U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2011–5164 

Prepared in cooperation with the City of Wilmington, Delaware 

Pathogenic Bacteria and Microbial-Source Tracking 

Markers in Brandywine Creek Basin, Pennsylvania and 

Delaware, 2009–10 
Cover: View from the U.S. Geological Survey streamflow-gaging station at West Branch Brandywine Creek at Coatesville, 

Pennsylvania, during high flow. (Photograph by Andrew Reif, U.S. Geological Survey.) 
Pathogenic Bacteria and Microbial-Source 

Tracking Markers in Brandywine Creek 

Basin, Pennsylvania and Delaware, 

2009–10 

By Joseph W. Duris, Andrew G. Reif, Leif E. Olson, Heather E. Johnson

Prepared in cooperation with the City of Wilmington, Delaware 

Scientific Investigations Report 2011–5164

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2011 

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Suggested citation:

Duris, J.W., Reif, A.R., Olson, L.E., and Johnson, H.E., 2011, Pathogenic bacteria and microbial-source tracking 

markers in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10: U.S. Geological Survey Scientific 

Investigations Report 2011–5164, 27 p. 
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Purpose and Scope ..............................................................................................................................3

Description of Study Area ...................................................................................................................3

Physical Setting.....................................................................................................................................3

Methods...........................................................................................................................................................3

Water-Sample Collection.....................................................................................................................3

Bacterial Enumeration, Enrichment, and Preservation..................................................................5

DNA Extraction......................................................................................................................................6

Polymerase Chain Reaction to Detect Pathogens and Microbial-Source 

Tracking Markers ....................................................................................................................6

Quantitative Polymerase Chain Reaction for Microbial-Source Tracking Markers..................7

Quantitative PCR Data Analysis .........................................................................................................7

Data Analysis.........................................................................................................................................8

Microbiological Water Quality of Brandywine Creek..............................................................................8

Results of Indicator Organism Sampling ..........................................................................................9

Results of Pathogen Testing..............................................................................................................11

Results of Microbial-Source Tracking.............................................................................................15

Results of Qualitative Microbial-Source Tracking ...............................................................16

Results of Quantitative Microbial-Source Tracking.............................................................17

Quality Control.....................................................................................................................................18

Summary and Conclusions.........................................................................................................................19

References Cited..........................................................................................................................................20

Appendix 1. Concentrations of fecal-indicator bacteria, detections of pathogen and 

microbial-source tracking markers, and concentrations of microbial-source 

tracking markers from water collected in the Brandywine Creek Basin in 

Pennsylvania and Delaware, 2009–10 ............................................................................23

Appendix 2. Concentrations of fecal-indicator bacteria, detections of pathogen and 

microbial-source tracking markers, and concentrations of microbial-source 

tracking markers from quality-control samples collected in the Brandywine 

Creek Basin in Pennsylvania and Delaware, 2009–10 .................................................27
iv

Figures

1. Map showing location of sampling sites used in the Brandywine Creek 

Basin study, 2009–10 ....................................................................................................................4

2. Boxplots showing concentrations of fecal-indicator bacteria, by sample 

location, from samples collected at high mean daily flow or normal mean 

daily flow during the Brandywine Creek Basin study, 2009–10...........................................10

3. Bar graphs showing frequency of all samples testing positive for pathogen 

and microbial-source tracking (MST) markers in the Brandywine Creek Basin 

study, 2009–10..............................................................................................................................12

4. Bar graph showing detection frequency of selected gene markers at routinely 

sampled sites at high flow in the Brandywine Creek Basin study, 2009–10 .....................14

5. Bar graph showing percent contribution of AllBac, BoBac, and qHF183 microbialsource tracking marker load from East and West Branches of Brandywine Creek 

during targeted high-flow event sampling, 2009–10 .............................................................17

Tables

1. Sites sampled in the Brandywine Creek Basin study, 2009–10.............................................5

2. Growth medium and gene targets for pathogens and microbial-source 

tracking organisms.......................................................................................................................6

3. Standard-curve characteristics for AllBac, BoBac, and qHF183 microbial-source 

tracking (MST) markers used in the Brandywine Creek Basin study, 2009–10..................8

4. Comparison of median fecal-indicator bacteria concentrations in East Branch 

and West Branch Brandywine Creek, 2009–10, by flow condition.......................................9

5. Detection frequency of pathogen and microbial-source tracking gene markers 

in the Brandywine Creek Basin study, 2009–10, based on relation to recreational 

water-quality criteria..................................................................................................................13

6. Detection-frequency comparison of pathogen and microbial-source tracking 

gene markers at routinely sampled sites during high and normal flow in the 

Brandywine Creek Basin study, 2009–10 ................................................................................13

7. Number of concurrently positive samples collected at selected sites in the 

Brandywine Creek Basin study, 2009–10 ................................................................................14

8. Relative standard deviation of field replicates for quantitative microbial 

methods in the Brandywine Creek Basin study, 2009–10.....................................................18

9. Frequency of agreement between field replicates for qualitative microbial 

methods in the Brandywine Creek Basin study, 2009–10.....................................................18
v

Conversion Factors

Multiply By To obtain

Length

inch (in.) 2.54 centimeter (cm)

mile (mi) 1.609 kilometer (km)

yard (yd) 0.9144 meter (m)

Volume

gallon (gal) 3.785 liter (L) 

milliliter (mL) 1,000 microliter (μL)

Flow rate

cubic foot per second (ft3

/s) 0.02832 cubic meter per second (m3/s)

cubic foot per second (ft3

/s) 283.168 deciliters per second (dL/s)

Mass

pound, avoirdupois (lb) 0.4536 kilogram (kg) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

°C=(°F-32)/1.8

Concentrations of fecal-indicator bacteria are given in colony-forming units per 100 milliliters 

(CFU/100 mL). 

Concentrations of quantitative microbial-source tracking gene markers are given in copies per 

100 milliliters (copies/100 mL). 
vi

Abbreviations used in report

BMP best-management practices

C Celsius

CDC Centers for Disease Control and Prevention

CSO combined sewer overflow

DNA Deoxyribonucleic acid

E. coli Escherichia coli

EPA U.S. Environmental Protection Agency

FIB fecal-indicator bacteria

LLQ lower limit of quantification

LOD limit of detection

MST microbial-source tracking

NTNC no-template negative control

PCR polymerase chain reaction

qPCR quantitative polymerase chain reaction

RSD relative standard deviation

rRNA ribosomal ribonucleic acid

RWQC recreational water-quality criteria

STEC Shiga-toxin producing Escherichia coli 

ULQ upper limit of quantification

USGS U.S. Geological Survey
Pathogenic Bacteria and Microbial-Source Tracking 

Markers in Brandywine Creek Basin, Pennsylvania and 

Delaware, 2009–10 

By Joseph W. Duris, Andrew G. Reif, Leif E. Olson, Heather E. Johnson 

Abstract

The City of Wilmington, Delaware, is in the downstream 

part of the Brandywine Creek Basin, on the main stem of 

Brandywine Creek. Wilmington uses this stream, which drains 

a mixed-land-use area upstream, for its main drinking-water 

supply. Because the stream is used for drinking water, Wilmington is in need of information about the occurrence and 

distribution of specific fecally derived pathogenic bacteria 

(disease-causing bacteria) and their relations to commonly 

measured fecal-indicator bacteria (FIB), as well as information regarding the potential sources of the fecal pollution and 

pathogens in the basin.

This study focused on five routinely sampled sites within 

the basin, one each on the West Branch and the East Branch 

of Brandywine Creek and at three on the main stem below the 

confluence of the West and East Branches. These sites were 

sampled monthly for 1 year. Targeted event samples were collected on two occasions during high flow and two occasions 

during normal flow.

On the basis of this study, high flows in the Brandywine 

Creek Basin were related to increases in FIB densities, and 

in the frequency of selected pathogen and source markers, in 

the West Branch and main stem of Brandywine Creek, but 

not in the East Branch. Water exceeding the moderate fullbody-contact single-sample recreational water-quality criteria 

(RWQC) for Escherichia coli (E. coli) was more likely to contain selected markers for pathogenic E. coli (eaeA, stx1

, and 

rfbO157 gene markers) and bovine fecal sources (E. hirae and 

LTIIa gene markers), whereas samples exceeding the enterococci RWQC were more likely to contain the same pathogenic 

markers but also were more likely to carry a marker indicative 

of human source (esp gene marker). 

On four sample dates, during high flow between October 

and March, the West Branch was the only observed potential 

contributor of selected pathogen and bovine source markers to 

the main stem of Brandywine Creek. Indeed, the stx2

 marker, 

which indicates a highly virulent type of pathogenic E. coli,

was found only in the West Branch and main stem at high flow 

but was not found in the East Branch under similar conditions. 

However, it must be noted that throughout the entire year of 

sampling there were occasions, during both high and normal 

flows, when both the East and West Branches were potential 

contributors of pathogen and microbial-source tracking markers to the main stem. Therefore, this study indicates that under 

selected conditions (high flow, October through March), West 

Branch Brandywine Creek Basin was the most likely source of 

elevated FIB densities in the main stem. These elevated densities are associated with more frequent detection of selected 

pathogenic E. coli markers (rfbO157, stx1

) and are associated 

with MST markers of bovine source. However, during other 

times of the year, both the West Branch and East Branch 

Basins are acting as potential sources of FIB and fecally 

derived pathogens. 

Introduction

Brandywine Creek drains a mixed-land-use basin that 

contains agricultural, urban, and suburban areas and has 

within it several wastewater-treatment discharges and industrial and public-supply withdrawals. The City of Wilmington, 

Delaware, which is in the downstream part of the basin on the 

main stem of Brandywine Creek, uses the stream for its main 

drinking-water supply. Because of the stream’s use as drinking water, Wilmington is in need of information about the 

occurrence and distribution of specific fecally derived pathogenic bacteria (disease-causing bacteria) and their relations to 

commonly measured fecal-indicator bacteria (FIB), as well as 

information regarding the potential sources of the fecal pollution and pathogens in the basin. Potential contaminant sources 

include waste from humans, domestic animals, farm animals, 

and wildlife. This information could be used by the City of 

Wilmington, Del., to develop a best-management-practice 

(BMP) strategy based on the fecal pathogens and potential 

fecal sources within the Brandywine Creek Basin. Since 2005, 

Wilmington has been investing in BMP projects in the Brandywine headwaters to control sediments and pollutants, including 

bacteria that could adversely affect the drinking-water supply.
2 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

Water-quality studies by the U.S. Geological Survey 

(USGS) Pennsylvania Water Science Center demonstrated 

that in certain reaches of West Branch Brandywine Creek near 

Coatesville, Pennsylvania, elevated concentrations of FIB 

were related to leaking sewer pipes, failed septic systems, 

combined sewer overflows, and illegal discharges. In addition, resuspension of fluvial sediments was cited as a potential 

source for elevated concentrations of FIB, but neither the origin of the FIB in sediments nor the potential for redeposition 

of this sediment in the stream system was assessed (Cinotto, 

2005). A study of the historical trends in FIB concentrations in the Brandywine Creek Basin showed higher median 

concentrations of both fecal coliform bacteria and Escherichia 

coli (E. coli) in West Branch Brandywine Creek than in East 

Branch Brandywine Creek and the main stem (Town, 2001).

Although the USGS did preliminary surveys of FIB 

sources along select sections of the Brandywine Creek Basin 

and has done routine monitoring of the basin for FIB, allowing 

for historical perspectives on concentrations, no work has been 

done to assess the specific fecal sources of FIB or the occurrence of fecally derived pathogenic bacteria within the basin. 

Recently issued reports indicate that commonly measured FIB 

have a variable relation with pathogenic bacteria, suggesting 

sampling location and spatial-temporal differences in distributions of FIB and pathogens are primary influencing factors in 

the relation (Duris and others, 2009; Smith and others, 2009). 

Current (2011) methods used to assess microbial water 

quality rely on commonly measured FIB (fecal coliform bacteria, E. coli, and enterococci). The presence of FIB indicates 

possible contamination by fecal waste, which may also contain 

pathogens from human or animal fecal sources. Most of these 

indicator organisms are not pathogens themselves; they simply 

indicate the possible presence of pathogens. There are, however, specific groups of bacteria within the broader FIB group 

that are known human pathogens. Among the fecal coliform 

bacteria is a class of E. coli known as the Shiga-toxin producing E. coli (STEC), including E. coli O157:H7. The genus 

Shigella, which includes the pathogen Shigella sonnei, is also 

in the broad fecal coliform bacteria group. The total coliform indicator bacteria group contains the well-known fecal 

pathogen Salmonella enterica. Campylobacter jejuni and other 

Campylobacter species are also fecally derived pathogens but 

are not part of any commonly measured FIB group. 

The four fecally derived pathogens STEC, Shigella, 

Salmonella, and Campylobacter are noted by the Centers for 

Disease Control and Prevention (CDC) to be dominant bacterial causes of fecal-associated waterborne disease (Lee and 

others, 2002). All these organisms can be easily enriched from 

water samples via selective media, including common FIB 

culturing media, and this approach serves as a simple platform 

for detection of these organisms from water samples. Once a 

water sample is enriched for the specific bacterial group, the 

bacterial growth can be used as a basis for specific pathogen 

detection with the DNA-based tool polymerase chain reaction 

(PCR). Additionally, bacterial DNA can be extracted directly 

from water samples without an enrichment step and used in 

quantitative PCR (qPCR) reactions. 

