U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2005-5058

Preliminary Survey of Antibiotic-Resistant Fecal Indicator 

Bacteria and Pathogenic Escherichia coli from River-Water 

Samples Collected in Oakland County, Mich., 2003

In cooperation with Oakland County, Michigan
Preliminary Survey of Antibiotic-Resistant 

Fecal Indicator Bacteria and Pathogenic 

Escherichia coli from River-Water Samples 

Collected in Oakland County, Mich., 2003 

By Lisa R. Fogarty, Joseph W. Duris, and Stephen S. Aichele

In cooperation with Oakland County, Michigan

Scientific Investigations Report 2005-5058

U.S. Department of the Interior

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

Gale A. Norton, Secretary

U.S. Geological Survey

P. Patrick Leahy, Acting Director

U.S. Geological Survey, Reston, Virginia: 2005

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Suggested citation:

Fogarty, L.R., Duris, J.W., Aichele, S.S., 2005, Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and 

Pathogenic Escherichia coli from River-Water Samples Collected in Oakland County, Mich., 2003: U.S. Geological 

Survey Scientific Investigations Report 2005-5058, 34 p.
CONTENTS

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

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

Purpose and scope ................................................................................. 2

Description of study area ........................................................................ 2

Background ............................................................................................. 3

Bacteria groups studied and their relation to pathogens ................................. 3

Fecal coliform bacteria and Escherichia coli .......................................... 3

Enterococci ................................................................................. 4

Overview of antibiotic resistance in bacteria ................................................ 4

Intrinsic versus acquired resistance ................................................... 4

Environmental versus clinical bacteria antibiotic resistance ..................... 5

Clinically significant antibiotic resistance ................................................... 5

Extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae ...... 6

Vancomycin-resistant enterococci (VRE) ............................................. 6

Using antibiotic resistances to determine sources of fecal contamination ............ 6

Methods and approach ................................................................................. 7

Sample collection ................................................................................. 7

Quantification of fecal indicator bacteria ................................................... 7

Determination of antibiotic resistance ...................................................... 7

Determination of the presence of antibiotic-resistant E. coli ........................... 8

Determination of the presence of vancomycin-resistant enterococci .................. 8

Determination of the presence of antibiotic-resistance genetic elements ............... 8

Determination of the presence of pathogenic E. coli ....................................... 9

Results of bacterial analyses ........................................................................ 9

Fecal indicator bacteria enumeration ......................................................... 10

Antibiotic resistances ........................................................................... 12

Antibiotic-resistant fecal coliform bacteria .......................................... 12

Antibiotic-resistant E. coli ............................................................... 14

Antibiotic-resistant enterococci ......................................................... 15

Potential sources of resistance ............................................................ 17

Pathogenic bacteria .............................................................................. 18

Summary and conclusions ........................................................................... 21

Suggestions for future studies ........................................................................ 22

Acknowledgments ....................................................................................... 23

References ................................................................................................ 23

Glossary ................................................................................................... 26

Appendix A: Fecal indicator bacteria concentrations in river-water samples collected 

from Oakland County, Mich. in August and September 2003. ........................ 27

Appendix B: Field measurements at sites in Oakland County, Mich., in August 

 and September 2003. ........................................................................... 29

iii
Appendix C: Number of fecal coliform and enterococci isolates resistant to tested 

 antibiotics that were isolated in August 2003 from samples collected in 

 Oakland County, Mich. ..................................................................... 31

Appendix D: Percentage of fecal coliform and enterococci isolates resistant to tested 

 antibiotics that were isolated in September 2003 from samples collected in 

 Oakland County, Mich. ..................................................................... 33

FIGURES

1. Map showing locations of Oakland County, Mich., study sites ........................... 2

2−4. Graph showing: 

 2. Escherichia coli concentrations for Oakland County and reference samples 

 collected in August and September 2003. .......................................... 11

 3. Fecal coliform concentrations for Oakland County and reference samples 

 collected in August and September 2003. .......................................... 11

 4. Enterococci concentrations for Oakland County and reference samples 

 collected in August and September 2003. .......................................... 12

5−6. Map showing Oakland County, Mich., sites that contain: 

5. Fecal coliform bacteria with antibiotic resistance profiles indicative of 

 extended-spectrum-β-lactamases (ESBLs) [cephalothin and ceftriaxone; 

cephalothin, ceftriaxone, and ampicillin; cephalothin, ceftriaxone, 

 ampicillin, and one or more non-β-lactam antibiotics (tetracycline, 

 streptomycin, and/or gentamicin)] ................................................... 15

