Final Report: Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in Water

EPA Grant Number: R827639
Title: Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in Water
Investigators: Field, Katharine G.
Institution: Oregon State University
EPA Project Officer: Hahn, Intaek
Project Period: November 1, 1999 through October 31, 2002
Project Amount: $223,829
RFA: Ecological Indicators (1999) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Ecosystems

Objective:

Fecal contamination of water is widespread in the United States, causing illness and beach closures, impacting shellfish harvest, and degrading habitat. Often the problem cannot be corrected, because the specific source of the fecal contamination cannot be identified. Potential sources include leaking septic and sewage systems, stormwater runoff, runoff from agricultural wastes, and wildlife. Federal legislation requires total maximum daily loads (TMDLs) for bacteria to be established in watersheds to achieve or maintain water quality. To mitigate pollution and establish TMDLs, water managers must be able to identify the sources of bacterial contamination. The objective of this research project was to develop a new method to identify the source of fecal contamination in water, based on polymerase chain reaction (PCR) amplification of 16S rDNA markers from uncultured members of the Bacteroidetes group of fecal anaerobes. We concentrated on markers for more species and quantitative methods. Our field study site was Tillamook Bay, Oregon, a major shellfish-growing area with significant bacterial impairment throughout the watershed. Past research found that some groups of Bacteroidetes bacteria have a host-species-specific or host-group-specific distribution. Thus, molecular markers from these groups can be used diagnostically. This method differs from other methods of fecal source identification in that it does not require culturing bacteria, and is therefore rapid, not subject to culture bias, and does not require a "library" of representative isolates from various kinds of feces.

Summary/Accomplishments (Outputs/Outcomes):

We constructed 16S rDNA clone libraries of uncultured Bacteroidetes from the feces of elk, pig, dog, cat, gull, and horse. We screened the clone libraries by restriction analysis, sequenced representatives of each restriction pattern, and performed phylogenetic analysis on these sequences along with sequences from previous studies. Most of the sequences fell into two major branches representing the genera Bacteroides and Prevotella. Many of the human, dog, cat, and gull sequences were closely related to each other. Some were closely related to known Bacteroides species, including B. vulgatus, B. uniformis, B. thetaiotaomicron, and B. stercoris. This means that, in general, known Bacteroides species are not good indicators to distinguish animal and human fecal pollution. None of the cloned sequences was closely related to any cultivated Prevotella species. A group of human, cat, and dog Prevotella sequences was very closely related to each other. The majority of the pig clones were most closely related to oral Prevotellas. Horse sequences produced two unique clusters, one within the Prevotella branch, and the other of less certain lineage. All but one of the ruminant sequences fell outside either the Bacteroides or Prevotella branches of the tree and did not cluster with any database sequences other than those recovered from our previous work in Tillamook Bay, OR. The sequence analyses were used to design PCR primers from each of the host species. Although there was a large amount of overlap among human, dog, and cat sequences, both of our original PCR human-specific primers did not amplify any sequences from dog and cat. Two new primers, PF163F (pig) and HoF597F (horse), when paired with a general Bacteroidetes primer, specifically amplified fecal DNAs from pig and horse, respectively, and did not amplify DNAs from any other feces tested. Primers designed from gull, cat, dog, and elk sequences were not specific. The most likely reason for this is that our clone libraries did not recover all of the genetic diversity of uncultured Bacteroidetes in feces (coverage was incomplete). We concluded that it is not practical to construct clone libraries large enough to sample the entire range of Bacteroidetes sequence diversity found within each host species, because of the large cost associated with sequencing and the diminishing return of unique groups as more sequences were analyzed. Nevertheless, the patterns of diversity indicated that there was substantial endemism within the Bacteroidetes group; sequence variability within this group was very great.

We adapted the technique of subtractive hybridization to find new host-specific markers without the generation of a clone library for each new host species. This is the first time this method has been applied to the problem of fecal source identification. We hybridized Bacteroidetes rDNA from a source of interest for primer design (target) with one or more reference sources (subtracters) in microplate wells, resulting in an enrichment of unique (nonhybridized) sequences. Because our clone library analyses proved unable to design primers to distinguish wild (elk) feces from domestic (cattle) ruminant feces, and likewise proved to be unable to distinguish dog feces from human feces, we chose these groups for our subtractive hybridization experiments. Experiments with dog fecal DNA (hybridized with cat and human subtracters) and elk fecal DNA (hybridized with human and cattle subtracters) resulted in the recovery of unique sequences that were used for primer design. A new elk primer, EF990, successfully distinguished elk from cattle and human feces, the subtracters used in the experiment. The primer's specificity extended to some host sources not used in the experiment (it did not amplify pig and deer fecal DNAs), but not to all hosts (sheep amplified with the primer). A new dog primer, DF475, did not amplify fecal DNAs from human, cat, cattle, pig, or gull feces. Thus, microplate subtractive hybridization was successful as a method of generating unique source-specific markers. The capacity to characterize markers that distinguish sources of fecal pollution without obtaining large numbers of clones for each new host will expedite the addition of new source markers for fecal pollution.