STEC can cause a range of intestinal illnesses, from common watery “traveler’s diarrhea” to severe bloody diarrhea 

(hemorrhagic colitis) that can lead to kidney failure (hemolytic 

uremic syndrome) and death. The E. coli O157 serotype can 

be identified by the presence of the rfbO157 gene (Maurer and 

others, 1999). Genes found in pathogenic E. coli include eaeA, 

stx1, and stx2 (Fagan and others, 1999). In addition, specific 

genes that encode the heat-labile toxin IIa (LTIIa gene) and 

the heat-stable toxin (STh gene) have been demonstrated to be 

specific for pathogenic E. coli from cattle and human sources, 

respectively (Duris and Beeler, 2008; Jiang and others, 2007). 

Shigella sonnei is another common bacterial pathogen 

belonging to the fecal coliform bacteria group. Many Shigella

species can cause shigellosis, an intestinal infection in humans 

that manifests as disease ranging from mild watery diarrhea to 

severe hemorrhagic colitis. Symptoms are similar to, but typically less severe than, diarrhea resulting from STEC infection 

(Schroeder and Hilbi, 2008). 

Salmonella species are one of the most prevalent causes 

of recreational illness in the United States and are occasionally associated with drinking-water-related illness (Lee and 

others, 2002). Salmonella enterica serotypes typically cause 

self-limiting enteritis, but extreme cases can lead to severe 

complications including blood and heart infections (Chiu and 

others, 2004). 

Campylobacter jejuni (C. jejuni) and Campylobacter 

coli (C. coli) are emerging enteric pathogens that have been 

increasingly observed in drinking-water-related illness (Lee 

and others, 2002). Campylobacter causes diarrheal disease 

similar to that resulting from Shigella and STEC (Centers for 

Disease Control and Prevention, 2008). 

In addition to determining what fecal pathogens are 

present, knowing the source of the fecal contamination to a 

water system is useful. Microbial source tracking (MST) is a 

widely used tool for identifying the probable sources of fecal 

pollution that are found in surface waters (Santo Domingo and 

others, 2007). This technology identifies specific differences 

among bacteria present in the feces of different animal species. 

Time, diet, environment, and many other factors may have 

contributed to produce these evolutionary distinctions; MST 

uses these species-specific distinctions to identify potential 

animal (including human) sources of fecal pollution. No MST 

method is 100 percent accurate, owing to variations (seasonal, 

ecological, and others) in occurrence and concentrations of 

the species-specific bacteria tested by most MST approaches. 

It has been documented that MST methods are most effective 

when they are part of a tiered approach that employs multiple 

lines of evidence (Santo Domingo and others, 2007).
Methods 3

Because the City of Wilmington, Del., uses Brandywine 

Creek as its primary source of drinking water, the City was 

in need of information about the occurrence and distribution 

of specific fecally derived pathogenic bacteria, their relations 

to commonly measured FIB, and the potential sources of the 

fecal pollution and pathogens in the basin. In cooperation 

with the City of Wilmington, the USGS did a 1-year study 

in the Brandywine Creek Basin to aid in understanding the 

occurrence and distribution of fecal-indicator bacteria, fecally 

derived pathogens, and potential fecal sources that contribute 

to microbial water-quality impairments. Information obtained 

in this study could be used by the City of Wilmington to 

develop more effective best management practices to control 

fecal contamination within the basin. The pathogen and fecalsource-tracking data collected during the study also support 

the USGS mission to provide scientific information to help 

manage the Nation’s water resources by contributing to the 

nationwide water-quality data program. 

Purpose and Scope

The purpose of this report was to document methodology and the results of analysis done to determine the patterns 

of occurrence of specific fecally derived pathogenic bacteria 

and their relations to commonly measured FIB and to provide information regarding the potential sources of the fecal 

pollution and pathogens in the Brandywine Creek Basin. 

FIB concentration, pathogen and MST marker occurrence, 

and MST marker concentration results were analyzed in the 

context of streamflow conditions both at and between sampling locations. The relation of pathogen and MST markers 

to recreational water quality criteria, and concurrent detection 

of pathogen and MST markers at upstream and downstream 

sampling locations was also analyzed. A detailed examination 

of the study results is provided to illustrate the implications of 

the study findings.

Description of Study Area

Brandywine Creek drains 327 mi2 in southeastern Pennsylvania and northern Delaware. The headwaters of Brandywine Creek are in Chester County, Pa. The stream flows south 

into New Castle County, Del., where it is a tributary to the 

Christina River (fig. 1). The Christina River is a tributary to 

the Delaware River. Population centers in the upstream parts 

of the Brandywine Creek Basin in Pennsylvania include the 

city of Coatesville and the boroughs of Downingtown and 

West Chester. Wilmington, Del., is the largest population center and is in the downstream part of the basin.

Physical Setting

The Brandywine Creek Basin encompasses areas 

in the Piedmont Physiographic Province in southeastern 

Pennsylvania (Sevon, 2000) and the Piedmont and Coastal 

Plain Physiographic Provinces in northern Delaware. 

Land use in the Brandywine Creek Basin is predominantly 

agriculture (39 percent), forested (32 percent), and residential 

(17 percent), with lesser amounts of open (4 percent), urban 

(6 percent), and other land use (2 percent) including industrial 

and commercial uses (Senior and Koerkle, 2002).

The Brandywine Creek Basin has a moderate humid 

continental climate with mild to moderately cold winters and 

warm and humid summers. Mean annual air temperature at the 

National Oceanic and Atmospheric Administration (NOAA) 

weather station in West Chester, Pa. (fig. 1), for 1971–2001 is 

52.2°F (11.2°C). Mean annual precipitation (1971–2000) at 

West Chester is 48.96 in. (National Oceanic and Atmospheric 

Administration, 2010). Precipitation is distributed evenly 

throughout the year.

Major water-withdrawal locations for drinking-water 

supplies are near Wilmington, Del., and Coatesville, Downingtown, and West Chester, Pa. Major wastewater discharges 

in the Brandywine Creek Basin are at Honey Brook, Pa., 

and below Coatesville, Downingtown, and West Chester, Pa. 

(fig. 1).

Methods

Water-Sample Collection

Stream-water samples were collected both routinely and 

for targeted hydrologic events between June 1, 2009, and 

May 30, 2010. Routine samples were collected monthly (on or 

about the 15th of each month) from five sites within the Brandywine Creek Basin (table 1, fig. 1). These samplings covered 

a range of seasonal and hydrologic conditions. Targeted samples were collected quarterly from the five routinely sampled 

sites plus three additional sites to provide more resolution for 

determining potentially important source areas (table 1, fig. 1). 

Two of the targeted sampling events were collected at high 

flow, and two other samplings were collected at normal flow. 

To isolate the influence of combined sewer overflows (CSOs), 

samples were collected at the Baynard Street Bridge, which is 

downstream of several CSO outfalls. All wading samples (12 

of 16 samples) were collected proximal to the Baynard Street 

Bridge except during very high flows, when sampling from the 

stream itself would have been unsafe. On these occasions, the 

sampling location was moved to the Van Buren Street Bridge, 

which was upstream of most CSO outfalls (Colleen Arnold, 

Asst. Water Division Director, City of Wilmington, written 

commun., 2010). These storm and high-flow events occurred 

on October 19, 2009, December 9, 2009, December 15, 2009, 

and May 3, 2010, and were times when the CSOs may have 

been flowing. 
4 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

Honey Brook

Coatesville

Downingtown

West Chester

Wilmington

DELAWARE

PENNSYLVANIA

01481000

01480870

01480700

01480617

01480500

01480300

01481500

01481540

01481555

75°50'0"W

75°50'0"W

75°40'0"W

40°0'0"N 40°0'0"N

39°50'0"N 39°50'0"N

75°40'0"W

•

0 2 4 8 Miles

0 2 4 8 Kilometers

East

Branch

Brandywine Creek

West

Branch

01480617

01481500

01481555

U.S. Geological Survey (USGS) streamgage

and bacteria sample site and station number

Bacteria sample site and station number

USGS streamgage and station number

Borough or city

EXPLANATION

PENNSYLVANIA

DELAWARE

STUDY AREA

Figure 1. Location of sampling sites used in the Brandywine Creek Basin study, 2009–10. 
Methods 5

Table 1. Sites sampled in the Brandywine Creek Basin study, 2009–10. 

[USGS, U.S. Geological Survey; Pa., Pennsylvania; Del., Delaware; CSO, combined sewer overflow]

USGS

station Field 

number identification Station name

Routinely monitored and targeted event survey stations1

01480617 West Branch West Branch Brandywine Creek at Modena, Pa.

01480870 East Branch East Branch Brandywine Creek below Downingtown, Pa.

01481000 Main Brandywine Creek at Chadds Ford, Pa.

01481540 Intake Brandywine Creek near Wilmington, Del.

01481555 CSO Brandywine Creek at Baynard Blvd near Wilmington, Del.

Additional stations for targeted event surveys only2

01480300 Honey Brook West Branch Brandywine Creek near Honey Brook, Pa.

01480500 Coatesville West Branch Brandywine Creek at Coatesville, Pa.

01480700 Downingtown East Branch Brandywine Creek near Downingtown, Pa.

1

Routinely monitored stations were sampled monthly, plus four additional targeted event samples 

(16 total samples).

2

Additional stations were sampled on four occasions only targeting flow events (4 total samples).

All samples were collected by USGS personnel using 

consistent protocols and procedures designed to obtain a sample representative of the stream waters; standard depth- and 

width-integrating techniques were employed (Shelton, 1994). 

At each site, raw composite water samples were collected 

from three to seven vertical profiles. The composites were 

subsequently split into appropriate containers for shipment 

to the USGS laboratory in Lansing, Mich. All samples were 

collected in sterilized plastic containers and stored on ice. A 

total of 100 samples (60 routine, 32 targeted, 4 field replicates, 

and 4 field blanks) were collected. All samples were shipped 

overnight on ice to the USGS laboratory in Lansing, Mich. 

Field measurements of discharge (using automated or manual 

measurements), pH, dissolved oxygen, specific conductance, 

temperature, and turbidity were made each time samples were 

collected (U.S. Geological Survey, 2010 and 2011).

Bacterial Enumeration, Enrichment, and 

Preservation

Samples were examined for the presence of FIB by 

use of standard membrane filtration and serial dilution 

methods as indicated in Britton and Greeson (1989). E. coli

were enumerated according to EPA method 1603 (2006a), 

enterococci were enumerated according to EPA method 

1600 (2006b), and fecal coliform bacteria were enumerated 

according to standard EPA methods (1989). In addition, for 

each sample, 50 mL of sample water was passed through one 

0.45-μm nylon-membrane filter but was not enriched in a 

growth medium (non-enriched). The non-enriched filter, which 

served as the material for qPCR analysis for the quantitative 

MST part of the study, was folded in half four times and 

inserted open side down into a MoBio power soil DNA 

extraction bead beating tube and frozen at −70°C until all 

samples had been collected. 

Selective growth enrichments for pathogenic bacteria 

were done by inoculating growth medium enrichments with 

a standard membrane filter through which 50 mL of sample 

water was passed. To enrich for Campylobacter, one filter 

was added to 14 mL of Bolton Broth with Preston supplement (Oxoid, Cambridge, United Kingdom) in a sterile 15-mL 

polypropylene tube (Baylis and others, 2000). To enrich for 

Salmonella species, a separate filter was added to 10 mL of 

Rappaport-Vassiliadis R10 media (BD, Franklin Lakes, N.J.) 

in a sterile 15-mL polypropylene tube (Zimbro and Power, 

2003). Both enrichments were incubated for 4 hours at 37°C 

and then incubated for 48 hours at 41.5°C. 

Preservation stocks were made from E. coli, enterococci, 

and fecal coliform bacteria growth from the enriched filter 

that received 50 mL of sample water, according to procedures 

in Duris and others (2009). Growth from the Bolton Broth 

and Rappaport-Vassiliadis R10 enrichments was collected 

after incubation, the filter was removed aseptically from the 

broth, and the culture was centrifuged to form a pellet. The 

supernatant was decanted, and the pellet was resuspended in 

1 mL of phosphate buffered saline with a final concentration 

of 15 percent glycerol to create a freezer stock. These stocks 

were preserved at −70°C until further analyzed.
6 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

DNA Extraction

Stocks of enrichment cultures were removed from −70°C 

storage and allowed to thaw. The stocks were homogenized, 

and 50 μL of stock was added to 450 μL of alkaline polyethylene glycol reagent (pH ≈13.3) in a sterile cryotube and mixed 

(Chomczynski and Rymaszewski, 2006). The mixture was 

incubated at 70°C for 10 minutes and then stored at –20°C 

until analyzed by PCR. 

The non-enriched filters were removed from −70°C 

storage and allowed to thaw. MoBio bead beating tubes were 

placed into a Mini Beadbeater-8 (Biospec Products, Bartlesville, Okla.) and homogenized at full speed for 2 minutes. 