6. Positive results for the following: IA, GA and rfb0157 for the detection of 

E. coli O157; and genes (eaeA, stx1, and stx2) responsible for 

 pathogenicity .............................................................................. 20

7−8. Photographs showing polymerase chain reaction detection of toxin genes 

 (eaeA, stx1, and stx2) and an Escherichia coli (E. coli) specific gene, in 

 samples collected:

7. August 2003 in Oakland County watersheds, Mich. ................................. 20

8. September 2003 in Oakland County watersheds, Mich. .............................. 21

TABLES

1. Sample-collection sites .............................................................................. 3

iv
2. Selected data from the National Antimicrobial Resistance Monitoring System 

(NARMS): Percentage of antibiotic-resistant E. coli O157:H7 from human 

 clinical samples and E. coli from diagnostic veterinary, dairy cattle, fruit 

 and vegetables, and environmental samples ............................................. 6

3. Primers and polymerase chain reaction conditions for integrons and vancomycin 

 resistance genes ................................................................................. 8

4. Primers and polymerase chain reaction (PCR) conditions for the rfb0157 gene, 

 and multiplex PCR for the virulence genes ............................................. 10

5. Percentage of fecal coliform bacteria resistant to multiple antibiotics in 

 September 2003 ................................................................................. 13

6. Resistances of individual fecal coliform bacteria to cephalosporin antibiotics, 

 September 2003 ................................................................................. 14

7. Escherichia coli antibiotic-resistance profiles and integron-detection results ............ 16

8. Percentage of enterococci isolates resistant to tested antibiotics ........................... 17

9. Results of pathogenic Escherichia coli tests ................................................... 19

Conversion Factors and Abbreviated Water-Quality Units

Multiply By To obtain

square mile (mi2) 2.590 square kilometer (km2)

gallon (gal) 3.785 liter (L)

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) 1000 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

Concentration of bacteria in water is given in colony forming units per 100 milliliters (CFU/100 mL).

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

v

Preliminary Survey of Antibiotic-Resistant Fecal Indicator 

Bacteria and Pathogenic Escherichia coli from RiverWater Samples Collected in Oakland County, Mich., 2003 

By Lisa R. Fogarty, Joseph W. Duris, and Stephen S. Aichele

Abstract

A preliminary study was done in Oakland County, Michigan, to determine the concentration of fecal indicator bacteria 

(fecal coliform bacteria and enterococci), antibiotic resistance 

patterns of these two groups, and the presence of potentially 

pathogenic Escherichia coli (E. coli). For selected sites, specific members of these groups [E. coli, Enterococcus faecium

(E. faecium) and Enterococcus faecalis (E. faecalis)] were isolated and tested for levels of resistance to specific antibiotics 

used to treat human infections by pathogens in these groups 

and for their potential to transfer these resistances. In addition, water samples from all sites were tested for indicators of 

potentially pathogenic E. coli by three assays: a growth-based 

assay for sorbitol-negative E. coli, an immunological assay for 

E. coli O157, and a molecular assay for three virulence and 

two serotype genes. Samples were also collected from two 

non-urbanized sites outside of Oakland County. Results from 

the urbanized Oakland County area were compared to those 

from these two non-urbanized sites. 

Fecal indicator bacteria concentrations exceeded State of 

Michigan recreational water-quality standards and (or) recommended U.S. Environmental Protection Agency (USEPA) 

standards in samples from all but two Oakland County sites. 

Multiple-antibiotic-resistant fecal coliform bacteria were 

found at all sites, including two reference sites from outside 

the county. Two sites (Stony Creek and Paint Creek) yielded 

fecal coliform isolates resistant to all tested antibiotics. Patterns indicative of extended-spectrum-β-lactamase (ESBL)-

producing fecal coliform bacteria were found at eight sites in 

Oakland County and E. coli resistant to clinically significant 

antibiotics were recovered from the River Rouge, Clinton 

River, and Paint Creek. Vancomycin-resistant presumptive 

enterococci were found at six sites in Oakland County and 

were not found at the reference sites. Evidence of acquired 

antibiotic resistances was detected in bacteria from multiple 

sites in Oakland County but not detected in bacteria from the 

reference sites. Integrons capable of transferring resistance 

were detected in isolates from the River Rouge and Clinton 

River. E. faecium and E. faecalis identified in samples collected from Kearsley Creek and Evans Ditch were resistant 

to high levels of vancomycin and carried transferable genes 

responsible for resistance. 

