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.     

Antimicrobial resistance of fecal indicators in municipal wastewater treatment plant

Antimicrobial resistance of fecal coliforms (n = 153) and enterococci (n = 199) isolates was investigated in municipal wastewater treatment plant (WWTP) based on activated sludge system. The number of fecal indicators (in influent and effluent as well as in the aeration chamber and in return activated sludge mixture) was determined using selective media. Susceptibility of selected strains was tested against 19 (aminoglycosides, aztreonam, carbapenems, cephalosporins, β-lactam/β-lactamase inhibitors, fluoroquinolones, penicillines, tetracycline and trimethoprim/sulfamethoxazole) and 17 (high-level aminoglycosides, ampicillin, chloramphenicol, erythromycin, fluoroquinolones, glycopeptides, linezolid, lincosamides, nitrofuration, streptogramins, tetracycline) antimicrobial agents respectively. Among enterococci the predominant species were Enterococcus faecium (60.8%) and Enterococcus faecalis (22.1%), while remaining isolates belonged to Enterococcus hirae (12.1%), Enterococcus casseliflavus/gallinarum (4.5%), and Enterococcus durans (0.5%). Resistance to nitrofuration and erythromycin was common among enterococci (53% and 44%, respectively), and followed by resistance to ciprofloxacin (29%) and tetracycline (20%). The resistance phenotypes related to glycopeptides (up to 3.2%) and high-level aminoglycosides (up to 5.4%) were also observed. Most frequently, among Escherichia coli isolates the resistance patterns were found for ampicillin (34%), piperacillin (24%) and tetracycline (23%). Extended-spectrum β-lactamase producing E. coli was detected once, in the aeration chamber. In the study the applied wastewater treatment processes considerably reduced the number of fecal indicators. Nevertheless their number in the WWTP effluent was higher than 10⁴ CFU per 100 ml and periodically contained 90% of bacteria with antimicrobial resistance patterns. The positive selection of isolates with antimicrobial resistance patterns was observed during the treatment processes. Substantial concern should be paid to the isolates resistant to 3 or more chemical classes of antimicrobials (MAR). In treated wastewater MAR E. coli and MAR enterococci constituted respectively 9% and 29% of tested isolates.     

Identification of the sources of fecal coliforms in an urban watershed using antibiotic resistance analysis

Bacteria such as fecal coliforms are used as indicators of fecal pollution in natural waters. These bacteria are found in the feces of most wild and domestic animals and thus provide no information as to the source of fecal contamination, yet identification of indicator bacteria sources allows improved risk assessment, remediation, and total daily maximum load (TDML) assessment of environmental waters. This bacterial source tracking study was initiated in order to identify the dominant source(s) of fecal contamination in the urban watershed of Stevenson Creek in Clearwater, Florida. Five sites that represent areas where routine monitoring has previously shown high levels of fecal coliforms were sampled over 7 months. Fecal coliforms were enumerated by membrane filtration, and antibiotic resistance analysis was used to "fingerprint" a subset of randomly selected isolates and statistically match them to fingerprints of fecal coliforms from known sources (the library). A field test of the classification accuracy of the library was carried out by isolating fecal coliforms from the soil and waters surrounding a failing onsite wastewater treatment and disposal system (OSTDS). The vast majority of the isolates were classified into the human category. The major sources of fecal pollution in Stevenson Creek over the course of the study were wild animal, human, and, to a lesser extent, dog. Overall, wild animal feces were identified as the dominant source when fecal coliform levels were high, but when fecal coliform levels were low, the dominant source was identified as human. The results of this study demonstrate that the sources of fecal indicator bacteria within one urban watershed can vary substantially over temporal and spatial distances.     