After homogenization, DNA extraction was completed according to manufacturer’s instructions.

Polymerase Chain Reaction to Detect Pathogens 

and Microbial-Source Tracking Markers

A volume of 1 μL of DNA solution, representing 

1–100 ng of DNA, was isolated from the appropriate growth 

enrichment and used as a template to conduct PCR analysis 

for selected gene targets (table 2). Results from these analyses indicate only the presence or absence of the target genes; 

they do not indicate the quantity of the genes or the source 

(whether the target genes are all contained in the same organism), only that they were present in the original water sample.

The DNA from the modified mTEC stocks was analyzed 

for the genes required to confirm the presence of enterohemorrhagic E. coli, including the genes eaeA, stx2

, stx1, and a gene 

common to all E. coli, (EC), which was used as an internal 

positive control to confirm the presence of E. coli (Duris 

and others, 2009). E. coli O157 was detected as indicated by 

targeting the rfbO157 gene that is responsible for the production 

of the specific antigen found on the surface of an E. coli O157 

cell (Osek, 2003). The LTIIa gene, which is carried by some 

pathogenic E. coli strains common to bovine fecal sources, 

was analyzed according to methods of Jiang and others (2007). 

The STh gene that is carried by some pathogenic E. coli

strains common to human fecal sources also was detected by 

using methods of Jiang and others (2007). 

Table 2. Growth medium and gene targets for pathogens and microbial-source tracking organisms. 

[E. coli, Escherichia coli]

Gene 

target

Gene 

product

Growth 

medium

Medium 

purpose Organism Reference

eaeA Intimin protein Modified mTEC agar Enumeration/Enrichment E. coli Duris and others, 2009

stx2 Shiga toxin 2 Modified mTEC agar Enumeration/Enrichment E. coli Duris and others, 2009

stx1 Shiga toxin 1 Modified mTEC agar Enumeration/Enrichment E. coli Duris and others, 2009

EC 16s RNA

(common to all E. coli)

Modified mTEC agar Enumeration/Enrichment E. coli Duris and others, 2009

rfbO157 O157 surface protein Modified mTEC agar Enumeration/Enrichment E. coli Osek, 2003

spvC Salmonella plasmid of 

virulence

Rappaport-Vassilidais 

R10 broth

Enrichment only Salmonella Chiu and Ou, 1996

invA Invasion determinant A Rappaport-Vassiliadis 

R10 broth

Enrichment only Salmonella Chiu and Ou, 1996

ipaH Invasion plasmid 

antigen H

mFC agar Enumeration/Enrichment Shigella Islam and others, 1993

16s rDNA 

(Campy)

16s RNA 

(common to indicated 

organisms)

Bolton Broth Enrichment only Campylobacter 

jejuni and coli

Inglis and Kalischuk, 

2003

esp Enterococcus surface 

protein (human sources)

mEI agar Enumeration/Enrichment Enterococci Haack and others, 

2009

E. hirae Putative helicase 

(bovine sources)

mEI agar Enumeration/Enrichment Enterococci Soule and others, 2006

LTIIa Heat labile toxin 

(bovine sources)

Modified mTEC agar Enumeration/Enrichment E. coli Chern and others , 

2004

STh Heat stable toxin

(human sources)

Modified mTEC agar Enumeration/Enrichment E. coli Jiang and others, 2007
Methods 7

The DNA from the mEI stocks was analyzed for MST 

gene markers that indicate enterococci from a human source 

and enterococci from a bovine source. The esp gene is a 

commonly carried by pathogenic strains of Enterococcus that 

infect humans (Shankar and others, 1999) and as a result has 

since been found to be a representative indicator of Enterococcus from a human fecal source (Scott and others, 2005). 

A putative helicase gene in the organism Enterococcus hirae

has been found to represent Enterococcus from a bovine fecal 

source (Soule and others, 2006).

The DNA from mFC agar enrichments was analyzed for 

a pathogenic marker of Shigella sonnei. S. sonnei was then 

detected by using PCR to target a specific variant of the ipaH

gene, which codes for the invasion plasmid antigen of Shigella

species (Islam and others, 1993).

The DNA from Rappaport-Vassiliadis R10 broth enrichments was analyzed for two genes from Salmonella. Salmonella enterica was detected by using PCR detection of the 

invA (invasion gene A) and the spvC (Salmonella virulence 

plasmid). These two genes have been demonstrated to be 

efficient at detection of a broad range of Salmonella enterica 

(Chiu and Ou, 1996).

The DNA from Bolton Broth enrichments was analyzed 

for a gene representing both Campylobacter coli and Campylobacter jejuni. Detection of C. jejuni and C. coli (Campy) 

was achieved by using PCR analysis for the 16S rDNA, which 

encodes the ribosomal RNA specific to C. jejuni and C. coli

(Inglis and Kalischuk, 2003).

A summary of culture enrichments for detection of pathogen and MST genes can be found in table 2. All PCR assays 

followed standard protocols (U.S. Environmental Protection 

Agency, 2004). All methods included laboratory positive 

controls, consisting of DNA extracted from bacteria known to 

contain the target gene(s), and no-template (negative) controls.

Quantitative Polymerase Chain Reaction for 

Microbial-Source Tracking Markers

Quantitative polymerase chain reaction for AllBac (general feces-associated marker), BoBac (ruminant feces-associated marker) and qHF183 (human feces-associated marker) 

was done on an iCycler IQ5 thermal cycler and detector, with 

sample DNA placed in a semi-skirted, 96-well qPCR plate 

and sealed with an optically clear microseal B adhesive seal 

machine (Bio-Rad Laboratories, Inc., Hercules, Calif.). Reactions were carried out in 25 μL total volume, and the reaction 

mixture contained a final concentration of 1X Applied Biosystems Power SYBR Green PCR master mix (Life Technologies, 

Carlsbad, Calif.), 0.25 μM AllBac, BoBac, or HF183 forward 

and reverse primers (Seurinck and others, 2005; Layton and 

others, 2006), 2 μL of neat (undiluted) or 10-fold diluted 

DNA, representing <1 to 60 ng of DNA, with a balance of 

sterile ultrapure water. AllBac and BoBac PCR reactions were 

done by using a 50°C hold for 2 minutes, a 95°C hold for 

10 minutes, and then 50 cycles of 95°C for 30 seconds and 

57°C for 45 seconds (Layton and others, 2006). The HF183 

qPCR was done by using a 50°C hold for 2 minutes, a 95°C 

hold for 10 minutes, and then 40 cycles of 95°C for 30 seconds, 53°C for 60 seconds, and 60°C for 60 seconds (Seurinck 

and others, 2005). All samples were run in triplicate, and a 

no-template (negative) control and standard curve (positive 

controls) were run for each reaction plate. 

Quantitative PCR Data Analysis

For each run of the qPCR assay, a set of standards 

at known concentrations (from 10 copies to 109 copies of 

DNA) were run in triplicate concurrently with environmental 

samples. Raw fluorescence data from each qPCR assay were 

analyzed by using default data analysis on the iQ5 optical 

system software (Bio-Rad Laboratories, Inc., Hercules, Calif.). 

Default data processing consists of subtracting baseline 

fluorescence (determined by the iQ5 instrument) from total 

fluorescence detected by the instrument for each reaction well. 

A threshold value was set that accounted for fluorescence 

increases above baseline that fell within the exponential phase 

of the concurrently run standard curve. All thresholds were 

then checked manually after the automated data processing 

routine, and some were adjusted manually as needed. The iQ5 

software calculated the cycle threshold (Ct) values for each 

sample in the reaction. The Ct value is the cycle number where 

the fluorescence emitted by the sample passes the threshold 

value (that is, it becomes greater than the background fluorescence). Higher concentrations of MST DNA markers in a 

sample will result in lower Ct values; lower concentrations of 

MST DNA markers in a sample will result in higher Ct values. 

Ct values were then converted to copies of DNA per microliter 

of extracted DNA by applying the standard curve relating Ct to 

known copy number of the target gene for each reaction. After 

conversion, Ct values are reported as copies of target DNA per 

100 mL water (copies/100 mL). 

Detection limits and data qualification were done in 

accordance with the methods of Bushon and others (2009). A 

limit of detection (LOD) for each reaction was established by 

using no-template negative control (NTNC) reactions. These 

reactions occasionally show fluorescence not related to target 

DNA late in the final cycles of thermal cycling. In the event 

that this occurred, the 99th-percentile confidence interval was 

calculated for NTNC for the specific reactions. To protect 

against false-positive results, the target concentration at the 

lower 99th percentile, transformed to a relevant concentration (copies/100 mL), was used as the limit of detection. Any 

reported concentration that was lower than this number was 

considered unreliable and was reported as a nondetect (“ND” 

in appendixes 1 and 2).

The lower and upper limits of quantification (LLQ and 

ULQ) for each reaction correspond to the lowest and highest 

concentration of positive-control DNA used to construct the 

standard curve. All DNA concentrations used to develop the 

standard curves fell within the linear range of that curve.
8 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

The LOD and LLQ were used to qualify low-concentration data from qPCR analysis. In all cases, the LOD was 

greater than the LLQ, therefore values that were above the 

LOD were not qualified, whereas values below the LOD were 

reported as nondetects. Quality-control information for all 

qPCR analysis is summarized in table 3.

Data Analysis

Instantaneous discharge measured or recorded from continuous discharge monitors during sample collection was compared to the daily mean flow values for the period of record 

at the corresponding USGS station. Flows at all main-stem 

sampling sites were compared to the flow statistics for USGS 

station 01481500. If the measured discharge was greater than 

the 75th percentile of mean daily flow for that station on 

that date, then the sample was considered to be a high-flow 

sample. If the measured discharge was between the 25th and 

75th percentile of mean daily flow for that station on that date, 

the sample was categorized as a normal-flow sample. Samples 

with measured discharges less than the 25th percentile of 

mean daily flow for that station on that date were to have been 

categorized as low-flow samples; however, flows were higher 

than the 25th percentile at all sites and for all sampling dates 

during the study. 

• For each site, differences in measured microbiological 

parameters were compared between samples collected 

at high flow and at normal flow. Differences in measured microbiological parameters were also compared 

between sites sampled at the same flow conditions. 

Statistical comparisons between sites were made only 

on samples from the five routinely sampled sites. 

• To achieve the study objective, comparisons among all 

five routinely sampled sites were made to determine 

whether there were differences in measured parameters 

at these five locations within the basin. Refined comparisons were made between the routine-sampling sites 

on the East and West Branch, and separate comparisons were made between the three routine-sampling 

sites (Main, Intake, and CSO) on the main stem. 

• Differences in median densities of fecal-indicator bacteria and median densities of MST markers were evaluated using the non-parametric Kruskal-Wallis test on 

log transformed values (Helsel and Hirsch, 2002). Frequencies of gene detections in water samples between 

different sampling sites, flow conditions, or in comparison to water-quality criteria were done using the 

Fisher Exact Test of independence (Helsel and Hirsch, 

2002). All statistical comparisons were computed using 

TIBCO Spotfire S+ 8.1 for Windows statistics package 

(Tibco Software Inc., Somerville, Mass.) and Microsoft Excel 2007. A statistic was considered significant 

when the p-value was less than 0.05.

Microbiological Water Quality of 

Brandywine Creek

The three distinct methods used in this study provided 

multiple perspectives on the microbial quality of the Brandywine Creek Basin. The first method was traditional FIB 

quantification. FIB densities can yield a coarse differentiation of the microbial water quality across a range of sites and 

potentially help to identify “hot spot” areas where exogenous 

factors, such as point sources, could be contributing to fecalindicator bacteria concentrations. The second method was the 

use of selective enrichment combined with presence/absence 

PCR assays for both common fecally derived pathogens and 

for selected MST markers for human and bovine source from 

both E. coli and enterococci. The third method was quantitative MST by means of quantitative PCR from direct (not 

enriched) samples. These three methods provide a multiplelines-of-evidence approach that is suggested as a means to 

demonstrate real differences in the microbial quality of surface 

water (Santo Domingo and others, 2007).

Table 3. Standard-curve characteristics for AllBac, BoBac, and qHF183 microbialsource tracking (MST) markers used in the Brandywine Creek Basin study, 2009–10. 

[R2, coefficient of determination]

MST 

marker

Number of 

compiled 

curves Dynamic range R2 range

Efficiency 

range

(in percent)

Detection 

limit1

AllBac 6 4.31×101–1.00×109 0.966–0.997 78.9–105.4 160

BoBac 6 1.00×102–1.00×109 .951–.998 79.0–94.9 205

qHF183 4 1.00×102–1.00×109 .971–.986 81.5–92.3 274

1 Gene copies per 100 milliliters of water. 
Microbiological Water Quality of Brandywine Creek 9

Results of Indicator Organism Sampling

During the study, log E. coli densities ranged from 0.60 

to 4.87 colony forming units per 100 mL (CFU/100 mL), with 

a log geometric mean of 2.37 CFU/100 mL. Log enterococci 

densities ranged from nondetection to 4.63 CFU/100 mL, 

with a log geometric mean of 2.12 CFU/100 mL. Log fecal 

coliform bacteria densities ranged from 0.60 to 

4.72 CFU/100 mL, with a log geometric mean of 

2.41 CFU/100 mL. Log FIB densities for each site for samples 

collected at high flow and normal flow are displayed by site in 

figure 2.

