U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2007–5242

In cooperation with the Oakland County Health Department, Michigan

Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury 

in Surface Waters of Oakland County, Michigan, 2005–2006
Cover image: View of River Rouge near Birmingham, Mich.
Antibiotic-Resistant Fecal Bacteria, 

Antibiotics, and Mercury in Surface Waters 

of Oakland County, Michigan, 2005–2006

By Lisa R. Fogarty, Joseph W. Duris, Suzanne L. Crowley, and Nicole Hardigan

In cooperation with the Oakland County Health Department, Michigan

Scientific Investigations Report 2007–5242

U.S. Department of the Interior

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

DIRK KEMPTHORNE, Secretary

U.S. Geological Survey

Mark D. Myers, Director

U.S. Geological Survey, Reston, Virginia: 2007

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Suggested citation:

Fogarty, L.R., Duris, J.W., Crowley, S.L., and Hardigan, Nicole, 2007, Antibiotic-resistant fecal bacteria, antibiotics, 

and mercury in surface waters of Oakland County, Michigan, 2005–2006: U.S. Geological Survey Scientific 

Investigations Report 2007–5242, 30 p.
iii

Contents

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

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

Purpose and Scope ..............................................................................................................................2

Description of Study Area ...................................................................................................................2

Antibiotic-Resistant Bacteria in the Environment...........................................................................2

Analytical and Sampling Methods..............................................................................................................6

Field Measurements.............................................................................................................................7

Collection of Samples for Chemical Analyses .................................................................................7

Collection of Samples for Bacteria Analyses ..................................................................................7

Quality Assurance and Quality Control .............................................................................................9

Results of Data Analyses..............................................................................................................................9

Fecal Indicator Bacteria Enumeration ..............................................................................................9

Vancomycin-Resistant Enterococci and Human-Fecal-Enterococci Gene..............................13

Occurrence of Cephalosporin-Resistant E. coli ............................................................................13

Spatial and Temporal Patterns in the Percentage of Cephalosporin-Resistant E. coli...........15

Antibiotic Concentrations and Cephalosporin-Resistant E. coli.................................................15

Mercury- and Cephalosporin-Resistant E. coli..............................................................................15

Integron Detection..............................................................................................................................17

Results From Clinton River Watershed Study ................................................................................22

Future Studies...............................................................................................................................................26

Summary and Conclusions.........................................................................................................................26

References Cited..........................................................................................................................................28

Figures

1. Map showing sampling locations for study sites and land use/land cover for 

Oakland County, Mich. .................................................................................................................3

2. Map showing sampling locations for study sites and land use/land cover for 

Clinton River Watershed study...................................................................................................5

3–6. Graphs showing:

3. Fecal indicator bacteria concentrations for samples collected September 19, 

2005, and April 28, June 28, August 9, and August 23, 2006, at Oakland County 

study sites ...........................................................................................................................12

4. Percentage of cephalosporin-resistant E. coli isolates and daily mean 

streamflow on five sampling dates at study sites, Oakland County..........................16

5. Precipitation totals on the day of sample collection and in the preceding 24 

and 72 hours at locations in Oakland County................................................................17

6. Percentage of cephalosporin-resistant E. coli isolates and total mercury 

concentration for samples collected on five sampling dates at Oakland 

County study sites..............................................................................................................19

7. Schematic diagram of the relation of transferable genetic elements: plasmid, 

transposon, integron, and resistance genes .........................................................................20
iv

8–10. Graphs showing:

8. E. coli concentrations for samples collected April 26 and August 23, 2006, 

at Clinton River Watershed study sites..........................................................................23

9. Enterococci concentrations for samples collected April 26 and August 23, 

2006, at Clinton River Watershed study sites................................................................23

10. Percentage of E. coli isolates resistant to cefoxitin and ceftriaxone with 

respect to total mercury concentration for Clinton River Watershed 

study sites ...........................................................................................................................24

11. Map showing percentage of E. coli isolates resistant to cefoxitin for samples 

collected April 26 and August 23, 2006, at the Clinton River Watershed 

study sites ....................................................................................................................................25

12. Map showing percentage of E. coli isolates resistant to ceftriaxone for samples 

collected April 26 and August 23, 2006, at the Clinton River Watershed study sites ......25

Tables

1. Sample-collection sites for the Oakland County, Mich., study, 2006 ...................................2

2. Land-cover percentages for Oakland County study sites, 2000 ...........................................4

3. Clinton River Watershed study sites, 2006 ...............................................................................4

4. Summary of field measurements (dissolved oxygen, pH, specific conductance, 

and streamflow) and total mercury, antibiotic, and bacteria samples collected 

September 19, 2005, through August 23, 2006, in Oakland and Macomb Counties ...........7

5. List of antibiotics for which samples were analyzed in the Oakland County study...........8

6. Concentrations of antibiotics detected in replicate samples collected at Clinton 

River at Auburn Hills, Mich., August 9, 2006.............................................................................9

7. Field measurements for samples collected at Oakland County study sites, 

September 2005 through August 2006.....................................................................................10

8. Fecal indicator bacteria concentrations for samples collected at Oakland County 

study sites, September 2005 through August 2006................................................................11

9. Percentage of E. coli isolates resistant to tested antibiotics for samples 

collected at Oakland County study sites, September 2005 through August 2006 ............14

10. Antibiotics detected in samples collected September 2005 through August 2006 

at Oakland County study sites ..................................................................................................18

11. Mercury concentration in samples collected September 2005 through 

August 2006 at Oakland County study sites............................................................................18

12. Results of polymerase chain reactions (PCR) for integron structures in 

samples collected September 2005 through August 2006 at Oakland County 

study sites ....................................................................................................................................21
v

Conversion Factors and Abbreviated Water-Quality Units

Multiply By To obtain

square mile (mi2

) 2.590 square kilometer (km2)

ounce, fluid (fl. oz) 29.57 milliliter (mL)

microliter (μL) 0.001 milliliter (mL)

cubic foot per second (ft3

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

ounce, avoirdupois (oz) 28.35 gram (g)

milligram per liter (mg/L) 1,000 micrograms per microliter (μg/μL)

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

°F = (1.8 x °C) + 32

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

(CFU/100 mL).

Concentrations of antibiotics are given in micrograms per liter (μg/L) and micrograms per 

milliliter (μg/mL).

Concentrations of mercury are given in nanograms per liter (ng/L).

Concentrations of analytical reagents are given in millimolar (mM) or micromolar (μM).

Concentrations of other chemical constituents of water are given in milligrams per liter (mg/L).
vi

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Antibiotic-Resistant Fecal Bacteria, Antibiotics, and 

Mercury in Surface Waters of Oakland County, Michigan, 

2005–2006

By Lisa R. Fogarty, Joseph W. Duris, Suzanne L. Crowley, and Nicole Hardigan

Abstract

Water samples collected from 20 stream sites in Oakland 

and Macomb Counties, Mich., were analyzed to learn more 

about the occurrence of cephalosporin-resistant Escherichia 

coli (E. coli) and vancomycin-resistant enterococci (VRE) 

and the co-occurrence of antibiotics and mercury in area 

streams. Fecal indicator bacteria concentrations exceeded the 

Michigan recreational water-quality standard of 300 E. coli 

colony forming units (CFU) per 100 milliliters of water in 

19 of 35 stream-water samples collected in Oakland County. 

A gene commonly associated with enterococci from humans 

was detected in samples from Paint Creek at Rochester and 

Evans Ditch at Southfield, indicating that human fecal waste 

is a possible source of fecal contamination at these sites. E. 

coli resistant to the cephalosporin antibiotics (cefoxitin and/

or ceftriaxone) were found at all sites on at least one occasion. The highest percentages of E. coli isolates resistant to 

cefoxitin and ceftriaxone were 71 percent (Clinton River at 

Auburn Hills) and 19 percent (Sashabaw Creek near Drayton Plains), respectively. Cephalosporin-resistant E. coli was 

detected more frequently in samples from intensively urbanized or industrialized areas than in samples from less urbanized areas. VRE were not detected in any sample collected in 

this study. Multiple antibiotics (azithromycin, erythromycin, 

ofloxacin, sulfamethoxazole, and trimethoprim) were detected 

in water samples from the Clinton River at Auburn Hills, and 

tylosin (an antibiotic used in veterinary medicine and livestock 

production that belongs to the macrolide group, along with 

erythromycin) was detected in one water sample from Paint 

Creek at Rochester. Concentrations of total mercury were as 

high as 19.8 nanograms per liter (Evans Ditch at Southfield). 

There was no relation among percentage of antibiotic-resistant 

bacteria and measured concentrations of antibiotics or mercury 

in the water. Genetic elements capable of exchanging multiple 

antibiotic-resistance genes (class I integrons) were detected in 

several samples, indicating that the resistance carried by these 

organisms may be transferable to other bacteria, including 

disease-causing bacteria. 

Introduction

The effects of urbanization on water quality in streams in 

Oakland County were evaluated during 2001–2003 by the U.S. 

Geological Survey (USGS), in cooperation with the county 

(Aichele, 2005). Results from that study showed that concentrations of fecal indicator bacteria in many streams throughout 

the county exceeded Michigan’s recreational water-quality 

standards. An initial followup investigation of the fecal indicator bacteria showed the presence of multiple-antibiotic-resistant fecal coliform bacteria, vancomycin-resistant enterococci 

(VRE), and indicators of pathogenic Escherichia coli (E. coli) 

(Fogarty and others, 2005). This initial bacteria study was 

done in August and September 2003, at a time of very low 

streamflows. 

Antibiotic-resistant bacteria have become of increasing 

public health concern because of the emergence of multiple 

antibiotic-resistant pathogens, such as multiple-drug-resistant 

E. coli and Salmonella spp. (Walsh, 2003) and VRE (Cetinkaya and others, 2000). Recent studies have identified antibiotic-resistant bacteria and genes responsible for antibiotic 

resistances in surface waters in the United States (McArthur 

and Tuckfield, 2000; Ash and others, 2002; Pruden and others, 2006), but very little is known about the occurrence and 

patterns of these resistant bacteria during recreational months 

in Oakland County. Because the enteric bacteria E. coli and 

enterococci have previously been detected in high concentrations and have been isolated with resistance to several human 

antibiotics, the USGS, in cooperation with the Oakland 

County Health Department, further evaluated the spatial and 

temporal distribution of cephalosporin-resistant E. coli, VRE, 

and an enterococci gene that is commonly associated with 

disease-causing enterococci and also is used as a marker for 

human fecal pollution (Shankar and others, 1999; Scott and 

others, 2005). The co-occurrence of antibiotics and mercury 

concentrations in these waters also was evaluated in the current study to determine whether bacterial antibiotic resistances 

were related to these chemicals and whether these chemicals 

might aid in the transfer and maintenance of antibiotic resistances in the environment. 
2 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

The purpose of this study was to learn more about the 

occurrence of resistant bacteria and their relation to antibiotics or metals in streams during typical recreational months 

in Michigan (April through September) in order to provide 

important information to the public and communities that use 

these waters.

