United StatesEnvironmental ProtectionAgencyWastewater Technology Fact - PDF document

1 United StatesEnvironmental ProtectionAge
United StatesEnvironmental ProtectionAgencyWastewater Technology Fact SheetBacterial Source TrackingINTRODUCTIONPathogens are a major pollutant of water bodiesnationwide according to many states’ Clean Water Act303(d) reports. Various sources contribute pathogensto contaminated waters, including fecal pollution fromhumans, wildlife, and livestock. Besides being potentialpathogens, fecal bacteria (such as Escherichia coli)can indicate the presence of other waterbornepathogens. Bacteria from human sources may indicatethe presence of human viruses, while bacteria from wildand domestic animals may indicate the presence of theparasites or Cryptosporidia. The presenceof any fecal bacteria in drinking water is considered ahealth hazard. Knowing the source(s) of bacteria in awater body or water supply is of great value in theremediation and prevention of further bacterialcontamination. However, it can be difficult to addresswater quality impairment effectively without a reliablemethod to determine the source of contamination.Bacterial Source Tracking (BST) is new methodologyused to determine the source of fecal pathogencontamination in environmental samples.There are many BST methods available and more areunder development. Interest in applying thesetechniques stems from EPA’s recent implementation ofthe Total Maximum Daily Load (TMDL) study, asBST techniques appear to provide the best method todetermine the origins of fecal contamination in waterbodies. Projects to develop TMDLs for fecal coliformsand to design and implement best managementpractices (BMPs) to reduce fecal loading in water maybenefit from BST technology (Hager, 2001). This factsheet discusses BST methods and presents examplesof BST application to TMDL development andimplementation. sources of fecal bacteria are generallygrouped into three major categories: human, livestock,or wildlife. In more urban watersheds, a fourthcategory of pets or dogs may be added. Each sourceproduces unique, identifiable strains of fecal bacteriabecause the intestinal environments and selectivepressures to which the bacteria are subjected differfrom source to source.BST may use one of several methods to differentiatebetween potential sources of fecal contamination, all ofwhich follow a common sequence of analysis. First, adifferentiable characteristic, or fingerprint (such asantibiotic resistance or DNA), must be selected toidentify various strains of bacteria. A representativelibrary of bacterial strains and their fingerprints mustthen be generated from the human and animal sourcesthat may impact the water body. Indicator bacteriafingerprints from the polluted water body are comparedto those in the library and assigned to the appropriatesource category based on fingerprint similarity. BSTmethods can be grouped as molecular or non-molecular methods, according to the characteristicused to identify or fingerprint the bacteria. Table 1summarizes the classification of various BST methods.Molecular methods are also referred to as "DNAfingerprinting" and are based on the unique geneticmakeup of different strains of fecal bacteria. Molecularmethods rely on genetic v

2 ariation as the fingerprint toidentify t
ariation as the fingerprint toidentify the source of fecal contamination. Threemolecular BST methods are commonly used, includingribotyping (RT), polymerase chain reaction (PCR), andpulsed-field gel electrophoresis (PFGE). Proceduresfor the RT and PFGE methods are relatively similaramong multiple studies, but substantially differentvariations are reported when using PCR methods(Hagedorn, 2001). TABLE 1 CLASSIFICATION OF BSTMETHODSMolecular methods (DNA fingerprinting)Ribotyping (RT)Polymerase chain reaction (PCR)Pulsed-field gel electrophoresis (PFGE)Non-molecular methodsBiochemical methodsAntibiotic resistance analysis (ARA)Cell wall fatty acid methyl ester (FAME)F-specific coliphage typingCarbon utilization (BIOLOG)Chemical methodsCaffeine detectionOptical brightener detectionSource: Parsons, 2001.Non-molecular methods use non-geneticcharacteristics as the fingerprint or basis to differentiatethe source of fecal bacteria, and may be furthersubdivided between biochemical and chemicalmethods. Biochemical methods are based on theability of an organism's genes to actively produce abiochemical substance. The type and quantity of thesubstance(s) produced form the bacterial fingerprint.Antibiotic resistance analysis (ARA) is the mostcommonly used biochemical BST method. Otherbiochemical methods, such as cell wall analysis of fattyacid methyl ester (FAME), F-specific coliphage typing,and carbon source utilization (BIOLOG system), areunder development. Chemical methods do not