At routinely sampled sites on the West Branch and all 

sites on the main stem of Brandywine Creek (Main, Intake, 

CSO sites), median densities of FIB (E. coli, enterococci, and 

fecal coliform bacteria) were significantly higher in samples 

collected during high flow than in those collected during normal flow at the same site. However, no significant differences 

were found between FIB densities in high-flow and normalflow samples collected in East Branch Brandywine Creek 

(table 4, fig. 2).

Median densities of FIB were not significantly different 

among all five routinely sampled sites for samples collected 

during normal flow. However, during high flow, the difference in median E. coli densities among all routinely sampled 

sites was statistically significant (Kruskal-Wallis test, p<0.05). 

Finer scale comparisons were made between the East and West 

Branches of Brandywine Creek and among the three sites on 

the main stem. 

In comparisons of median densities of FIB between the 

West Branch and East Branch, no significant differences were 

observed at normal flows, but during high flows significantly 

higher counts of E. coli and enterococci (but not fecal coliform 

bacteria) were found on the West Branch than on the East 

Branch (table 4). No significant differences were observed 

in median densities of FIB among the three sites on the main 

stem of Brandywine Creek at either high or normal flow.

Because there are only two high-flow observations at the 

event-targeted sites, no statistical comparison of FIB densities 

can be made. However, the highest FIB counts were measured 

during event samples at West Branch Brandywine Creek near 

Honey Brook, Pa., site (USGS station number 01480300, 

appendix 1). 

Table 4. Comparison of median fecal-indicator bacteria concentrations in East Branch and 

West Branch Brandywine Creek, 2009–10, by flow condition. 

[CFU/100 mL, colony-forming units per 100 milliliters]

Fecal-indicator 

organism Flow condition1

West Branch2 East Branch3

p-value4

Median

concentration

(CFU/100 mL)

Median 

concentration 

(CFU/ 100 mL)

Escherichia coli High 4.60E+03 1.53E+02 p<0.05

Normal 1.75E+02 3.75E+01 Not significant

p-value4 p<0.05 Not significant

Enterococci High 5.30E+02 7.50E+01 p<0.05

Normal 5.00E+01 1.70E+01 Not significant

p-value4 p<0.05 Not significant

1

Flow condition in stream at time of sample.

2

West Branch, U.S. Geological Survey (USGS) station number, 01480617.

3

East Branch, USGS station number, 01480870.

4

p-value of Kruskal-Wallis rank sum test on log-transformed bacteria concentrations, p-value <0.05 indicates 

a significant difference in the comparison.
10 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

01480300 (Honey Brook)

01480500 (Coatesville)

01480617 (West Branch)

01480700 (Downingtown)

01480870 (East Branch)

01481000 (Main)

01481540 (Intake)

01481555 (CSO)

01480300 (Honey Brook)

01480500 (Coatesville)

01480617 (West Branch)

01480700 (Downingtown)

01480870 (East Branch)

01481000 (Main)

01481540 (Intake)

01481555 (CSO)

01480300 (Honey Brook)

01480500 (Coatesville)

01480617 (West Branch)

01480700 (Downingtown)

01480870 (East Branch)

01481000 (Main)

01481540 (Intake)

01481555 (CSO)

0

1

2

3

4

5

0

1

2

3

4

5

0

1

2

3

4

5

log Enterococci densities,

in colony forming units per 100 milliliters

log Escherichia coli densities,

in colony forming units per 100 milliliters

log Fecal coliform densities,

in colony forming units per 100 milliliters

A. Enterococci

B. Escherichia coli C. Fecal coliform

EXPLANATION

Flow condition—Red indicates high flow;

 blue indicates normal flow

Density of fecal-indicator bacteria

 Extreme outlier

 Outlier

 Largest data value within 1.5 times the

 interquartile range above the box

 75th percentile

 Median (50th percentile)

 25th percentile

 Smallest data value within 1.5 times the

 interquartile range below the box

Significant difference

CSO, combined sewer overflow

Interquartile range

Figure 2. Concentrations of fecal-indicator bacteria, by sample location, from samples collected at high mean 

daily flow or normal mean daily flow during the Brandywine Creek Basin study, 2009–10. A yellow triangle 

above the x-axis label indicates a significant difference in median concentrations at that site between samples 

collected at high flow and normal flow. 
Microbiological Water Quality of Brandywine Creek 11

Results of Pathogen Testing

All pathogen gene markers were detected on at least one 

occasion, from at least one site, during the course of the study. 

The internal positive control EC gene marker was detected 

in 98 percent of samples. It was not detected on two occasions when densities of E. coli were near detection limits 

(appendix 1). The overall frequency of pathogen gene markers 

ranged from 84 percent for the eaeA gene of pathogenic E. coli

to 6.5 percent for the spvC gene marker of pathogenic Salmonella. A summary of the detection frequencies of pathogen and 

MST gene markers across all sites can be found in figure 3.

Table 5 shows the frequency of gene detections in 

samples meeting or exceeding the moderate full-body-contact 

single-sample recreational water-quality criteria (RWQC) for 

E. coli and enterococci FIB (Dufour and Ballantine, 1986). 

Of the 92 regular samples collected, 46 samples exceeded 

both the RWQC for E. coli and enterococci and 35 samples 

exceeded neither the RWQC for E. coli or enterococci. Four 

samples exceeded the RWQC for E. coli but not enterococci, 

and seven samples exceeded the RWQC for enterococci 

but not E. coli. Samples exceeding the E. coli RWQC of 

298 CFU/100 mL more frequently contained the eaeA, stx1, 

and rfbO157 pathogen marker genes. Pathogen marker frequencies also were compared in samples meeting or exceeding the 

enterococci RWQC of 78 CFU/100 mL; as with the E. coli

RWQC, samples exceeding the enterococci RWQC also more 

frequently contained the eaeA, stx1, and rfbO157 pathogen genes 

(table 5). 

The frequency of pathogen marker detection in samples 

collected at high flow and normal flow at each routine site was 

compared (table 6). Detection of the stx1 and rfbO157 pathogen 

markers were significantly more frequent in high-flow samples 

compared to normal-flow samples in the West Branch. In the 

East Branch, no difference was found in the detection frequencies of any pathogen marker between high and normal 

flow. Differences in the frequency of stx1 and rfbO157 at the 

Main site were also observed between high and normal flow. 

The frequency of rfbO157 pathogen marker was also greater in 

high-flow samples than in normal-flow samples at the Intake 

and CSO sites, reflecting differences observed at the Main and 

West upstream sampling locations (table 6).

The frequency of pathogen gene occurrence specifically in the high-flow samples was compared among all five 

routinely sampled sites. Only the frequency of rfbO157 pathogen 

marker was significantly different among the five sites (Fisher 

Exact Test, p<0.05). The largest difference in rfbO157 frequency 

(when comparing all sites) was found to be between the East 

Branch and the Intake/CSO sites (fig. 4). When frequencies 

of gene detection from the same five sites were compared for 

samples collected at normal flow, no differences were found 

among the five sites. 

A comparison of the gene detection frequency that 

was restricted to the West Branch and East Branch samples 

collected at high flow revealed significantly more frequent 

detections of stx1 marker in the West Branch but not rfbO157, for 

which significant differences were found in the comparison of 

all routine sites.

The stx2 gene was found at the routinely sampled Main 

site on only two occasions (December 9, 2010, and March 15, 

2010). Samples on both dates represented high flow for all 

sites, and on both occasions the gene was also found upstream 

only in West Branch Brandywine Creek, not in East Branch 

Brandywine Creek (table 7, appendix 1). The sample collected on December 9, 2010, was a targeted high-flow event 

sample, and in addition to the routinely sampled site being 

positive for the stx2 gene, the Coatesville and Honey Brook 

sites on the West Branch also were positive on that occasion. 

The exclusive detection of this gene indicates that at high flow, 

West Branch Brandywine Creek is the most probable source 

for fecally derived pathogenic E. coli bacteria possessing the 

stx2 gene marker that are being transported to the main stem. 

It should also be noted that other gene markers found at the 

main stem on December 9, 2010, including eaeA and stx1 gene 

markers, were also found upstream in both the East and West 

Branches, indicating that pathogenic E. coli containing the 

eaeA and stx1 genes could be originating from either the West 

or East Branch subbasins. 

When the frequency of gene detection was compared 

between the East and West Branches at normal flow, no significant differences were observed. However, these data show 

that at high flow, the stx1 gene was more frequently detected 

in the West Branch than East Branch and that on selected 

sampling dates; the West Branch was acting as the only potential observed source for the stx2

 containing E. coli pathogens 

(table 7) at the Main site (Brandywine Creek at Chadds Ford, 

Pa., 01481000).

The frequency of pathogen markers was also specifically 

compared among the three main-stem sites (Main, Intake, 

and CSO), and no significant differences were found between 

high-flow and normal-flow samples. Four samples at the CSO 

site were collected from the Van Buren Street Bridge upstream 

of the CSO outfalls. In the four remaining samples collected at 

high flow that were downstream of the CSO outfalls (Baynard 

Street Bridge), pathogen markers detected at the CSO site 

were also detected upstream on the main stem, so no determination of CSO impact could be made. 

Although frequency analysis is informative about the 

general condition of the surface-water system in the basin, it 

does not explain connections between sites or variation that is 

taking place over time. In an effort to address specific effects 

that upstream water quality had on downstream water quality, 

as indicated by frequency analysis, each pathogen gene marker 

was categorized on the basis of its relation with the upstream 

sample locations on the same date of sampling. For instance, 

if a pathogen marker was detected at the West Branch site and 

Main site on a particular sample date but not detected in the 

East Branch site on the same date, then that condition was 

noted as West and Main, and the number of samples meeting 

this condition was noted. The results from this categorization 

are displayed in table 7. 
12 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

Gene marker Gene marker

eaeA

stx2

stx1

spvC

invA

Shigella

Campy

esp-human

STh-human

E. hirae-bovine

LTlla-bovine

rfb0157

E. coli (control)

0

20

40

60

80

100

Positive samples, in percent

Pathogen markers Microbial-source tracking markers

eaeA

stx2

stx1

E. coli (control)

rfb0157

spvC

invA

Shigella

Campy

esp-human

E. hirae-bovine

STh-human

LTlla-bovine

Gene marker

Intimin protein

Shiga toxin 2

Shiga toxin 1

Escherichia coli

O157 surface protein

Salmonella plasmid of virulence

Invasion determinant A

Shigella invasion plasmid antigen H gene

16S ribosomal deoxyribonucleic acid gene of Campylobacter coli or Campylobacter jejuni

Enterococcal surface protein (human sources)

Enterococcus hirae (bovine sources)

Heat stable toxin (human sources)

Heat labile toxin (bovine sources)

EXPLANATION

Figure 3. Frequency of all samples testing positive for pathogen and microbial-source tracking markers in the Brandywine Creek 

Basin study, 2009–10. 
Table 5. Detection frequency of pathogen and microbial-source tracking gene markers in the Brandywine Creek Basin study, 2009–10, based on relarecreational water-quality criteria. [RWQC, recreational water-quality criteria; n, number of samples; MST, microbial-source tracking; E. hirae, Enterococcus hirae; E. coli, Escherichia col;, --, not relevant; <, lens, not significant; CFU/100 mL, colony-forming units per 100 milliliters] 

RWQC n

Pathogen gene markers MST gene mark

eaeA stx2 stx1 EC rfbO157 spvC invA ipaH Campy

esp

human 

E. hirae

bovine

ST

hum

Meet E. coli1 42 0.64 0.12 0.048 0.95 0.095 0.071 0.24 0.024 0.048 0.024 0.048 0.1Exceed E. coli1 50 1 .12 .5 1 .64 .06 .34 .12 .12 .16 .62 .1p-value2 -- <.05 ns <.05 ns <.05 ns ns ns ns ns <.05 ns Meet enterococci3 40 .62 .12 .05 .95 .075 .075 .2 .025 .075 0 .075 .2Exceed enterococci3 52 1 .12 .48 1 .63 .058 .37 .12 .096 .17 .58 .3p-value2 -- <.05 ns <.05 ns <.05 ns ns ns ns <.05 <.05 ns 

1E. coli moderate full-body contact recreational water-quality criteria of 298 CFU/100 mL (Dufour and Ballantine, 1986).2Fisher’s exact test p-value, significant difference in frequency between groups when p<0.05.3Enterococci moderate full-body contact recreational water quality criteria of 78 CFU/100 mL (Dufour and Ballantine, 1986).