Purpose and Scope

This report describes the occurrence and distribution of 

antibiotic-resistant fecal bacteria, antibiotics, and mercury 

concentrations in surface-water samples collected in Oakland 

and Macomb Counties, Mich. This report is a followup to 

the previous USGS-Oakland County study (Fogarty and others, 2005) and includes information on (1) concentrations of 

fecal indicator bacteria (fecal coliform bacteria, E. coli, and 

enterococci), antibiotics, and mercury in river-water samples 

collected from September 2005 through August 2006, (2) percentage of E. coli isolates resistant to cephalosporin antibiotics 

and percentage of enterococci resistant to vancomycin, (3) 

detection of transferable genetic elements (class I integrons 

and vancomycin-resistance genes), and (4) presence of an 

enterococci gene used to indicate human fecal waste. In addition, this report includes a comparison of data obtained from 

Oakland County to results obtained from analyses of samples 

collected throughout the Clinton River Watershed as part of 

the USGS National Water-Quality Assessment Program mercury topical study for Lake Erie and Lake St. Clair (Brigham 

and others, 2003). 

Description of Study Area

Oakland County, Mich., comprises 910 mi2 in southeastern Michigan. The county is largely suburban, with a population of approximately 1.2 million in 2005. Approximately 

42 percent of the land in Oakland County is residential, 6.3 

percent commercial and industrial, and 3.2 percent agricultural. The heaviest residential and commercial land use is 

in the southeastern part of the county (fig. 1). Seven of the 

fourteen sites studied in 2003 (Fogarty and others, 2005) were 

sampled again in 2005 and 2006 (fig. 1, table 1) for followup 

microbial and chemical analysis. These sites are in the Clinton 

River, Flint River, and River Rouge Watersheds (table 1 and 

fig. 1). Land-use characteristics for waters draining to these 

sites were reported by Aichele (2005) and are summarized 

in table 2. Four of the seven sampling sites are in the Clinton 

River Watershed. The Clinton River at Auburn Hills (USGS 

station number 04161000) is the only Oakland County study 

site downstream from a known wastewater discharge. 

The Clinton River Watershed extends from Oakland 

County into Macomb County and includes small parts of 

Genesee and Lapeer Counties. A total of 16 sites in the Clinton River Watershed (table 3 and fig. 2) were studied as part of 

the USGS National Water-Quality Assessment (NAWQA) for 

Lake Erie and Lake St. Clair. Similar to Oakland County, the 

southern part of Macomb County is a predominantly urbanized area with residential areas extending north. Land in the 

northeast part of the watershed is mainly agricultural (fig. 2). 

Antibiotic-Resistant Bacteria in the Environment

Antibiotics have been detected at low concentrations (in 

the nanograms per liter range) in waters across the United 

States (Kolpin and others, 2002). Little is known about the 

effects of nontherapeutic levels of antibiotics in the environment on human health, aquatic ecology, or microbial communities. Antibiotic-resistant bacteria have been well documented 

in the environment (Ash and others, 2002; Park and others, 

2003; Roe and others, 2003), but antibiotic resistance to newer 

antibiotics that are currently being used to treat humans has 

not been. Little is known about the presence of antibiotics in 

stream waters in Oakland County. The initial 2003 study did 

not investigate the presence of antibiotics, nor were the authors 

able to find any information regarding other studies that measured antibiotics in the environment in this area of Michigan. 

Table 1. Sample-collection sites for the Oakland County, Mich., study, 2006.

[USGS, U.S. Geological Survey]

Map 

number

USGS 

station 

number Station name County Watershed

1 04148035 Kearsley Creek at Mill Street at Ortonville Oakland Flint

2 04160800 Sashabaw Creek near Drayton Plains Oakland Clinton

3 04161000 Clinton River at Auburn Hills Oakland Clinton

4 04161540 Paint Creek at Rochester Oakland Clinton

5 04161580 Stony Creek near Romeo Macomb Clinton

6 04166000 River Rouge at Birmingham Oakland Rouge

7 04166200 Evans Ditch at Southfield Oakland Rouge
Introduction 3

42 °30 ’

42 °45 ’

83 °45 ’ 83 °30 ’ 83 °15 ’ 83 °0 ’

Political boundaries from Oakland County GIS Utility, 2003.

Hydrographic data from USGS 1:100,000 scale Digital Line Graphs.

Land-use data from Michigan Department of Natural Resources IFMAP, 2003.

Oakland

County

Michigan

0 3 6 KILOMETERS

0 3 6 MILES

Watershed boundary

Stream water-quality sample locations

EXPLANATION

Flint River

Watershed

Shiawassee River 

Watershed

Huron River

Watershed River Rouge

Watershed

Clinton River

Watershed

Rivers and streams

County boundary

Residential

Commercial or transportation

Agriculture

Open land

Forest

Water

Wetland

Barren

Land use/land cover

2

1

3

4

5

6

7

1

Figure 1. Sampling locations for study sites and land use/land cover for Oakland County, Mich.
4 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Table 2. Land-cover percentages for Oakland County study sites, 20001.

[USGS, U.S. Geological Survey]

USGS 

station 

number Built2

ResidentialCommercialAgriculturalOpen 

space Forest Water Wetland

Total

Built Unbuilt

4148035 1 32.8 1.6 12.1 17.8 15.5 3.9 15.4 35.3 64.7

4160800 6.8 36.1 7.3 3.9 15.7 5.1 6 19.1 50.2 49.8

4161000 6.4 44.3 12.5 2.5 9.3 4.8 10.3 10 63.1 36.9

4161540 5.4 43.9 3.1 9.2 15.3 7.1 4 11.9 52.4 47.6

4161580a 3 24.7 2.4 13.2 22.1 16 4.3 14.2 30.2 69.8

4166000 5.7 70 10.7 0.4 2.8 2.3 4.8 3.4 86.3 13.7

4166200 8.1 65.7 21 0 1.8 2.8 0 0.7 94.8 5.2

1

Data summarized from Aichele (2005).

2

Undifferentiated urban land, not clearly identified as commercial or residential.

a Site location is in Macomb County, Mich.

Table 3. Clinton River Watershed study sites, 2006.

[USGS, U.S. Geological Survey; map number shown in fig. 2]

Map 

number

USGS 

station 

number Site name County

2 04160800 Sashabaw Creek near Drayton Plains Oakland

8 04160900 Clinton River near Drayton Plains Oakland

3 04161000 Clinton River at Auburn Hills Oakland

4 04161540 Paint Creek at Rochester Oakland

5 04161580 Stony Creek near Romeo Macomb

9 04161800 Stony Creek near Washington Macomb

10 04161820 Clinton River at Sterling Heights Macomb

11 04163030 Red Run at Warren Macomb

12 04163400 Plum Brook at Utica Macomb

13 04164000 Clinton River near Fraser Macomb

14 04164100 East Pond Creek near Romeo Macomb

15 04164110 East Pond Creek at Powell Macomb

16 04164300 East Branch Coon Creek at Armada Macomb

17 04164350 Highbank Creek at 32 Mile Road near Armada Macomb

18 04164500 North Branch Clinton River near Mt. Clemens Macomb

19 04164800 Middle Branch Clinton River at Macomb Macomb
Introduction 5

0 5 1 0 MILES

0 5 10 KILOMETERS

Oakland

County

Macomb

County

County linework data from Michigan Resource Information System , 1998.

Hydrographic data from USGS 1:100,000 scale Digital Line Graphs.

Land-use data from Michigan Department of Natural Resources IFMAP, 2003.

13

1 0

1 2

1 1

18

1 9

1 7

1 6

1 5

1 4

5

9

4

3

2

8

EXPLANATION

County boundary

Clinton River Watershed

Rivers

1

Land use/land cover

Residential

Commercial and transportation

Agriculture

Open land

Forest

Water

Wetland

One means by which an organism may develop antibiotic 

resistance is through gene transfer. Bacteria originally sensitive to an antibiotic may acquire resistance genes from resistant organisms that have been exposed to antibiotics. Genes 

responsible for antibiotic resistance are commonly carried on 

transferable genetic elements (such as plasmids and class I 

integrons, described in a review by Roy, 1999) that can carry 

multiple genes—including not only genes for antibiotic resistance but also genes responsible for resistance to heavy metals, 

detergents, solvents, or other quaternary ammonia compounds. 

Thus, it is possible that antibiotic-resistance genes may be 

selectively maintained and transferred in the environment as 

a result of exposure of microorganisms to either antibiotics, 

heavy metals, or other chemicals constituents (Bass and others, 1999; Ug and Ceylan, 2003). Previous studies identified 

several wastewater organic compounds, including detergents 

and solvents, in stream waters in Oakland County (Aichele, 

2005). Study limitations prevented further investigation of any 

potential link of these chemicals to antibiotic resistances in 

this current 2005–2006 study, but it should be noted that such 

compounds also may influence antibiotic resistance and gene 

transfer in stream-water samples.

In this study, cephalosporin-resistant E. coli and VRE 

were targeted for analysis in selected stream waters of Oakland County. E. coli is typically sensitive to all cephalosporin 

antibiotics, and resistance is often a result of an acquired gene 

that is responsible for resistance to a large group of similar 

antibiotics (beta-lactam (β-lactam) antibiotics). β-lactam 

resistance in E. coli has been identified as a resistance problem 

for patients suffering with infectious diseases (Levy, 2005). 

This study evaluated the environmental occurrence of E. coli 

resistant to levels of cefoxitin and ceftriaxone, which have 

been determined to be clinically significant. In other words, 

organisms causing infections that are resistant to those levels 

tested would be difficult to treat with conventional antibiotics. 

Cefoxitin and ceftriaxone are antibiotics that are typically prescribed only for humans, but they may have some veterinary 

uses. They belong to the cephalosporin antibiotic group, which 

has widespread applications (Hornish and Kotarski, 2002; 

Aarestrup, 2006). Cephalosporin antibiotics break down relatively quickly and therefore are not typically found in environmental waters. However, microorganisms may develop resistance at their source, where antibiotic concentrations might be 

higher, or by acquisition of an antibiotic-resistance gene that 

Figure 2. Sampling locations for study sites and land use/land cover for Clinton River Watershed study, Mich.
6 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

was carried on a genetic element and transferred to that organism via exposure to a different chemical. For example, if a 

microorganism is in an environment contaminated with heavy 

metals, the organism may obtain a genetic element from other 

organisms that survive because they carry a gene responsible 

for resistance to those metals. If a gene responsible for antibiotic resistance also was present on the same element, it too 

would be transferred to the organism. 

Mercury has been identified as a problem in several 

streams and lakes in southeast Michigan. Several lakes in Oakland County and parts of the Clinton River and River Rouge 

have been listed as Areas of Concern or have a total maximum 

daily loads (TMDLs) for mercury either because of accumulation of mercury in fish tissues or because waters fail to meet 

the Michigan water-quality standard (Michigan Department of 

Environmental Quality, 2006). Mercury-resistance genes are 

commonly associated with elements that bacteria can readily 

transfer among each other. In addition to carrying genes for 

mercury resistance, the elements that can transfer between 

organisms also may contain genes for antibiotic resistance (Ug 

and Ceylan, 2003; Baker-Austin and others, 2006). The presence of mercury may encourage the transfer of the element 

(which contains the mercury-resistance gene) to other organisms for survival in a mercury-contaminated environment. 

The organism also may acquire the antibiotic-resistance gene 

also being carried on this element, resulting in a resistance 

to antibiotic without being exposed to it. This implies that 

the presence of mercury may be involved in the maintenance 

and transfer of antibiotic resistance in these environments as 

well. In particular, Baker-Austin and others (2006) discuss 

shared structural and functional characteristics of bacteria and 

antibiotic- and metal-resistance systems, including a resistance 

mechanism for mercury and β-lactam antibiotics (including 

the cephalosporins). 

