Sharing of Ribotype Patterns of Escherichia 

Coli Isolates During Baseflow and 

Stormflow Conditions

By Peter G. Hartel, Elizabeth A. Frick, Adrienne L. Funk, Jennifer L. Hill,

Jacob D. Summer, and M. Brian Gregory

Prepared in cooperation with the 

U.S. DEPARTMENT OF THE INTERIOR

 NATIONAL PARK SERVICE

UNIVERSITY OF GEORGIA

Scientific Investigations Report 2004-5004

U.S. Department of the Interior

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

Gale A. Norton, Secretary

U.S. Geological Survey

Charles G. Groat, Director

U.S. Geological Survey, Reston, Virginia, 2004

For more information about the USGS and its products:

Telephone: 1-888-ASK-USGS

World Wide Web: http://www.usgs.gov/

Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Hartel, Peter G., Frick, Elizabeth A., Funk, Adrienne L., Hill, Jennifer L., Summer, Jacob D., and Gregory, M. Brian, 2004, 

Sharing of Ribotype Patterns of Escherichia Coli Isolates During Baseflow and Stormflow Conditions, U.S. Geological 

Survey, Scientific Investigations Report 2004-5004, p. 10.
Illustrations

Figures

Figure 1. Map showing location of the sampling sites, Upper Chattahoochee River 

watershed, 2000 ................................................................................. 3

Figure 2. Graphs showing (A) mean-daily streamflow and (B) streamflow on primary sampling 

dates at selected gages on the Chattahoochee River and tributary streams, 

January–April 2000 .............................................................................. 4

Figure 3. Graph showing the percentage of ribotype patterns shared plotted against distance 

between sites for each of 66 possible site pairs in the Chattahoochee River watershed, 

February–April 2000 .............................................................................. 8

Tables

Table 1. Streamflow and colony-forming units of presumptive E. coli during baseflow and 

stormflow conditions at four mainstem and eight tributary stream sampling sites, 

Upper Chattahoochee River watershed ...................................................... 6

Table 2. Number of presumptive E. coli isolates obtained after initial isolation on mTEC 

agar that were subsequently confirmed as E. coli (oxidase negative, citrate 

negative, and urea hydrolysis negative) ...................................................... 7

Table 3. Shared and unshared ribotype patterns and isolates obtained from four mainstem 

and eight tributary stream sampling sites on the Chattahoochee River during 

baseflow and stormflow conditions ............................................................ 8 

iii

Contents

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

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

Materials and Methods ................................................................................. 2

Study area .......................................................................................... 2

Water sampling .......................................................................................... 2

Selection and identification of E. coli isolates from water .................................... 2

DNA extraction, quantification, and ribotyping of E. coli isolates ........................... 3

Statistical analysis ................................................................................. 5

Results ...................................................................................................... 5

Discussion ................................................................................................ 5

Conclusions ................................................................................................ 7

Acknowledgments ....................................................................................... 8

References ................................................................................................ 9

Abstract

Factors affecting bacterial source tracking are important to 

understand because they affect the amount of sampling needed 

to describe fecal sources in a watershed adequately. The study 

area was a 76-kilometer reach of the Chattahoochee River and 

its tributaries in Metropolitan Atlanta, Georgia. Escherichia 

coli was isolated from water samples collected during 

baseflow and stormflow conditions from four mainstem and 

eight tributary sites; 262 isolates were ribotyped and assessed 

for their similarity. The vast majority of the E. coli ribotype 

patterns were unshared, whether the comparisons were 

between baseflow and stormflow conditions at one location, 

or between one location and another. The data suggest that 

either baseflow and stormflow conditions affected sharing 

of ribotype patterns, or that the sample size was too small to 

characterize the sharing adequately. Regardless, the results 

suggest that a large sampling of E. coli isolates is needed 

during various flow conditions from watersheds with complex 

land-use patterns for adequate bacterial source tracking. 