Table 6. Detection-frequency comparison of pathogen and microbial-source tracking gene markers at routinely sampled sites during high and normBrandywine Creek Basin study, 2009–10. [USGS, U.S. Geological Survey; Field ID, station identifier from table 1; MST, microbial-source tracking; E. hirae, Enterococcus hirae] 

USGS 

station

number

Field 

ID

Pathogen gene markers MST gene marke

eaeA stx2 stx1 EC rfbO157 spvC invA ipaH Campy

esp

human 

E. hirae

bovine

STh

huma

01480617 West ns1 ns high2 ns high ns ns ns ns ns ns ns 01480870 East ns ns ns ns ns ns ns ns ns ns ns ns 01481000 Main ns ns high ns high ns ns ns ns ns high ns 01481540 Intake ns ns ns ns high ns ns ns ns ns high ns 01481555 CSO ns ns ns ns high ns ns ns ns ns high ns 

1ns, no significant difference in frequency of marker detection between high- and normal-flow conditions.2high, significantly higher frequency of marker detection under high-flow conditions when compared to normal-flow conditions.
14 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

West (7) East (12) Main (8) Intake (8) CSO (8)

Routine station (number of samples)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Frequency of detection

rfbO157 (O157 surface protein)

E. hirae (Enterococcus hirae)

stx1 (Shiga toxin 1)

stx2 (Shiga toxin 2)

Pathogen marker

EXPLANATION

CSO, combined sewer overflow

Figure 4. Detection frequency of selected gene markers at routinely sampled sites at high flow in the 

Brandywine Creek Basin study, 2009–10. 

Table 7. Number of concurrently positive samples collected at selected sites in the Brandywine Creek Basin study, 2009–10. 

[MST, microbial-source tracking; E. hirae, Enterococcus hirae; --, no concurrently positive samples]

Positive Pathogen gene markers MST gene markers

sample 

locations1 eaeA stx2 stx1 rfb spvC invA O157 ipaH Campy

esp

human 

E. hirae STh

bovine human

LTIIa

bovine

High flow

West and Main 1 2 2 2 -- -- 1 -- -- 4 -- 1

East and Main -- -- -- -- -- -- -- -- -- -- -- --

West, East, and Main 7 -- 2 3 -- 2 -- -- -- 1 -- 2

Main only -- -- 1 1 -- 2 -- -- -- 1 1 --

East or West, but 

not Main

-- -- -- -- 1 2 2 -- -- -- 1 2

Intake only -- -- -- -- -- 1 -- -- 3 -- 1 --

Intake and CSO -- -- -- 1 -- -- -- -- -- -- -- 1

CSO, no other 

main stem

-- -- 1 -- -- -- -- -- -- -- 1 --

Normal flow

West and Main 2 -- -- -- -- -- -- -- -- -- -- --

East and Main 1 -- -- 1 -- -- -- -- -- -- 1 --

West, East, and Main 3 -- -- -- -- -- -- 1 -- -- -- --

Main only -- -- -- -- 1 2 1 -- -- -- 1 1

East or West, but 

not Main

1 1 1 1 2 1 -- 1 1 3 2 1

Intake site only -- -- -- 2 -- 1 -- 1 -- -- -- 1

Intake and CSO -- -- -- -- -- -- -- -- 1 -- -- --

CSO, no other 

main stem

-- 2 2 -- -- 1 -- -- -- -- 1 --

1

Sample location names refer to field names in table 1.
Microbiological Water Quality of Brandywine Creek 15

At high flow, there were eight separate occasions when 

five different pathogen markers (eaeA, stx2, stx1, rfbO157, and 

ipaH) were detected in the West Branch samples but not the 

East Branch samples, and these same genes were also detected 

in the Main samples on the same day; this pattern indicates 

a likely connection between the West Branch and Main sites 

(table 7). These samples were all collected from October 

through March (appendix 1). At no time under high-flow sampling conditions did an East Branch sample exclusively contain a gene that was concurrently found in the Main sample, 

indicating the East Branch was an unlikely sole source of 

increased FIB densities in the main stem on those occasions. 

However, it should be noted that there were 14 other occasions 

covering 4 pathogen gene markers (eaeA, stx1, rfbO157, and 

invA) when the marker was found at both the East and West 

sites and also at the Main site. Therefore on select occasions 

at high flow the West Branch was the only measured source of 

stx2, stx1, rfbO157, and ipaH to the Main site. The East Branch 

was never observed to be the sole contributor of pathogen 

genes to the Main site at high flow. However, on select occasions, both the East and West Branch sites at high flow could 

have been contributing pathogen gene markers to the Main 

site, making determination of the probable subbasin delivering 

markers and FIB difficult on those occasions. 

There were several occasions where Salmonella markers (invA and spvC) and the Shigella marker (ipaH) were 

detected upstream at East and West Branch but not at the 

Main site downstream. Flow at the time of these samples was 

greater than the 75th percentile of daily mean flow at each 

corresponding USGS site, which should have been sufficient 

to transport these markers downstream. The markers were possibly present in low (but detectable) concentrations in the East 

and West Branches but were diluted to nondetectable amounts 

as they were moved downstream. 

The stx1 pathogen marker was detected at the CSO site on 

one high-flow occasion when it was not concurrently detected 

upstream at either the Main or the Intake site. On this occasion (December 15, 2009) the site was sampled at the Van 

Buren Street Bridge, which is upstream of the CSOs (Colleen 

Arnold, Asst. Water Division Director, City of Wilmington, 

written commun., 2010). So, the occurrence of the marker on 

that date was likely unrelated to the CSOs. The stx1, invA, and 

stx2 genes also were found under normal flow conditions at the 

CSO site (but not at the Main or Intake sites) at normal flow 

(table 7). Finding elevated pathogens and FIB downstream of 

CSOs that are not actively discharging has been reported by 

Donovan and others (2008) in a study of the Lower Passaic 

River, and could explain gene detections downstream of the 

CSO during normal flow in this study.

At normal flow, there were two instances when the eaeA

pathogen marker was detected in the West Branch and concurrently detected at the Main site (table 7). Also at normal flow, 

there were two occasions, one each for eaeA and rfbO157, when 

pathogen markers were detected in the East Branch and concurrently at the Main site. However, on three occasions, the 

eaeA pathogen marker was detected at the Main site when it 

was detected concurrently in both the East and West Branches, 

a pattern that indicates that both subbasins are contributing 

to the movement of the eaeA gene to the main stem. There 

were several normal and high flow samples when genes were 

detected in the East or the West Branch but were not detected 

downstream at the Main site. This result could be indicative of 

specific point sources affecting the occurrence of these genes 

on the East and West Branches. 

The combined pathogen analysis indicates that certain 

of the pathogen markers are significantly more likely to occur 

when densities of FIB exceed RWQC. The stx2 gene on two 

occasions was exclusively detected at upstream locations in 

the West Branch and was also observed on the same dates at 

the Main site downstream. The stx2 gene was never detected 

on the East Branch, indicating that the West Branch is serving 

as the only measured source of stx2-carrying pathogens at high 

flows in the Brandywine Creek Basin. In addition, there were 

six other observations when pathogen genes were measured at 

the West Branch and Main sites but not at the East Branch site 

at high flow. This finding also supports the conclusion that the 

West Branch is a likely source for fecally derived pathogens at 

high flows in the Brandywine Creek Basin. However, several 

observations of matching pathogen gene markers at the East 

Branch, West Branch, and Main sites were also made. Determining the source of the markers at the Main site is difficult on 

every occasion, but these findings indicate that there are most 

likely specific conditions and times when the West Branch is 

the exclusive contributor of pathogen genes to the main stem 

of Brandywine Creek. This study found the West Branch to 

be the only potential measured contributor on eight occasions. 

These samples were collected at high flow between October 

and March. 

Results of Microbial-Source Tracking

All MST gene markers were detected on at least one 

occasion, from at least one site, during the course of the 

study. The overall frequency of MST gene markers ranged 

from 35.9 percent for the E. hirae bovine MST marker to 

9.8 percent for the esp human MST marker. A summary of the 

detection frequencies can be found in figure 3. 

Quantitative analyses for general (AllBac), bovine 

(BoBac), and human (qHF183) Bacteroides MST markers 

were also done. The AllBac marker ranged in detection 

from a maximum of 4,210,000 copies/100 mL to a 

minimum of nondetection, with a mean concentration of 

162,000 copies/100 mL, and a median concentration of 

800 copies/100 mL. The BoBac marker ranged from a 

maximum of 3,650,000 copies/100 mL to minimum of 

nondetection, with a mean concentration of 

89,900 copies/100 mL and a median of 103 copies/100 mL 

(nondetection). The qHF183 marker ranged from a 

maximum concentration of 171,000 copies/100 mL to a 

minimum of nondetection, with a mean concentration of 

16,200 copies/100 mL, and a median concentration of 

2,500 copies/100 mL.
16 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

Results of Qualitative Microbial-Source Tracking

Table 5 shows the frequency of MST marker detections in 

samples meeting or exceeding the moderate-full-body-contact 

single-sample recreational water-quality criteria (RWQC) for 

E. coli and enterococci FIB (Dufour and Ballantine, 1986). 

Samples exceeding the E. coli RWQC more frequently contained the E. hirae and LTIIa MST gene markers that represent 

enterococci and E. coli from a bovine source. MST marker frequencies were also compared in samples meeting or exceeding the enterococci RWQC of 78 CFU/100 mL. Samples 

exceeding this criterion more frequently contained the esp 

(human sources), E. hirae (bovine sources), and LTIIa (bovine 

sources) MST markers (table 5). Results indicate a relation 

between samples exceeding the E. coli RWQC and bovine 

fecal pollution, but exceedance of the enterococci RWQC was 

related to both human and bovine fecal sources.

The frequency of MST marker detection in samples collected during high flow and normal flow at each routine site 

was compared (table 6). Significantly more frequent detections of LTIIa bovine MST marker were found in high-flow 

samples than in normal-flow samples in the West Branch. In 

the East Branch, there was no significant difference in the 

frequencies of any MST marker between high and normal 

flow. Interestingly, the E. hirae bovine MST marker was more 

frequent at the Main site, in contrast to the LTIIa bovine MST 

marker, which was more frequent in the West Branch. Differences in frequency of E. hirae bovine MST marker between 

high and normal flow were also observed at the Intake and 

CSO sites, again reflecting differences observed at upstream 

sampling locations; however, the occurrences of E. hirae at the 

main stem sites appear unrelated to those on the East or West 

Branch.

The frequency of MST marker occurrence during high 

flow was compared among all five routinely sampled sites. 

Only the frequency of the E. hirae bovine MST marker was 

different among the five sites (fig 4), with the largest difference occurring between the Intake site and the East Branch 

site. When the frequency of gene detection from the same five 

sites was compared for normal flow, the frequency of E. hirae

marker detection was again found to be significantly different among the sites (not shown). At normal flow, the E. hirae 

MST marker was detected on one of four occasions in the East 

Branch and three of nine occasions in West Branch Brandywine Creek. This difference between the East and West Branch 

samples was never translated downstream because the E. hirae

marker was never found on the main stem at normal flow, even 

when found upstream.

A comparison of the MST marker detection frequency 

only between the routinely monitored sites in West Branch 

and East Branch Brandywine Creek revealed that at high flow, 

detections of the E. hirae bovine MST marker were more 

frequent in the West Branch, a result that parallels observations when simultaneously comparing all five routinely monitored sites at high flow (fig. 4). When the frequency of gene 

detection was compared between the East and West Branches 

at normal flow, no significant differences in the frequency 

of gene detection were observed. These results indicate that 

West Branch of Brandywine Creek is the most likely source of 

increased frequencies of the E. hirae MST bovine marker at 

high flow in the basin. 

The frequency of MST markers was also specifically 

compared among the three main-stem sites (Main, Intake, and 

CSO). No differences were found in the frequency of MST 

marker detection among the main-stem sites at high flow 

(fig. 4) or normal flow (not shown). Four samples at the CSO 

site were collected from the Van Buren Street Bridge upstream 

of the CSO outfalls. In the four other samples collected at high 

flow downstream of the CSO outfalls (Baynard Street Bridge), 

only one sample (June 15, 2009) was found to contain a MST 

marker (STh) that was not also found upstream. Although 

this indicates a potential effect on the microbial water quality 

of the stream from the CSO outfalls, the lack of downstream 

samples during the most appropriate conditions to observe a 

CSO effect substantially limits interpretation. 

In an effort to understand the specific relations that 

upstream water quality had on downstream water quality on 

each sample date, each MST marker was categorized on the 

basis of its relation with the upstream sample locations, as 

was done for pathogen gene markers (table 7). On a total of 

five sampling occasions at high flow, E. hirae (four occasions) 

and LTIIa (one occasion) were detected upstream in the West 

Branch and concurrently at the Main site (table 7). These five 

occasions were spread throughout the year (June, October, 

December, and March) (appendix 1). The East Branch was 

never the exclusive contributor of either E. hirae or LTIIa 

gene at high flow, but there were three occasions when the 

genes were observed at both the East and West Branches and 

downstream at Main, making determination of the subbasin 

delivering these markers to the main stem difficult on those 

occasions. On other sampling occasions at high flow, MST 

markers were detected at the Main site but not upstream at 

the East or West Branch sites. This pattern could indicate that, 

under certain conditions, there may be sources that are closer 

to the Main site that also are contributing the fecal load (and 

thus MST markers) to the stream. There were few occasions 

when MST markers (two LTIIa, one STh) were detected 

upstream in the East and West Branches but not downstream 

at the Main site, suggesting that the markers may have been 

diluted or reduced past detectable densities at the Main site.

