Persistence of fecal indicator bacteria and associated genetic markers from wastewater treatment plant effluents in freshwater microcosms.

Limited information exists on the environmental persistence of genetic markers for fecal indicator bacteria (FIB) in treated wastewaters. Here, the decay rate constants of culturable cells and genetic markers for four diverse groups of FIBs, such as enterococci, Clostridium, Escherichia coli, and Bacteroides, were investigated in freshwater microcosms seeded with disinfected and non-disinfected secondary-treated wastewaters. Decay rate constants of genetic markers and culturable cells varied significantly among the different FIB groups. Water temperatures (winter vs. fall/spring/summer) significantly affected the decay of all genetic marker and cell types; however, genetic marker decay were not found to be significantly different in disinfected (chlorination/ultraviolet) and non-disinfected wastewater-seeded microcosms or, for example, lake- and river-receiving waters. No evidence was seen that decay rate constants of FIB genetic markers from treated wastewater were substantially different from those observed in similar, previously reported microcosm studies using raw sewage. Unexpected relationships between decay rate constants of genetic markers and culturable cells of Bacteroides were observed. Results suggest that decay rate constants of FIB genetic markers determined from other studies may be applicable to treated wastewaters. Results of this study should be informative for ongoing efforts to determine the persistence of FIB genetic markers relative to surviving pathogens after wastewater treatment.

Frequent detection of a human fecal indicator in the urban ocean: environmental drivers and covariation with enterococci.

Fecal pollution of surface waters presents a global human health threat. New molecular indicators of fecal pollution have been developed to address shortcomings of traditional culturable fecal indicators. However, there is still little information on their fate and transport in the environment. The present study uses spatially and temporally extensive data on traditional (culturable enterococci, cENT) and molecular (qPCR-enterococci, qENT and human-associated marker, HF183/BacR287) indicator concentrations in marine water surrounding highly-urbanized San Francisco, California, USA to investigate environmental and anthropogenic processes that impact fecal pollution. We constructed multivariable regression models for fecal indicator bacteria at 14 sampling stations. The human marker was detected more frequently in our study than in many other published studies, with detection frequency at some stations as high as 97%. The odds of cENT, qENT, and HF183/BacR287 exceeding health-relevant thresholds were statistically elevated immediately following discharges of partially treated combined sewage, and cENT levels dissipated after approximately 1 day. However, combined sewer discharges were not important predictors of indicator levels typically measured in weekly monitoring samples. Instead, precipitation and solar insolation were important predictors of cENT in weekly samples, while precipitation and water temperature were important predictors of HF183/BacR287 and qENT. The importance of precipitation highlights the significance of untreated storm water as a source of fecal pollution to the urban ocean, even for a city served by a combined sewage system. Sunlight and water temperature likely control persistence of the indicators via photoinactivation and dark decay processes, respectively.

Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

Influence of wastewater disinfection on densities of culturable fecal indicator bacteria and genetic markers.

The US Environmental Protection Agency has proposed the use of quantitative polymerase chain reaction (qPCR) as a rapid alternative analytical method for monitoring recreational water quality at beaches. For qPCR to be considered for other Clean Water Act purposes, such as inclusion in discharge permits and use in Total Maximum Daily Load calculations, it is necessary to understand how qPCR detectable genetic markers are influenced by wastewater disinfection. This study investigated genetic markers for Escherichia coli, Enterococcus, Clostridium spp., Bacteroides, total Bacteroidales, as well as the human-associated Bacteroides markers, HF183 and HumM2, to determine which, if any, were influenced by disinfection (chlorination or ultraviolet light) of effluents from secondary wastewater treatment in different seasons. The effects of disinfection on culturable enterococci, E. coli, Bacteroides, and C. perfringens were also compared to their associated genetic markers. Disinfection of secondary treatment effluents significantly reduced culturable fecal indicator bacteria (FIB) but not genetic marker densities. No significant differences were observed in the responses of FIB culture and genetic marker densities to type of disinfection (chlorination vs UV) or season. Results of this study provide evidence that qPCR may not be suitable for monitoring efficacy of wastewater disinfection on the inactivation of bacterial pathogens.

Water quality, weather and environmental factors associated with fecal indicator organism density in beach sand at two recreational marine beaches.