Vancomycin-resistant enterococci (VRE) were evaluated 

in this study. Vancomycin is a potent antibiotic used strictly in 

human medicine and is considered an antibiotic of last resort 

when other treatments fail. Vancomycin was never approved 

for use in livestock production, but avoparcin, another antibiotic similar to vancomycin, was used in European countries 

for livestock production for many years. VRE has become a 

significant problem in some European countries, where it is 

believed the use of avoparcin may be partially responsible 

(Kruse and others, 1999; Kühn and others, 2005). Avoparcin 

was never approved for use in the United States; however, 

VRE has recently been identified as a major health issue in 

U.S. hospitals. VRE has also been detected in enterococci 

isolates obtained from canines, poultry, and wastewaters in the 

United States (Harwood and others, 2001; Simjee and others, 

2002; Poole and others, 2005). Resistance to vancomycin is 

often attributed to genes that can be transferred among similar 

organisms, and molecular assays can often be used to identify 

the presence of these genes in a given sample (Dutka-Malen 

and others, 1995). Other studies have identified a gene in 

enterococci isolates that has been demonstrated to be associated solely with human fecal waste (Shankar and others, 

1999; Scott and others 2005). This gene is commonly found 

in isolates that have caused infections in humans; therefore, it 

may be not only an indicator of human fecal pollution but also 

a marker for a human pathogen. 

Microbial antibiotic resistance is a recognized clinical 

problem because of limited treatment options (McGowan 

and Tenover, 2004; Levy, 2005; Centers for Disease Control 

and Prevention, 2007), but little is known about the role the 

environment also may have on antibiotic resistances of clinical 

concern. Improving the understanding of the distribution of 

antibiotic-resistant bacteria, as well as the genes responsible 

for antibiotic resistance in bacteria isolated from the environment, will improve the ability to assess the effects these 

waters may have on human health. In addition, relating these 

resistance patterns to other environmental contaminants will 

improve our understanding of the sources and (or) maintenance of resistance in the environment. 

Analytical and Sampling Methods

Physical, chemical, and bacterial water quality were 

determined for five different sampling dates (September 19, 

2005, and April 26, June 28, August 9, and August 23, 2006) 

at 7 sites in Oakland County and an additional 12 sites in 

the Clinton River Watershed for two of those sampling dates 

(April 26 and August 23, 2006). In addition, during a rainstorm (on June 28, 2006), a sample was collected for bacteria 

analyses from a drain that discharges into Evans Ditch at 

Southfield. Not including replicates or blanks, a total of 59 

samples were collected for total mercury determinations, 60 

for bacterial analyses, and 17 for antibiotic analyses. A summary of samples collected and analyzed is presented in table 4. 

E. coli and enterococci were isolated from stream water 

and were tested for the ability to grow in the presence of 

antibiotics. E. coli and enterococci isolates that were capable 

of growing on media with antibiotic concentrations considered significant for human health (Clinical Laboratory and 

Standards Institute, 2002) were reported as a percentage of 

isolates resistant to the antibiotic. Isolates were further tested 

for growth on media containing mercury to determine resistance (organisms capable of growth at 50 μM of mercury 

were considered resistant). Molecular analyses were used to 

identify antibiotic-resistance genes, or elements that may carry 

resistance genes, in isolates from stream-water samples. These 

results were then compared to antibiotic and mercury concentrations that were measured in similar stream-water samples. 

More detailed explanations of these methods are given in the 

following sections.
Analytical and Sampling Methods 7

Field Measurements

Field measurements of water temperature, dissolved 

oxygen, specific conductance, and pH were made near the 

sampling point with a multiparameter water-quality meter. 

USGS streamgages at the sampling site were used to calculate 

streamflow as described in Rantz and others (1982) with the 

exception of Kearsley Creek at Ortonville. Streamflow at the 

Kearsley Creek site was measured by means of an acoustic 

Doppler velocimeter (Kraus and others, 1994).

Collection of Samples for Chemical Analyses

For total mercury and antibiotic analyses, grab samples 

were collected according to USGS protocols (Wilde and others, 1999). Water samples collected for total mercury analysis 

were collected in an acid-rinsed Teflon bottle and kept on ice 

until all samples had been collected for the day, at which time 

samples were preserved with 10 mL of a 6 N hydrochloric acid 

solution (Olson and DeWild, 1999) and shipped to the USGS 

Wisconsin Mercury Research Laboratory for analysis. Samples for antibiotic analysis were collected in a Teflon bottle 

cleaned in the laboratory prior to sample collection, as specified in Wilde (2004). The sample was placed on ice and kept 

cool until the next day, when it was filtered through a 0.7-μm 

glass fiber filter into three 150-mL glass amber bottles and 

packed on ice for shipment to the USGS Organic Geochemistry Research Laboratory for analysis (laboratory schedule 

LCAN (Lindsey and others, 2001; Kolpin and others, 2002)). 

The antibiotics that were analyzed for are listed in table 5.

Collection of Samples for Bacteria Analyses

Samples were collected and processed for fecal coliform 

bacteria, E. coli, and enterococci, following protocols of the 

U.S. Environmental Protection Agency (2000) and American 

Public Health Association and others (1998). A single grab 

sample was collected from the center of river flow in a 1-L 

sterile bottle and kept on ice or cooled at 4°C until samples 

could be processed (18–24 hours following collection). Water 

samples were filtered through a 0.45-μm membrane filter in 

100-, 10-, and 1-mL volumes. Fecal coliform bacteria were 

enumerated on mFC medium; E. coli were differentiated by 

transferring the fecal coliform colonies to NA-MUG medium. 

Enterococci were enumerated on mEI medium. Growth on 

filters from the 100-mL sample for both mFC and mEI was 

rinsed from the filters with a sterile phosphate buffered saline 

solution (except for samples collected on April 26, 2006, in 

which all colonies from 100 mL were isolated and preserved). 

The samples were spun in a centrifuge to separate the cells 

from the liquid. The cells were then resuspended in 1 mL of 

phosphate-buffered saline containing 20 percent glycerol and 

stored at -80oC. Those preserved cells are hereafter referred 

to as the “FC or MEI culture stocks.” DNA was extracted from 

these cultures by a process of freezing and thawing a pellet 

of cells from 100 μL of the FC or MEI culture stock that had 

been resuspended in 100 μL of TE buffer (10 mM tris and 1 

mM EDTA, pH 8.0).

E. coli and enterococci isolates were transferred to 

Mueller-Hinton and brain-heart infusion broth, respectively, in 

96-well plates, grown overnight, preserved with glycerol, and 

stored at -80oC. Isolates were transferred to fresh media and 

Table 4. Summary of field measurements (dissolved oxygen, pH, specific conductance, and streamflow) and total mercury, antibiotic, 

and bacteria samples collected September 19, 2005, through August 23, 2006, in Oakland and Macomb Counties, Mich. 

[Shading indicates that measurements were made or samples were collected on the corresponding date]

USGS 

station 

number

Field measurements Total mercury Antibiotics Bacteria/antibiotic 

resistance

9/19 4/26 6/28 8/9 8/23 9/19 4/26 6/28 8/9 8/23 9/19 4/26 6/28 8/9 8/23 9/19 4/26 6/28 8/9 8/23

04148035

04160800

04161000

04161540

04161580

04166000

04166200

Drain

04160900

04161800

04161820

04163030

04163400

04164000

04164100

04164110

04164300

04164350

04164500

04164800
8 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

grown overnight prior to antibiotic resistance testing. Antibiotic solutions were made up according to the Clinical Laboratory Standards Institute protocols (2002) in the following 

concentrations: cefoxitin, 16 and 32 μg/mL; ceftriaxone, 32 

and 64 μg/mL; erythromycin, 8 μg/mL; ofloxacin, 8 μg/mL; 

and vancomycin, 16 μg/mL. Growth medium containing antibiotic solutions was dispensed into 96-well plates in 100-μL 

volumes. All E. coli isolates were transferred to 96-well plates 

containing 100 μL of Mueller-Hinton media with cefoxitin 

or ceftriaxone antibiotic solutions. Enterococci isolates were 

transferred to 96-well plates containing brain-heart infusion 

broth with vancomycin. E. coli isolates from samples collected 

April 26 and August 23 were also tested against erythromycin and ofloxacin. Isolates were incubated at 37oC for 18–24 

hours (48 hours for vancomycin), after which any growth was 

recorded as a resistant isolate. American Type Culture Collection (ATCC) strains E. coli ATCC 25922 and Enterococcus 

faecalis (E. faecalis) ATTC 29212 were used as controls for 

antibiotic analyses, as described by the Clinical Laboratory 

and Standards Institute (2002).

E. coli isolates were tested for resistance to mercury by 

plating isolates to Luria-Bertani (LB) media containing 50 μM 

mercuric chloride. Isolates were incubated overnight at 37oC. 

Any growth was recorded as a resistant isolate. 

E. coli isolates resistant to mercury, erythromycin, or 

ofloxacin (from April and August 23, 2006, samples) were 

chosen for molecular analyses to identify genes responsible 

for antibiotic resistance. Polymerase chain reaction (PCR) 

was performed on all mFC culture stocks and selected isolates 

from April 26 and August 23, 2006, to detect three different components of class I integrons, the 5 prime to 3 prime 

conserved region containing gene cassettes (Lévesque and 

others, 1995), the integrase gene (Koeleman and others, 2001), 

and the sulfonamide-resistance gene (Lévesque and others, 

1995). A positive control (ECOR-03, E. coli reference collection, Michigan State University, East Lansing, Mich.) and 

negative control E. coli strain (ATCC 25922) were run with 

each PCR reaction. All enterococci isolates resistant to 16 μg/

mL vancomycin were tested by PCR for the presence of vanA, 

a transferable gene responsible for high-level vancomycin 

resistance, following protocols described in Fogarty and others 

(2005). DNA extracted from MEI culture stocks was analyzed 

for the presence of the vanA following protocols similar to 

those previously described (Fogarty and others, 2005) by 

Table 5. List of antibiotics for which samples were analyzed in the Oakland County study.

[Analyzed by U.S. Geological Survey Geochemistry Organics Research Laboratory]

Beta-lactams Sulfonamides Others

Amoxicillin Sulfachloropyridazine Carbadox 

Ampicillin Sulfadiazine Lincomycin 

Cefotaxime Sulfadimethoxine Trimethoprim 

Cloxacillin Sulfamerazine Ormetoprim 

Oxacillin Sulfamethazine 

Penicillin G Sulfamethoxazole 

Penicillin V Sulfathiazole 

Macrolides Tetracyclines and degradation products

Azithromycin Chlortetracycline 

Erythromycin Anhydro-chlortetracycline 

Erythromycin-H2

O Demeclocycline 

Roxithromycin Doxycycline 

Tylosin Minocycline 

Virginiamycin Oxytetracycline 

Tetracycline 

Quinolines Anhydro-tetracycline 

Ciprofloxacin Alpha apo-oxytetracycline 

Clinafloxacin Beta apo-oxytetracycline 

Flumequine Epi-anhydro-chlortetracycline 

Lomefloxacin Epi-chlortetracycline 

Norfloxacin Iso-chlortetracycline 

Ofloxacin Epi-Iso-chlortetracycline 

Oxolinic acid Epi-oxytetracycline 

Sarafloxacin Epi-anhydro-tetracycline 

Epi-tetracycline 
Results of Data Analyses 9

using100 μL of the stock culture instead of an isolate. A 

positive-control enterococci strain (ATCC 700221) containing 

the vanA gene and a negative control (no DNA) was included 

with each PCR reaction. MEI cultures were also analyzed for 

the esp gene from Enterococcus faecium and E. faecalis. The 

esp gene encodes a surface protein that is found on human 

pathogenic strains of Enterococcus, and it is commonly used 

to identify sources of human fecal pollution. All reagents 

were from Applied Biosystems (Foster City, Calif.). Primers 

and the positive control strain (E53) used for the esp analyses are described in Shankar and others (1999). Each 15-μL 

reaction contained (final concentration) 1× Gold buffer, 2.5 

mM MgCl2, 0.2 mM dNTP’s, 0.5 μg/μL BSA, 0.5 μM of the 

forward and reverse primers, 0.05 unit/μL of Amplitaq Gold 

Polymerase and between 1 and 100 ng of template DNA. 