On the main stem of Brandywine Creek at high flows, 

the esp human MST marker was detected exclusively on three 

occasions at the Intake site. On a fourth occasion (independent of the esp detections), another human marker—the STh 

marker—was found at the Intake site but not upstream or 

downstream on the main stem of Brandywine Creek. Neither 

marker was found at normal flow at the Intake site; however, 

the rfbO157, invA, and Campy pathogen markers were found 

exclusively at the Intake site at normal flow on separate occasions (table 7). These four separate detections of human MST 
Microbiological Water Quality of Brandywine Creek 17

markers at high flow in the absence of immediate upstream or 

downstream detection of the observed genes, and the exclusive 

detection of selected pathogen genes at normal flow, likely 

indicate a potential proximal human source that is contributing human fecal markers as well as Salmonella and pathogenic 

E. coli. Storm-water runoff from non-sanitary storm-water 

drainage systems has been implicated as a source of pathogens 

in urban environments and could explain detection of human 

MST markers at high flow (Arnone and Walling, 2007).

Results of Quantitative Microbial-Source 

Tracking

Analysis for selected MST markers from the fecal anaerobe Bacteroides also was done for general (AllBac), human 

(qHF183), and bovine (BoBac) fecal sources. The median of 

log transformed Bacteroides concentrations were compared 

among all routinely sampled sites. No significant differences 

were found between samples collected at high and normal 

flows. Comparisons were also made between median Bacteroides concentrations in the East and West Branches for high and 

normal flows. During high flow, the median AllBac concentration was significantly greater in the West Branch, which 

parallels the finding, that median FIB densities were also 

greater in the West Branch during high flow. No differences 

were observed between median MST marker concentrations 

in samples collected at the West and East Branches at normal 

flow. Comparison of samples collected from the main-stem 

sites revealed no significant differences in median concentrations for high and normal flows.

The highest AllBac, BoBac, and qHF183 concentrations 

were measured in samples collected during high-flow event 

sampling. Instantaneous loads were calculated for the three 

MST markers and the percent contribution of load from the 

West and East Branch was computed at the Main site. Percent 

contribution data are presented in figure 5. Instantaneous loads 

of qHF183 were excluded for the December 9, 2009, sample 

because no qHF183 markers were detected at the Main site 

on this day, even though there were high concentrations in the 

contributing tributaries. In some samples collected during the 

study, the measured qHF183 and BoBac concentrations exceed 

the concentration of the AllBac measurement. This phenomenon has been observed in previous studies, and it is typically 

attributed to differences in the performance of qPCR assay 

primers (Layton and others, 2006).

During both high-flow event samples, the West Branch 

was found to contribute a greater percentage of the load of AllBac and BoBac markers to the Main site than the East Branch 

did. The qHF183 contributions were found to be roughly similar for the high-flow sample where the analysis was possible. 

It is important to note that differences in the quantity of MST 

markers in different fecal sources (even from individual animals) can vary greatly, making these types of direct comparisons useful guides—but not necessarily indications of absolute 

differences—in the amount of fecal contamination (Field and 

Samadpour, 2007; Stoeckel and Harwood, 2007).

AllBac BoBac qHF183

12/9/2009

5/3/2010

12/9/2009

5/3/2010

12/9/2009

5/3/2010

Percent Bacteroides marker load

0

50

100

150

200

250

300

*

West Branch

East Branch

Fecal source

 General

 Bovine

 Human

EXPLANATION

AllBac

BoBac

qHF183

* Percent contribution calculation was not possible on this date

 because of a nondetection for qHF183 at Main site

Figure 5. Percent contribution of 

AllBac, BoBac, and qHF183 microbialsource tracking marker load from East 

and West Branches of Brandywine 

Creek during targeted high-flow event 

sampling, 2009–10. The asterisk indicates 

that the percent contribution calculation 

was not possible on this date because of 

a nondetect for qHF183 at the Main site. 
18 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

Quality Control

Four field blanks and four field replicates were collected 

and run in the same manner as regular field samples. A routine 

laboratory no-template (negative) control was processed 

for every 25 samples analyzed for each assay, and positive 

controls were run at 2–3 concentrations ranging from near the 

detection limit of the assay to three orders of magnitude above 

the detection limit. All no-template controls were found to be 

negative as expected and all positive assay controls yielded 

appropriate responses (data not shown). 

All field blanks were negative (below detection limits) for 

all FIB, for all qualitative pathogen markers and MST markers, and for the AllBac and BoBac analysis. However, on two 

of four occasions, the qHF183 marker was found at concentrations just above the qHF183 method limit of detection of 

272 copies/100 mL. This result suggests that a very low level 

of field contamination was possible but, because of the low 

magnitude of the contamination, the impact on data interpretation was judged to be negligible. All laboratory blanks were 

found to be negative.

Results from field replicates were assessed in two ways. 

For quantitative FIB assays, a relative standard deviation was 

calculated and a mean was computed. In all cases, the average 

RSD fell below the recommended 35 percent (U.S. Environmental Protection Agency, 2006a). For quantitative MST 

assays, a relative standard deviation was also calculated and 

a mean computed. There is no currently recommended RSD 

standard for field replicates for Bacteroides MST qPCR methods. A summary of the RSD replicate analysis can be found in 

table 8. 

For qualitative pathogen and MST assays, which yield 

only a presence or absence result, the frequency of agreement 

between the replicates was determined by dividing the number 

of paired replicates with identical values by the total number of replicates. Only two assays showed any variation: the 

eaeA, and stx1 gene assays both showed variable results in the 

same replicate. This result is suggestive of a quantity of target 

DNA very near the detection limit and was considered to have 

a negligible effect on data interpretation. A summary of the 

frequency of agreement field replicate analysis can be found in 

table 9.

Table 8. Relative standard deviation of field replicates for quantitative microbial methods in the 

Brandywine Creek Basin study, 2009–10. 

[E. coli, Escherichia coli; RSD, relative standard deviation] 

Statistic E. coli Enterococci

Fecal coliform 

bacteria AllBac BoBac1 HuBac

Maximum RSD, in percent 29 70 6 141 0 141

Minimum RSD, in percent 18 10 33 16 0 18

Average RSD, in percent 24 28 19 110 0 72

Number of field replicates 4 4 4 4 4 4

1

All four field replicate samples fell below the BoBac assay limit of detection.

Table 9. Frequency of agreement between field replicates for qualitative microbial methods in the Brandywine Creek Basin study, 

2009–10. 

[MST, microbial-source tracking; E. hirae, Enterococcus hirae] 

Statistic

Pathogen gene markers MST gene markers

eaeA stx2 stx1 EC rfbO157 spvC invA ipaH Campy

esp

human 

E. hirae

bovine

STh

human

LTIIa

bovine

Percent 

agreement

75 100 75 100 100 100 100 100 100 100 100 100 100

Number of field 

replicates

4 4 4 4 4 4 4 4 4 4 4 4 4
Summary and Conclusions 19

Summary and Conclusions

The City of Wilmington, Delaware, is in the downstream 

part of the Brandywine Creek Basin, on the main stem of 

Brandywine Creek. Wilmington uses this stream, which drains 

a mixed land-use area upstream, for its main drinking-water 

supply. Because the stream is used as a drinking-water source, 

Wilmington is in need of information about the occurrence of 

specific fecally derived pathogenic bacteria (disease-causing 

organisms) and their relations to commonly measured fecalindicator bacteria (FIB), as well as information regarding the 

potential sources of the fecal pollution and pathogens in the 

basin. Since 2005, Wilmington has been investing in bestmanagement-practices (BMP) projects in the Brandywine 

Creek headwaters to control sediments and pollutants, including bacteria that could affect the drinking-water supply. In 

cooperation with the City of Wilmington, the U.S. Geological 

Survey did a 1-year study in the Brandywine Creek Basin to 

aid in understanding the occurrence and distribution of FIB, 

fecally derived pathogens, and potential fecal sources that contribute to microbial water-quality impairments. Information 

obtained in this study could be used by the City of Wilmington 

to target BMP strategies within the Brandywine Creek Basin. 

The study focused on five routinely sampled sites within 

the basin, one each on the West Branch and the East Branch 

and three on the main stem of Brandywine Creek below the 

confluence of the West and East Branches. These sites were 

sampled monthly for 1 year. Targeted event samples were 

collected on two occasions during high flow (greater than the 

75th percentile of mean daily flow at the site) and two occasions during normal flow (between the 25th and 75th percentiles of mean daily flow). During these targeted events, three 

additional sites were sampled upstream of the routine sampling sites on the West and East Branches. In addition, four 

field blanks and four field replicates were collected throughout 

the study period.

The collected samples were analyzed for the densities 

of the FIB, Escherichia coli (E. coli), enterococci, and fecal 

coliform bacteria. Samples were enriched for growth of the 

fecally derived pathogens Campylobacter and Salmonella,

and total DNA was extracted from each water sample. FIB 

cultures, plus enrichments originating from each water sample, 

were analyzed for the presence of the selected genes that indicated the presence of pathogenic types of E. coli, Salmonella, 

Shigella, and Campylobacter bacteria, as well as the presence 

of specific microbial-source tracking (MST) genes indicating bovine or human fecal pollution. DNA extracted directly 

from each water sample was analyzed for the quantity of each 

of three types of Bacteroides MST markers indicating general, bovine, and human fecal pollution. In addition, routine 

chemical measurements, turbidity measurements, and streamdischarge measurements were made with each sample.

Median concentrations of FIB in Brandywine Creek 

Basin were more elevated at the West Branch, Main, Intake, 

and combined sewer overflow sites (the latter three on the 

main stem) in samples collected during high flow than in those 

collected at normal flow. There was no difference in median 

FIB density in samples collected during high and normal flow 

in the East Branch. Concentrations of E. coli and enterococci 

were higher in samples collected from the West Branch than 

from the East Branch during high flow. No differences in FIB 

densities were observed among the three sites on the main 

stem under any flow condition. The highest concentrations of 

FIB were found in the West Branch samples during high flow.

Samples that exceeded the U.S. Environmental Protection Agency’s recreational water quality criteria (RWQC) for 

E. coli and enterococci FIB were found to more frequently 

contain the eaeA, stx1, and rfbO157 pathogen markers than those 

meeting RWQC. However, there was no difference in the 

frequency of the stx2 E. coli pathogen marker or any measured 

marker for Salmonella, Shigella, or Campylobacter when 

comparing samples that met, versus exceeded, the RWQC for 

either E. coli or enterococci FIB. Water samples exceeding 

the FIB RWQC were more likely to contain selected E. coli

pathogens, but the occurrence of other E. coli pathogens (with 

stx2), Salmonella, Shigella, and Campylobacter was unrelated 

to the FIB RWQC in this study.

The data clearly demonstrate that there are specific 

instances during high flow where the West Branch is the only 

observed source of selected pathogenic E. coli and Shigella

to the main stem, whereas the East Branch was never found 

to be the exclusive contributing source of any measured 

marker. During high flow, the stx1 marker was more frequently 

detected in the West Branch than the East Branch. On two 

occasions during high flow, the West Branch was the only 

observed source of the stx2 gene to the main stem. On other 

occasions during high flow, the West Branch was the only 

observed source of the eaeA, stx1, rfbO157, and ipaH genes 

to the main stem. There were occasions when both the East 

Branch and West Branch were likely delivering markers to 

the main stem; however, results from this study indicate that 

increased discharge was a likely factor related to pathogen 

delivery from the West Branch to main stem. There are likely 

seasonal, spatial, ecological, and hydrological factors not measured during this study that would help refine the understanding of fecally derived pathogens in the basin. Indeed, on some 

occasions at normal flow, we observed markers of pathogenic 

E. coli and Salmonella near the Intake site on the main stem 

but did not observe these markers immediately upstream; this 

pattern suggests diverse transport pathways and alternative 

sources.
20 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

Samples exceeding the E. coli RWQC more frequently 

contained markers of bovine feces, suggesting a relation 

between high E. coli densities and bovine fecal pollution. 

Samples exceeding the enterococci RWQC were found to 

more frequently contain markers for both bovine and human 

feces, suggesting that high enterococci densities are related to 

both bovine and human fecal pollution. The E. hirae bovine 

fecal marker was found more frequently in the West Branch 

than the East Branch during high flow, indicating that elevated 

FIB densities in the West Branch are more frequently related 

to bovine sources than in the East Branch. Matching bovine 

markers were found in the West Branch and main stem on four 

sample dates, indicating the most likely source of FIB in the 

main stem on those dates was water from the West Branch that 

was carrying FIB of bovine origin. The finding of the West 

Branch as the most likely source of FIB was supported by 

the similar increases in frequency of stx1 and rfbO157 markers 

during high flows in the West Branch as well as in the main 

stem, whereas there was no parallel increase in the frequency 

of pathogen or MST marker detection during high flows in the 

East Branch. In addition to having similar patterns of detection 

frequency (which do not account for temporal differences in 

detection), the West Branch and main stem had concurrently 

matching pathogen and MST markers on several occasions, 

whereas the markers were absent from the concurrent East 

Branch samples. It should be noted that there were other 

occasions at high flow when the markers found in East Branch 

samples (both MST and pathogen markers) matched those in 

main-stem samples; but on those occasions, the markers were 

also found in the West Branch, so no clear determination of 

source can be made. There was never an occasion at high flow 

when the East Branch markers exclusively matched main-stem 

markers, indicating that the East Branch is less likely to be a 

source for FIB in the main stem.

