Characterization of fecal concentrations in human and other animal sources by physical,culture-based,and quantitative real-time PCR methods

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.     

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were collected during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples collected during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both Campylobacter jejuni mapA and Salmonella invA genes detected in 10% samples collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 10² and 4.3 × 10² genomic copies per 500 ml of water Giardia lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.     

Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide

We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24 d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T₉₉ (two-log reduction) of host-specific Bacteroidales cells was 28 h, whereas that of host-specific Bacteroidales DNA was 177 h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24 d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70 h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450 h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality.     

Evaluation of molecular community analysis methods for discerning fecal sources and human waste

Molecular microbial community analyses provide information on thousands of microorganisms simultaneously, and integrate biotic and abiotic perturbations caused by fecal contamination entering water bodies. A few studies have explored community methods as emerging approaches for microbial source tracking (MST), however, an evaluation of the current state of this approach is lacking. Here, we utilized three types of community-based methods with 64 blind, single- or dual-source, challenge samples generated from 12 sources, including: humans (feces), sewage, septage, dogs, pigs, deer, horses, cows, chickens, gulls, pigeons, and geese. Each source was a composite from multiple donors from four representative geographical regions in California. Methods evaluated included terminal restriction fragment polymorphism (TRFLP), phylogenetic microarray (PhyloChip), and next generation (Illumina) sequencing. These methods correctly identified dominant (or sole) sources in over 90% of the challenge samples, and exhibited excellent specificity regardless of source, rarely detecting a source that was not present in the challenge sample. Sensitivity, however, varied with source and community analysis method. All three methods distinguished septage from human feces and sewage, and identified deer and horse with 100% sensitivity and 100% specificity. Method performance improved if the composition of blind dual-source reference samples were defined by DNA contribution of each single source within the mixture, instead of by Enterococcus colony forming units. Data analysis approach also influenced method performance, indicating the need to standardize data interpretation. Overall, results of this study indicate that community analysis methods hold great promise as they may be used to identify any source, and they are particularly useful for sources that currently do not have, and may never have, a source-specific single marker gene.     

Microbial source tracking using host specific FAME profiles of fecal coliforms

The objective of this study was to investigate the host-specific differences in fatty acid methyl ester (FAME) profiles of fecal coliforms (FC). A known-source library was constructed with 314 FC isolates cultured from 6 possible sources of fecal pollution; 99 isolates from sewage; 29 from bovine; 29 from poultry; 50 from swine; 46 from waterfowl; and 61 from deer. It was found that the hydroxy FAMEs 12:0 2 OH, 12:03 OH, and 14:02 OH were exclusively associated with isolates of human origin. On the other hand, 3 saturated FAMEs, 10:0, 15:0, and 18:0 were found only in isolates from non-human sources, 15:0 being associated with livestock samples only. In addition to the presence of these signature FAMEs, the mean relative masses of 16:1 omega7c and 16:1 ISO/14:03 OH were significantly different between the isolates of human and non-human origins. A linear discriminant function differentiated FC isolates of human origin from those of livestock and wildlife origin at 99% accuracy. These results strongly suggest that the FAME profiles of FC show statistically significant host specificity and may have the potential to be used as a phenotypic microbial source tracking tool.     

Performance of forty-one microbial source tracking methods: A twenty-seven lab evaluation study