Characterization of Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution in recreational waters

Enterophages are a novel group of phages that specifically infect Enterococcus faecalis and have been recently isolated from environmental water samples. Although enterophages have not been conclusively linked to human fecal pollution, we are currently characterizing enterophages to propose them as viral indicators and possible surrogates of enteric viruses in recreational waters. Little is known about the morphological or genetic diversity which will have an impact on their potential as markers of human fecal contamination. In the present study we are determining if enterophages can be grouped by their ability to replicate at different temperatures, and if different groups are present in the feces of different animals. As one of the main objectives is to determine if these phages can be used as indicators of the presence of enteric viruses, the survival rate under different conditions was also determined as was their prevalence in sewage and a large watershed. Coliphages were used as a means of comparison in the prevalence and survival studies. Results indicated that the isolates are mainly DNA viruses. Their morphology as well as their ability to form viral plaques at different temperatures indicates that several groups of enterophages are present in the environment. Coliphage and enterophage concentrations throughout the watershed were lower than those of thermotolerant coliforms and enterococci. Enterophage concentrations were lower than coliphages at all sampling points. Enterophages showed diverse inactivation rates and T₉₀ values across different incubation temperatures in both fresh and marine waters and sand. Further molecular characterization of enterophages may allow us to develop probes for the real-time detection of these alternative indicators of human fecal pollution.     

Using an intervening sequence of Faecalibacterium 16S rDNA to identify poultry feces

This study was designed to identify poultry feces-specific marker(s) within sequences of Faecalibacterium 16S rDNA for detecting poultry fecal pollution in water. Bioinformatics tools were used in the comparative analysis of 7,458 sequences of Faecalibacterium 16S rDNA, reportedly associated with various poultry (chicken and turkey) and animal species. One intervening sequence (IVS) within between the hypervariable region 1 and the conserved region 2, designated as IVS-p, was found to be unique to poultry feces. Based on this sequence, a PCR assay (PCR-p) was developed. The PCR-p produced an amplicon of 132 bp only in the test when fecal or wastewater samples from poultry were used, but not when using fecal or wastewater samples from other sources. The non-poultry sources included feces of beef or dairy cattle, dog, horse, human, domestic or wild geese, seagull, sheep, swine, and wild turkey. These data indicate that IVS-p may prove to be a useful genetic marker for the specific identification of poultry fecal pollution in environmental waterways. Furthermore, results of data mining and PCR assay indicate that the IVS-p may have a broad geographic distribution. This report represents initial evidence of the potential utility of ribosomal intervening sequences as genetic markers for tracking host sources of fecal pollution in waterways.     

Deer diet affects ribotype diversity of Escherichia coli for bacterial source tracking

Ribotyping is one of a number of genotypic methods for bacterial source tracking. This method requires a host origin database of one bacterial species be established in order to identify environmental isolates. Researchers establishing these databases have observed considerable ribotype diversity within a specific bacterial species. One source of this diversity may be diet. We determined the effect of diet on ribotype diversity for Escherichia coli in penned and wild deer (Odocoileus virginianus) in a 13-ha forested watershed. A total of 298 E. coli isolates was obtained, 100 from penned deer, 100 from wild deer, and 98 from the stream in the watershed to which all deer had access. The wild deer had significantly more ribotypes (35) than the penned deer (11 ribotypes, p = 0.05). This result suggests that diet affected ribotype diversity, and that a host origin database for bacterial source tracking should contain bacterial isolates from wild rather than from captive animals. Also, 42 of 98 (42.9%) environmental isolates matched penned and wild deer ribotypes. If bacterial source tracking determines that fecal contamination is predominantly from wildlife, then it may be unnecessary to monitor these watersheds because control over wildlife is difficult.     

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.     

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.     