Introduction

In recent years, a number of phenotypic and genotypic 

methods have been developed for bacterial source tracking, 

which are ways to determine the host origin of fecal bacteria 

in contaminated waters. Most of the recent research on 

phenotypic methods has concentrated on multiple antibiotic 

analysis (Wiggins et al., 1999), whereas most of the research 

on genotypic methods has concentrated on ribotyping (Parveen 

et al., 1999), pulsed field gel electrophoresis (Kariuki et al., 

1999), and various polymerase chain reaction (PCR)-based 

(Dombek et al., 2000) methods. All of these methods are 

based on the assumption that specific markers or strains of 

bacteria are associated with specific animal species (Amor et 

al., 2000), and most require a host origin database consisting 

of one bacterial species isolated from the feces of a number 

of warm-blooded animals (including humans). By matching 

environmental isolates to isolates contained in the host origin 

database, the host source of the fecal contamination can be 

identified.

In conducting bacterial source tracking, it is important 

to understand the factors affecting changes in a bacterial 

subspecies hosted by a population of warm-blooded animals 

because these factors affect the amount of sampling needed to 

describe the bacterial population in the watershed adequately. 

One of these factors is flow conditions. During baseflow 

conditions, the contributing geographic area and feces from 

different host animal species are likely relatively constant over 

time. This contribution contrasts with stormflow conditions, 

when the contributing geographic area and feces from different 

host animal species are constantly changing over time, 

depending on the antecedent rainfall and type of infrastructure 

present (such as the percent and distribution of impervious 

areas, stormwater drainage network, settling basins, and the 

conditions and capacities of sanitary sewer lines).

Whitlock et al. (2002) used antibiotic resistance analysis 

as the bacterial source tracking method to determine that 

sources of fecal indicator bacteria varied geographically and 

temporally in an urban watershed during a 7-month period. 

In terms of geographic changes, Hartel et al. (2002) noted 

that the percentage of ribotype patterns matching each other 

increased with decreased distance between geographic 

locations for cattle and horses, but not for swine and 

chickens. The shortest distance between two tested geographic 

locations was 175 kilometers (km). In a study in which the 

two geographic locations were closer together, Buchan et al. 

(2001) used denaturing gradient gel electrophoresis of the 

16S-23S intergenic spacer region to obtain 45 unique banding 

patterns from 51 E. coli isolates from two waterways in 

Georgia, which were 11 km apart. None of the E. coli banding 

patterns from the two sites overlapped. Finally, in studies of 

temporal changes, little temporal stability was observed in 

E. coli isolates obtained from feral house mice (Gordon, 

1997) or cattle (Jenkins et al., 2003).

Sharing of Ribotype Patterns of Escherichia Coli Isolates 

During Baseflow and Stormflow Conditions

By Peter G. Hartel1, Elizabeth A. Frick2, Adrienne L. Funk2, Jennifer L. Hill1, 

Jacob D. Summer1, and M. Brian Gregory2

1Department of Crop and Soil Sciences, 3111 Plant Sciences, University of Georgia, Athens, GA 30602-7272

2U.S. Geological Survey, 3039 Amwiler Road, Suite 130, Atlanta, GA 30360-2824
To determine the effect of flow conditions on changes of 

E. coli subspecies, isolates of this fecal bacterium were 

obtained from water samples collected in the Upper 

Chattahoochee River watershed in Georgia during baseflow 

and stormflow conditions. Subspecies composition was 

evaluated by the extent of sharing of ribotype patterns among 

E. coli isolates. Sharing of ribotype patterns was defined 

as the DNA banding patterns (representing DNA encoding 

for ribosomal RNA) of two or more environmental isolates 

matching at or greater than 90 percent similarity. 