detectthe presence of fecal bacteria, but rely on theidentification of compounds that co-occur with fecalbacteria in human wastewater to differentiate thesource of fecal pollution. Thus, chemical methods canonly determine whether or not the source of fecalpathogens is human (Hagedorn, 2001). Examples ofcompounds used in chemical BST include caffeine andoptical brighteners commonly used in laundrydetergents. APPLICABILITY is intended to aid in identifying sources (e.g.,human, livestock, or wildlife) of fecal contamination inwater bodies. Several states have started to use DNAfingerprinting to target water quality problems andformulate a mitigation strategy (Pelley, 1998;Blankenship, 1996). These techniques can also beused to direct implementation of effective BMPs toremove or reduce fecal contamination. For example,two New Hampshire communities are performing BSTsurveys (using the RT method) to determine thecontribution of bacterial contamination from severalspecific sources so that BMPs may be put in place tohelp rehabilitate water quality. The following is asummary of one representative survey.Hampton Harbor, New HampshireHampton Harbor is a tidally dominated, shallowestuary located at the extreme southeast corner of NewHampshire. The Hampton Harbor clamflats are closedfor clam harvesting during September and October dueto elevated fecal coliform levels. The flats are openfrom November through May but close temporarily ifthe rainfall exceeds 0.25 inches. These clamflats arepopular, productive, and accessible to the public.Despite the construction of a new wastewatertreatment facility in the Town of Seabrook, the bacterialevels often exce

3 ed the limits set by the NewHampshire Sh
ed the limits set by the NewHampshire Shellfish Program, resulting in flat closuresand frustrated clam diggers. The potential sources ofbacterial contamination include birds (cormorants,starlings, gulls), domestic animals (cats, dogs, goats,horses), sanitary wastewater from wastewatertreatment plant failures, and wildlife. The intent of thesurvey was to provide information to supportimplementation of specific source controls and toreduce the bacterial contamination to a level thatincreases the number of days that the clamflats areopen for recreational harvesting.Source classification provided by BST is often used inthe development and implementation of TMDLprojects. The information can be used to assign loadreduction allocations to sources in a watershed. Forexample, BST techniques have been very useful toregulatory officials in Virginia, where the ARA method has been used in seven TMDL watershed projects todate.Virginia Department of Conservation andRecreation TMDLsOver 300 stream segments were listed on theCommonwealth of Virginia’s 303(d) list for fecalcoliform bacteria violations. The uncertainty inherent inidentifying specific sources of fecal coliform bacteria inthe streams has hindered development of the TMDLs.BST studies were applied to three stream segments(Accotink Creek, Blacks Run, and Christians Creek)to provide more accurate waste-load allocations andenhance the development and defensibility of theTMDLs. In each, the RT method was used todetermine the dominant sources of fecal coliform in theimpaired stream segments. The source-trackingdistribution determined in each segment were used tomodify and strengthen the waste-load allocations in theTMDL watershed model. In addition, DNA testing isunderway in the Muddy Creek, Lower Dry River, MillCreek, and Pleasant Valley watersheds as part of theirTMDL implementation plans.Cedar Creek, Hall Creek, Byers Creek, HuttonCreek, and Lower Blackwater River were also placedon the Commonwealth of Virginia's 303(d) list becauseof violations of the fecal coliform bacteria water qualitystandard. In fulfilling the state requirement to developa TMDL Implementation Plan, a framework wasestablished to reduce fecal coliform levels and achievethe water quality goals for which TMDL allocationswere developed. BST analysis using the ARA methodwas performed as part of the TMDL implementation.Results indicated contributions of fecal coliform fromlivestock, human, and wildlife sources. The wildlifecontribution alone was enough to push fecal coliformlevels beyond the standard at five sampling sites, whilehuman sources alone were high enough to violate thestandard at five sampling sites. Livestock sources weresufficient to violate the standard at eight of elevensampling sites. In the Cedar and Hutton Creekwatersheds, livestock appeared to be an issuethroughout the watershed, while in the Hall/ByersCreek watershed, livestock problems appeared limitedto smaller tributaries (e.g. Indian Run and TattleBranch). Human sources seemed most significant inthe Hall/Byers and Hutton Creek watersheds. Thequantity of control measures required duringimplementation was determined an