Recent studies showing an association between fecal indicator organisms (FIOs) in sand and gastrointestinal (GI) illness among beachgoers with sand contact have important public health implications because of the large numbers of people who recreate at beaches and engage in sand contact activities. Yet, factors that influence fecal pollution in beach sand remain unclear. During the 2007 National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study, sand samples were collected at three locations (60 m apart) on weekend days (Sat, Sun) and holidays between June and September at two marine beaches - Fairhope Beach, AL and Goddard Beach, RI - with nearby publicly-owned treatment works (POTWs) outfalls. F(+) coliphage, enterococci, Bacteroidales, fecal Bacteroides spp., and Clostridium spp. were measured in sand using culture and qPCR-based calibrator-cell equivalent methods. Water samples were also collected on the same days, times and transects as the 144 sand samples and were assayed using the same FIO measurements. Weather and environmental data were collected at the time of sample collection. Mean FIO concentrations in sand varied over time, but not space. Enterococci CFU and CCE densities in sand were not correlated, although other FIOs in sand were. The strongest correlation between FIO density in sand and water was fecal Bacteroides CCE, followed by enterococci CFU, Clostridium spp. CCE, and Bacteroidales CCE. Overall, the factors associated with FIO concentrations in sand were related to the sand-water interface (i.e., sand-wetting) and included daily average densities of FIOs in water, rainfall, and wave height. Targeted monitoring that focuses on daily trends of sand FIO variability, combined with information about specific water quality, weather, and environmental factors may inform beach monitoring and management decisions to reduce microbial burdens in beach sand. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.

Human fecal source identification with real-time quantitative PCR.

Waterborne diseases represent a significant public health risk worldwide and can originate from contact with water contaminated with human fecal material. We describe a real-time quantitative PCR (qPCR) method that targets a genetic marker of the human-associated Bacteroides dorei for identification of human fecal pollution in ambient water samples. The following protocol includes water sample collection, filtration, DNA isolation with a sample processing control, qPCR amplification with an internal amplification control, and quality control data analysis.

Fecal indicators in sand, sand contact, and risk of enteric illness among beachgoers.

BACKGROUND: Beach sand can harbor fecal indicator organisms and pathogens, but enteric illness risk associated with sand contact remains unclear. METHODS: In 2007, visitors at 2 recreational marine beaches were asked on the day of their visit about sand contact. Ten to 12 days later, participants answered questions about health symptoms since the visit. F+ coliphage, Enterococcus, Bacteroidales, fecal Bacteroides, and Clostridium spp. in wet sand were measured using culture and molecular methods. RESULTS: We analyzed 144 wet sand samples and completed 4999 interviews. Adjusted odds ratios (aORs) were computed, comparing those in the highest tertile of fecal indicator exposure with those who reported no sand contact. Among those digging in sand compared with those not digging in sand, a molecular measure of Enterococcus spp. (calibrator cell equivalents/g) in sand was positively associated with gastrointestinal (GI) illness (aOR = 2.0 [95% confidence interval (CI) = 1.2-3.2]) and diarrhea (2.4 [1.4-4.2]). Among those buried in sand, point estimates were greater for GI illness (3.3 [1.3-7.9]) and diarrhea (4.9 [1.8-13]). Positive associations were also observed for culture-based Enterococcus (colony-forming units/g) with GI illness (aOR digging = 1.7 [1.1-2.7]) and diarrhea (2.1 [1.3-3.4]). Associations were not found among nonswimmers with sand exposure. CONCLUSIONS: We observed a positive relationship between sand-contact activities and enteric illness as a function of concentrations of fecal microbial pollution in beach sand.

Rapidly measured indicators of recreational water quality and swimming-associated illness at marine beaches: a prospective cohort study.

INTRODUCTION: In the United States and elsewhere, recreational water quality is monitored for fecal indicator bacteria to help prevent swimming-associated illnesses. Standard methods to measure these bacteria take at least 24 hours to obtain results. Molecular approaches such as quantitative polymerase chain reaction (qPCR) can estimate these bacteria faster, in under 3 hours. Previously, we demonstrated that measurements of the fecal indicator bacteria Enterococcus using qPCR were associated with gastrointestinal (GI) illness among swimmers at freshwater beaches. In this paper, we report on results from three marine beach sites. METHODS: We interviewed beach-goers and collected water samples at marine beaches affected by treated sewage discharges in Mississippi in 2005, and Rhode Island and Alabama in 2007. Ten to twelve days later, we obtained information about gastrointestinal, respiratory, eye, ear and skin symptoms by telephone. We tested water samples for fecal indicator organisms using qPCR and other methods. RESULTS: We enrolled 6,350 beach-goers. The occurrence of GI illness among swimmers was associated with a log10-increase in exposure to qPCR-determined estimates of fecal indicator organisms in the genus Enterococcus (AOR = 2.6, 95% CI 1.3-5.1) and order Bacteroidales (AOR = 1.9, 95% CI 1.3-2.9). Estimates of organisms related to Clostridium perfringens and a subgroup of organisms in the genus Bacteroides were also determined by qPCR in 2007, as was F+ coliphage, but relationships between these indicators and illness were not statistically significant. CONCLUSIONS: This study provides the first evidence of a relationship between gastrointestinal illness and estimates of fecal indicator organisms determined by qPCR at marine beaches.

Improved strategies and optimization of calibration models for real-time PCR absolute quantification.