Materials and Methods

Study area

The study area was a 76-km reach of the Upper 

Chattahoochee River watershed (fig. 1). The 12 sampling 

sites comprised 4 mainstem and 8 tributary sites. This reach 

of the Chattahoochee River was selected because it is one of 

the most heavily used rivers in Georgia, serving as a resource 

for drinking water, recreation, and wastewater assimilation 

for much of Metropolitan Atlanta. During the early 1990s, 

urban land use in watersheds of tributary streams ranged from 

approximately 21 percent in James Creek, near the upstream 

end of the study area, to 80 percent in Rottenwood Creek, near 

the downstream end of the study area (Evelyn H. Hopkins, 

Geographer, written commun., U.S. Geological Survey, 

Atlanta, Georgia, 2000). According to the Georgia Department 

of Natural Resources (1997), 67 of 77 stream reaches assessed 

in Metropolitan Atlanta did not meet or only partially met 

State and Federal water quality standards. Excessive numbers 

of fecal coliform bacteria were a contributing factor in 63 

of these 67 streams. From May to October 1994 and 1995, 

median numbers of fecal coliforms in the Chattahoochee 

River increased steadily downstream from less than 20 MostProbable-Number (MPN) per 100 milliliters (mL) below 

Buford Dam to 790 MPN per 100 mL downstream from 

Metropolitan Atlanta (Gregory and Frick, 2000).

Water sampling

Water samples were collected on February 22, 2000, at all 

sites during winter baseflow conditions during a multiyear 

drought (fig. 2). For sites where insufficient E. coli isolates 

were obtained, supplemental baseflow samples were collected 

on March 1, 2000. Water samples were collected on April 2, 

2000, at near-peak-flow conditions for one of the larger storms 

during early 2000. The Chattahoochee River is a regulated 

river with upstream releases primarily for hydroelectric power 

generation, water supply for Metropolitan Atlanta, and flood 

control. Chattahoochee River samples were collected from a 

bridge or boat. Tributary stream samples were collected by 

wading during baseflow conditions and from bridges during 

stormflow conditions. The one exception was the stormflow 

sample from James Creek, which was a grab sample collected 

from the side of the creek because flow conditions made it 

unsafe to collect from a bridge. 

Except for the grab sample from James Creek, surfacewater samples were collected according to U.S. Geological 

Survey (USGS) protocols for depth-integrated, equal-width 

increment samples (Myers and Sylvester, 1999; Wilde et 

al., 1999). The 1- to 3-L samples collected at each site were 

composites of isokinetic, depth-integrated samples normally 

collected from five, approximately equal-width increments. 

The combination of the constant sampling rate for each 

vertical transit and the isokinetic property of the samplers 

results in a discharge-weighted sample that is proportional to 

total streamflow.

Selection and identification of E. coli

isolates from water

Samples were transported from the sites to the laboratory 

on ice in coolers and were processed within 6 hours (h). 

Each water sample was serially diluted and passed through 

separate 0.45-micrometer filters. Each filter was placed on 

a 5-centimeter petri dish containing mTEC medium (Difco 

Laboratories, Sparks, Md.), and the bacteria were resuscitated 

at 35.0 ± 0.5 degrees Centigrade (°C) for 2 h before incubating 

at 44.5 ± 0.2°C for 22 to 24 h according to USGS protocols 

(Myers and Sylvester, 1999). 

The study objective was to obtain 50 E. coli isolates from 

each of the 12 locations during baseflow and stormflow 

conditions. However, this was not possible because the 

membrane filters were often contaminated with non-E. 

coli bacteria, particularly during stormflow conditions. If the 

composite of the membrane filters from a serial dilution had 

more than 50 yellow to yellowish-brown colonies, then the 

isolates were arbitrarily selected, streaked onto tryptic soy 

agar (Difco Laboratories) and incubated at 35°C for 24 h. If 

the composite of the membrane filters from a serial dilution 

had 50 or fewer yellow to yellowish-brown colonies, then 

all of these isolates were selected. All isolates were streaked 

twice on tryptic soy agar to ensure purity. Each isolate was 

inoculated into a 24-multiwell tissue culture plate containing 

separate 1-mL slants of Simmons citrate and urea agar (both 

from Difco Laboratories). Three bacterial species from the 

American Type Culture Collection (ATCC; Manassas, Va.) 

were used as controls: Escherichia coli ATCC #11775 (citrate 

negative, urea hydrolysis negative), Klebsiella pneumoniae

ATCC # 13883 (citrate positive, urea hydrolysis positive), and 

Enterobacter aerogenes ATCC # 13048 (citrate positive, urea 

hydrolysis negative). Isolates that were both citrate negative 

on Simmons citrate agar and urea hydrolysis negative on 

urea agar were subjected to an oxidase test. Isolates that were 

oxidase negative were considered as confirmed E. coli isolates 

and kept for long-term storage.