4 d progress towardend goals will be asses
d progress towardend goals will be assessed during implementationthrough tracking control measure installations andcontinued water quality monitoring. Water qualitymonitoring will include fecal coliform enumeration andBST analysis. BST will provide an indication of theeffectiveness of specific groups of control measures,specifically agricultural and urban. Implementation wasscheduled to begin in July 2001, with the final goalbeing the delisting of the impaired segments from theCommonwealth of Virginia’s 303(d) List of ImpairedWaters by 2011.ADVANTAGES AND DISADVANTAGESIn general, molecular BST methods may offer the mostprecise identification of specific types of sources, butare limited by high per-isolate costs and detailed, time-consuming procedures. They are also not yet suitablefor assaying large numbers of samples in a reasonabletime frame. Biochemical BST methods are simpler,faster, less expensive, and allow large numbers ofsamples to be assayed in a short period of time.BST development is so new that little researchcomparing individual methods is complete. Results ofinitial studies should become available over the nextfew years.The United States Department of Agriculture recentlyfunded a two-year study to compare three BSTmethods using E. coli and ARA,PFGE, and RT. The merits of these methods will becompared by a) accuracy, cost, and processing time;b) determining the geographic range of the libraries;and c) assessing the utility of each method in fieldexperiments. This comparison and development ofBST methodology is intended to refine BMPimplementation and focus resources on pollutionsources for water quality impairments.The United States Geologic Survey is developing aprogram to identify sources of fecal bacteria in thewaters of Berkeley County, West Virginia. At leastfive methods will be tested for their ability to determineanimal sources of fecal bacteria in water samples (RT, PFGE, ARA, PCR, and BIOLOG carbon-utilization).The three objectives of this project include buildingsource libraries for the five methods, comparingmethods to see which is best to determine sources, andusing the best method to identify sources of bacteria inwater resources of Berkeley County. This study willprovide source libraries for five promising methods toidentify bacteria sources, quantitative information onwhich method(s) works best, determination of bacteriasources for ten domestic wells that contain bacteria,and determination of bacteria sources for five largepublic-supply springs. The libraries and methods willbe applicable to both surface and ground water inBerkeley County and surrounding areas.PERFORMANCE BST techniques are undergoing intensiveresearch that leads to rapid change in existing methodsand the creation of new methods. BST technologiesare quickly becoming proven and should be used byfederal and state regulatory agencies to addresssources of fecal bacterial pollution in water. Althoughthey are still experimental, BST methods represent thebest tools available to determine pathogen TMDL loadallocations and TMDL implementation plandevelopment. The following are examples of BSTtechnique performance i

5 n specific watershed studies.Antibiotic
n specific watershed studies.Antibiotic Resistance Analysis (ARA) MethodHolmans Creek, VirginiaHolmans Creek watershed was listed on theCommonwealth of Virginia's 1998 303(d) TMDLPriority List of Impaired Waters based on violations ofthe fecal coliform bacteria water quality standard.There are several potential fecal coliform sources in thiswatershed, including the non-point sources of wildlife,livestock, individual residential sewerage systems, andland application of manure and litter. Beef cattle,poultry and dairy are the major livestock operations inthe Holmans Creek watershed. Residential seweragein the watershed consists of direct discharges fromstraight pipes (homes without facilities to treat theirwaste discharge), privies, and failing septic systems.BST analysis using the ARA method was used toclassify sources of the fecal bacteria found in thepolluted water. Results of the BST analysis suggestthat the primary source of fecal pollution is human,constituting just under half of the total fecal coliformdeposited into the waters of Holmans Creek. Wildlifeand cattle sources each contribute approximately one-fourth of the total fecal coliform loads in the watershed.Poultry were determined to be a minor contributor tofecal coliform pollution in Holmans Creek, contributingone-tenth of the total fecal load.Stevenson Creek, FloridaThe Stevenson Creek basin encompassesapproximately 6,000 acres in central Pinellas County,Florida. In keeping with the objectives of theStevenson Creek Watershed Management Plan, a