Quality Assurance and Quality Control

Additional samples were collected for quality assurance 

and quality control at all steps for all analyses in this study. A 

sequential replicate sample was collected for antibiotics, mercury, and bacterial analyses on one occasion during this study. 

Field blanks were collected at one site for antibiotic analyses, two sites for mercury analyses, and one site for bacterial 

analyses in accordance with Wilde and others (1999). In addition, laboratory blanks (sterilized phosphate buffer saline filter 

in 100-mL volumes) were also analyzed for each sampling 

event for all bacterial analyses. A laboratory split replicate 

was also processed by filtering equal volumes of sample water 

onto two different filters and processing as described above. 

Concentrations of all constituents of interest in the field blanks 

were less than the method detection limits for all constituents. Concentrations in all bacterial-analysis lab blanks also 

were less than the method detection limit (less than 1 cell per 

100 mL). Analytical results for mercury were identical for 

replicate samples collected at the Clinton River Auburn Hills 

on August 9, 2007 (1.17 ng/L). Analytical results for replicate 

samples collected at the Clinton River at Auburn Hills on 

August 9, 2006, for antibiotics were fairly consistent (table 6). 

Differences detected in both the bacterial field and laboratory 

replicates are discussed in the following section.

Results of Data Analyses

Fecal indicator bacteria were enumerated and the percentages of cephalosporin-resistant E. coli and VRE were determined and related to antibiotic and mercury concentrations in 

the water. Field measurements made for each of the seven sites 

on all five sampling dates are listed in table 7. 

Fecal Indicator Bacteria Enumeration

Fecal coliform bacteria, E. coli, and enterococci were 

quantified for samples collected on five separate occasions 

from September 2005 to August 2006. These results are 

shown in table 8. State of Michigan recreational-water quality standards for all surface waters in the State are in effect 

from May 1 to October 31 (Michigan’s water-quality standard 

R323.1062, Rule 62). The Michigan water-quality standard for 

a single sampling event specifies three or more samples from 

which a geometric mean is calculated. 

Bacteria concentrations reported in this study resulted 

from enumeration of a single sample rather than a calculation of the geometric mean of three samples. However, the 

Michigan recreational-water quality standard of 300 colonyforming units (CFU) per 100 mL and partial contact-water 

quality standard of 1,000 CFU/100 mL for a single sampling 

event can be used as a frame of reference for samples collected in this study. Figure 3 shows the concentrations for all 

the indicator bacteria measured and the recreational standard 

of 300 CFU/100 mL for E. coli. Of the 35 samples collected 

for Oakland County, 19 exceeded this standard. Every site 

exceeded this standard on at least one sampling date. No sample collected on April 26, 2006, exceeded 300 CFU/100 mL. 

Two samples (Evans Ditch and Kearsley Creek) collected June 

28, 2006, exceeded 1,000 CFU/100 mL. The high levels of 

E. coli and enterococci frequently measured in these samples 

are indicative of fecal pollution. High levels of these indicator 

organisms have been shown to be correlated with increased 

risk of illness (U.S. Environmental Protection Agency, 1986). 

Table 6. Concentrations of antibiotics detected in replicate samples collected at Clinton River at Auburn Hills, 

Mich., August 9, 2006.

[Concentrations are in micrograms per liter]

Sample Azithromycin Erythromycin

ErythromycinH2

O Ofloxacin Trimethoprim

Clinton River at 

Auburn Hills <0.005 0.064 0.063 0.138 0.013

Replicate 0.108 0.067 0.064 0.136 0.014
10 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Table 7. Field measurements for samples collected at Oakland County, Mich., study sites, September 2005 through August 

2006.

[USGS, U.S. Geological Survey; ft3/s, cubic feet per second; °C, degrees Celsius; mg/L, milligram per liter; μS/cm, microsiemens per centimeter; 

NA, not available; **, instantaneous discharge measurement, daily mean value not available]

USGS station 

number Site name

Samplecollection 

date

Daily mean 

discharge 

(ft3

/s ) pH

Water 

temperature

(°C)

Dissolved 

oxygen 

(mg/L)

Specific 

conductance 

(μS/cm)

04148035 Kearsley Creek at Mill 

Street at Ortonville

09/19/05 4.1** 7.9 14.2 8.5 842

04/26/06 25** 8.4 8.2 9.6 446

06/28/06 11** 8.2 17.6 7.4 739

08/09/06 8.3** 8.3 17.0 8.2 751

08/23/06 4.2** 8.3 18.0 NA 862

04160800 Sashabaw Creek near 

Drayton Plains

09/19/05 2.0 7.9 16.5 7.8 708

04/26/06 21 8.7 9.7 9.2 579

06/28/06 10 8.0 23.1 8.5 713

08/09/06 3.2 8.0 22.0 8.0 697

08/23/06 2.6 8.3 17.6 6.4 682

04161000 Clinton River at Auburn 

Hills

09/19/05 25 7.6 19.0 7.7 1,108

04/26/06 124 8.4 15.3 10.0 657

06/28/06 131 8.1 23.4 6.9 838

08/09/06 39 8.3 22.1 7.5 904

08/23/06 26 7.8 21.0 9.0 1,110

04161540 Paint Creek at 

Rochester

09/19/05 14 8.0 15.5 9.5 794

04/26/06 69 8.4 13.0 10.0 499

06/28/06 38 8.4 19.1 8.3 707

08/09/06 20 8.7 17.2 8.4 746

08/23/06 12 8.4 19.0 NA 885

04161580 Stony Creek near 

Romeo

09/19/05 2.2 8.0 15.1 9.3 713

04/26/06 21 7.5 7.7 10.0 626

06/28/06 5.3 8.5 20.0 7.8 640

08/09/06 3.3 8.4 17.6 8.0 637

08/23/06 3.3 8.0 17.5 8.0 724

04166000 River Rouge at 

Birmingham

09/19/05 7.8 8.0 18.9 8.4 1,086

04/26/06 30 8.4 13.8 9.7 788

06/28/06 14 8.1 22.8 8.1 1,030

08/09/06 6.4 8.2 23.4 7.0 1,020

08/23/06 6.4 8.1 21.9 7.9 1,070

04166200 Evans Ditch at 

Southfield

09/19/05 1.6 7.7 17.5 5.2 1,690

04/26/06 5.0 8.4 9.9 11.3 1,570

06/28/06 5.6 8.0 20.0 7.4 1,790

08/09/06 1.2 8.1 20.5 5.1 2,780

08/23/06 1.3 8.2 19.4 5.7 2,340

Because samples were collected as a single sample, it is 

important to understand the bacteria variation that may occur 

in these samples. Sequential replicate samples collected from 

the Clinton River on August 9, 2006, show very little difference in fecal coliform bacteria or E. coli concentrations (table 

8). There was a greater difference in the enterococci concentration from this sample. Single samples were also processed 

as laboratory split replicates (table 8). Again, differences were 

detected, but they do not change the interpretation in regards 

to the recreational water-quality standards. It is important, 

however, to understand these are single samples that may not 

be a full representation of the conditions at the site. Yet the 

analyses give an indication of how high fecal bacteria concentrations may be in these waters. 
Results of Data Analyses 11

Table 8. Fecal indicator bacteria concentrations for samples collected at Oakland County, Mich., study sites, 

September 2005 through August 2006.

[USGS, U.S. Geological Survey; CFU/100 mL, colony forming units per 100 milliliters]

USGS 

station 

number Site name

Samplecollection 

date

Fecal coliform bacteria 

(CFU/100 mL)

E. coli

(CFU/100 mL)

Enterococci 

(CFU/100 mL)

04148035 Kearsley Creek at Mill Street 

at Ortonville

09/19/05 330 310 750

04/26/06 128 98 50

06/28/06 3,200 1,300 720

08/09/06 208 208 390

08/23/06 809 357 280

04160800 Sashabaw Creek near Drayton 

Plains

09/19/05 850 830 360

04/26/06 45 36 20

06/28/06 410 410 176

08/09/06 250 204 243

08/23/06 982/720a 483/520a 370/260a

04161000 Clinton River at Auburn Hills 09/19/05 300 140 280

04/26/06 68 42 29

06/28/06 1,173 847 590

08/09/06 78/79b 72/68b 230/146b

08/23/06 1,282 278 99

04161540 Paint Creek at Rochester 09/19/05 510 410 540

04/26/06 39 24 19

06/28/06 760 330 410

08/09/06 280 204 330

08/23/06 490 300 300

04161580 Stony Creek near Romeo 09/19/05 340 310 560

04/26/06 48 42 21

06/28/06 290 150 250

08/09/06 430 430 590

08/23/06 29 2 6

04166000 River Rouge at Birmingham 09/19/05 570 200 460

04/26/06 27 24 22

06/28/06 1,673 540 680

08/09/06 690 640 360

08/23/06 1,535 935 370

04166200 Evans Ditch near Southfield 09/19/05 840 830 360

04/26/06 71 51 37

06/28/06 5,600 1,400 8,600

08/09/06 720 60 542

08/23/06 2,600/1,000a 700/639b 2,300/1,600b

Drain at Evans Ditch 06/28/06 4,100 1,900

a

 Replicate lab sample—single sample filtered in replicate.

b

 Replicate field sample—replicate samples collected in field.
12 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Kearsley Creek

1

10

100

1000

10000

Sept 19

Apr 26

Jun 28

Aug 9

Aug 23

0

10

20

30

Sashabaw Creek

1

10

100

1000

10000

Sept 19

Apr 26

Jun 28

Aug 9

Aug 23

Rep.

0

10

20

30

Clinton River at Auburn Hills

1

10

100

1000

10000

Sept 19

Apr 26

Jun 28

Aug 9

Rep.

Aug 23

0

30

60

90

120

150

Paint Creek

1

10

100

1000

10000

Sept 19

Apr 26

Jun 28

Aug 9

Aug 23

0

20

40

60

80

Stony Creek

1

10

100

1000

10000

Sept 19

Apr 26

Jun 28

Aug 9

Aug 23

0

10

20

30

River Rouge

1

10

100

1000

10000

Sept 19

Apr 26

Jun 28

Aug 9

Aug 23

0

10

20

30

40

Evans Ditch

1

10

100

1000

10000

Sept 19

Apr 26

Jun 28

Aug 9

Aug 23

Rep.