Real-time PCR absolute quantification applications are becoming more common in the recreational and drinking water quality industries. Many methods rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, the generation of a standard curve for each qPCR experiment set-up can be expensive and time consuming, especially for studies with large numbers of unknown samples. As a result, many researchers have adopted a master calibration strategy where a single curve is derived from DNA standard measurements generated from multiple instrument runs. However, a master curve can inflate uncertainty associated with intercept and slope parameters and decrease the accuracy of unknown sample DNA target concentration estimates. Here we report two alternative strategies termed 'pooled' and 'mixed' for the generation of calibration equations from absolute standard curves which can help reduce the cost and time of laboratory testing, as well as the uncertainty in calibration model parameter estimates. In this study, four different strategies for generating calibration models were compared based on a series of repeated experiments for two different qPCR assays using a Monte Carlo Markov Chain method. The hierarchical Bayesian approach allowed for the comparison of uncertainty in intercept and slope model parameters and the optimization of experiment design. Data suggests that the 'pooled' model can reduce uncertainty in both slope and intercept parameter estimates compared to the traditional single curve approach. In addition, the 'mixed' model achieved uncertainty estimates similar to the 'single' model while increasing the number of available reaction wells per instrument run.

Relationship and variation of qPCR and culturable Enterococci estimates in ambient surface waters are predictable.

The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P < 0.05) at most sites. A linearly decreasing variance of CCE with increasing CFU levels was significant (P < 0.05) or evident for all sites. Both marine and freshwater sites under continuous influence of point-source contamination tended to reveal a relatively constant proportion of CCE to CFU. The consistency in the mean and variance patterns of CCE versus CFU indicates that the relationship of results based on these two methods is more predictable at high CFU levels (e.g., log(10)CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

Deposition of glomalin-related soil protein and sequestered toxic metals into watersheds.

We hypothesized that glomalin-related soil proteins (GRSP) are eroded in sufficient quantities to influence metal loading into watersheds. We tested correlations among GRSP, Glomeromycota fungi, and metals (cadmium, iron, lead, and manganese) in proteins extracted from embankment soils at seven locations along an urban/coastal watershed. Immunoreactive (IRSP) and easily extractable (EE-IRSP) glomalin ranged from 0.007 to 2.9 mg g(-1) and from 0.006 to 0.63 mg g(-1) of soils, respectively. Glomalin-bound metals (microg mg(-1) protein) were Cd = 0.00-0.338; Fe = 0.5-227.7; Pb = 0.11-188.95; Mn = 2.23-784.42). Glomeromycota fungi were detected in 24% of all samples tested with PCR targeting the 18S and 28S ribosomal DNA extracted from soils. Specific assay for G. intraradices showed 3.08 x 10(7) copies g(-1) and 1.96 x 10(3) copies g(-1) of soil at two sites. Estimated annual glomalin loading into the watershed ranged from 5.48 x 10(2) to 7.22 x 10(4) kg of IRSP and from 2.57 x 102 to 2.86 x 10(4) kg of EE-IRSP; including 2.7 kg (Cd), 6.3 x 10(3) kg (Fe), 5.06 x 10(2) kg (Pb), and 1.80 x 10(3) kg (Mn). These data provide insights into the sources and ecological fate of a ubiquitous soil protein and its metals content.

Occurrence of genes associated with enterotoxigenic and enterohemorrhagic Escherichia coli in agricultural waste lagoons.

The prevalence among all Escherichia coli bacteria of the LTIIa toxin gene and STII toxin gene, both associated with enterotoxigenic E. coli, and of three genes (stxI, stxII, and eaeA) associated with enterohemorrhagic E. coli was determined in farm waste disposal systems seasonally for 1 year. Single- and nested-PCR results for the number of E. coli isolates carrying each toxin gene trait were compared with a five-replicate most-probable-number (MPN) method. The STII and LTIIa toxin genes were present continuously at all farms and downstream waters that were tested. Nested-MPN-PCR manifested sensitivity increased over that of single-MPN-PCR by a factor of 32 for LTIIa, 10 for STII, and 2 for the stxI, stxII, and eaeA genes. The geometric mean prevalence of each toxin gene within the E. coli community in waste disposal site waters after nested MPN-PCR was 1:8.5 E. coli isolates (1:8.5 E. coli) for the LTIIa toxin gene and 1:4 E. coli for the STII toxin gene. The geometric mean prevalence for the simultaneous occurrence of toxin genes stxI, stxII, and eaeA, was 1:182 E. coli. These findings based on total population analysis suggest that prevalence rates for these genes are higher than previously reported in studies based on surveys of single isolates. With a population-based approach, the frequency of each toxin gene at the corresponding disposal sites and the endemic nature of diseases on farms can be easily assessed, allowing farmers and public health officials to evaluate the risk of infection to animals or humans.

A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking.

Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal samples. Researchers who used community terminal restriction fragment length polymorphism (T-RFLP) were limited by the amount of DNA recovered from samples, but they correctly identified human and cattle fecal contamination when sufficient DNA was obtained. Culture independent methods show considerable promise; further research is needed to develop markers for additional fecal sources and to understand the correlation of these source-tracking indicators to measures of human and environmental health.