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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.)

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.)

Background: Traditional fecal indicator bacteria (FIB) measurement is too slow (>18 h) for timely swimmer warnings., Objectives: Assess relationship of rapid indicator methods (qPCR) to illness at a marine beach impacted by urban runoff., Methods: We measured baseline and two-week health in 9525 individuals visiting Doheny Beach 2007-08. Illness rates were compared (swimmers vs. non-swimmers). FIB measured by traditional (Enterococcus spp. by EPA Method 1600 or Enterolert™, fecal coliforms, total coliforms) and three rapid qPCR assays for Enterococcus spp. (Taqman, Scorpion-1, Scorpion-2) were compared to health. Primary bacterial source was a creek flowing untreated into ocean; the creek did not reach the ocean when a sand berm formed. This provided a natural experiment for examining FIB-health relationships under varying conditions., Results: We observed significant increases in diarrhea (OR 1.90, 95% CI 1.29-2.80 for swallowing water) and other outcomes in swimmers compared to non-swimmers. Exposure (body immersion, head immersion, swallowed water) was associated with increasing risk of gastrointestinal illness (GI). Daily GI incidence patterns were different: swimmers (2-day peak) and non-swimmers (no peak). With berm-open, we observed associations between GI and traditional and rapid methods for Enterococcus; fewer associations occurred when berm status was not considered., Conclusions: We found increased risk of GI at this urban runoff beach. When FIB source flowed freely (berm-open), several traditional and rapid indicators were related to illness. When FIB source was weak (berm-closed) fewer illness associations were seen. These different relationships under different conditions at a single beach demonstrate the difficulties using these indicators to predict health risk., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Several studies have examined how fecal indicator bacteria (FIB) measurements compare between quantitative PCR (qPCR) and the culture methods it is intended to replace. Here, we extend those studies by examining the stability of that relationship within a beach, as affected by time of day and seasonal variations in source. Enterococcus spp. were quantified at three southern California beaches in the morning and afternoon using two qPCR assays, membrane filtration, and defined-substrate testing. While qPCR and culture-based measurements were consistently and significantly correlated, strength of the correlation varied both among and within beaches. Correlations were higher in the morning (0.45 < ρ < 0.74 [P < 0.002]) than in the afternoon (0.18 < ρ < 0.45 [P < 0.021]) and higher when the fecal contamination was concentrated (0.38 < ρ < 0.83 [P < 0.001]) than when it was diffuse (0.19 < ρ < 0.34 [P < 0.003]). The ratios of culture-based and qPCR results (CFU or most probable number [MPN] per calibrator cell equivalents [CCE]) also varied spatially and temporally. Ratios ranged between 0.04 and 0.85 CFU or MPN per CCE and were lowest at the beach affected by diffuse pollution. Patterns in the ratios over the course of the day were dissimilar across beaches, increasing with time at one beach and decreasing at another. The spatial and temporal variability we observed indicate that the empirical relationship between culture-based and qPCR results is not universal, even within a beach.

Aims: The objective was to assess the impacts of repairing a failing onsite wastewater treatment system (OWTS, i.e., septic system) as related to coastal microbial water quality., Methods and Results: Wastewater, groundwater and surface water were monitored for environmental parameters, faecal indicator bacteria (total coliforms, Escherichia coli, enterococci) and the viral tracer MS2 before and after repairing a failing OWTS. MS2 results using plaque enumeration and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) often agreed, but inhibition limited the qRT-PCR assay sensitivity. Prerepair, MS2 persisted in groundwater and was detected in the nearby creek; postrepair, it was not detected. In groundwater, total coliform concentrations were lower and E. coli was not detected, while enterococci concentrations were similar to prerepair levels. E. coli and enterococci surface water concentrations were elevated both before and after the repair., Conclusions: Repairing the failing OWTS improved groundwater microbial water quality, although persistence of bacteria in surface water suggests that the OWTS was not the singular faecal contributor to adjacent coastal waters. A suite of tracers is needed to fully assess OWTS performance in treating microbial contaminants and related impacts on receiving waters. Molecular methods like qRT-PCR have potential but require optimization., Significance and Impact of Study: This is the first before and after study of a failing OWTS and provides guidance on selection of microbial tracers and methods., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Aims: To examine microbial transport through properly functioning and failing onsite wastewater treatment systems (OWTS) and its implication in surrounding water quality., Methods and Results: Water samples were collected from monitoring wells near leach lines of OWTS and nearby ditches and receiving surface waters to analyse for Escherichia coli and enterococci. Tracer studies with Rhodamine WT (RWT) and coliphage MS2 were also carried out to understand the fate and transport of contaminants through each OWTS. Escherichia coli and enterococci concentrations were higher around failing than properly functioning OWTS by as much as 85-fold. A storm event resulting in 9·5 cm of rainfall increased E. coli and enterococci concentrations by averages of 4·1 × 10³ and 7·9 × 10³ MPN per 100 ml, respectively, in wells close to the OWTS. MS2 persisted in the wastewater distribution boxes of the OWTS for several months and was detected in some outer perimeter wells., Conclusions: Properly functioning OWTS in eastern North Carolina were effective in treating wastewater, whereas the failing OWTS affected the groundwater quality more adversely, especially after a rain storm, but had minor impact on the nearby coastal surface water., Significance and Impact of the Study: The study is the first description of the microbial contaminant signature stemming from properly functioning and failing systems under regular use in a high-priority coastal area., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (r²) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r², 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR.