Current methods of detecting fecal contamination in water based on cultures of indicator bacteria are slow, taking a minimum of 1 to 2 days. As a result, measures to protect public health, such as beach closures and shellfish harvest advisories, are implemented considerably after the fact of fecal pollution, and people inadvertently may be exposed during the interval between the occurrence of fecal pollution and its detection. In addition, by the time these measures are put into effect, the water may no longer be contaminated, resulting in unnecessary beach closures and shellfish advisories and causing economic implications. We have designed real-time quantitative PCR (Q-PCR) taq nuclease (TaqMan) assays for both general Bacteroidetes (to measure total fecal pollution) and human fecal Bacteroidetes. Our objectives were to develop a rapid method of water quality assessment and to develop a method of quantifying the contribution of different sources to fecal pollution in water. We tested our Q-PCR assay for Bacteroidetes using serial dilutions of human sewage influent. There was a linear decrease in Bacteroidetes 16S rDNA copy numbers as a function of serially diluting the sewage influent. The decrease remained linear over seven orders of magnitude. The linearity of the dilution curve showed that neither PCR inhibitors nor the presence of large amounts of heterologous DNA inhibited amplification, even at the highest copy numbers. Bacteroidetes 16S rDNA copy numbers were compared to coliform and Escherichia coli most probable numbers (MPNs) in sewage dilutions. Simple linear correlation analysis of the 10-4 to 10-7 dilutions showed that mean Bacteroidetes concentrations were highly correlated with both coliform and E. coli mean concentrations (r values of 0.999 for both comparisons). This dilution range contains the U.S. Environmental Protection Agency (EPA) threshold concentration of E. coli in ambient water, above which the health risk from waterborne illness is deemed unacceptably high (5-day geometric mean of 126 organisms/100 mL). The Q-PCR assay for Bacteroidetes 16S rDNA was rapid, sensitive, and reproducible. It correlated with current standard indicators, but took only 3 to 4 hours to complete. The quantitative range spanned eight orders of magnitude, compared with approximately four orders for E. coli enumeration. This Q-PCR assay provides a framework for expanding the use of Bacteroidetes PCR indicators beyond fecal source tracking and into a health risk-based analysis of fecal pollution.

To test our methods, we collected water samples from sites in Tillamook Bay and its tributaries, from agricultural, urban, or rural land use. We analyzed DNA extracted from filters with five PCR primer pairs specific for human, ruminant, or general Bacteroidetes markers. All but one of the urban sites and sites near sewage treatment plants tested positive for human feces. Human markers were absent from sites not influenced by sewage treatment plants or urban land use. Ruminant feces occurred at all of the sites in dairy farming areas, and in the upper and middle estuary, but not in the lower estuary and mouth of the bay. We detected one of the markers for ruminant feces more frequently than the second in saline waters; differential survival of the bacterial groups carrying the two markers is the most likely explanation. Triplicate water samples were analyzed for total coliforms (TC), fecal coliforms (FC), E. coli (EC), and the presence/absence of Bacteroidetes PCR markers. The molecular approach was less variable than the standard public health indicators. We tested whether adding duplicate or triplicate samples provided significant improvements in the detection of Bacteroidetes at or above the bacterial threshold levels (14 colony forming units [CFU] FC in shellfish waters; 200 CFU FC in recreational waters; and 126 CFU EC in recreational waters). At an EC cutoff of 126 CFU, (the estimate of the proportion of the sample sets that would have been improved by adding another sample) was equal to 4.3 ± 0.8 percent. Adding a second EC sample would have brought the mean number of EC CFU above the threshold to 2.1 ± 0.6 percent. Adding a third sample gave no improvement. Results were similar for FC and Bacteroidetes. Thus, the improvement from adding replicate samples was less than 5 percent; balancing cost against improvement, we concluded that single samples were sufficient. We noted, however, that was negatively correlated with the number of samples; thus, in studies where the total number of samples will be low, it will remain important to take replicate samples. In a 2-year analysis of water samples from 28 sites in the Tillamook Bay watershed, we found that the watershed had human fecal contamination in a few sites concentrated around the town of Tillamook that were strongly correlated with the distance from point sources of human contamination. Ruminant fecal pollution was present throughout the watershed, especially in the rivers where dairy farms were concentrated, and in the bay. Our study provided evidence for extensive downstream transport of ruminant fecal contamination, and identified areas where human and ruminant fecal pollution could be mitigated. This will improve water quality in Tillamook Bay and reduce the number of days the bay is closed for shellfish harvest.