0

2

4

6

FECAL INDICATOR BACTERIA CONCENTRATION, IN COLONY-FORMING UNITS PER 100 MILLILITERS

Fecal coliform bacteria

E. coli 

Enterococci

Michigan recreational water quality 

standard for E. coli (300 CFU/100 mL)

Daily mean streamflow

DAILY MEAN STREAMFLOW, IN CUBIC FEET PER SECOND

Figure 3. Fecal indicator bacteria concentrations for samples collected September 19, 2005, and April 28, 

June 28, August 9, and August 23, 2006, at Oakland County study sites.
Results of Data Analyses 13

Vancomycin-Resistant Enterococci and HumanFecal-Enterococci Gene

Vancomycin-resistant enterococci were previously 

detected in surface-water samples collected in Oakland County 

in 2003 (Fogarty and others, 2005). In the current 2005–2006 

study, no enterococci isolated out of the stream water were 

resistant to vancomycin at the same concentration tested in 

2003 (16 μg/mL). The mEI cultures were tested for the presence of two genes responsible for vancomycin resistance, 

vanA and vanB. These cultures presumably represent a larger 

enterococci population than the number of single isolates 

tested (ranging from 3 to 96 isolates per sample, depending 

on the number of well-isolated colonies that grew on a single 

plate). The vanA gene was not detected in any of the MEI cultures, whereas in 2003 the vanA gene was identified is several 

enterococci isolates from Kearsley Creek and Evans Ditch. 

The source or sources of the vancomycin-resistant isolates 

in 2003 are unknown. Owing to the absence of vancomycinresistant organisms in 2005–2006, it does not appear that the 

source is a persistent problem at those sites where VRE was 

once detected. 

All mEI cultures were analyzed for the presence of a 

human fecal enterococci gene (esp). The esp gene is commonly associated with enterococci known to cause human illness and has also been used to identify human fecal pollution 

in the environment (Betancourt and Fujioka, 2006). The esp

gene was detected in two stream samples collected June 28, 

2006—Paint Creek and Evans Ditch—and in the Evans Ditch 

drain sample collected during a rain event on that day. Samples in which the esp gene was found had enterococci concentrations greater than 400 CFU/100 mL. However, this gene 

was not detected in all samples with concentration greater than 

400 CFU/100 mL. The presence of this gene indicates a source 

of human fecal pollution at these two sites.

Occurrence of Cephalosporin-Resistant E. coli 

In the study conducted in 2003, cephalosporin-resistant 

fecal coliform bacteria and E. coli were frequently isolated 

from streams in Oakland County (Fogarty and others, 2005). 

As a result, two cephalosporin antibiotics tested in that study 

were chosen for analysis in the 2005–2006 study: cefoxitin, a 

second-generation cephalosporin antibiotic, and ceftriaxone, 

a third-generation cephalosporin antibiotic. Both antibiotics 

are approved for human use only; however, cefoxitin is legally 

used to treat infections in cats and dogs, and similar antibiotics 

are given to livestock. E. coli isolates were grown on antibiotic 

concentrations representing intermediate resistance (16 μg/

mL for cefoxitin, 32 μg/mL for ceftriaxone) and high-level 

resistance (32 μg/mL for cefoxitin, 64 μg/mL for ceftriaxone). 

Levels of resistance are often used to determine severity of 

resistance, which is sometimes controlled by different types 

of genes. The greater the level of resistance, the more difficult 

treatment could be. “Intermediate-” and “high-level” resistance is defined by the Clinical Laboratory Standards Institute 

(Clinical Laboratory and Standards Institute, 2002).

E. coli resistant to cefoxitin and/or ceftriaxone were isolated at all sites (table 9). Out of the 35 samples collected, 24 

samples contained E. coli resistant to high-level cefoxitin, and 

13 samples contained E. coli resistant to high-level ceftriaxone. Isolates resistant to high-level cefoxitin were detected at 

all seven Oakland County study sites. High-level ceftriaxone 

resistance was detected at all sites except Paint Creek and 

Stony Creek. High level resistance to cefoxitin was detected 

in 50 percent of E. coli isolates, whereas intermediate-level 

resistance was detected in 75 percent of the isolates. Highlevel and intermediate ceftriaxone resistance was detected 

in 6 percent and 41 percent of E. coli isolates, respectively. 

Isolates resistant to both ceftriaxone and cefoxitin were 

detected in 10 of the 35 samples analyzed. Samples from all 

sites except Paint Creek and Stony Creek contained E. coli 

resistant to both cephalosporin antibiotics. E. coli resistant to 

one or more cephalosporin antibiotics commonly carry a gene 

that results in resistance to not only the cephalosporin antibiotics but also a large class of antibiotics (β-lactam antibiotics) 

commonly used to treat infections. Infections caused by organisms with this type of resistance may be very difficult to treat 

because of a lack of treatment options. 

Because E. coli are not typically resistant to these antibiotics, it is interesting to see such widespread dissemination of 

these resistances in this area. Further studies would need to be 

done to determine whether the E. coli that was isolated out of 

the environment resembles E. coli that are common to human 

or animal intestinal systems or that cause human illnesses. 

This would provide a better understanding of the potential 

effects these organisms have on human health. 

Significant differences in the percentage of resistant isolates between replicates samples were observed in the sample 

set (table 9). Difference in the percentage of antibiotic-resistant isolates was as high as 45 percent. This may be a reflection of heterogeneity of the microbial community in water 

samples. It is important to understand that, like the fecal-indicator bacteria concentrations, the sample used to determine the 

percentage of resistant isolates is just a snapshot of the stream 

site and may not be a true representation of conditions at the 

stream site over time. 

Studies by the National Antimicrobial Resistance 

Monitoring System (NARMS) have detected very few human 

pathogenic E. coli isolates with resistance to either cefoxitin 

or ceftriaxone (Centers for Disease Control and Prevention, 

2004). Despite being approved only for human medicine, a 

greater percentage of E. coli isolated from veterinary samples 

were found to be resistant to either cefoxitin or ceftriaxone 

(U.S. Department of Agriculture, 2007) than in the human 

pathogenic E. coli study. The results shown in the NARMS 

studies do not rule out human fecal waste as a source of cephalosporin-resistant E .coli, because only pathogenic E. coli were 

tested in the NARMS studies. However, the NARMS studies 

do indicate E. coli from fecal sources other than humans may 

be a source of cephalosporin-resistant E. coli.
14 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Table 9. Percentage of E. coli isolates resistant to tested antibiotics for samples collected at Oakland County, Mich., study sites, 

September 2005 through August 2006.

[USGS, U.S. Geological Survey; CFX, cefoxitin; AXO, ceftriaxone; HLR, high-level resistance to both cefoxitin and ceftriaxone resistant; ERY, erythromycin; OFL, oflaxicin; int., intermediate resistance; NT, not tested]

USGS 

station 

number Site name

Sample 

collection date

Number of 

isolates 

tested

Percentage of E. coli isolates resistant

CFX 

int.

CFX 

high

AXO 

int.

AXO 

high HLR 

ERY 

high

OFL 

high

04148035 Kearsley Creek at Mill 

Street at Ortonville

09/19/05 31 0 0 3 0 0 NT NT

04/26/06 74 11 0 26 0 0 0 0

06/28/06 29 17 17 3 3 3 NT NT

08/09/06 16 25 6 0 0 0 NT NT

08/23/06 31 23 10 6 3 0 13 0

04160800 Sashabaw Creek near 

Drayton Plains

09/19/05 83 8 0 13 0 0 NT NT

04/26/06 30 7 0 53 0 0 0 0

06/28/06 16 38 31 19 19 19 NT NT

08/09/06 27 7 7 0 0 0 NT NT

08/23/06 37/47a 54/13a 49/13a 0/0a 0/0a 0/0a 5 0

04161000 Clinton River at Auburn 

Hills

09/19/05 14 14 0 43 7 0 NT NT

04/26/06 36 11 3 31 3 0 0 0

06/28/06 7 71 71 0 0 0 NT NT

08/09/06 72/68b 39/3b 29/3b 11/0b 3/0b 3/0b NT NT

08/23/06 16 44 38 13 6 6 13 0

04161540 Paint Creek at Rochester 09/19/05 41 5 0 17 0 0 NT NT

04/26/06 22 5 0 32 0 0 0 0

06/28/06 33 21 18 0 0 0 NT NT

08/09/06 30 13 10 10 0 0 NT NT

08/23/06 30 3 3 0 0 0 0 0

04161580 Stony Creek near Romeo 09/19/05 31 19 0 32 0 0 NT NT

04/26/06 39 3 0 38 0 0 0 3

06/28/06 36 19 14 3 0 0 NT NT

08/09/06 43 19 2 0 0 0 NT NT

08/23/06 2 50 0 0 0 0 0 0

04166000 River Rouge at 

Birmingham

09/19/05 19 42 5 21 16 5 NT NT

04/26/06 23 26 4 17 4 4 0 0

06/28/06 54 31 30 11 7 7 NT NT

08/09/06 56 4 4 0 0 0 NT NT

08/23/06 45 31 31 0 0 0 4 0

04166200 Evans Ditch at 

Southfield

09/19/05 61 15 0 34 2 0 NT NT

04/26/06 45 13 2 42 4 2 0 0

06/28/06 14 71 64 7 0 0 NT NT

08/09/06 62 5 5 5 2 2 NT NT

08/23/06 46/7a 74/29a 67/29a 24/0a 0/0a 0/0a 13 0

Drain at Evans Ditch 06/28/06 4 100 100 0 0 0 NT NT

a

 Replicate lab sample—single sample filtered in replicate.

b

 Replicate field sample—replicate samples collected in field.
Results of Data Analyses 15

The resistance seen in the veterinary isolates was not directly 

attributed to cephalosporin antibiotic use in that animal. Therefore, it is possible that other environmental or natural factors 

may result in the presence of cephalosporin-resistant bacteria 

in the environment. 

Spatial and Temporal Patterns in the Percentage 

of Cephalosporin-Resistant E. coli

Despite the difficulties in interpreting the value of the 

percentage resistance due to the results in replicate samples, 

there were still interesting patterns in the percentage of cephalosporin-resistant E. coli isolates temporally and spatially. 

Sites in less urbanized areas (sites with less than or 51 percent 

developed area (table 2): Kearsley Creek, Sashabaw Creek, 

Paint Creek, and Stony Creek) had no detectable cefoxitin- or 

ceftriaxone-resistant E. coli in September 2005 and April 

2006, but cefoxitin- and/or ceftriaxone-resistant E. coli were 

detected in summer months (June through August 2006) (fig. 

4). This pattern did not hold true for the more urbanized sites 

(sites with greater than 51 percent developed area (table 2): 

Clinton River, River Rouge, and Evans Ditch), at which there 

appeared to be more consistent detection of resistant E. coli 

throughout the study period (fig. 4). 

Rainfall varied slightly throughout the county prior to 

sample collections (fig. 5), and only one sample was collected 

during a precipitation event, on June 28, at Evans Ditch. There 

was no correlation with 24- or 72-hour preceding rainfall and 

the percentage of cephalosporin-resistant E. coli detected, 

nor was there was any linear relation between discharge and 

percent resistant E. coli at any site. In other words, it does not 

appear that rainfall controls the levels at which cephalosporinresistant E. coli are found in streams in the study area. 