Multiple lines of evidence to identify the sources of fecal pollution at a freshwater beach in Hamilton Harbour, Lake Ontario

Multiple microbial source-tracking methods were investigated to determine the source of elevated Escherichia coli levels at Bayfront Park Beach in Hamilton Harbour, Lake Ontario. E. coli concentrations were highest in wet foreshore sand (114,000 CFU/g dry sand) and ankle-depth water (177,000 CFU/100mL), declining rapidly in deeper waters. Many gull and geese droppings were enumerated each week on the foreshore sand within 2m of the waterline. Both antimicrobial resistance analysis and rep-PCR DNA fingerprinting of E. coli collected at the beach and nearby fecal pollution sources indicated that E. coli in sand and water samples were predominantly from bird droppings rather than from pet droppings or municipal wastewater. Both methods indicated a trend of decreasing bird contamination, and increasing wastewater contamination, moving offshore from the beach. When foreshore sand was treated as a reservoir and secondary source of E. coli, waterborne E. coli were found to be more similar to sand isolates than bird or wastewater isolates out to 150 m offshore. Multiple lines of evidence indicated the importance of bird droppings and foreshore sand as primary and secondary sources of E. coli contamination in beach water at Bayfront Park.     

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.     

Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed

Our ability to identify and eliminate fecal contamination of water, now and in the future, is essential to reduce incidences of waterborne disease. Bacterial source tracking is a recently developed approach for identifying sources of fecal pollution. PCR primers designed by Bernhard and Field [Bernhard, A.E., Field, K.G., 2000a. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66(10), 4571-4574] and Dick et al. [Dick, L.K., Bernhard, A.E., Brodeur, T.J., Santo Domingo, J.W., Simpson, J.M., Walters, S.P., Field, K.G., 2005. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71(6), 3184-3191] for the detection of human (HF183), pig (PF163) and ruminant (CF128) specific Bacteroidales 16s rRNA genetic markers were tested for their suitability in detecting fecal pollution in Saskatchewan, Canada. The sensitivity and specificity of these primers were assessed by testing eight raw human sewage samples and 265 feces from 12 different species in Saskatchewan. The specificity of each primer set was ≥94%. The accuracy of HF183 and PF163 to distinguish between the different species was 100%, whereas CF128 cross-reacted with 22% of the pig feces. Occurrence of the host-specific Bacteroidales markers and the conventional indicator Escherichia coli in relation to several enteropathogens was investigated in 70 water samples collected from different sites along the Qu'Appelle River (Saskatchewan, Canada). Human and ruminant fecal markers were identified in 41 and 14% of the water samples, respectively, whereas the pig marker was never detected in the river water. The largest concentrations in E. coli counts were concomitant to the simultaneous detection of HF183 and CF128. Thermotolerant Campylobacter spp., Salmonella spp. and Shiga toxin genes (stx1 and stx2)-positive E. coli (STEC) were detected in 6, 7 and 63% of the water samples, respectively. However, none of the stx positive water samples were positive for the E. coli O157:H7 gene marker (uidA). Odds ratios analysis suggests that CF128 may be predictive for the presence of Salmonella spp. in the river investigated. None of the fecal indicators were able to confidently predict the presence of thermotolerant Campylobacter spp. and STEC.     

Variability of fecal indicator bacteria in flowing and ponded waters in southern California: implications for bacterial TMDL development and implementation

Recreational water quality is assessed by using water quality objectives for fecal indicator bacteria (FIB) including total coliform, fecal coliform (or E. coli), and/or Enterococcus. It is required under the Clean Water Act that a TMDL be developed for a bacteria-impaired water body. The development and implementation of bacterial TMDLs has proven challenging and often difficult due to unknown source(s) of FIB. This study found that FIB levels varied significantly in flowing water, ponded water, and associated sediment. FIB levels in isolated ponded water in waterways were significantly higher than in flowing water. Sediment under ponded water contained a great amount of FIB. Furthermore, FIB concentrations in ponded water tended to increase with increasing water temperature and to decrease with increasing water salinity. The result provides the field evidence of survival/growth of FIB in water and sediment under ambient conditions in southern California. A holistic approach including natural sources (e.g., a reference system) should be considered for practical and applicable purposes while developing and implementing bacterial TMDLs for pathogen-impaired waterbodies.     

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.     