The Southern California Coastal Water Research Project and the U.S. EPA sponsored a comparative source identification study. Study participants, using a variety of phenotypic and genotypic methods currently in use, were asked to identify the fecal source(s) in water samples containing human, cattle, dog, or gull feces; sewage; or a mixture. Methods were assessed according to their ability to identify whether samples contained or did not contain human feces, to identify each fecal source, and handle freshwater and saltwater samples as well as samples with humic acids. Most of the methods had a significant number of false positives and/or failed to correctly identify the samples. The methods that performed the best were our methods based on PCR of Bacteroidetes markers, ribotyping, and pulse field gel electrophoresis (PFGE). Of these three, the Bacteroidetes method is by far the most rapid and least expensive.

We wrote and tested a quality assurance/quality control procedure for water testing using our approach and communicated our methods to several user groups around the world, including collaborators from the Cincinnati EPA, Health Canada, and the European Union and helped several laboratories establish our methods. We are collaborating with the U.S. EPA (Cincinnati, OH) in an epidemiological study using our rapid quantitative assay for fecal pollution based on Bacteroidetes 16S rDNA markers.

Overall, this project has been very productive, resulting in 13 publications from personnel involved in the project, more than 20 public presentations, and the training of one Ph.D. student (Linda K. Dick) and seven undergraduates (Amanda Barry, Michael Pester, Kristine Gerbus, Michael Acevedo, Melissa Younger, Rebecca Cooper, and Thierry Goyard). In addition, other graduate students (Sarah Walters, Thomas Jones, Caragwen Bracken) and postdoctorates (Michael Simonich and Orin Shanks), although not directly supported by this grant, were involved in and benefited from the research.

Impact

Fecal contamination of water is widespread in the United States. It affects public health and causes streams and rivers to be listed, public beaches to be closed, and shellfisheries to be closed for harvest. Often the problem cannot be corrected because the specific cause of the contamination cannot be identified. Potential sources of fecal contamination may include sewage and septic system leaks, agricultural runoff, and wildlife. Federal legislation requires TMDLs for bacteria to be established in watersheds to achieve or maintain water quality. To mitigate pollution and establish TMDLs, water managers must be able to identify the sources of bacterial contamination. The objectives of this project were to further develop and apply a method of fecal source identification based on PCR amplification of genetic markers from fecal anaerobic bacteria. Laboratory research established assays for feces from several additional species. A study comparing this method with other methods currently in use or under development showed it to be one of the two most accurate methods available; in addition, it was rapid and inexpensive. In a collaboration between scientists and local volunteers and staff, the method was applied in Tillamook Bay, OR, a major shellfish growing area. The resulting information was used in education. Results of the study provided data on specific sources of fecal pollution in Tillamook Bay, which will enable specific corrective measures to be applied. In addition, the project developed a rapid method (2 to 4 hours) of detecting fecal contamination, which could replace the slow methods (1 to 2 days) currently in use. This will allow beach closures, shellfish harvest advisories, and other measures of protecting public health to be applied and lifted in an appropriate timeframe. The quantitative assay developed in this project has been included in an ongoing major epidemiological study sponsored by the U.S. EPA, which will correlate the results of several rapid methods of detecting fecal contamination with human health outcomes. The results of this epidemiological study may change the way that bacterial pollution is detected from slow, culture-based methods, to rapid molecular methods. The methods developed during the course of this project already have had a major impact on the way the sources of fecal contamination are identified in the United States and elsewhere. Results will save money for farmers, homeowners, municipalities, industries, and water quality managers as they become able to target mitigation efforts toward only those areas where they are needed. In addition, the use of rapid, PCR-based molecular methods for fecal source detection have spurred other researchers to develop similar approaches, changing and improving the entire field of fecal source identification.