2 Ribotype Sharing of Escherichia Coli Isolates
Material and Methods 3

DNA extraction, quantification, and 

ribotyping of E. coli isolates

A standard protocol for extracting, quantifying, and 

ribotyping the DNA was followed as described by Hartel et 

al. (2002). Briefly, E. coli isolates were streaked on tryptic 

soy agar and incubated at 35°C for 24 h. A single colony 

was inoculated into 10 mL of Luria-Bertani broth and was 

incubated on a rotary shaker at 75 revolutions per minute 

(rpm) at 35°C. After 18 h, a 2.0-mL sample was removed 

and the DNA extracted with a commercially available kit. 

A portion of the DNA was mixed with a fluorescent dye and 

quantified with a fluorometer using DNA from E. coli as 

a standard.

For ribotyping, two 1-microgram samples of DNA from 

each isolate were each separately digested overnight with 

the restriction enzymes EcoRI and PvuII. The digested DNA 

was stained and loaded into a 1-percent agarose gel. The gel 

was electrophoresed at 58 volts for 3 h using a horizontal gel 

system. A commercial digoxigenin-labeled (DIG-labeled) 

molecular weight marker occupied every fifth lane of the gel. 

Control lanes consisted of a lane with no DNA and a lane

Buford Dam

River

Chattahoochee

Lake Sidney Lanier

Chattahoochee River watershed

Morgan Falls Dam

285

75

85

0

0 4 2

2 4 6 MILES

6 KILOMETERS

Base modifed from U.S. Geological Survey

1:500,000-scale digital data

N

EXPLANATION

Sampling site and name

Chattahoochee River 

 National Recreation 

 Area

Chattahoochee River

Tributary

James Creek

Johns Creek Suwanee Creek

Crooked Creek

Big

Creek

Willeo Creek

Sope Creek

Rottenwood

Creek

Paces Ferry

State 

Route 280

Johnsons Ferry

Settles Bridge

Study area

GEORGIA

Atlanta

Figure 1. Location of the sampling sites, Upper Chattahoochee River watershed, 2000.
with DNA from E. coli ATCC # 11775. DNA was transferred 

by Southern blotting to a nylon membrane using a vacuum 

blotting system and the DNA on the membrane was crosslinked with ultraviolet light. Following a 2-h prehybridization 

at 42°C, the membrane was hybridized at the same temperature overnight to DIG-labeled cDNA from E. coli total 

ribosomal RNA. Membranes were prepared for chemiluminescence by a series of washing steps before a chemiluminescent 

substrate for alkaline phosphatase was added. Membranes 

were placed in an imager and images saved as TIFF files, 

which were imported into BioNumerics® software (Version 

3.0, Applied Maths, Kortrijk, Belgium) for analysis. Typically, 

gels showed 9 to 11 bands for EcoRI and 11 to 13 bands for 

PvuII digestion. DNA fragments with less than 1,375 base 

pairs were ignored because they were often indistinct. Lanes 

were normalized within the gel with the molecular weight 

marker and variations among the gels were assessed with the 

E. coli ATCC # 11775 strain. Optimization (maximum percentage shift allowed between two different patterns for the 

patterns to still be considered a match) and tolerance (maximum percentage shift allowed between two bands on different 

patterns for the bands to still be considered a match) were each 

4 Ribotype Sharing of Escherichia Coli Isolates

FEBRUARY 22, 2000

JAN 1

FEB 22

MAR 1

APR 2

APR 30

2000

APRIL 2, 2000

0.1

1

10

100

1,000

0.1

1

10

100

1,000

STREAMFLOW, IN CUBIC

METERS PER SECOND

MEAN-DAILY STREAMFLOW, IN

CUBIC METERS PER SECOND

Chattahoochee River 

 at State Road 280 

Sope Creek

Suwanee Creek

Chattahoochee River 

 at Buford Dam

Sample

Settles

Bridge

sample

Power generation release

A.