Fecal bacteria and sex hormones in soil and runoff from cropped watersheds amended with poultry litter

The application of poultry litter to agricultural fields can provide plant nutrients for crops and forage production, but fecal bacteria and the sex hormones estradiol and testosterone are components of litter that can be detrimental to the environment. Our objective was to determine if applications of poultry litter to small watersheds would contribute to the load of fecal bacteria and sex hormones to soil and runoff. We, therefore, investigated the fate and transport of fecal bacteria, estradiol and testosterone from surface applied poultry litter to four small cropped watersheds. Poultry litter was applied to meet the nitrogen requirements of pearl millet (Pennisetum glaucum L.) in 2000 and grain sorghum [Sorghum bicolor (L.) Moench] in 2001. Neither Salmonella nor Campylobacter were detected in the litter but the fecal indicator bacteria were. The average load of total coliforms, Escherichia coli, and fecal enterococci applied with the litter was 12.2, 11.9, and 12.7 log(10) cells ha(-1), respectively. The average load of estradiol and testosterone was 3.1 and 0.09 mg ha(-1), respectively. Runoff events first occurred 7 months after the first litter application in 2000, and 3 weeks after the second application in 2001. Only for the 25 July 2001 runoff event 3 weeks after the second litter application were the concentrations of total coliforms, E. coli, and fecal enterococci in runoff greater than background concentrations which were on average 5.2, 2.9, and 1.1 log(10) MPN 100 ml(-1), respectively. Average background levels of total coliforms, fecal enterococci, and E. coli in surface soil were 8.2, 7.9, and 3.5 log(10) cells kg(-1) soil. At the rate of litter application the concentrations of estradiol and testosterone in the litter did not appear to impact the background levels in the soil and runoff. Because concentrations of sex hormones in litter from other broiler operations are known to be greater than in the litter we applied, further study on the connection between concentrations of sex hormones in poultry litter and operational practices is recommended.     

Identification of chicken-specific fecal microbial sequences using a metagenomic approach

In this study, we applied a genome fragment enrichment (GFE) method to select for genomic regions that differ among different fecal metagenomes. Competitive DNA hybridizations were performed between chicken fecal DNA and pig fecal DNA (CP) and between chicken fecal DNA and an avian DNA composite consisting of turkey, goose, and seagull fecal DNA extracts (CB) to enrich for chicken-specific DNA fragments. A total of 471 non-redundant chicken metagenomic sequences were retrieved and analyzed. All of the clone sequences were similar to prokaryotic genes, of which more than 60% could not be assigned to previously characterized functional roles. In general terms, sequences assigned characterized functional roles were associated with cellular processes (11.7%), metabolism (11.0%) and information storage and processing (13.4%). Approximately 53% of the non-redundant sequences are similar to genes present in intestinal bacteria belonging to Clostridia (20.9%), Bacteroidetes (15.0%), and Bacilli (17.3%). Twenty-five sequences from the CP and CB clone libraries were selected to develop chicken fecal-specific PCR assays. These assays were challenged against fecal DNA extracted from 21 different animal species, including mammals and birds. The results from the host-specificity studies showed that 12 of the assays had a high degree of specificity to chicken feces. In addition, three assays were specific to chicken and turkey while another four assays tested positive to more than two avian species, suggesting a broader distribution of some of the enriched gene fragments among different avian fecal microbial communities. Fecal pollution signals were detected using chicken-specific assays in contaminated water samples, although the PCR assays showed different detection limits. These results indicate the need for multiple assays to detect poultry fecal sources of pollution. The competitive DNA hybridization approach used in this study can rapidly select for numerous chicken fecal metagenomic regions that can be used as potential genetic markers for fecal source tracking.     

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory,comparative study

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10–50 gene copies or plaques × 50 ml⁻¹) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40–70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent.     

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.     