Conclusions:

Upon completion of this research project, we have concluded that:

• A method of fecal source identification based on PCR amplification of marker sequences from uncultured Bacteroidetes fecal anaerobic bacteria can rapidly and accurately identify sewage and feces from humans, cattle and other ruminants, dogs, pigs, horses, and elk.

• Although clone library analysis yielded unique Bacteroidetes sequences and markers for some host species (pig and horse), it is not practical to construct clone libraries large enough to sample the entire range of Bacteroidetes sequence diversity found within some host species. This is especially true for closely related species because of the large cost associated with sequencing and the diminishing return of unique groups as more sequences are analyzed.

• Few or no known cultured species of Bacteroides and Prevotella will serve to identify the source of feces or distinguish human from animal feces, because humans, dogs, cats, and gulls share closely related sequences, some of which match sequences from known species.

• Subtractive hybridization in microtiter dishes provides a rapid method of enriching for unique fecal-source-specific sequences for marker development and led to the design of markers for dog and elk.

• The Bacteroidetes method of fecal source identification compared favorably with other methods in a comparative study. Most other methods were inaccurate or had very high levels of false positives. Of three methods that performed well, including ribotyping and PFGE, the Bacteroidetes method is the least expensive and most rapid.

• Real-time Q-PCR TaqMan assays for Bacteroidetes provided a rapid method of water quality assessment that spanned eight orders of magnitude, compared with approximately four orders for E. coli enumeration. Q-PCR results were highly correlated with both coliform and E. coli mean concentrations. This Q-PCR assay provides a framework for expanding the use of Bacteroidetes PCR indicators beyond fecal source tracking and into a health risk-based analysis of fecal pollution.


Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other project views: All 38 publications 10 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Bernhard AE, Goyard T, Simonich MT, Field KG. Application of a rapid method for identifying fecal pollution sources in a multi-use estuary. Water Research 2003;37(4):909-913. R827639 (2001)
R827639 (2002)
R827639 (Final)
CR830396 (Final)
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  • Journal Article Bernhard AE, Colbert D, McManus J, Field KG. Microbial community dynamics based on 16S rRNA gene profiles in a Pacific Northwest estuary and its tributaries. FEMS Microbiology Ecology 2005;52(1):115-128. R827639 (2002)
    R827639 (Final)
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  • Journal Article Dick LK, Field KG. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Applied and Environmental Microbiology 2004;70(9):5695-5697. R827639 (Final)
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  • Journal Article Dick LK, Bernhard AE, Brodeur TJ, Santo Domingo JW, Simpson JM, Walters SP, Field KG. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Applied and Environmental Microbiology 2005;71(6):3184-3191. R827639 (Final)
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  • Journal Article Dick LK, Simonich MT, Field KG. Microplate subtractive hybridization to enrich for Bacteroidales genetic markers for fecal source identification. Applied and Environmental Microbiology 2005;71(6):3179-3183. R827639 (Final)
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  • Journal Article Field KG, Chern EC, Dick LK, Fuhrman J, Griffith J, Holden PA, LaMontagne MG, Le J, Olson B, Simonich MT. A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking. Journal of Water and Health 2003;1(4):181-194. R827639 (Final)
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    R828676C003 (Final)
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  • Other: So. California Coastal Water Research Project - Full Text PDF
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  • Supplemental Keywords:

    water, drinking water, watersheds, groundwater, marine, estuary, risk assessment, health effects, ecological effects, human health, animal, pathogens, viruses, bacteria, effluent, discharge, ecosystem, indicators, aquatic, innovative technology, conservation, environmental, biology, molecular, Northwest, Pacific Coast, Pacific Northwest, Oregon, OR, agriculture., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Nutrients, Ecology, Wastewater, Environmental Chemistry, Ecosystem/Assessment/Indicators, Ecosystem Protection, Ecological Effects - Environmental Exposure & Risk, Ecological Indicators, risk assessment, ecological exposure, aquatic, health indicator, aquatic ecosystem, environmental monitoring, microbial indicators, nutrient transport, sewage treatment, bacteria, anaerobic bacteria, coliform, waterfowl, ecosystem indicators, estuarine ecosystems, water quality, coliforms, sewage treratment, water treatment, fecal pollution

    Relevant Websites:

    Center for Genome Research & Biocomputing Exit
    http://oregonstate.edu/dept/microbiology/faculty/field/field.html Exit
    A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking Exit

    Progress and Final Reports:

    Original Abstract
  • 2000 Progress Report
  • 2001 Progress Report