study was initiated to identify the dominantsource(s) of fecal contamination to Stevenson Creek inClearwater, Florida. The ARA method was chosenbecause it can assess the source of indicator organismsbased on a much larger subset of the bacterialpopulation than molecular methods can. The dominantsources of fecal coliform over the course of the studywere wild animal, dog, and human, with the overalltrend indicating that wild animal isolates comprised themajority of fecal coliforms obtained when colonyforming units (CFU) counts exceeded the acceptablelimit of 200 CFU per 100 mL. While human input wasnot the major cause of elevated fecal coliform levels formost of the samples analyzed for this study, thedomination of some small populations by humanisolates suggests that human sources contribute tolow-level background contamination. This occurswhen fecal coliform populations are low, near thetransition to dry season, and perhaps few isolates arewashed into surface waters from draining storm water.Lowering water tables may also draw wastewater fromsmall, otherwise innocuous leaks. Overall, there waslittle evidence of acute human fecal contamination on alarge scale; however, human sources may influence twosampling sites, detectable despite the presence of fecalcoliforms from other sources. The human input alonefor these two sites in one month was high enough toviolate water quality standards. Pulsed-Field Gel Electrophoresis (PFGE)MethodEastern Shore, VirginiaDNA fingerprinting using PFGE proved helpful whenan oyster farmer on Virginia's Eastern Shore was facedwith the closure of his shellfish beds due to elevat

6 edlevels of E. coli. Failing septic tan
edlevels of E. coli. Failing septic tanks were assumed tobe the primary source of the fecal pollution, but asurvey of septic systems in the sparsely populatedwatershed indicated that they were not the cause, andit became necessary to identify other potential sources.The highest levels of coliform bacteria were measuredin the small tidal inlets and rivulets of the wetlandslocated upstream of local houses, shifting suspectedsources from human to other sources. Researcherscollected fecal samples from raccoon, waterfowl, otter,muskrat, deer, and humans in the area and used DNAfingerprinting to confirm the suspicion that the sourcewas not anthropogenic in nature. Comparing E. colifrom the shellfish beds against the fingerprints of knownstrains in the DNA library, the researchers linked thein-stream coli to deer and raccoon (mostlyraccoon). Several hundred animals, including 180raccoon, were removed from areas adjacent to thewetlands. E. coli levels subsequently declined by 1 to2 orders of magnitude throughout the watershed,allowing threatened areas of the tidal creeks to bereopened to shellfishing.Four Mile Run, VirginiaFour Mile Run is listed on the Commonwealth ofVirginia’s 303(d) listing for elevated levels of fecalcoliform bacteria. The Northern Virginia RegionalCommission is currently developing a TMDL for theFour Mile Run watershed, with the final draft to besubmitted to Virginia Department of EnvironmentalQuality by March 1, 2002. Four Mile Run is an urbanstream with no agricultural runoff. The watershed ishome to 183,000 people, just over 9,000 per squaremile. The dominant land use in the watershed ismedium to high density residential housing. Sevencentral business districts exist within this 20 square milewatershed, and two high-capacity interstates passthrough the watershed along with numerous primaryand secondary roadways. The watershed isapproximately 40 percent impervious. A large petpopulation accompanies the dense human population inthe watershed. As to potential fecal sources, there islittle manufacturing industry to generate point sourcedischarges and there are no combined sewers in themajority of the watershed. Sanitary sewers serve morethan 99.9 percent of the watershed population. Thenumber of septic systems in the watershed is believedto be less than 50. The PFGE method of BST analysiswas conducted on E. coli DNA from seasonally variedstream and sediment samples in the watershed. Resultsof the analysis show that waterfowl contribute overone-third (38 percent) of the bacteria, humans and petstogether account for over one-fourth (26 percent), andraccoons account for 15 percent of the contamination,with deer (9 percent) and rats (11 percent) alsocontributing. The predominant non-human sourcesinclude wildlife species with intimate association withthe waterways.Ribotyping (RT) methodLittle Soos Creek, WashingtonA BST survey was designed to help characterizesources of fecal coliform bacterial contamination inLittle Soos Creek in southeast King County,Washington, in response to the impact of existing andanticipated urban development in the area. Little SoosCreek has historically been cate