Quantitative analysis of MST markers revealed that 

during targeted high-flow event samples, the West Branch 

accounted for a larger proportion of the load of both general 

and bovine source markers at the main stem when compared 

to the East Branch, but care should be taken in direct comparisons of MST quantities because these data are often highly 

variable.

On the basis of this study, high flow in the Brandywine 

Creek Basin was related to increases in FIB densities and 

increases in the detection frequency of selected pathogens and 

MST gene markers in West Branch Brandywine Creek and at 

sites on the main stem of Brandywine Creek, but not in East 

Branch Brandywine Creek. Water exceeding the RWQC for 

E. coli was more likely to contain the selected markers for 

pathogenic E. coli and bovine fecal sources, whereas samples 

exceeding the enterococci RWQC were more likely to contain 

the same pathogenic markers but also carried indications of 

human sources. 

On four sampling dates, during high flow between 

October and March, the West Branch was the only observed 

contributor of selected pathogen and bovine source markers to 

the main stem of Brandywine Creek. Indeed, the stx2 marker, 

which indicates a highly virulent type of pathogenic E. coli,

was found only in the West Branch and main stem at high 

flow—never in the East Branch. However, it must be noted 

that throughout the entire year of sampling there were occasions, during both high and normal flows, when both the East 

and West Branches were potential contributors of pathogen 

and MST markers to the main stem. The major conclusion 

from this study is that under selected conditions (high flow, 

October through March), West Branch Brandywine Creek is 

the most likely source of elevated FIB densities in the main 

stem. These elevated densities are associated with more frequent detection of the stx1

 pathogenic E. coli marker and are 

associated with the E. hirae MST marker of bovine source. 

However, during other times of the year, both the West Branch 

and East Branch are likely acting as potential sources of FIB 

and fecally derived pathogens on the main stem. 

References Cited

Arnone R.D., and Walling J.P., 2007, Waterborne pathogens 

in urban watersheds: Journal of Water and Health, v. 5, 

p. 149–162.

Baylis, C.L., MacPhee, S., Martin, K.W., Humphrey, T.J., and 

Betts, R.P., 2000, Comparison of three enrichment media 

for the isolation of Campylobacter spp. from foods: Journal 

of Applied Microbiology, v. 89, p. 884 – 891.

Britton, L.J., and Greeson, P.E., eds., 1989, Methods for collection and analysis of aquatic biological and microbiological samples: U.S. Geological Survey Techniques of WaterResources Investigations, book 5, chap. A4, 363 p.

Bushon, R.N., Stelzer, E.A., and Stoeckel, D.M., 2009, Results 

from a microbial source-tracking study at Villa Angela 

Beach, Cleveland, Ohio, 2007: U.S. Geological Survey 

Open-File Report 2009 –1160, 9 p.

Centers for Disease Control and Prevention, 2008, The 

National Center for Zoonotic, Vector-Borne, and Enteric 

Diseases, Division of Foodborne, Bacterial and Mycotic 

Diseases, accessed February, 4 2009, at http://www.cdc.gov/

nczved/divisions/dfbmd/diseases/campylobacter/.

Chern E.C., Tsai, Y.L., and Olson B.H. 2004, Occurrence 

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

lagoons. Applied and Environmental Microbiology, v. 70, 

p. 356 –362.
References Cited 21

Chiu, C.H., and Ou, J.T., 1996, Rapid identification of Salmonella serovars in feces by specific detection in virulence 

genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay: Journal of Clinical Microbiology, v. 34, p. 2619 –2622.

Chiu, C.-H., Su, L.-H., and Chu, Chishih, 2004, Salmonella 

enterica serotype Choleraesuis—Epidemiology, pathogenesis, clinical disease, and treatment: Clinical Microbiology 

Reviews, v. 17, p. 311–322.

Chomczynski, Piotr, and Rymaszewski, Michal, 2006, 

Alkaline polyethylene glycol-based method for direct PCR 

from bacteria, eukaryotic tissue samples, and whole blood: 

BioTechniques, v. 40, p. 454 – 458.

Cinotto, P.J., 2005, Occurrence of fecal-indicator bacteria and 

protocols for identification of fecal-contamination sources 

in selected reaches of the West Branch Brandywine Creek, 

Chester County, Pennsylvania: U.S. Geological Survey 

Scientific Investigations Report 2005–5039, 91 p.

Donovan, E., Unice, Ken, Roberts, J.D., Harris, Mark, and 

Finley, Brent, 2008, Risk of gastrointestinal disease associated with exposure to pathogens in the water of the Lower 

Passaic River: Applied and Environmental Microbiology, 

v. 74, p. 994 –1003.

Dufour, A.P., and Ballantine, R.K., 1986, Ambient water 

quality criteria for bacteria, 1986—Bacteriological ambient 

water quality criteria for marine and freshwater recreational 

waters: Washington, D.C., EPA 440/5-84-002.

Duris, J.W., and Beeler, Stephanie, 2008, Fecal-indicator 

bacteria and Escherichia coli pathogen data collected near 

a novel sub-irrigation water-treatment system in Lenawee 

County, Michigan, June–November 2007: U.S. Geological 

Survey Open-File Report 2008 –1025, 13 p.

Duris, J.W., Fogarty, L.R., and Haack, S.K., 2009, Gene and 

antigen markers of Shiga-toxin producing E. coli from 

Michigan and Indiana river water—Occurrence and relation 

to recreational water quality criteria: Journal of Environmental Quality, v. 38, p. 1878 –1886.

Fagan, P.K., Hornitzky, M.A., Bettelheim, K.A., and Djordjevic, S.P., 1999, Detection of Shiga-like toxin (stx1

 and 

stx2), intimin (eaeA), and enterohemorrhagic Escherichia 

coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces 

by multiplex PCR: Applied and Environmental Microbiology, v. 65, p. 868 – 872.

Field, K.G., and Samadpour, Mansour, 2007, Fecal source 

tracking, the indicator paradigm, and managing water quality: Water Research, v. 41, p. 3517–3538.

Haack, S.K., Duris, J.W., Fogarty, L.R., Kolpin, D.W., Focazio, M.J., Furlong, E.T., and Meyer, M.T., 2009, Comparing 

wastewater chemicals, indicator bacteria concentrations, 

and bacterial pathogen genes as fecal pollution indicators: 

Journal of Environmental Quality, v. 38, p. 248 –258.

Helsel, D.R., and Hirsch, R.M., 2002, Statistical Methods in 

Water Resources: U.S. Geological Survey Techniques of 

Water-Resource Investigation, book 4, chap. A3, 522 p.

Inglis, G.D., and Kalischuk, L.D., 2003, Use of PCR for 

direct detection of Campylobacter species in bovine feces: 

Applied and Environmental Microbiology, v. 69, p. 3435–

3447.

Islam, M.S., Hasan, M.K., Miah, M.A., Sur, G.C., Felsenstein, 

A., Venkatesan, M., Sack, R.B., and Albert, M.J., 1993, Use 

of the polymerase chain reaction and fluorescent-antibody 

methods for detection of viable but nonculturable Shigella 

dysenteriae type 1 in laboratory microcosms: Applied and 

Environmental Microbiology, v. 59, p. 536 –540.

Jiang, S.C.; Chu, Weiping; Olson, B.H.; He, J.-W.; Choi, 

Samuel; Zhang, Jenny; Le, J.Y.; and Gedalanga, P.B., 2007, 

Microbial source tracking in a small southern California 

urban watershed indicates wild animals and growth as the 

source of fecal bacteria: Applied Microbiology and Biotechnology, v. 76, p. 927– 934.

Layton, Alice; McKay, Larry; Williams, Dan; Garrett, Victoria; Gentry, Randall; and Sayler, Gary, 2006, Development 

of Bacteroides 16S rRNA gene TaqMan-based real-time 

PCR assays for estimation of total, human, and bovine fecal 

pollution in water: Applied and Environmental Microbiology, v. 72, p. 4214 – 4224.

Lee, S.H., Levy, D.A., Craun, G.F., Beach, M.J., and Caleron, 

R.L., 2002, Surveillance for waterborne-disease outbreaks—United States, 1999–2000: Centers for MMWR 

Surveillance Summaries, v. 51(SS08), p. 1–28.

Maurer, J.J.; Schmidt, Denise; Petrosko, Patricia; Sanchez, 

Susan; Bolton, Lance; and Lee, M.D., 1999, Development 

of primers to O-antigen biosynthesis genes for specific 

detection of Escherichia coli O157 by PCR: Applied and 

Environmental Microbiology, v. 65, p. 2954 –2960.

National Oceanic and Atmospheric Administration, 2010, 

NOWData-NOAA Online Data, 1971–2000, accessed 

August 26, 2010, at http://www.weather.gov/climate/local_

data.php.

Osek, Jacek, 2003, Development of a multiplex PCR approach 

for the identification of Shiga toxin-producing Escherichia 

coli strains and their major virulence factor genes: Journal 

of Applied Microbiology, v. 95, p. 1217–1225.
22 Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pennsylvania and Delaware, 2009–10

Santo Domingo, J.W., Bambic, D.G., Edge, T.A., and Wuertz, 

Stefan, 2007, Quo vadis source tracking?—Towards a 

strategic framework for environmental monitoring of fecal 

pollution: Water Research, v. 41, p. 3539 –3552.

Schroeder, G.N., and Hilbi, Hubert, 2008, Molecular pathogenesis of Shigella spp.—Controlling host cell signaling, 

invasion, and death by type III secretion: Clinical Microbiology Reviews, v. 21, p. 134 –156. 

Scott, T.M., Jenkins, T.M., Lukasik, Jerzy, and Rose, J.B., 

2005, Potential use of a host associated molecular marker in 

Enterococcus faecium as an index of human fecal pollution: 

Environmental Science & Technology, v. 39, p. 283–287.

Senior, L.A., and Koerkle, E.H., 2002, Simulation of streamflow and water quality in the Brandywine Creek subbasin 

of the Christina River basin, Pennsylvania and Delaware, 

1994–98: U.S. Geological Survey Water-Resources Investigations Report 2002– 4279, 207 p.

Seurinck, Sylvie; Defoirdt, Tom; Verstraete, Willy; and 

Siciliano, S.D., 2005, Detection and quantification of the 

human-specific HF183 Bacteroides 16S rRNA genetic 

marker with real-time PCR for assessment of human faecal 

pollution in freshwater: Environmental Microbiology, v. 7, 

p. 249 –259.

Sevon, W.D., comp., 2000, Physiographic provinces of Pennsylvania (4th ed.): Pennsylvania Geological Survey Map 13, 

2 p.

Shankar, Viswanathan; Baghdayan, A.S.; Huycke, M.M.; Lindahl, Gunnar; and Gilmore, M.S., 1999, Infection-derived 

Enterococcus faecalis strains are enriched in esp, a gene 

encoding a novel surface protein: Infection and Immunity, 

v. 67, p. 193–200.

Shelton, L.R., 1994, Field guide for collecting and processing 

stream-water samples of the National Water Quality Assessment Program: U.S. Geological Survey Open-File Report 

94 – 455, 42 p.

Smith, C.J., Olszewski, A.M., and Mauro, S.A., 2009, Correlation of Shiga toxin gene frequency with commonly used 

microbial indicators of recreational water quality: Applied 

and Environmental Microbiology, v. 75, p. 316 –321.

Soule, Marilyn; Kuhn, Edward; Loge, Frank; Gay, John; 

and Call, D.R., 2006, Using DNA microarrays to identify 

library-independent markers for bacterial source tracking: 

Applied and Environmental Microbiology, v. 72, p. 1843–

1851.

Stoeckel, D.M., and Harwood, V.J., 2007, Performance, 

design, and analysis in microbial source tracking studies: 

Applied and Environmental Microbiology, v. 73, p. 2405–

2415.

Town, D.A., 2001, Historical trends and concentrations of 

fecal coliform bacteria in the Brandywine Creek Basin, 

Chester County, Pennsylvania: U.S. Geological Survey 

Water-Resources Investigations Report 2001– 4026, 46 p.

U.S. Geological Survey, 2010, Water-resources data for the 

United States, Water Year 2009: U.S. Geological Survey 

Water-Data Report WDR-US-2007, accessed February 1, 

2010, at http://wdr.water.usgs.gov/.

U.S. Geological Survey, 2011, Water-resources data for the 

United States, Water Year 2010: U.S. Geological Survey 

Water-Data Report WDR-US-2007, accessed February 1, 

2010, at http://wdr.water.usgs.gov/.

U.S. Environmental Protection Agency, 1989, Drinking 

water—National Primary Drinking Water Regulations—

Total coliforms (including fecal coliforms and E. coli): 

Federal Register, v. 54, no. 124, p. 27544 –27568.

U.S. Environmental Protection Agency, 2004, Quality assurance/quality control guidance for laboratories performing 

PCR analysis on environmental samples: Office of Water, 

EPA 815–B–04–001, 56 p.