The last decade has seen development of numerous new microbial source tracking (MST) methodologies, but many of these have been tested in just a few laboratories with a limited number of fecal samples. This method evaluation study examined the specificity and sensitivity of 41 MST methodologies by analyzing data generated in 27 laboratories. MST methodologies that targeted human, cow, ruminant, dog, gull, pig, horse, and sheep were tested against sewage, septage, human, cow, dog, deer, pig, chicken, pigeon, gull, horse, and goose fecal samples. Each laboratory received 64 blind samples containing a single source (singletons) or two sources (doubletons), as well as diluted singleton samples to assess method sensitivity. Laboratories utilized their own protocols when performing the methods and data were deposited in a central database before samples were unblinded. Between one and seven laboratories tested each method. The most sensitive and specific assays, based on an analysis of presence/absence of each marker in target and non-target fecal samples, were HF183 endpoint and HF183SYBR (human), CF193 and Rum2Bac (ruminant), CowM2 and CowM3 (cow), BacCan (dog), Gull2SYBR and LeeSeaGull (gull), PF163 and pigmtDNA (pig), HoF597 (horse), PhyloChip (pig, horse, chicken, deer), Universal 16S TRFLP (deer), and Bacteroidales 16S TRFLP (pig, horse, chicken, deer); all had sensitivity and specificity higher than 80% in all or the majority of laboratories. When the abundance of MST markers in target and non-target fecal samples was examined, some assays that performed well in the binary analysis were found to not be sensitive enough as median concentrations fell below a minimum abundance criterion (set at 50 copies per colony forming units of enterococci) in target fecal samples. Similarly, some assays that cross-reacted with non-target fecal sources in the binary analysis were found to perform well in a quantitative analysis because the cross-reaction occurred at very low levels. Based on a quantitative analysis, the best performing methods were HF183Taqman and BacH (human), Rum2Bac and BacR (ruminant), LeeSeaGull (gull), and Pig2Bac (pig); no cow or dog-specific assay met the quantitative specificity and sensitivity criteria. Some of the best performing assays in the study were run by just one laboratory so further testing of assay portability is needed. While this study evaluated the marker performance in defined samples, further field testing as well as development of frameworks for fecal source allocation and risk assessment are needed.     

Assessment of sources of human pathogens and fecal contamination in a Florida freshwater lake

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.     

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1–3.3% and 1.9–7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1–2 log₁₀copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.     

Phenotypic characterization of Escherichia coli through whole-cell fatty acid profiling to investigate host specificity

The objective of the study was to investigate whole-cell fatty acid methyl ester (FAME) profiles of 605 Escherichia coli isolates to determine their host specificity. The isolates were cultured from six possible sources of fecal pollution; 180 isolates from sewage, 85 from dairy cow, 98 from chicken, 76 from swine, 94 from deer, and 72 from waterfowl, mostly geese and ducks. The FAME profiles were presented as the relative masses of 12 FAMEs identified in the isolates and it was found that none of the six hosts carried a "signature" FAME, a FAME that is uniquely associated with a particular host category. However, two-sample t-test analyses indicated that the mean relative masses of seven FAMEs out of the 12 identified showed statistically significant differences (95% confidence interval) between isolates of human and non-human origins. In addition, a linear discriminant function based on mean relative mass variations in individual FAMEs classified the known-source isolates into their respective host categories with a 47.6% average rate of correct classification (ARCC) in a six-way discriminant analysis. The ARCC increased to 61.3% when the individual hosts were pooled into larger categories of human, livestock, and wildlife. The accuracy was 75.5% when isolates of human origin were discriminated against those of non-human origins. Random cluster formation analysis indicated that the library size was sufficient to prevent random grouping among the isolates.     

Specificity and sensitivity evaluation of novel and existing Bacteroidales and Bifidobacteria-specific PCR assays on feces and sewage samples and their application for microbial source tracking in Ireland

Three novel ruminant-specific PCR assays, an existing ruminant-specific PCR assay and five existing human-specific PCR assays, which target 16S rDNA from Bacteroidales or Bifidobacteria, were evaluated. The assays were tested on DNA extracted from ruminant (n = 74), human (n = 59) and non-ruminant animal (n = 44) sewage/fecal samples collected in Ireland. The three novel PCR assays compared favourably to the existing ruminant-specific assay, exhibiting sensitivities of 91-100% and specificities of 95-100% as compared to a sensitivity of 95% and specificity of 94%, for the existing ruminant-specific assay. Of the five human-specific PCR assays, the assay targeting the Bifidobacterium catenulatum group was the most promising, exhibiting a sensitivity of 100% (with human sewage samples) and a specificity of 87%. When tested on rural water samples that were naturally contaminated by ruminant feces, the three novel PCR assays tested positive with a much greater percentage (52-87%) of samples than the existing ruminant-specific assay (17%). These novel ruminant-specific assays show promise for microbial source tracking and merit further field testing and specificity evaluation.     