7 gorized as a Class Astream, but violates
gorized as a Class Astream, but violates fecal coliform standards for thisclassification. The goal of the BST survey was to helpdetermine the contribution to contamination of thestream from two potential sources: livestock on hobbyfarms and ranches adjacent to the stream and on-siteseptic systems close to the stream in highly permeablesoils. Other animal sources were also considered.Genetic fingerprinting (using ribosomal RNA typing orRT) was performed on E. coli isolates to effectivelymatch specific strains of E. coli from a contaminatedsite in the stream to its source. The intent was toprovide information to support implementation ofspecific source controls. The study identified thesources of approximately three-fourths of the fecalcoliform contamination, with the primary sourcesdetermined to be cows, dogs, and horses. Althoughseptage was identified as a contributor to thecontamination problem, it was not indicated as a major source. However, even low levels of contribution fromseptage suggest the potential for Little Soos Creek toharbor a number of human viral, bacterial, and parasiticpathogens associated with human sources. For thisreason, further investigation of the contribution fromseptic systems and of human exposure (particularlychildren) to the stream may be warranted.Lower Boise River, IdahoThe Lower Boise River watershed from Lucky PeakReservoir to the Snake River near Parma containsalmost one-third of Idaho's population and four majormunicipalities, including the city of Boise. An aridclimate (approximately 10 inches of annual rainfall)makes irrigation a requirement on most farmland. Thisirrigation coupled with reuse of pasture water onirrigated fields results in the contribution of non-pointdischarge of fecal coliform bacteria to the Lower BoiseRiver. In 1994, the Idaho Department ofEnvironmental Quality (IDEQ) placed the Lower BoiseRiver on the 303(d) list for impairment of primary andsecondary contact designated uses because fecalcoliform levels exceeded state standards. A draftTMDL was completed and submitted to the USEPAon December 1998 and approved on January 2001with implementation plan due July 2001. The TMDLindicates that bacteria discharge loads will require morethan 95 percent reductions from non-point sourcebacteria loadings to meet the primary contact bacteriastandard. A DNA fingerprinting of coliform bacteriawas conducted to focus bacteria reductionimprovement. E. coli cultures were grown from fecalsamples of cows, sheep, humans, ducks, and geese,and DNA from these samples was identified. Themajor bacteria sources in the watershed identified usingthe RT method were waterfowl, humans, pets, andcattle/horses. Waterfowl were clearly the largestsource. The major advantage of using the DNAfingerprinting tool is the ability to develop accuratecontrol measures (BMPs) in terms of bacterial sources.Prior to this study, IDEQ knew there were bacteriaproblems, but did not know where to focus controlmeasures. The results of the BST analysis identify themajor sources, allowing IDEQ to strategically placeBMPs.University of Georgia/USDA RT comparisonBST methods, including RT, rely on a data

8 base ofknown source fingerprints to iden
base ofknown source fingerprints to identify environmentalisolates of fecal bacteria. It is not well understood towhat degree these known source fingerprints arebiogeographically variable. This is important becausea fingerprint database developed for one state or regionmay or may not be applicable to another. Theobjective of a University of Georgia/USDA study(Hartel et al, 2002) was to use the RT method of BSTanalysis to determine the geographic variability of thefecal bacterium, E. coli, from one location in Idaho andthree locations in Georgia for four animals: cattle,horse, swine, and poultry. The study identified distancefrom the source sample to the watershed as a keyvariable for cattle and horses, but not for swine andpoultry. When the E. coli ribotypes among the animalswere compared at one location, the relative percentdifference between them was 86, 89, 81, and 79 foreach of the four locations, suggesting good ribotypeseparation among host animal species at one location.Achieving a high degree of accuracy in matchingenvironmental isolates of fecal bacteria to a host origindatabase depends on having a large number of isolatesfor comparison and using a distance of 175 km or less(at least for certain host animal species).COSTSGiven the fact that many BST methods are still in theresearch and development phase, there is greatvariation for cost per sample (or per isolate) amongdifferent laboratories. Factors that affect cost includethe following:Analytical method - Molecular BST methods (e.g.RT) are generally more expensive than