B.

Figure 2. (A) Mean-daily streamflow and (B) streamflow on primary sampling dates at selected gages on the 

Chattahoochee River and tributary streams, January–April 2000. The Chattahoochee River at Buford Dam 

(short dashes) is 7.2 kilometers upstream from Settles Bridge.
set at 1 percent. The normalized banding patterns for both 

enzymes were stacked with EcoRI on the top and PvuII on 

the bottom to create one combined ribotype pattern for each 

isolate, which was used to discriminate among ribotype 

patterns. Indices were determined using Dice’s coincidence 

index (Dice, 1945) and the distance between clusters calculated using the unweighted pair-group method using arithmetic averages (UPGMA). The banding pattern of the control E. coli ATCC # 11775 strain varied from gel to gel, and 

all the banding patterns of the control strain were similar at 

the 90-percent level. Because the control strain was a single 

ribotype, the banding patterns of two or more environmental 

isolates had to have a similarity index greater than or equal 

to 90 percent to be considered the same ribotype.

Statistical analysis

Significant differences (p = 0.01) between the number of 

presumptive and confirmed E. coli isolates obtained during 

baseflow and stormflow conditions were determined with a 

binomial proportion comparison test.

To compare isolates during the different flow conditions, 

isolates obtained at each location were analyzed with 

the BioNumerics® software, and the sharing of ribotype 

patterns and isolate sharing were arrayed in a table. To 

compare isolates obtained from different locations, isolates 

obtained during baseflow and stormflow conditions at 

each location were combined and compared to combined 

isolates at each of the other locations (66 total comparisons). 

The sharing of ribotype patterns and isolate sharing were 

converted to percentages (to normalize unequal sample 

sizes) and were arrayed over distance in a graph. The 

significance of the correlation coefficient between percent 

sharing of ribotype patterns and distance, or the percent 

isolate sharing and distance, was determined using standard 

statistical tables.

Results

At four mainstem and eight tributary streams sampling 

sites, numbers of presumptive E. coli ranged from 9 to 320 

colonies per 100 mL during baseflow conditions, and from 

1,300 to 24,000 colonies per 100 mL during stormflow 

conditions (table 1). The increase in numbers of presumptive 

E. coli during stormflow conditions ranged from about 

10-fold higher at Suwanee Creek to more than 2,000-fold 

higher at the Chattahoochee River at State Route 280 

compared numbers of presumptive E. coli during baseflow 

conditions. The numbers of fecal coliforms were 10- to 

100-fold higher during stormflow conditions at the same 

sites in the Chattahoochee River watershed compared to 

baseflow conditions (Gregory and Frick, 2001). Similar 

increases have also been observed for E. coli on other rivers 

(Solo-Gabriele et al., 2000).

In general, a higher percentage of presumptive E. coli

colonies were confirmed to be E. coli from water samples 

collected during baseflow conditions compared with water 

samples collected during stormflow conditions (table 2). Of 

550 presumptive E. coli isolates obtained during baseflow 

conditions, 490 (89%) were subsequently confirmed as E. coli. 

During stormflow conditions, the number of confirmed E. coli 

isolates decreased significantly, and only 174 of 275 presumed 

E. coli isolates (63%) were confirmed as E. coli. Therefore, 

11 percent and 37 percent of the presumptive E. coli isolates 

on mTEC medium were false positives during baseflow and 

stormflow conditions, respectively.

