Elsevier B.V.)

There is a growing interest for the use of coliphage as an alternative indicator to assess fecal pollution in recreational waters. Coliphage are a group of viruses that infect Escherichia coli and are considered as potential surrogates to infer the likely presence of enteric viral pathogens. We report the use of a dead-end hollow fiber ultrafiltration single agar layer method to enumerate F+ and somatic coliphage from surface waters collected from three Great Lake areas. At each location, three sites (two beaches; one river) were sampled five days a week over the 2015 beach season (n = 609 total samples). In addition, culturable E. coli and enterococci concentrations, as well as 16 water quality and recreational area parameters were assessed such as rainfall, turbidity, dissolved oxygen, pH, and ultra violet absorbance. Overall, somatic coliphage levels ranged from non-detectable to 4.39 log 10 plaque forming units per liter and were consistently higher compared to F+ (non-detectable to 3.15 log 10  PFU/L), regardless of sampling site. Coliphage concentrations weakly correlated with cultivated fecal indicator bacteria levels ( E. coli and enterococci) at 75% of beach sites tested in study (r = 0.28 to 0.40). In addition, ultraviolet light absorption and water temperature were closely associated with coliphage concentrations, but not fecal indicator bacteria levels suggesting different persistence trends in Great Lake waters between indicator types (bacteria versus virus). Finally, implications for coliphage water quality management and future research directions are discussed. [ABSTRACT FROM AUTHOR]

Summary Genetic markers from Bacteroides and other faecal bacteria are being tested for inclusion in regulations to quantify aquatic faecal contamination and estimate public health risk. For the method to be used quantitatively across environments, persistence and decay of markers must be understood. We measured concentrations of contaminant molecular markers targeting Enterococcus and Bacteroides spp. in marine and freshwater microcosms spiked with human sewage and exposed to either sunlight or dark treatments. We used Bayesian statistics with a delayed Chick-Watson model to estimate kinetic parameters for target decay. DNA- and RNA-based targets decayed at approximately the same rate. Molecular markers persisted (could be detected) longer in marine water. Sunlight increased the decay rates of cultured indicators more than those of molecular markers; sunlight also limited persistence of molecular markers. Within each treatment, Bacteroides markers had similar decay profiles, but some Bacteroides markers significantly differed in decay rates. The role of extracellular DNA in persistence appeared unimportant in the microcosms. Because conditions were controlled, microcosms allowed the effects of specific environmental variables on marker persistence and decay to be measured. While marker decay profiles in more complex environments would be expected to vary from those observed here, the differences we measured suggest that water matrix is an important factor affecting quantitative source tracking and microbial risk assessment applications. [ABSTRACT FROM AUTHOR]

Cultivated fecal indicator bacteria such as Escherichia coli and enterococci are typically used to assess the sanitary quality of recreational waters. However, these indicators suffer from several limitations, such as the length of time needed to obtain results and the fact that they are commensal inhabitants of the gastrointestinal tract of many animals and have fate and transport characteristics dissimilar to pathogenic viruses. Numerous emerging technologies that offer same-day water quality results or pollution source information or that more closely mimic persistence patterns of disease-causing pathogens that may improve water quality management are now available, but data detailing geospatial trends in wastewater across the United States are sparse. We report geospatial trends of cultivated bacteriophage (somatic, F, and total coliphages and GB-124 phage), as well as genetic markers targeting polyomavirus, enterococci, E. coli, Bacteroidetes, and human-associated Bacteroides spp. (HF183/BacR287 and HumM2) in 49 primary influent sewage samples collected from facilities across the contiguous United States. Samples were selected from rural and urban facilities spanning broad latitude, longitude, elevation, and air temperature gradients by using a geographic information system stratified random site selection procedure. Most indicators in sewage demonstrated a remarkable similarity in concentration regardless of location. However, some exhibited predictable shifts in concentration based on either facility elevation or local air temperature. Geospatial patterns identified in this study, or the absence of such patterns, may have several impacts on the direction of future water quality management research, as well as the selection of alternative metrics to estimate sewage pollution on a national scale. [ABSTRACT FROM AUTHOR]

Abstract: The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers. [Copyright &y& Elsevier]