non-molecularmethods. In addition, automated techniques are moreexpensive but less labor-intensive than manualtechniques for the same method (such as RT).Size of the database - It is not known what sizedatabase or library of bacterial isolates from knownfecal sources is required for accurate source predictionin a given watershed. Considerations in thedevelopment of the BST library include the size of thewatershed, the diversity of animal species and human sources that may significantly impact water quality, andthe heterogeneity of the population within a givensource species. In many studies, the number of isolatesrequired to develop the known source database maymake up the majority of total isolates analyzed,constituting a large fraction of the total cost for thestudy. of environmental isolates - The number ofisolates that must be analyzed from the water body ofinterest varies among study sites. There may bemultiple isolates from each water sample taken, withcosts generally calculated per isolate.Level of accuracy - Cost increases in proportion withaccuracy or the percentage of isolates classifiedcorrectly. In some cases 80 percent is considered thelowest acceptable level of accuracy. More studies areneeded to determine the level of accuracy achievableby each BST method.The cost for BST analysis ranges from $25 to $100per isolate using molecular methods and from $10 to$30 per isolate for non-molecular methods. Thesecosts are based on classifying a sample within anaccuracy range of 70 to 90 percent or higher.However, there is little firm guidance on the requirednumber of reference f

9 ecal samples and isolatesextracted from
ecal samples and isolatesextracted from each sample, causes wide variance inthe total cost for a fecal source tracking project. Forexample, the cost for TMDL developments forAccotink Creek, Blacks Run and Christians Creek inVirginia by the USGS Richmond office wasapproximately $617,000 (total for the three TMDLs),while the New Hampshire Department ofEnvironmental Services spent approximately $225,000to establish the ribotyping laboratory and partiallysupport the two source tracking surveys. In twoongoing comparison studies, the cost of the San JuanCreek Watershed Bacteria Study (California) is$274,000 (excluding the expenses for laboratoryanalysis), while the USDA grant to compare three BSTmethods (RT, PFGE and ARA) is $310,000.REFERENCESOther Related Fact SheetsOther EPA Fact Sheets can be found at the followingweb address:http://www.epa.gov/owm/mtbfact.htmhip, K. 1996. DNA library wouldgive investigators inside poop on pollutionsources. Bay Journal 6(6),http://www.bayjournal.com/96-09/ K. 1996. Masked bandituncovered in water quality theft / Team tailspollution to unlikely culprit. Bay Journal 6(6),http://www.bayjournal.com/ M. C., 2001. Stormwater Magazine,Vol. 2 No. 3, p16-25, http://www.forester.net/sw_0105_detecting.html, Vol. 2 No. 4 / June, p22-27, http://www.forester.net/sw_0106_detecting.html. Virginia Regional Commission(NVRC), 2000. Bacteria Research Menu,http://www.novaregion.org/4milerun/ S. and Tamplin, M. L., 2002.Sources of fecal contamination, inEncyclopedia of Environmental Microbiology(Editor: Bitton, G.), John Wiley and Sons, Inc.,New York, NY.6. J. 1998. DNA fingerprinting holdspromise for identifying nonpoint sources ofpollution. Environmental Science andTechnology. 32(21): 486A.7. D., 1999. Fecal contaminationsource identification methods in surface water,Washington Department of Ecology, Report #99-345. 8. S., Satokari, R., Saarela, M.,Mattila-Sandholm, T., and Saxelin, M., 1999.Comparison of Ribotyping, RandomlyAmplified Polymorphic DNA Analysis, andPulsed-Field Gel Electrophoresis in Typing ofLactobacillus rhamnosus and caserStrains, Appl. Environ. Microbiol.65:3908-3914. EPA, 1997. DNA fingerprinting aidsinvestigation-fecal coliform sources traced tounlikely suspects. Nonpoint Source NewsNotes April/May 48:19-20.http://www.epa.gov/owow/info/N EPA, 2001. Protocol for developingpathogen TMDLs, EPA 841-R-00-002,Washington, D.C.11. EPA, 2002. Workshop on MicrobialSource Tracking (February 5, 2002; Irvine,CA). http://www.sccwrp.org/tools/workshop/source_tracking_workshop.html. 2001. Identifying Sources of FecalColiform Bacteria in Selected Streams onVirginia's TMDL Priority List,http://va.water.usgs.gov/projects/va129. html13. Polytechnical University, 2001.Bacterial Source Tracking (BST), IdentifyingSources of Fecal Pollution,http://www.bsi.vt.edu/Antibiotic Resistance Analysis (ARA) Peer-reviewed Journal Publications14. C., S. L. Robinson, J. R. Filtz, S.M. Grubbs, T. A. Angier, and R. B. Reneau,Jr. 1999. Using antibiotic resistance patterns inthe fecal streptococci to determine sources offecal pollution in a rural Virginia watershed.Appl. Environ. Microbiol. 65:5522-5531.15.d, V. J., J.