It is important to remember that the percentage of resistance is inherently variable in the sample (as seen in the replicate samples); therefore, these results only indicate possible 

patterns in such resistance and would require more intensive 

sampling to confirm.

Antibiotic Concentrations and CephalosporinResistant E. coli

Antibiotic concentrations were measured in water 

samples collected on multiple occasions during this study 

(table 10). There was no apparent relation between the antibiotics detected in the water to the percentage of cephalosporinresistant E. coli in the sample. Cephalosporin antibiotics 

were not detected in any sample. Cephalosporin antibiotics 

degrade quite quickly in the environment and are not frequently detected in environmental samples. However, multiple 

antibiotics were detected in samples from the Clinton River 

at Auburn Hills: erythromycin, ofloxacin, sulfamethoxazole, 

and trimethroprim were detected in all three of the samples, 

and azithromycin was also detected in two of the five samples 

from this site. The only other water sample collected that had 

detectable antibiotics was the August 9, 2006, Paint Creek 

sample (tylosin). 

All E. coli isolates from April 26 and August 23, 2006, 

samples were tested for resistance to high levels of erythromycin (8 μg/mL) and oflaxicin (8 μg/mL), because both 

antibiotics were detected in the Clinton River at Auburn Hills 

samples. Erythromycin-resistant E. coli were not detected in 

any of the April samples but were detected at five of the seven 

sites in August. Erythromycin-resistant E. coli was detected at 

the Clinton River at Auburn Hills site, but no difference in frequency of occurrence was observed compared to other sites at 

which erythromycin was not detected in the water (table 10). 

Although no antibiotic tested was detected at the Stony Creek 

site, ofloxacin-resistant E. coli isolates were detected only at 

Stony Creek near Romeo in April. 

Antibiotics that are detected in the water are not related 

to the antibiotic-resistant E. coli isolated from these waters.

Therefore, antibiotics in the water are not a reliable indictor 

for the co-occurrence of antibiotic-resistant E. coli.

Mercury- and Cephalosporin-Resistant E. coli

Several studies have linked mercury pollution to antibiotic resistance (McArthur and Tuckfield, 2000; Baker-Austin 

and others, 2006). Mercury concentrations were measured 

in this study to determine whether there was any apparent 

relation between mercury concentrations in the area and 

antibiotic-resistant bacteria. Mercury concentrations in the 

water are listed in table 11. Samples collected from Clinton 

River at Auburn Hills and Evans Ditch frequently exceeded 

the Michigan water-quality criterion for mercury of 1.3 ng/L. 

The Clinton River site is just downstream from a wastewatertreatment-plant discharge, and the Evans Ditch site is in a 

heavily industrialized area.

Samples from seven study sites (Kearsley Creek, Sashabaw Creek, Paint Creek, Clinton River, River Rouge, and 

Evans Ditch) contained E. coli isolates resistant to mercury in 

either September or April. Mercury-resistant E. coli were not 

detected in samples collected in June or August. In September, 

mercury-resistant E. coli isolates were detected at Paint Creek 

(2 percent), Sashabaw Creek (1 percent), and River Rouge (26 

percent). In April, resistant isolates were detected at Kearsley 

Creek (4 percent), Stony Creek (3 percent), Clinton River (3 

percent), and Evans Ditch (7 percent). In September, 1 and 

26 percent of E. coli isolates from Sashabaw Creek and River 

Rouge, respectively, were resistant to both mercury and cephalosporin antibiotics. In April, isolates from the Clinton River 

(3 percent) and Evans Ditch sites (2 percent) were resistant 

to both mercury and cephalosporin antibiotics. Overall, the 

percentage of mercury-resistant isolates is not related to the 

mercury concentrations detected in that sample (fig. 6), nor 

does it seem as if this percentage is related to the median or 

mean concentrations of mercury over the study period. There 

are, however, some patterns at individual sites. 
16 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.STREAMFLOW, IN CUBIC FEET PER SECOND

Kearsley Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

10

20

30

Clinton River at Auburn Hills

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 9-R

Aug 23

0

50

100

150

Evans Ditch

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

2

4

6

Paint Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

20

40

60

80

River Rouge 

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

10

20

30

40

Stony Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

10

20

30

Sashabaw Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

Aug 23-R

0

10

20

30

PERCENTAGE OF E.COLI ISOLATES RESISTATNT TO CEPHALOSPORIN ANTIBIOTIC

Cefoxitin resistance

Ceftriaxone resistance

Cefoxitin + ceftriaxone resistance

Daily mean streamflow

Figure 4. Percentage of cephalosporin-resistant E. coli isolates and daily mean streamflow on five sampling dates at 

study sites, Oakland County, Mich.
Results of Data Analyses 17

Auburn Hills

0

0.1

0.2

0.3

0.4

0.5

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

RAINFALL, IN INCHES

Total rainfall on day of sampling

Total rainfall preceding 24 hours

Total rainfall preceding 72 hours

 Rochester Hills

0

0.1

0.2

0.3

0.4

0.5

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

Southfield

0

0.1

0.2

0.3

0.4

0.5

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

Birmingham

0

0.1

0.2

0.3

0.4

0.5

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

For example, at River Rouge at Birmingham, ceftriaxoneresistant E. coli was detected only on September 19, April 26, 

and June 28, in which mercury concentrations were greatest; 

and at Evans Ditch near Southfield, the highest percentage 

of cefoxitin-resistant E. coli occurred along with the highest 

concentrations of mercury. 

Although the results of this study cannot confirm a relation between mercury concentrations in the water to the percentage of mercury- or cephalosporin-resistant E. coli in the 

water, there are some indications that mercury may be related 

to resistances under specific conditions in areas with greater 

urban or industrialization. This result agrees with results seen 

in a study by McArthur and Tuckfield (2000), who found 

antibiotic-resistant bacteria at an industrially perturbed and 

an undisturbed stream; however, in that study, the industrially 

perturbed stream had much higher proportion of antibioticresistant bacteria. There was no significant difference between 

the Oakland County industrialized and nonindustrialized 

samples. 

Integron Detection

Class I integrons are genetic structures that may contain

several different resistance genes that could be transferred 

among similar bacteria. A schematic of this structure is shown 

in figure 7. Class I integrons have been implicated in the transmission and spread of multidrug resistance in disease-causing 

organisms (Lévesque and others, 1995; Rosser and Young 

1999; Gruteke and others 2003; Singh and others, 2005). 

The presence of this element in organisms isolated from the 

environment suggests a possible mechanism for the transfer 

of antibiotic resistances in stream waters to disease-causing 

organisms. 

Figure 5. Precipitation totals on the day of sample collection and in the preceding 24 and 72 hours at locations 

in Oakland County, Mich.
18 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Table 10 Antibiotics detected in samples collected September 2005 through August 2006 at Oakland County study sites. 

[USGS, U.S. Geological Survey; μg/L; micrograms per liter; <, less than; bold, detectable concentration]

USGS 

station 

number Site name

Samplecollection 

date

Antibiotic concentration (μg/L)

AzithromycinErythromycin

ErythromycinH2

O Ofloxacin

Sulfamethoxazole

Trimethoprim Tylosin

04148035 Kearsley Creek at 

Mill Street at 

Ortonville

04/26/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

08/23/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

04160800 Sashabaw Creek near 

Drayton Plains

04/26/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

08/23/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

04161000 Clinton River at 

Auburn Hills

04/26/06 0.151 0.052 0.019 0.008 0.046 0.013 <0.005

08/09/06 0.108 0.067 0.064 0.026 0.136 0.014 <0.005

08/23/06 <0.005 0.055 0.053 0.043 0.277 0.006 <0.005

04161540 Paint Creek at 

Rochester

04/26/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

08/09/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 0.006

08/23/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

04161580 Stony Creek near 

Romeo

04/26/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

08/23/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

04166000 River Rouge at 

Birmingham

04/26/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

08/09/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

08/23/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

04166200 Evans Ditch at 

Southfield

04/26/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

08/23/06 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005

Table 11. Mercury concentration in samples collected September 2005 through August 2006 at Oakland County study sites.

[USGS, U.S. Geological Survey; ng/L, nanograms per liter; bold, concentration exceeds Michigan’s Rule 57 water-quality criterion of 1.3 ng/L ]

USGS 

station 

number Site name

Mercury concentration (ng/L)

Median Mean 9/21/2005 4/26/2006 6/28/2006 8/9/2006 8/23/2006

04148035 Kearsley Creek at Mill Street at Ortonville 0.53 0.68 0.49 0.68 1.36 0.53 0.34

04160800 Sashabaw Creek near Drayton Plains 0.99 0.90 0.99 1.31 1.12 0.6 0.46

04161000 Clinton River at Auburn Hills 1.82 1.76 1.82 1.95 2.66 1.17 1.18

04161540 Paint Creek at Rochester 0.57 0.69 0.57 0.95 1.07 0.45 0.40

04161580 Stony Creek near Romeo 0.6 0.46 0.6 1.22 0.68 0.47 0.42

04166000 River Rouge at Birmingham 1.04 1.10 1.04 1.41 1.28 1 0.79

04166200 Evans Ditch at Southfield 3.83 7.78 3.68 3.42 19.8 8.18 3.83
Results of Data Analyses 19

Kearsley Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

1

2

3

4

5

Clinton River at Auburn Hills

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 9-R

Aug 23

0

1

2

3

4

5

Evans Ditch

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

Aug 23-R

0

5

10

15

20

25

Paint Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

1

2

3

4

5

River Rouge 

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

1

2

3

4

5

Stony Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

0

1

2

3

4

5

Sashabaw Creek

0%

20%

40%

60%

80%

100%

Sep 19

Apr 26

Jun 28

Aug 9

Aug 23

Aug 23-R

0

1

2

3

4

5

PERCENTAGE OF E.COLI ISOLATES RESISTANT TO CEPHALOSPORIN ANTIBIOTIC

TOTAL MERCURY, IN NANOGRAMS PER LITER

Cefoxitin resistance

Ceftriaxone resistance

Cefoxitin + ceftriaxone resistance

Total mercury

Figure 6. Percentage of cephalosporin-resistant E. coli isolates and total mercury concentration for 

samples collected on five sampling dates at Oakland County study sites.
20 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Plasmid

Transposon

Integron

ABr

ABr

MTr

EXPLANATION

Generic antibiotic 

resistance gene

Generic metal resistance 

gene

Quaternary compound 

resistance gene

Sulfonamide resistance 

gene

Integrase gene

Region on the integron

that contains resistance

gene cassettes

Regions analyzed using 

polymerase chain 

reactions

CR

ABr ABr ABr ABr ABr ABr ABr ABr sulr qacr qac sul int

ABr

MTr

qac

sul

int

CR

ABr

MTr

E. coli

cell

chromosomal 

DNA

Plasmid

Transposon

Integron

ABr

ABr

MTr

EXPLANATION

Generic antibiotic 

resistance gene

Generic metal resistance 

gene

Quaternary compound 

resistance gene

Sulfonamide resistance 

gene

Integrase gene

Region on the integron

that contains resistance

gene cassettes

Regions analyzed using 

polymerase chain 

reactions

CR

ABr ABr ABr ABr ABr ABr ABr ABr sulr qacr qac sul int

ABr

MTr

qac

sul

int

CR

ABr

MTr

E. coli

cell

chromosomal 

DNA

The presence of class I integrons at study sites was determined by use of PCR to detect key components of the integron: the integrase gene (int1) required for incorporation of 

gene cassettes, a sulfonamide resistance gene (sul1) commonly 

found at the end of integrons, and the cassette region (CR) in 

which resistance genes are incorporated. All FC cultures from 

September, June, and August were analyzed for the presence 

of these three components of the integron (CR, int1, and sul1). 