U.S. Environmental Protection Agency, 2006a, Method 

1603—Escherichia coli in water by membrane filtration 

using modified membrane-thermotolerant Escherichia coli

agar: Washington, D.C., EPA–821–R–06–011[variously 

paged].

U.S. Environmental Protection Agency, 2006b, Method 

1600—Enterococci in water by membrane filtration using 

membrane-Enterococcus indoxyl-β-D-glucoside agar (mEI): 

Washington, D.C., EPA–821–R–06–009 [variously paged].

Zimbro, M.J., and Power, D.A., 2003, DifcoTM & BBLTM 

Manual—Manual of microbiological culture media: Sparks, 

Md., Beckton, Dickenson and Company, p. 467, 468. 
Appendix 1. Concentrations of fecal-indicator bacteria, detections of pathogen and microbial-source tracking markers, and concentrations of micrmarkers from water collected in the Brandywine Creek Basin in Pennsylvania and Delaware, 2009–10.—Continued[USGS, U.S. Geological Survey; EC, Escherichia coli; ENT, enterococci; FC, fecal coliform bacteria; E. coli; Escherichia coli marker; E. hirae, Enterococcus hirae marker; CFper 100 milliliters; +, marker detected; -, marker not detected; ND, marker concentration below detection limit; E, estimated concentration; k, concentration based on calculated 

USGS

station

number

Sample

date

EC ENT FC eaeA stx2 stx1 E. coli rfb spvC invA ipaH Campy esp E. hirae LTIIa STh Al

CFU/100 mL Presence/absence 

01480300 10/6/2009 380 210 453Ek + - + + - - - - - - - - - 01480300 12/9/2009 13,225Ek 10,025Ek 16,550Ek + + + + + - + - - - + + - 4,2101480300 4/21/2010 560 74 640Ek + - - + - - - - + - - - - N01480300 5/3/2010 74,400Ek 42,800Ek 52,800Ek + - + + + - + + - - + + - 2,4801480500 10/6/2009 38Ek 60 46 - + - + - - - - - - - - - N01480500 12/9/2009 4,600 12,700Ek 4,400 + + + + - - - - - + + + - 8301480500 4/21/2010 58 6Ek 42 + - - + - - - - - - - - - 01480500 5/3/2010 8,300Ek 9,175Ek 4,400Ek + - - + - + + - - - - - - 4101480617 6/15/2009 4,600 3,700 5,800 + - + + + - - - - - + + - 01480617 7/15/2009 577 52 1,233 + - - + - - - - - - - - + 01480617 8/17/2009 340 112 360 + - - + - - + - - - - - - N01480617 9/15/2009 410 470 700 + - - + - - - - - - - + + 01480617 10/6/2009 68 50 66 + + - + - - - + - - - - - 01480617 10/19/2009 7,800 530 2,900 + - + + + - - - - - + + - 01480617 11/16/2009 175Ek 40 178Ek - - - + - - - - - - + - - N01480617 12/9/2009 5,800 14,100Ek 5,925Ek + + + + + - - + - - + + - 5601480617 12/15/2009 430 450 480 + - + + + - - - - - + - - N01480617 1/13/2010 82 52 62 + - - + - - - - - - - - - 301480617 2/16/2010 5Ek 18Ek 4Ek - - - - - - - - - - + - + 101480617 3/15/2010 350 390 480 + + + + - - - + - - + - - 3601480617 4/12/2010 46 20Ek 40 + - + + - - - - - - - - - 01480617 4/21/2010 290 29Ek 310 + - - + - + + - + - - - - N01480617 5/3/2010 21,600Ek 20,800Ek 12,800Ek + - - + + + + - - - - + - 1,1401480617 5/12/2010 1,200 1,500 1,000 + - - + - - - - + + + + - 01480700 10/6/2009 42 104 54 + - - + - - - - - - - - - 01480700 12/9/2009 4,000 8,975Ek 5,100 + - + + + - - - - + + + - 4101480700 4/21/2010 33Ek 8Ek 44 + - - + - - + - - - - - - 01480700 5/3/2010 14,700Ek 22,800Ek 12,500Ek + - - + + - + + - - - + - 3201480870 6/15/2009 800 353Ek 1,167 + - + + + - - - - - - + - N01480870 7/15/2009 300 34Ek 330 + - - + - - - - - - + - + 01480870 8/17/2009 200 58 290 + - - + - - + - - - - - + N
Appendix 1. Concentrations of fecal-indicator bacteria, detections of pathogen and microbial-source tracking markers, and concentrations of micrmarkers from water collected in the Brandywine Creek Basin in Pennsylvania and Delaware, 2009–10.—Continued [USGS, U.S. Geological Survey; EC, Escherichia coli; ENT, enterococci; FC, fecal coliform bacteria; E. coli; Escherichia coli marker; E. hirae, Enterococcus hirae marker; CFper 100 milliliters; +, marker detected; -, marker not detected; ND, marker concentration below detection limit; E, estimated concentration; k, concentration based on calculated 

USGS station number Sample date EC ENT FC eaeA stx2 stx1 E. coli rfb spvC invA ipaH Campy esp E. hirae LTIIa STh Al

CFU/100 mL Presence/absence 

01480870 9/15/2009 230 280 400 + - - + - - - - - - - - + 01480870 10/6/2009 50 30Ek 40 - + - + + - - - - - - - - N01480870 10/19/2009 4Ek 2< 10Ek - - - - - - - - - - - - - 01480870 11/16/2009 22Ek 4Ek 48 + - - + - - - - - - - - - N01480870 12/9/2009 3,600 10,650Ek 5,900 + - + + - - + - - - + + - N01480870 12/15/2009 106 92 230 + - - + - - - - - - - - - 01480870 1/13/2010 5Ek 2< 10Ek + - - + - - - - - - - - + N01480870 2/16/2010 8Ek 2< 7Ek - - - + - - - - - - - - + N01480870 3/15/2010 84 42 90 + - - + - + + - - - - + - N01480870 4/12/2010 13Ek 6Ek 23Ek - - - + - - - - - - - - - N01480870 4/21/2010 25Ek 4Ek 50 - - - + - - - - - - - - - N01480870 5/3/2010 13,200Ek 19,600Ek 22,400Ek + - - + + - + + - - - + - 2701480870 5/12/2010 1,333 4,300 3,000 + - - + + + - - + + - + - 01481000 6/15/2009 6,800 2,000 6,000 + - + + + - - - - - + - - N01481000 7/15/2009 4,500 30Ek 290 + - - + - - - - - - - + - 01481000 8/17/2009 152 100 227Ek + - - + - - + - - - - - - N01481000 9/15/2009 200 98 484Ek + - - + - - + - - - - - - N01481000 10/6/2009 38Ek 29Ek 30Ek + - - + + - - - - - - - + N01481000 10/19/2009 6,900 5,325Ek 10,525Ek + - + + + - + - - - + + - 601481000 11/16/2009 37Ek 12Ek 35Ek + - - + - - - - - - - - - 01481000 12/9/2009 3,500 22,900Ek 4,200 + + + + + - - - - - + + + 7201481000 12/15/2009 1,000 1,600 698Ek + - - + + - - - - - + - - 2601481000 1/13/2010 22Ek 21Ek 23Ek + - - + - - - - - - - - - 01481000 2/16/2010 40 8Ek 40 - - - + - + + - - - - - + 01481000 3/15/2010 330 1,079Ek 550 + + + + + - - + - - + - - 5901481000 4/12/2010 40 14Ek 58 - - - + - - - - - - - - - 01481000 4/21/2010 48 6Ek 52 + - - + - - - - - - - - - 01481000 5/3/2010 9,700Ek 14,900Ek 10,400Ek + - + + + - + - - - + + - 3001481000 5/12/2010 600 667 470 + - - + - - + - + - - - - 01481540 6/15/2009 5,500 2,900 6,650Ek + - + + + - - - - - + + - N01481540 7/15/2009 230 1,500 6,875Ek + - - + + - - - - + - - - 2
Appendix 1. Concentrations of fecal-indicator bacteria, detections of pathogen and microbial-source tracking markers, and concentrations of micrmarkers from water collected in the Brandywine Creek Basin in Pennsylvania and Delaware, 2009–10.—Continued[USGS, U.S. Geological Survey; EC, Escherichia coli; ENT, enterococci; FC, fecal coliform bacteria; E. coli; Escherichia coli marker; E. hirae, Enterococcus hirae marker; CFper 100 milliliters; +, marker detected; -, marker not detected; ND, marker concentration below detection limit; E, estimated concentration; k, concentration based on calculated 

USGS

station

number

Sample

date

EC ENT FC eaeA stx2 stx1 E. coli rfb spvC invA ipaH Campy esp E. hirae LTIIa STh Al

CFU/100 mL Presence/absence 

01481540 8/17/2009 10,625Ek 3,900 7,375Ek + - - + + - - - - - + - + 101481540 9/15/2009 11,600Ek 2,921Ek 11,200Ek + - - + + - + - - - - - - 901481540 10/6/2009 68 22Ek 66 + - - + - - - - - - - - - N01481540 10/19/2009 7,475Ek 5,300 2,000 + - + + + - - - - - + + - N01481540 11/16/2009 56 22Ek 50 + - - + - - - - + - - - - N01481540 12/9/2009 5,400 12,175Ek 5,975Ek + - + + + - - - - - + + - 89701481540 12/15/2009 1,833 3,700 1,867 + - - + + - + - - + + - - 61001481540 1/13/2010 50 78 28Ek + - - + - - - - - - - - - 501481540 2/16/2010 42Ek 8Ek 40 - - - + - - - - - - - - - 1601481540 3/15/2010 570 543Ek 845Ek + - - + + - - - - + + - - N01481540 4/12/2010 44 25 42 - - - + - - - - - - - - - 101481540 4/21/2010 38Ek 10Ek 24Ek + - - + + - + - - - - - - 01481540 5/3/2010 792 200 593Ek + - - + - - + - - + + - - 5401481540 5/12/2010 330 410 250 + - - + - - - - + - - + - N01481555 6/15/2009 6,300 1,900 5,300Ek + - + + + - - - - - + + + N01481555 7/15/2009 2,100 450 3,800 + - - + - - - - - + - - - 301481555 8/17/2009 7,700 1,667 6,350Ek + - - + + - + - - - - - - N01481555 9/15/2009 4,600 867 5,800 + - - + + - + - - - - - - 801481555 10/6/2009 24Ek 12Ek 28Ek + + + + - - - - - - - - - N01481555 10/19/2009 6,300 3,400 2,900 + - + + + - + - - - + + - N01481555 11/16/2009 58 19Ek 62 + - - + - - + - - - - - - N01481555 12/9/2009 5,800 13,350Ek 5,400 + - + + + - - - - - + + - N01481555 12/15/2009 2,000 2,200 1,567 + - + + + - - - - - + - - 01481555 1/13/2010 25Ek 54 12Ek + - - + - - - - - - - - - 3001481555 2/16/2010 32Ek 8Ek 22Ek - + - + - - + - - - - - + 2001481555 3/15/2010 560 400 631 + - - + + - - - - - + - - 101481555 4/12/2010 34Ek 10Ek 22Ek - - - + - - - - - - - - - N01481555 4/21/2010 32Ek 6Ek 16Ek - - - + - - - - - - - - - 101481555 5/3/2010 1,967 590 1,900 + - - + - - - - - - + - - 13601481555 5/12/2010 350 300 3,000 + - + + - - - - + - - - - N

Appendix 2. Concentrations of fecal-indicator bacteria, detections of pathogen and microbial-source tracking markers, and concentrations of micrmarkers from quality-control samples collected in the Brandywine Creek Basin in Pennsylvania and Delaware, 2009–10.[USGS, U.S. Geological Survey; QC, quality control; EC, Escherichia coli; ENT, enterococci; FC, fecal coliform bacteria; E. coli; Escherichia coli marker; E. hirae, EnterococCFU/100 mL, colony-forming units per 100 milliliters; +, marker detected; -, marker not detected; ND, marker concentration below detection limit; E, estimated concentration; lated value; <, less than]

USGS

station

number

Sample

date

QC

type

EC ENT FC eaeA stx2 stx1 E. coli rfb spvC invA ipaH Campy esp E. hirae LTIIa STh 

CFU/100 mL Presence/absence 

01480617 4/12/2010 Replicate 70 17Ek 64 - - - + - - - - - - - - - 01480700 4/21/2010 Replicate 24Ek 24Ek 48 + - - + - - - - - - - - - 01481555 5/3/2010 Replicate 1,533 450 1,500 + - - + - - + - + - - - - 01481000 5/12/2010 Replicate 420 767 350 + - - + - + - - + - - - - 01481540 4/12/2010 Blank 2< 2< 2< - - - - - - - - - - - - - 01480617 4/21/2010 Blank 2< 2< 2< - - - - - - - - - - - - - 01480500 5/3/2010 Blank 2< 2< 2< - - - - - - - - - - - - - 01481540 5/12/2010 Blank 2< 2< 2< - - - - - - - - - - - - - 


Duris and others—Pathogenic Bacteria and Microbial-Source Tracking in Brandywine Creek Basin, Pa. and Del., 2009–10—Scientific Investigations Report 2011–5164

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