10 Whitlock, and V. H.Withington. 2000. Cl
Whitlock, and V. H.Withington. 2000. Classification of theantibiotic resistance patterns of indicatorbacteria by discriminant analysis: use inpredicting the source of fecal contamination insubtropical Florida waters. Appl. Environ.Microbiol. 66:3698-3704.16. C.W., Burgess, J.L., Knight, I.T., andColwell, R.R., 1990. Antibiotic resistanceindexing of Escherichia coli to identifysources of fecal contamination in water, Can.J. Microbiol. 36:891-894.17. S., R., L. Murphree, L. Edmiston, C.W. Kaspar, K. M. Portier, and M. L.Tamplin. 1997. Association ofmultiple-antibiotic-resistance profiles with pointand nonpoint sources of Escherichia coli inApalachicola Bay. Appl. Environ. Microbiol.63:2607-2612. B. A., 1996. Discriminant analysis ofantibiotic resistance patterns in fecalstreptococci, a method to differentiate humanand animal sources of fecal pollution in naturalwaters. Appl. Environ. Microbiol.62:3997-4002. 19.Wiggins, B. A., R. W. Andrews, R. A.Conway, C. L. Corr, E. J. Dobratz, D. P.Dougherty, J. R. Eppard, S. R. Knupp, M. C.Limjoco, J. M. Mettenburg, J. M. Rinehardt,J. Sonsino, R. L. Torrijos, and M. E.Zimmerman. 1999. Identification of sources offecal pollution using discriminant analysis:supporting evidence from large datasets. Appl.Environ. Microbiol. 65:3483-3486.Non-Journal Publications20. R. J. 2000. M.S. Thesis. Sourceidentification of fecal pollution in the Tillamookwatershed: antibiotic discriminant analysis.Oregon State University, Corvallis, OR. 21.an, A. M., C. Hagedorn, and K. Hix.2000. Determining sources of fecal pollution inthe Blackwater River watershed. p. 44-54. InT. Younos and J. Poff (ed.), Abstracts,Virginia Water Research Symposium 2000,VWRRC Special Report SR-19-2000,Blacksburg, VA.22. A. K. 2000. M.S. Thesis. Determiningsources of fecal pollution in water for a ruralVirginia community. Virginia PolytechnicInstitute and State University, Blacksburg, VA.23. S., Tamplin, M. L., Portier, K. M.,Lukasik, G., Scott, T., Sheperd, S., Tobia, S.,Braun, K.R., Koo, P., and Farrah, S. R.,2001. Geographic variation in AntibioticResistance Patterns of Escherichia coliisolated from swine, poultry, beef and dairycattle farms in Florida, American Society forMicrobiology 101th General Meeting, May20-May 24, Orlando, FL.24.Wagner, V. and V.J. Harwood. 1999. Use ofantibiotic resistance patterns to discriminatebetween sources of fecal coliform bacteria insurface waters of northeast Florida. AmericanSociety for Microbiology 99th GeneralMeeting, May 30-June 3, Chicago, IL.Ribotyping (RT)Peer-reviewed Journal Publications25. A. C., B. L. Shear, M. R. Ellersieck,and A. Asfaw. 2001. Identification of fecalEscherichia coli from humans and animals byribotyping. Appl. Environ. Microbiol.67:1503-1507. P. G., Summer, J. D., Hill, J. L.,Collins, J. V., Entry, J. A., and Segars, W. I.,2002. Biogeographic variability ofEscherichia coli ribotypes from Idaho andGeorgia, Journal of Environmental Quality (inpress). T., Bopp, C., Olsvik, O, andWachsmuth, K., 1993. Epidemiologicapplication of a standardized ribotype schemefor cholerae O1, J. Clin. Microbiol,31(9): 2474-2082.28. S., K. M. Portier, K. Robinson, L.Edmiston, and M.

11 L. Tamplin. 1999.Discriminant analysis
L. Tamplin. 1999.Discriminant analysis of ribotype profiles ofEscherichia coli for differentiating human andnonhuman sources of fecal pollution. Appl.Environ. Microbiol. 65:3142-3147.29. W., Lambert, J., Smallwood, R.,Schembri, M., Ross B. C., and Dwyer, B.,1992. Ribotyping of Helicobacter pylorifrom clinical specimens, J. Clin. Microbiol,30(6): 1562-1567.30.Wheeler, A. L., Hartel, P. G., Godfrey, D. G.,Hill, J. L. and Segars, W. I., 2002. CombiningRibotyping and Limited Host Range ofEnterococcus faecalis for Microbial SourceTracking, Journal of American WaterResources Association (in press).Non-Journal Publications31. P. G., W. I. Segars, N. J. Stern, J.Steiner, and A. Buchan. 1999. Ribotyping todetermine host origin of Escherichia coliisolates in different water samples. p.377-382.In D. S. Olsen and J. P. Potyondy (Eds.),Wildland Hydrology. American WaterResources Association Technical PublicationSeries TPS-99-3, Herndon, VA.32. J. L., P. G. Hartel, W. I. Segars, and P.Bush. 2001. Ribotyping to determine thesource of fecal coliform contamination in threehousehold wells near Cochran, Georgia. p.743-746. In: K. J. Hatcher (ed.) Proceedingsof the 2001 Georgia Water ResourcesConference, March 26-27, University ofGeorgia, Athens, GA. 33. M., and N. Chechowitz. 