E. coli isolates tested were those from April and August 23 

that were resistant to either erythromycin or ofloxacin, and all 

mercury-resistant E. coli isolated from April were analyzed for 

the present of CR and int1. The sul1 gene was not analyzed for 

in these isolates. 

The presence of CR, int1, and sul1 is a clear indication 

of the presence of integrons in these samples. Of the 29 FC 

samples analyzed, 8 contained all three components of the 

integron (table 12). All three components were detected at 

least once from all sites but Sashabaw Creek. The presence of 

a sul1 gene has been shown to be commonly associated with 

integrons but is not critical to integron function; however, the 

int1 gene is required for the incorporation of gene cassettes 

onto the integron. Therefore, samples such as the Clinton 

River at Auburn Hills (Aug. 9), in which no sul1 gene was 

detected but the int1 and CR were detected, still indicate the 

potential for antibiotic resistance and transferability of resistance within fecal coliform populations. 

Because the FC cultures represent a large community 

of organisms, individual E. coli isolates were selected on the 

basis of resistance to erythromycin, ciprofloxacin, or mercury for integron analyses. Fourteen mercury-resistant E. coli 

isolated from April were tested for the presence of integrons; 

one resulted in a positive CR and int1 detection (Evans Ditch). 

Of the 16 erythromycin- or ofloxacin-resistant E. coli isolates 

tested, 3 isolates—all from Evans Ditch—were positive for the 

CR. No antibiotics were detected at the Evans Ditch site. 

Further analysis would be needed to determine which 

genes are present to determine if resistance to cephalosporin or 

other antibiotics is the result of a gene carried on the integrons 

or if the presence of the integrons is only a coincidence. These 

results indicate that integrons are present in these waters and 

could be a mechanism for transfer of resistance to other organisms, including pathogens. Further analyses would need to be 

done to determine precisely the resistance genes contained 

on the class I integron structure and the ability to transfer to 

pathogens. 

Figure 7. Schematic of the relation of transferable genetic elements: plasmid, transposon, integron, and resistance 

genes. Plasmids are circular pieces of DNA that can move from cell to cell. Plasmids commonly contain genes for antibiotic 

resistance or other resistance such as to heavy metals, as well as transposons, which also may contain resistance genes. In 

particular, integrons—located on the transposon—may carry multiple resistance genes and are commonly associated with 

a sulfonamide resistance gene (sul) and a gene for resistance to quaternary ammonia compounds (qac). The presence of the 

integrons can be detected by use of polymerase chain reaction (PCR) to amplify particular regions on the integrons.
Results of Data Analyses 21

Table 12. Results of polymerase chain reactions (PCR) for integron structures in samples 

collected September 2005 through August 2006 at Oakland County study sites.

[USGS, U.S. Geological Survey; PCR, polymerase chain reaction; int1, integrase gene; sul1, sulfonamide 

resistance gene; 5 ́-3 ́, 5 prime to 3 prime conserved region; +, positive reaction; -, negative reaction; NT, not 

tested]

USGS station 

number Site name

Samplecollection 

date

PCR products

int1 sul1

integron 

(5’-3’)

04148035 Kearsley Creek at Mill Street at 

Ortonville

09/19/05 - NT -

06/28/06 + + +

08/09/06 + + +

08/23/06 - - -

04160800 Sashabaw Creek near Drayton 

Plains

09/19/05 - NT -

06/28/06 + - -

08/09/06 - - -

08/23/06 - + -

04161000 Clinton River at Auburn Hills 09/19/05 + NT -

06/28/06 + + +

08/09/06 + - +

08/23/06 + - -

04161540 Paint Creek near Rochester 09/19/05 - NT -

06/28/06 + + +

08/09/06 + + -

08/23/06 + + +

04161580 Stony Creek near Romeo 09/19/05 + NT -

06/28/06 - - -

08/09/06 + - -

08/23/06 + + +

04166000 River Rouge at Birmingham 09/19/05 + NT -

06/28/06 + - +

08/09/06 - - -

08/23/06 + + +

04166200 Evans Ditch at Southfield 09/19/05 + NT +

06/28/06 + + -

08/09/06 + + +

08/23/06 + - -

Drain at Evans Ditch 06/28/01 + + +
22 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Results From Clinton River Watershed Study

To determine whether results from streams of Oakland 

County were representative of conditions across a broader 

region, samples were collected throughout the Clinton River 

Watershed on April 26 and August 23, 2006. Fecal indicator bacteria concentrations, mercury concentrations, and the 

percentage of cephalosporin-, vancomycin-, and mercuryresistant isolates were analyzed for an additional 12 sites in 

the watershed (fig. 2). E. coli and enterococci concentrations 

for sites in Oakland County were similar to concentrations at 

sites outside of Oakland County (figs. 8 and 9).

No VRE was detected in any water samples collected on 

these two dates. The percentage of cefoxitin- and ceftriaxoneresistant E. coli and total mercury concentration are shown 

in figure 10. At sites outside Oakland County, the percentage 

of cephalosporin-resistant E. coli and mercury concentrations were as high as or higher than those at sites in Oakland 

County, with the percentage of antibiotic resistance and 

mercury concentrations being higher in the more industrial or 

urbanized areas. 

The percentage of cefoxitin-resistant E. coli appears to 

be less in April then in August (fig. 11). April samples were 

collected from 24 to 72 hours after small rainfall events in the 

area (fig. 5), whereas August samples were collected during a very dry period. As a result, April streamflows were 

much higher than those in August. Wet weather (and as a 

result, more runoff into the streams) may tend to decrease the 

resistant E. coli populations. However, these results are not 

supported by the more intensive sampling done in Oakland 

County, in which the percentage of resistance was not correlated with rainfall or streamflow. 

Very few ceftriaxone-resistant E. coli were isolated in 

the larger watershed study (fig. 12). All resistant isolates 

were from sites in the more urban or industrialized areas in 

the watershed (figs. 2 and 12). The highest percentage of 

ceftriaxone-resistant E. coli was found at Red Run and Clinton 

River at Fraser (fig. 12). At Red Run, the ceftriaxone-resistant 

E. coli were detected only in April, and at Clinton River at 

Fraser, the percentage of ceftriaxone-resistant E. coli was 

greater in April than in August. These results are opposite to 

the percentage of cefoxitin-resistant E. coli results presented 

earlier, an indication of different sources of E. coli for these 

two time periods.

Results of this watershed-focused study demonstrate that 

the cephalosporin-resistant E. coli results for Oakland County 

are not highly local phenomena. In fact, similar results were 

found throughout a watershed spanning multiple counties. 

The results of the watershed-based study also confirm earlier 

findings that areas with greater urban or industrial development pose an increased probability of detecting cephalosporinresistant E. coli when compared to less developed areas. 

View of Sashabaw Creek near Drayton Plains, Mich.
Results of Data Analyses 23

1

10

100

1000

10000

Sashabaw Creek near Drayton Plains

Clinton River nr Drayton Plains

Clinton River at Auburn Hills

Paint Creek at Rochester

Stony Creek near Romeo

Stony Creek near Washington

Clinton River at Sterling Heights

Red Run nr Warren

Plum Brook at Utica

Clinton River near Fraser

East Pond Creek at Powell Rd

E Pond Creek nr Romeo

E Branch Coon Creek at Armada

Highbank Creek nr Armada

N Branch Clinton River nr Mt. Clemens

Middle Branch Clinton River at Macomb

Clinton River at Mt. Clemens

April

August

Water-quality standard

E. COLI CONCENTRATION, IN COLONY- 

 FORMING UNTIS PER 100 MILLILITERS

0

100

200

300

400

500

600

700

800

900

1000

Sashabaw Creek near Drayton Plains

Clinton River nr Drayton Plains

Clinton River at Auburn Hills

Paint Creek at Rochester

Stony Creek near Romeo

Stony Creek near Washington

Clinton River at Sterling Heights

Red Run nr Warren

Plum Brook at Utica

Clinton River near Fraser

East Pond Creek at Powell Rd

E Pond Creek nr Romeo

E Branch Coon Creek at Armada

Highbank Creek nr Armada

N Branch Clinton River nr Mt. Clemens

Middle Branch Clinton River at Macomb

Clinton River at Mt. Clemens

April

August

Water-quality standard

ENTEROCOCCI CONCENTRATION IN COLONY-  FORMING UNTIS PER 100 MILLILITERS

Figure 8. E. coli concentrations for samples collected April 26 and August 23, 2006, at Clinton River 

Watershed study sites.

Figure 9. Enterococci concentrations for samples collected April 26 and August 23, 2006, at Clinton 

River Watershed study sites.
24 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Cefoxitin

0%

25%

50%

75%

100%

Sashabaw Cr

Clinton River nr Drayton Plains

Clinton River at Auburn Hills

Paint Cr

Stony Cr nr Romeo

Stony Cr nr Washington

Clinton River at Sterling Heights

Red Run 

Plum Brook 

Clinton River nr Fraser

E Pond Cr nr Romeo

E Pond Cr at Powell

E Branch Coon Cr at Armada

Highbank Cr 

N Branch Clinton River nr Mt. Clemens

Middle Branch Clinton River at Macomb

Clinton River at Mt. Clemens

0.00

4.00

8.00

12.00

April

August

April Hg

August Hg

PERCENTAGE OF E. COLI ISOLATES 

RESISTANT TO CEFOXITIN

TOTAL MERCURY, IN NANOGRAMS 

PER LITER

Ceftriaxone

0%

25%

50%

75%

100%

Sashabaw Cr

Clinton River nr Drayton Plains

Clinton River at Auburn Hills

Paint Cr

Stony Cr nr Romeo

Stony Cr nr Washington

Clinton River at Sterling Heights

Red Run 

Plum Brook 

Clinton River nr Fraser

E Pond Cr nr Romeo

E Pond Cr at Powell

E Branch Coon Cr at Armada

Highbank Cr 

N Branch Clinton River nr Mt. Clemens

Middle Branch Clinton River at Macomb

Clinton River at Mt. Clemens

0.00

4.00

8.00

12.00

April

August

April Hg

August Hg

PERCENTAGE OF E. COLI ISOLATES 

RESISTANT TO CEFTRIAXONE

TOTAL MERCURY, IN NANOGRAMS 

PER LITER

Figure 10. Percentage of E. coli isolates resistant to cefoxitin (top) and ceftriaxone (bottom) with respect to 

total mercury concentration for Clinton River Watershed study sites.
Results of Data Analyses 25

EXPLANATION

County boundary

Clinton River Watershed

Rivers

0 5 10 MILES

0 5 10 KILOMETERS

Oakland

County

Macomb

County

25

50

0

Cefoxitin

April

August

Percentage

County linework data from Michigan Resource Information System , 1998.

Hydrographic data from USGS 1:100,000 scale Digital Line Graphs.

EXPLANATION

County boundary

Clinton River Watershed

Rivers

0 5 10 MILES

0 5 10 KILOMETERS

Oakland

County

Macomb

County CeftriaxoneApril August

County linework data from Michigan Resource Information System , 1998.

Hydrographic data from USGS 1:100,000 scale Digital Line Graphs.