Use of salinity mixing models to estimate the contribution of creek water fecal indicator bacteria to an estuarine environment: Newport Bay, California

The contribution of freshwater discharge to fecal indicator bacteria (FIB) impairment of an estuarine environment can be approximated from simple, two end-member mixing models using salinity as a tracer. We conducted a yearlong time series investigation of Newport Bay, a regionally important estuarine embayment in southern California, assessing the concentrations of FIB, specifically Escherichia coli and enterococci bacteria, and salinity. In total, eight within-bay stations and one offshore control site were sampled nearly once per week and the three tributaries draining into Newport Bay were sampled approximately daily. Using salinity as a conservative tracer for water mass mixing and determining the end-member values of FIB in both the creek sites and the offshore site, we created a linear, two end-member mixing model of FIB within Newport Bay. Deviations from the mixing model suggest either an additional source of FIB to the bay (e.g. bird feces, storm drain discharge) or regrowth and/or die-off of FIB within the bay. Our results indicate that salinity mixing models can be useful in predicting changes in FIB concentrations in the estuarine environments and can help narrow the search for sources of FIB to the bay and enhance our understanding of the fate of FIB within the bay.     

Evaluation of a quantitative H2S MPN test for fecal microbes analysis of water using biochemical and molecular identification

The sensitivity and specificity of the H₂S test to detect fecal bacteria in water has been variable and uncertain in previous studies, partly due to its presence-absence results. Furthermore, in groundwater samples false-positive results have been reported, with H₂S-positive samples containing no fecal coliforms or Escherichia coli. False-negative results also have been reported in other studies, with H₂S-negative samples found to contain E. coli. Using biochemical and molecular methods and a novel quantitative test format, this research identified the types and numbers of microbial community members present in natural water samples, including fecal indicators and pathogens as well as other bacteria. Representative water sources tested in this study included cistern rainwater, a protected lake, and wells in agricultural and forest settings. Samples from quantitative H₂S tests of water were further cultured for fecal bacteria by spread plating onto the selective media for detection and isolation of Aeromonas spp., E. coli, Clostridium spp., H₂S-producers, and species of Salmonella and Shigella. Isolates were then tested for H₂S production, and identified to the genus and species level using biochemical methods. Terminal Restriction Fragment Length Polymorphisms (TRFLP) was the molecular method employed to quantitatively characterize microbial community diversity. Overall, it was shown that water samples testing positive for H₂S bacteria also had bacteria of likely fecal origin and waters containing fecal pathogens also were positive for H₂S bacteria. Of the microorganisms isolated from natural water, greater than 70 percent were identified using TRFLP analysis to reveal a relatively stable group of organisms whose community composition differed with water source and over time. These results further document the validity of the H₂S test for detecting and quantifying fecal contamination of water.     

Spatial and temporal variability of ribotyping results at a small watershed in South Carolina

The utility of library-based ribotyping methods for a very small study area was evaluated through comparison of local results to libraries with differing spatial and temporal scales. Ribotyping of Escherichia coli isolates was used to evaluate sources of fecal pollution at a coastal golf course in Beaufort County, South Carolina. Thirty-five E. coli isolates were obtained from water samples from a detention pond for testing against several local and regional libraries of known-source isolate patterns. A library of 92 E. coli ribotype patterns was created from wildlife feces obtained on the site. Additional libraries were available for comparison, including a library from Morgan Island, a small, geographically isolated area (including a monkey colony), and a library from ongoing statewide assessments. Seventeen (49%) of the unknown E. coli isolates matched isolates from raccoon and deer scat from the local library. Two isolates (6%) were matched with monkey sources from Morgan Island, and 13 (37%) were matched to raccoon, deer, and cows from the statewide assessment. Evaluation of repeated ribotyping analyses at the study area revealed evidence of temporal variability of potential sources in the local library. Only one of the isolates from the second year of fecal samples successfully matched with a fecal isolate from the previous year. The results from this study suggest that source identification results were variable both spatially and temporally, and that local, temporally specific libraries are most appropriate for library-based MST studies in small watersheds. Results also suggest that it will be difficult to employ adequate sample sizes to satisfactorily address unknown pattern variability.