Of the 664 confirmed E. coli isolates, 262 isolates were 

ribotyped. When the isolates were compared between 

baseflow and stormflow conditions, they yielded a total of 163 

ribotypes, of which only 6 (3.6%) were shared (table 3). Only 

21 of 262 isolates (8.0%) shared ribotype patterns. Therefore, 

the vast majority of ribotype patterns were unshared, and the 

vast majority of isolates did not share ribotypes. In fact, 8 of 

the 12 sampling locations had ribotypes and isolates with no 

sharing whatsoever.

When the isolates from baseflow and stormflow conditions 

were combined at each sampling location and compared to 

combined isolates at each of the other locations, no significant 

correlation was observed either between the percentage of 

sharing of ribotype patterns and distance (from 4 to 76 km; r2

= 0.03; fig. 3) or between the percentage of isolates sharing the 

same ribotype and distance (r2 = 0.04; data not shown). The 

maximum percentage of sharing of ribotype patterns between 

any two sites, regardless of distance, was 29 percent. The vast 

majority of the comparisons (60 of 66; 91%) had less than 

15-percent sharing of ribotype patterns, and 10 comparisons 

(from 18 to 44 km apart) had no sharing of ribotype patterns. 

Therefore, the percentage of sharing of ribotype patterns did 

not depend on geographic location.

Discussion

The majority of E. coli ribotype patterns were unshared, 

whether the comparisons were between baseflow and 

stormflow conditions at one location, or between one location 

and another. There are two reasonable explanations for the 

data. First, it is possible that the sharing of E. coli ribotype 

patterns was affected by baseflow and stormflow conditions. 

The most likely sources to affect this sharing are sewage 

overflow (Richman, 1996) and overland flow (Entry et al., 

2000). Metropolitan Atlanta has well-documented problems 

with stormwater runoff and sewage overflow (Stevens, 2001). 

In terms of overland flow, it seems reasonable that fecal 

material on or near the riverbank would be transported to 

the river primarily during stormflow conditions. Whitlock et 

al. (2002) observed that humans and dogs were the dominant 

sources of fecal contamination in an urban watershed when 

fecal coliform numbers were low, and wild animal feces the 

dominant source when fecal coliform numbers were high.

Material and Methods 5
Table 1. Streamflow and colony-forming units of presumptive E. coli during baseflow and stormflow conditions at four mainstem and 

eight tributary stream sampling sites, Upper Chattahoochee River watershed (adapted from Gregory and Frick, 2001).

[USGS, U.S. Geological Survey; ID, identification; CFU 100 mL-1, colony-forming units per 100 milliliters; m3 sec-1, cubic meters per second; 

NA, not available]

Baseflow Stormflow

February 22, 2000 March 1, 2000 April 2, 2000

USGS 

site ID

Sampling 

site

Streamflow

(m3 sec-1)

Presumptive

E. coli

(CFU 100 mL-1)

Streamflow

(m3 sec-1)

Presumptive

E. coli

(CFU 100 mL-1)

Streamflow

(m3 sec-1)

Presumptive

E. coli

(CFU 100 mL-1)

02335420 James Creek 0.3 82 NA NA NA 2,300

02334550 Chattahoochee River—

Settles Bridge 192a, b 32 NA NA 23b 3,300

02334885 Suwanee Creek 0.9 320 0.9c NA 12 3,100

02335078 Johns Creek 0.2 100 NA NA 20 11,000

02335350 Crooked Creek 0.3 26 0.2c NA 21 10,000b

02335741 Big Creek 1.7 44 1.6b 44 25 4,800b

02335790 Willeo Creek 0.3 93 NA NA 7 3,800b

02335830 Chattahoochee River—

Johnsons Ferry 33 60 31 24 128 1,300b

02335870 Sope Creek 0.5 190 0.5c NA 23 9,300b

02335910 Rottenwood Creek 0.3 25b 0.3b 46b 27 7,400b

02336000 Chattahoochee River—

Paces Ferry 31 62b 30 21 150 3,500b

02336490 Chattahoochee River—

State Route 280 37 9b 33 40 334 24,000b

a

 Streamflow was high because of water released upstream for power generation rather than from rainfall (fig. 2b). 