25

50

0

Percentage

Figure 11. Percentage of E. coli isolates resistant to cefoxitin for samples collected April 26 and 

August 23, 2006, at the Clinton River Watershed study sites.

Figure 12. Percentage of E. coli isolates resistant to ceftriaxone for samples collected April 26 and 

August 23, 2006, at the Clinton River Watershed study sites.
26 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Future Studies

This study showed that cephalosporin-resistant E. coli 

could frequently be isolated from streams in Oakland County 

and the surrounding area. This study was not able to identify 

the source of resistant isolates. The prevalence of resistant 

bacteria appears to be highly variable and site dependent. 

To better understand the significance of the problem, future 

researchers would need to intensively monitor the sites for not 

only cephalosporin-resistant E. coli but also other potentially 

significant antibiotic resistance, such as flouroquinolone resistance. In addition, water-quality information, such as concentrations of organic constituents and/or trace metals other than 

mercury, may help to determine whether water-quality components may contribute to increased resistance. Sediment, which 

could be a source of mercury exposure to bacteria, was not 

sampled in this study. Organisms may develop resistance in 

the sediment and then be resuspended into the water column. 

A more thorough investigation of potential sources such as 

wastewater, animal feces, urban runoff, and stormwater is warranted in order to better understand the source and transport 

mechanisms of antibiotics, antibiotic-resistant bacteria, and 

other contaminants in Oakland County and surrounding areas. 

Finally, a better understanding of how cephalosporin-resistant 

E. coli isolated from the environment are related to isolates 

that cause human illness would help to define the significance 

these results have to human health.

Summary and Conclusions

Previous water-quality studies in Oakland County, Mich., 

revealed high fecal indicator bacteria concentrations and the 

presence of antibiotic-resistant fecal bacteria in stream water. 

Enterococci resistant to vancomycin and fecal coliform bacteria resistant to multiple antibiotics, including cephalosporin 

antibiotics, were isolated in stream waters throughout the 

county. 

Vancomycin and cephalosporin antibiotics are used in the 

clinical setting to treat serious human illnesses. Vancomycinresistant enterococci and cephalosporin-resistant E. coli have 

been listed as an emerging resistance of human health concern 

in hospitals. Bacteria normally sensitive to these antibiotics 

can become resistant by the acquisition of antibiotic-resistance 

genes by transfer from one organism to another. If bacteria that 

cause illness become resistant to antibiotics, then treatment of 

the illness becomes more difficult and treatment options more 

limited. Because the surface waters in Oakland County serve 

as an important resource of the county, antibiotic-resistant 

bacteria in stream waters may pose a risk to human health for 

those who use these resources. 

This current USGS study, in cooperation with the Oakland County Health Department, continued sampling to determine the presence of fecal indicator bacteria, cephalosporinresistant E. coli, and vancomycin-resistant enterococci. One 

objective of this current (2005–2006) study was to determine 

whether results obtained in the previous study could be replicated and whether water samples collected under different 

streamflow conditions and other times of the year would yield 

similar results. In addition, this current study expanded the 

sampling to measure antibiotic concentrations and mercury 

to determine whether these concentrations were related to 

antibiotic-resistant bacteria also found in these samples. This 

relation may help explain the link to the mechanism of transfer 

and maintenance of antibiotic-resistant bacteria in stream waters. 

The acquisition of genes responsible for antibiotic 

resistance may aid in the spread and maintenance of antibioticresistant bacteria. Genes responsible for resistance to chemicals, metals, or antibiotics are sometimes linked together on 

plasmids, transposons, and integrons. Therefore, exposure 

to any one of the compounds may result in transfer of these 

elements from organism in the environment that already have 

it to other organisms who would have otherwise been sensitive. This serves as a survival mechanism for bacteria in the 

environment. And because these genes are linked together, 

the receiving organisms could be resistant to all compounds 

in which there was a resistant gene on the element it received. 

Both antibiotics and mercury (a heavy metal that has been 

commonly associated with antibiotic resistance genes) were 

measured in this study. This is only a short list of compounds 

that could be linked to antibiotic resistance; therefore, there 

may be other compounds present at these site that are related 

to antibiotic-resistance results observed in this study. 

In the current study, fecal-indicator bacteria concentrations frequently exceeded the Michigan Recreation Water 

Quality Standard of 300 colony-forming units per 100 milliliters. High fecal bacteria concentrations are an indicator of 

possible fecal pollution that may carry harmful pathogens and 

pose a threat to human health. Fecal bacteria can be exposed 

to antibiotics at their source and in the environment; therefore, 

waters with increased fecal bacteria may also contain higher 

levels of antibiotic-resistant bacteria. Cephalosporin-resistant 

E. coli were detected in all surface waters tested in Oakland 

County. 

Interpretation of the percentage of the population 

resistant to antibiotics in this study is limited by the inherent 

variability in laboratory results and the number of samples 

available for analysis. Therefore, actual concentration at a 

given site is speculative and should be used only as guidance 

when interpreting the results of the study. 

Despite being detected in previous studies, vancomycinresistant enterococci (VRE) were not detected at any Oakland County site during this study. Intermediate resistance 

to vancomycin was detected at two Clinton River Watershed 

study sites on one sampling date, but the resistant bacteria did 

not carry genetic elements that could be transferred to other 

organisms. The source of VRE detected in 2003 is uncertain, 

and it is possible that specific instances of VRE may occur in 

the watershed at times and/or places that were not reflected in 

the current sampling.
Summary and Conclusions 27

E. coli isolated in this study were tested for resistance to 

two cephalosporin antibiotics used primarily for treatment of 

human disease (cefoxitin and ceftriaxone). E. coli resistant to 

one or both of these antibiotics were detected at all study sites 

in Oakland County and sites outside of the county within the 

Clinton River Watershed. However, there was no indication 

that an increase in fecal indicator bacteria led to increased 

percentages of antibiotic-resistant bacteria.

The percentage of cefoxitin- and ceftriaxone-resistant 

bacteria varied throughout the year at each site in this study. 

Although analytical variability makes it difficult to interpret 

the magnitude of variation in the proportion of antibiotic-resistant isolates in environmental samples, resistant E. coli were 

persistently present at the more urbanized sites but were less 

common at the less urbanized sites, both within in Oakland 

County and throughout the Clinton River Watershed. This pattern indicates the source of this resistance may be a result of 

human or urban impacts such as wastewater from wastewatertreatment plants or failed septic systems, urban runoff, industrial discharges, urban animal populations (domestic animals 

and birds), and so forth. 

Rainfall and seasonal patterns were examined to determine whether the percentage of antibiotic-resistance bacteria 

is a result of runoff or stormwater entering the stream or 

whether a level of resistance is fairly constant at a site at low 

flows but is changed by dilution from increased water inputs. 

The Clinton River Watershed results suggest a possible seasonal or rainfall-related effect on the cefoxitin and ceftriaxone 

resistance due to the dominant presence on only one sampling 

date (August for cefoxitin and April for ceftriaxone). However, 

this general pattern was not observed in the more intensively 

monitored Oakland County sites, where differences in the 

percentage of resistant isolates were site specific. 

Results of this study indicate that a more intensive, 

site-specific study would be required to address the source 

or sources of antibiotic-resistant bacteria in Oakland County 

streams. It cannot be determined from the current data whether 

antibiotic-resistant isolates found during the current study 

entered water bodies directly from wastewater discharge or 

acquired resistance after being exposed to chemicals or metals 

in the water or sediment. 

The only two sites with detectable antibiotic concentrations were the Clinton River at Auburn Hills, where several 

antibiotics were consistently detected, and Paint Creek at 

Rochester, at which only one antibiotic (tylosin) was detected 

on one sampling date at a very low concentration. Samples 

that contained detectable concentrations of antibiotics were 

not necessarily correlated with antibiotic-resistant E. coli. 

Although the percentage of cephalosporin-resistant E. coli 

was more consistent at Clinton River at Auburn Hills than at 

the Kearsley Creek, Sashabaw Creek, Paint Creek, and Stony 

Creek sites, the percentage was no greater or more consistent 

than for samples collected from the other urbanized locations 

(River Rouge and Evans Ditch). There was no strong evidence 

that antibiotics detected in the environment are related to 

cephalosporin, erythromycin, or ofloxacin-resistant E. coli in 

the same environment. 

Mercury was detected in samples from every site. Concentrations of total mercury ranged from a low of 0.34 ng/L at 

Kearsley Creek to a high of 19.8 ng/L at Evans Ditch. Mercury concentrations were greater than the Michigan Rule 57 

water-quality standard of 1.3 ng/L at two sites, Clinton River 

at Auburn Hills and Evans Ditch at Southfield. The highest 

percentage of cefoxitin-resistant E. coli occurred in the same 

sample as the highest detectable concentration of mercury 

(Evans Ditch at Southfield, June 28, 2006). Although some of 

the highest mercury concentrations were detected in samples 

with the highest percentage of cephalosporin-resistant E. coli,

there was no linear relation of mercury concentration to percentage of antibiotic-resistant E. coli. 

The absence of a relation between mercury and antibiotic 

concentrations in the water indicates that exposure to mercury 

and antibiotics in the water column may not be responsible for 

cephalosporin resistance in these samples. However, bacteria, 

metals, and antibiotics may co-occur in source material (such 

as a wastewater-treatment effluent) and encourage the transfer 

of resistance genes among organisms. Because the fate and 

transport mechanisms of bacteria, metals, and organics may 

vary, these varied constituents may be differentially dispersed 

when they enter the stream, which may explain the absence 

of observable relation between the percentage of antibioticresistant organisms and mercury or antibiotic concentration for 

samples collected in this study. 

The detection of genes that are part of the class I integron suggests that antibiotic resistances in bacteria in the 

environment may be transferred to other bacteria, including 

pathogens. Class I integron structures may carry and transfer 

multiple resistance genes from one organism to another. These 

structures were present in most samples, indicating that they 

may play a role in the transfer and maintenance of resistance 

in bacteria isolated from the environment. These structures 

would need to be further characterized to determine which 

resistance genes are present and how the environment would 

aid in the transfer of resistance among bacteria. Specifically, 

a better understanding on the relation between the presence of 

integrons and other organic contaminants or metals in both the 

water column and sediment would help to determine whether 

antibiotic resistance was being acquired by organisms as a 

result of contamination in the environment. 

Current results do not identify a source of high fecal bacteria or antibiotic-resistant bacteria concentration in Oakland 

County. There were indications that human activities may, in 

part, explain the detection of antibiotic resistance because of 

the more persistent detections in urbanized areas. In addition, 

a gene that has been used to identify human fecal pollution 

was detected at sites in the county, suggesting that the sites 

were being affected by human fecal waste. Nevertheless, effluent from a wastewater-treatment plant did not seem to increase 

the prevalence of antibiotic-resistant E. coli downstream compared to similar sites not directly downstream from a known 

wastewater input. 
28 Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters, Oakland County, Mich.

Studies by the National Antimicrobial Resistance Monitoring System (NARMS) have detected very few human pathogenic E. coli isolates with resistance to either cefoxitin or ceftriaxone. Despite being approved only for human medicine, a 

greater percentage of E. coli isolated from veterinary samples 

were found to be resistant to either cefoxitin or ceftriaxone 

than in the human pathogenic E. coli study. It is possible that 

other environmental or natural factors may result in the presence of cephalosporin-resistant bacteria in the environment. 

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