Host-specific 16S rRNA gene markers of Bacteroidales for source tracking of fecal pollution in the subtropical coastal seawater of Hong Kong

This study investigated the diversity of Bacteroidales communities in the feces of eight host species in Hong Kong (subtropical Asia), including human (in the form of sewage), cow, pig, horse, cat, dog, rabbit and rat. The analysis of terminal restriction fragment length polymorphism (TRFLP) in the 16S rRNA genes revealed significant differences in Bacteroidales communities among all host species, with the exception of dog and cat. Manual examination of TRFLP profiles resulted in six terminal restriction fragments (TRFs) that were potentially specific to the sewage (one TRF), cow (three TRFs) or pig (two TRFs) samples. All six TRFs were (1) present in 100% of the samples of the respective target host, (2) absent in other hosts or present only in low frequency and low intensity, and (3) verified for sizes using in silico digestion of DNA sequences in clone libraries. The six TRFs could reliably indicate the source of fecal contaminations in natural seawater amended with sewage, cow or pig fecal samples. In field tests conducted for two polluted and one unpolluted coastal site, the sewage-specific TRF was detected in all seawater samples of the sites known to be impacted by raw and treated sewage. However, only two of three cow-specific TRFs were detected for the two polluted sites, which also received fecal input from feral cows. No pig-specific TRF was detected, although one of the coastal sites was chronically polluted by pig farm run-offs. Nevertheless, the total absence of the six potentially host-specific TRFs in the seawater of an unpolluted site demonstrated the specificity of the TRFs as gene markers in indicating actual pollution.     

Microbial source tracking in a rural watershed dominated by cattle

Antibiotic resistance analysis (ARA), frequency of sampling, and seasonality were evaluated in a rural Virginia watershed dominated by cattle. The selected watershed (Mill Creek) was 3767 ha in size, included two small communities (one sewered and one unsewered), and several farms that when combined contained over 3800 beef and dairy cattle. Monthly monitoring of fecal coliforms at two sampling sites in Mill Creek from January to December, 2001, revealed that the recreational standard (1000 colony forming units, CFUs/100 ml) was exceeded a total of eight times for a 33% violation rate at each site. In addition, stream samples were collected weekly for 4 consecutive weeks during seasonal high flows (March) and seasonal low flows (September-October), plus daily for 7 consecutive days within the weekly schedules for a combined total of 60 stream samples (30 at each of two sites). The recreational standard was exceeded once during seasonal high flow and nine times during seasonal low flow. Microbial source tracking (MST) was performed by ARA to assess the impact of cattle on water quality within the different sampling routines. The resistance patterns of 2880 water isolates and 1158 known source (host-origin) isolates were determined with seven antibiotics at 28 different concentrations. The 1158 isolate database was reduced to 562 unique isolates when clonal ARA patterns were removed. This database of 562 unique isolates had an average rate of correct classification (ARCC) of 95.4%, and several statistical procedures confirmed the library as accurate and representative. Sixty-five percent of 50 challenge-set isolates from sources, but not samples, used in the library were correctly identified. The 562 unique pattern database was used to classify Escherichia coli isolates from water samples into six host source categories. The ARA results showed that cattle were the major source of pollution in the stream and cattle were the dominant source in over 60% of the water samples. Sampling frequency and seasonality had no effect on the MST results, as cattle dominated both seasons and samplings. Deer were a minor contributor in the summer (high water demand), and geese were a minor contributor in the winter when migratory flocks were observed moving through the watershed. An unexpected human allocation was found, especially under seasonal high flow conditions. The exact origin of this human allocation is not known. This project demonstrated that a host-origin library, based on a phenotypic method, could be developed for a well-defined watershed and was both representative of the sources in the watershed and performed reasonably well against a challenge set.     

Detection and remediation of human-origin pollution at two public beaches in Virginia using multiple source tracking methods

Two public beaches (Anderson and Hilton) in Newport News, Virginia, were frequently closed to swimming in 2004 due to high Enterococcus spp. counts that exceeded the regulatory standard. The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners) were used in the summer of 2004 to determine the origins of fecal pollution at the two beaches. Both MST methods detected substantial human-origin pollution at the two beaches, in locations producing consistently high levels of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of the mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE).     

Evaluation of multiple sewage-associated Bacteroides PCR markers for sewage pollution tracking

The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for the detection of sewage pollution in environmental waters in Australia. The HuBac (i.e., 63%) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.     

Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays

Microbial source tracking to distinguish between human, livestock and wildlife fecal pollution using molecular techniques is a rapidly evolving approach in many developed countries, but has not previously been applied on the African continent. DNA extracts from cow, donkey, and human fecal specimens and raw domestic sewage samples collected in Kenya were tested against five existing quantitative PCR assays designed to detect universal (2), human-specific (2), and cow-specific (1) fecal Bacteroidales genetic markers. Water samples from the River Njoro in Kenya were evaluated using the five tested Bacteroidales markers and a multi-species assay for Cryptosporidium in a preliminary exploration of fecal pollution sources and health risks in this watershed. Diagnostic sensitivity on the validation set varied from 18 to 100% for the five assays while diagnostic specificity was 100%. Of the 2 universal assays, Total Bacteroidales [Dick, L.K, Field, K.G., 2004. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Appl. Environ. Microbiol. 70, 5695-5697] showed lower generic fecal diagnostic sensitivity, at 55%, than BacUni-UCD, at 100%, in detecting fecal markers on the 42-sample validation set. Human-specific assay HF183 demonstrated 65% sensitivity overall, and 80% on the human sewage samples, compared to 18% overall and 0% sewage for human-specific assay BacHum-UCD. Cow-specific assay BacCow-UCD had 94% sensitivity. Testing of 18 water samples indicates cows are a likely predominant source of fecal contamination in the Njoro Watershed (78% prevailing rate). Probabilistic assessment of human assay results indicates at most three of the river water samples contained human Bacteroidales. Cryptosporidium spp. markers were detected in samples from nine of the 12 sampling locations. Evidence suggesting widespread contamination by cow feces and Cryptosporidium in the Njoro watershed raises serious concerns for human and animal health.     

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections—augmented with local observations and land-use patterns—indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination.     

Enterococcus and Escherichia coli fecal source apportionment with microbial source tracking genetic markers – Is it feasible?

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm.     

Concentrations of host-specific and generic fecal markers measured by quantitative PCR in raw sewage and fresh animal feces

We measured the concentrations of four host-specific (human, dog, cow, and horse Bacteroidales), four generic fecal (16S total Bacteroidales and Escherichia coli, 23S Enterococcus and uidA E. coli,) and two universal bacterial (16S universal and rpoB universal) DNA targets by qPCR in raw sewage and pooled fecal samples from dogs, cows, horses, and Canada Geese. A spiking protocol using the non-fecal bacterium Pseudomonas syringae pph6 was developed to estimate the recovery of DNA from fecal and environmental samples. The measured fecal marker concentrations were used to calculate baseline ratios and variability of host-specific to generic indicators for each host type. The host-specific markers were found in high concentrations (8-9 log₁₀ copies/g dry wt.) in their respective hosts' samples, which were equal to or greater than the concentrations of generic E. coli and Enterococcus markers, lending support to the use of host-specific and generic Bacteroidales as sensitive indicators of fecal pollution. The host-specific markers formed a consistent percentage of total Bacteroidales in target host feces and raw sewage, with human-specific comprising 82%, dog-specific 6%, cow-specific 4% and horse-specific 2%. Based on this limited data set, the measurement of host-specific indicators by qPCR has several promising applications. These applications include determining the percentage of total Bacteroidales contributed by a specific host type, using the ratios of host-specific markers to E. coli or Enterococcus to estimate the contribution of each source to these regulated fecal indicator bacteria, and estimating the mass of feces from each host type in environmental samples.     

Human-specific fecal bacteria in wastewater treatment plant effluents

The objective of this study was to identify fecal bacteria able to persist after wastewater treatment and that could be used as indicators of human fecal contamination. In a first step, the diversity of Bacteroidales, Clostridiaceae, Bifidobacterium, and Bacillus-Streptococcus-Lactobacillus cluster (BSL) was analysed using a fingerprint technique (CE-SSCP) and 16S rDNA libraries in waters collected at the end of the treatment process in different urban wastewater treatment plants. For each group, dominant bacteria present in most effluents were identified. Their origin (human feces, animal feces, non-fecal) was then analysed based on data of their closest relatives in public 16S rDNA databases. Among fecal bacteria recovered in the treated effluents analysed, phylotypes close to Bifidobacterium adolescentis and Bacteroides caccae seem to be specific to human beings. Phylotypes gathering only sequences of human fecal origin were also identified among the BSL and Clostridiaceae, two bacterial groups which have been poorly investigated for bacterial source-tracking purpose. Since these bacteria were detected post-treatment in most wastewater treatment plants, they may constitute potential new indicators of fecal contamination specific to humans that could be used to track fecal contamination of surface water by sewage.