b

 Estimated.

c

 Mean daily discharge

Furthermore, little sharing of ribotype patterns was 

observed between two different locations. A likely 

explanation for this lack of sharing is that land use varies 

from approximately 21 percent urban in James Creek, near 

the upstream end of the study area, to 80 percent urban in 

Rottenwood Creek, near the downstream end of the study 

area (Evelyn H. Hopkins, Geographer, written commun., 

U.S. Geological Survey, Atlanta, Georgia, 2000). Thus, the 

contribution from wildlife is more likely in the upper portion 

of the study area compared with the lower portion, which 

is more likely to have a greater contribution from human 

sewage. Given that specific markers or strains of bacteria are 

associated with specific animal species (Amor et al., 2000) 

and that these animal species likely change with land use, then 

the lack of ribotype sharing is not surprising. These data also 

are consistent with Buchan et al. (2001) who observed that 

none of the 45 banding patterns that they obtained from 51 

E. coli isolates from two water sources 11 km apart overlapped 

with each other.

A second explanation is that the two different flow 

conditions did not affect E. coli sharing of ribotype patterns. 

6 Ribotype Sharing of Escherichia Coli Isolates

Samadpour and Chechowitz (1995) suggested that the typical 

increase in fecal contamination observed after storm events 

may represent more of the same contributing sources rather 

than additional sources. Thus, all ribotypes were present in 

the water at all times, and only small sampling size made 

it appear as if the E. coli ribotype patterns were unshared. 

Unfortunately, it was not possible to know the diversity of 

the E. coli ribotype population before the experiment was 

conducted; the study was not designed to test this hypothesis.

Nevertheless, both explanations suggest a large 

environmental sampling is necessary for a watershed as 

complex as the Upper Chattahoochee River. Both Buchan 

et al. (2001), using 16S-23S ribosomal RNA intergenic 

spacer region analysis, and Hagedorn et al. (1999), using 

multiple antibiotic resistance analysis, concluded that many 

bacterial isolates are necessary for bacterial source tracking 

to describe a watershed. The environmental sampling 

should include both large numbers of isolates to represent 

subspecies diversity at a specific point in time, and sample 

collection during different flow conditions to represent 

different subspecies composition.
• The majority of E. coli ribotype patterns identified in 

this study were unshared, whether the comparisons 

were between baseflow and stormflow at one 

location, or between one location and another. One 

explanation is that land-use patterns and baseflow 

and stormflow conditions affected this sharing.

• An alternative explanation is that baseflow and 

stormflow conditions had no effect on E. coli sharing 

of ribotype patterns and only the relatively small 

sampling size made it appear as if the ribotype 

patterns were unshared.

• The results underscore the need for extensive 

environmental sampling in a watershed as complex as 

the Upper Chattahoochee River, and the need for an 

extensive host origin database for genotypic methods 

using such databases.

Table 2. Number of presumptive E. coli isolates obtained after initial isolation on mTEC agar that were subsequently confirmed 

as E. coli (oxidase negative, citrate negative, and urea hydrolysis negative). The isolates were obtained from water sampled 

during baseflow (February 22 and March 1, 2000) or stormflow conditions (April 2, 2000) from four mainstem sites and eight 

tributary streams of the Upper Chattahoochee River watershed.

[USGS, U.S. Geological Survey; ID, identification]

Baseflow Stormflow

USGS 

site ID

Location Confirmed E. coli / 

presumptive E. coli

(number)

False 

positives

(percent)

Confirmed E. coli / 

presumptive E. coli

(number)

False 

positives

(percent)