The Newport River Estuary (NPRE), an important North Carolina (NC) shellfish harvesting area, has been experiencing alterations to the land-water interface due to increasing population and coastal development. Water quality degradation in the estuary over the last decade has led to an increase of shellfish harvesting area closures, and has been postulated to be due to non-point source contamination in the form of stormwater. Water samples were taken in the NPRE (n =179) over a range of weather conditions and all seasons from August 2004 to September 2006. Fecal coliform (FC), as estimated by E. coli (EC), and Enterococcus (ENT) concentrations (MPN per 100 ml) were examined in relation to rainfall levels and distance from land. The relationships among the fecal indicator bacteria (FIB) and environmental parameters were also examined. The data revealed a significant increase in FC concentrations after measured rainfall amounts of 2.54 cm (general threshold) and 3.81 cm (management action threshold). However, higher than expected FIB concentrations existed during conditions of negligible rainfall (<0.25 cm), indicating a possible reservoir population in the sediment. Overall, stormwater runoff appears to be adversely impacting water quality in the NPRE., (Copyright IWA Publishing 2008.)

An assay based on transcription-mediated amplification (TMA) technology was used to quantitate Enterococcus fecal indicator bacteria in environmental water samples. The results generated by this and two growth-based methods relative to the 104 most-probable-number or CFU-per-100-ml threshold show that the three methods are in good qualitative agreement when tested against a range of water samples taken from different locations. The results demonstrate sensitive and rapid detection (approximately 4 h from sample collection to result) and quantitation of Enterococcus bacteria compared to the results with the growth-based methods.

In estuaries, frequent resuspension and deposition of sediment complicate bacterial transport model development by transporting particle-attached bacteria and possibly inducing bacterial responses, such as growth, degradation, or changes in attachment. In order to better characterize these dynamics, observations were made in the Neuse River Estuary (NRE) using the combination of an in situ sampler to monitor the water column and sediment cores to monitor sediment concentrations. Two allochthonous bacteria, Escherichia coli (EC) and Enterococcus sp. (ENT), were selected as proxies for fecal contamination from stormwater runoff. Vibrio sp. (VIB), native to the NRE, was also observed as an autochthonous bacterial group that includes potentially pathogenic species. Two sampling periods were identified as dominated by different suspension types: runoff and resuspension. Despite this difference, several bacterial measures remained comparable between sampling periods. In bottom water, VIB concentration was correlated with salinity and ENT concentration was correlated with turbidity. Differences were observed for EC, where higher concentrations were found in hypoxic waters and sediment during the resuspension period. In the sediment, EC and VIB concentrations significantly increased following the passage of Hurricane Ophelia in September 2005. Throughout this study, all bacterial groups showed evidence of persistence in sediment, suggesting that sediment resuspension represents a significant source of bacteria to the water column.

In July 1999, California's ocean recreational bacterial water quality standards were changed from a total coliform (TC) test to a standard requiring testing for all three bacterial indicators: TC, fecal coliforms (FC), and enterococci (EC). To compare the relationship between the bacterial indicators, and the effect that changing the standards would have on recreational water regulatory actions, three regional studies were conducted along the southern California shoreline from Santa Barbara to San Diego, California. Two studies were conducted during dry weather and one following a large storm event. In each study, samples were collected at over 200 sites which were selected using a stratified random design, with strata consisting of open beach areas and rocky shoreline, and areas near freshwater outlets that drain land-based runoff. During the dry weather studies, samples were collected once per week for 5 weeks. For the storm event study, sampling occurred on a single day about 24 h following the storm. The three indicator bacteria were measured at each site and the results were compared to the single sample standards (TC > 10,000; FC > 400 and EC > 104 MPN or cfu/100 ml). EC was the indicator that failed the single sample standards most often. During the wet weather study, 99% of all standard failures were detected using EC, compared with only 56% for FC, and 40% for TC. During the Summer Study, EC was again the indicator that failed the single sample standards most often, with 60% of the failures for EC alone. The increased failure of the EC standard occurred consistently regardless of whether the sample was collected at a beach or rocky shoreline site, or at a site near a freshwater outlet. Agreement among indicators was better during wet weather than during dry weather. During dry weather, agreement among indicators was better near freshwater outlets than along open shoreline. Cumulatively, our results suggest that replacement of a TC standard with an EC standard will lead to a five-fold increase in failures during dry weather and a doubling of failures during wet weather. Replacing a TC standard with one based on all three indicators will lead to an eight-fold increase in failures. Changes in the requirements for water quality testing have strong implications for increases in beach closures and restrictions., (Copyright 2002 Elsevier Science Ltd.)