1995.Little Soos Creek microbial source tracking: asurvey, University of Washington Departmentof Environmental Health, Seattle, WA, 49p.Pulsed-Field Gel Electrophoresis (PFGE)34., T. J., Lior, H., Green, J. H., Khakhria,R., Wells, J. G., Bell, B. P., Greene, K. D.,Lewis J., and Griffin, P.M., 1994. Laboratoryinvestigation of a multistate food-borneoutbreak of Escherichia coli O157:H7 byusing pulsed-field gel electrophoresis andphage typing, J. Clin. Microbiol, 32(12):3013-3017. C., Gangar, W., Murphree, R. L.,Tamplin, M. L., and Kaspar, C. W., 1995.Multiple vulnificus strains in oysters asdemonstrated by clamped homogeneouselectric field gel electrophoresis, Appl.Environ. Microbiol., 61(3): 1163-1168.36. J. M., Weagant, S. D., Jinneman, K.C., and Bryant, J. L., 1995. Use ofpulsed-field gel electrophoresis forepidemiological study of Escherichia coliO157:H7 during a food-borne outbreak, Appl.Environ. Microbiol., 61(7): 2806-2808.37.Kariuki,S., Gilks, C., Kimari, J., Obanda, A.,Muyodi, J., Waiyaki, P., and Hart1, C. A.,1999. Genotype Analysis of Escherichia coliStrains Isolated from Children and ChickensLiving in Close Contact, Appl. Environ.Microbiol. 65(2): 472-476.38. S., Hodge, N. C., Stall, R. E.,Farrah, S. R., and Tamplin, M. L., 2001.Phenotypic and genotypic characterization ofhuman and nonhuman Escherichia coli, WaterResearch, 35(2): 379-386.39.Simmons, G. M., Jr., S. A. Herbein, and C.M. James. 1995. Managing nonpoint fecalcoliform sources to tidal inlets. UniversitiesCouncil on Water Resources. WaterResources Update, Issue 100:64-74.40. G. M., Jr., and S. A. Herbein.1998. Potential sources of Escherichia coli tochildren's pool in La Jolla, CA. Final Reportfor the City of San Diego and the County ofSan Diego Department of EnvironmentalHealth.Simmons, G. M., D. F. Waye, S. Herbein, S.Myers, and E. Walker. 2000. Estimatingnonpoint fecal coliform source

12 s in NorthernVirginia's Four Mile Run wa
s in NorthernVirginia's Four Mile Run watershed. p.248-267. In T. Younos and J. Poff (ed.),Abstracts, Virginia Water ResearchSymposium 2000, VWRRC Special ReportSR-19-2000, Blacksburg, VA.Polymerase Chain Reaction (PCR)42. A. E., and K. G. Field. 2000. APCR assay to discriminate human and ruminantfeces based on host differences inBacteroides 16S ribosomal DNA.Appl. Environ. Microbiol. 66: 4571-4574.43. P. E., L. K. Johnson, S. T.Zimmerley, and M. J. Sadowsky. 2000. Useof repetitive DNA sequences and the PCR todifferentiate coli isolates fromhuman and animal sources. Appl. Environ.Microbiol. 66(6): 2572-2577.44.Farnleitner, A. H., Kreuzinger, N., Kavka, G.G., Grillenberger, S., Rath, J., and Mach1, R.L., 2000. Simultaneous Detection andDifferentiation of Escherichia coli Populationsfrom Environmental Freshwaters by Means ofSequence Variations in a Fragment of the-D-Glucuronidase Gene, Appl. Environ.Microbiol. 66(4): 1340-1346. 45.raad, P.M.F.J., F. M. Rombouts, andS.H.W. Notermans. 1997. Epidemiologicalaspects of thermophilic Campylobacter inwater-related environments: A review. WaterEnviron. Res. 69(1): 52-63.46. J.R., Edlind, T. D., LiPuma, J. J.,and Stull, T. L., 1992. Molecularepidemiology of Pseudomonas cepaciadetermined by polymerase chain reactionribotyping, J. Clin. Microbiol, 30(8):2084-2087.Other methods47. A. E., and K. G. Field. 2000.Identification of nonpoint sources of fecalpollution in coastal waters by usinghost-specific 16S ribosomal DNA geneticmarkers from fecal anaerobes. Appl. Environ.Microbiology 66(4): 1587-1594.48. M. W., and H. Kator. 1999.Sorbitol-fermenting bifidiobacteria asindicators of diffuse human faecal pollution inestuarine watersheds. J. Appl. Microbiol.87:528-535.Souza, V., Rocha, M., Valera, A, andEguiarte, L. E., 1999. Genetic Structure ofNatural Populations of Escherichia coli inWild Hosts on Different Continents, Appl.Environ. Microbiology 65(8): 3373-3385.ADDITIONAL INFORMATIONDr. C. Andrew CarsonDepartment of Veterinary PathobiologyCollege of Veterinary MedicineUniversity of Missouri201 Connaway HallColumbia, MO 65211-5120Dr. Charles HagedornDepartment of Crop and Soil Environmental SciencesVirginia Tech UniversityBlacksburg, VA 24061-0404Dr. Valerie J. HarwoodDepartment of BiologyUniversity of South Florida4202 East Fowler Ave.Tampa, FL 33620Dr. Peter G. HartelDepartment of Crop and Soil SciencesUniversity of Georgia3111 Plant Sciences BuildingAthens, GA 30602-7274Dr. Salina ParveenUSDA - Agricultural Research ServiceDelaware State University1200 N. DuPont HwyW.W. Baker CenterDover, DE 19901Dr. Michael J. SadowskyDepartment of Soil, Water, and ClimateUniversity of Minnesota258 Borlaug Hall 1991 Upper Buford Circle St. Paul, MN 55108 Dr. Mansour SamadpourDepartment of Environmental HealthSchool of Public Health & Community MedicineUniversity of Washington Health Sciences BuildingBox 357234Seattle, WA 98195-7234 Dr. Bruce A. WigginsDepartment of BiologyJames Madison UniversityHarrisonburg, VA 22807The mention of trade names or commercial productsdoes not constitute endorsement or recommendationfor use by the U. S. Environmental Protection Agency(EPA).Office of WaterEPA 832-F-02-0