02335420 James Creek 50 / 50 0 13 / 14 7 

02334550 Chattahoochee River—

Settles Bridge 42 / 42 0 24 / 24 0

02334885 Suwanee Creek 48 / 49 2 35 / 40 13 

02335078 Johns Creek 49 / 49 0 14 / 16 13 

02335350 Crooked Creek 45 / 45 0 17 / 28 39

02335741 Big Creek 23 / 41a 44 6 / 16 63 

02335790 Willeo Creek 48 / 48 0 7 / 15 53 

02335830 Chattahoochee River—

Johnsons Ferry 34 / 50 a 32 10 / 16 38 

02335870 Sope Creek 48 / 48 0 14 / 37 62 

02335910 Rottenwood Creek 44 / 44a 0 3 / 16 81 

02336000 Chattahoochee River—

Paces Ferry 36 / 45a 20 16 / 16 0 

02336490 Chattahoochee River—

State Route 280 23 / 39a 41 15 / 37 59

 Totals 490 / 550 11 174 / 275 37**

a

 Site was resampled on March 1, 2000, to obtain more isolates.

**Significant at the 0.01 probability level.

Discussion 7

The percentage of E. coli isolates that were false positive 

on mTEC agar increased significantly during stormflow 

conditions (37%) compared with baseflow conditions (11%). 

Typically, freshwater samples plated on the mTEC agar have 

a 15-percent false positive rate (Dufour et al., 1981). The 

relatively high percentage of false positive E. coli isolates 

observed when stormflow conditions were sampled suggests 

that some caution is needed in counting yellow and yellowbrown isolates as presumptive E. coli after isolation on 

mTEC agar from stormflow samples. The manufacturer of the 

medium notes that high fecal counts may not provide accurate 

urease results (Difco Laboratories, 1998).

Conclusions

• In bacterial source tracking, it is important to 

understand factors affecting changes in a bacterial 

subspecies that enter streams because these factors 

determine the amount of sampling needed to describe 

the watershed adequately.
Acknowledgments

This research was done in cooperation with the U.S. 

Department of the Interior, National Park Service and the 

University of Georgia. The work done by University of 

Georgia staff was partially supported by grants from the 

Georgia Water Resources Institute, the State of Georgia 

Environmental Protection Division through the U.S. 

Environmental Protection Agency, and the U.S. Department 

of Agriculture–Cooperative State Research, Education, and 

Extension Service through the Texas Agricultural Extension 

Service. We thank University of Georgia staff J. Victoria 

Collins and Robin Kuntz for their technical assistance. We 

also thank Dan Thomas for financial support of Jennifer Hill. 

8 Ribotype Sharing of Escherichia Coli Isolates

30

25

20

15

10

5

SHARING OF RIBOTYPE PATTERNS,

0

IN PERCENT

DISTANCE BETWEEN SITES, IN KILOMETERS

0 10 20 30 40 50 60 70 80

Table 3. Shared and unshared ribotype patterns and isolates obtained from four mainstem and eight tributary stream sampling sites 

on the Chattahoochee River during baseflow and stormflow conditions.

[USGS, U.S. Geological Survey; ID, identification]

USGS 

site ID Location

Number of isolates Number shared Number unshared

Baseflow Stormflow Ribotype patterns Isolates Ribotype patterns Isolates

02335420 James Creek 12 10 0 0 9 22

02334550 Chattahoochee River—

Settles Bridge 29 23 3 12 32 40

02334885 Suwanee Creek 11 15 1 2 17 24

02335078 Johns Creek 12 10 0 0 9 22

02335350 Crooked Creek 11 11 0 0 14 22

02335741 Big Creek 14 1 0 0 9 15

02335790 Willeo Creek 10 9 0 0 13 19

02335830 Chattahoochee River—

Johnsons Ferry 5 8 1 5 7 8

02335870 Sope Creek 6 4 1 2 8 8

02335910 Rottenwood Creek 27 2 0 0 14 29

02336000 Chattahoochee River—

Paces Ferry 10 13 0 0 18 23

02336490 Chattahoochee River—

State Route 280 4 5 0 0 7 9

Totals 151 111 6 21 157 241

Figure 3. Percentage of ribotype patterns shared 

plotted against distance between sites for each 

of 66 possible site pairs in the Chattahoochee 

River watershed, February–April 2000. Distances 

between sites were determined from Carter et al. 

(1989) and U.S. Army Corps of Engineers (1985).
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