Several sites in Oakland County had indicators of pathogenic E. coli in August and (or) September 2003. Two samples 

from the Clinton River in August tested positive for all three 

E. coli O157 tests. Both the August and September samples 

from one River Rouge site were positive for the immunological and molecular assay for E. coli O157. A combination of 

virulence genes commonly associated with human illness was 

detected at five sites in August and seven sites in September. 

Antibiotic-resistance profiles of clinical concern along with 

genes capable of transferring the resistance were found at 

several sites throughout Oakland County; samples from many 

of these sites also contained potentially pathogenic E. coli.

Introduction

Many rivers and streams in Michigan are affected by 

fecal contamination (Harrison, 2003). Pathogenic bacteria 

associated with fecal contamination can cause mild to serious 

illness. Pathogenic bacteria1 that may be associated with 

fecal contamination include: pathogenic strains of Escherichia 

coli (E. coli), Campylobacter, Salmonella species, Shigella, 

and Enterococcus species. In addition to these organisms 

causing human illnesses, resistance to antibiotics has made 

treatment more difficult. 

1 Terms defined in the glossary are in bold print where first used in the main body of the report.
As part of a larger study to assess the quality and quantity of waters in Oakland County, the U.S. Geological Survey 

(USGS), in cooperation with the Oakland County Department 

of Human Services, collaborated on a study to evaluate the 

microbiological quality of waters within the county. 

Purpose and scope

The purpose of this report is to describe the results of a 

preliminary study on the presence of antibiotic-resistant fecal 

indicator bacteria and potentially pathogenic E. coli in waters 

collected in Oakland County, Mich. This report includes 

(1) concentrations of fecal indicator bacteria (fecal coliform 

bacteria, E. coli, and enterococci) in river-water samples collected in August and September 2003, (2) fecal coliform and 

enterococci antibiotic resistances to selected antibiotics, (3) 

detection of transferable genetic elements (integrons and vancomycin resistance genes), and (4) indications of the presence 

of presumptive pathogenic E. coli. This report also includes a 

comparison to results obtained from two reference sites outside of Oakland County that represent very different land uses. 

Description of study area

Oakland County, Michigan, includes 910 mi2 in southeastern Michigan. The county is largely suburban, with a 

population of approximately 1.2 million in 2000. Oakland 

County is in the headwaters of five major rivers: the Shiawassee and Flint Rivers draining north towards Saginaw Bay, 

and the Clinton Rivers draining east towards Lake St. Clair, 

and the Huron and Rouge draining south towards Lake Erie. 

Developed land ranges from approximately 30 percent to 

almost 100 percent in some watersheds. Agricultural land use 

is typically less than 20 percent of total watershed area, and no 

known confined-feeding or other large-scale livestock producers operate in the county. Study sites include 14 sites in watersheds draining Oakland County (fig. 1, table 1); 13 sites were 

within the county, and 1 site was just outside of the county in 

the Clinton River watershed. These sites will be referred to as 

“Oakland County sites” hereafter. In addition to the Oakland 

County sites, two reference sites were also included: the Au 

Sable River in a national forest and Washington Creek in Isle 

Royale, Mich. (table 1).

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.

Oakland

County

Michigan

0 3 6 KILOMETERS

0 3 6 MILES

Watershed boundary

Township 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

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Holly Groveland Brandon Oxford Addison

Rose Springfield Independence Orion Oakland

Highland White Lake Waterford

Avon

Milford

Commerce West Bloomfield

Bloomfield Troy

Lyon Novi Farmington Southfield

Royal Oak

Pontiac

Figure 1. Locations of Oakland County, Mich., study sites. Map numbers correspond to U.S. Geological Survey station numbers in table 1.

2 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Table 1. Sample-collection sites. 

Background

Bacteria groups studied and their relation to 

pathogens 

Because pathogens are often difficult to detect in the 

environment, fecal indicator bacteria are commonly used to 

measure the sanitary quality of water (Rose and Grimes, 2001; 

U.S. Environmental Protection Agency, 1986, 2000). This 

study focuses on the fecal indicators fecal coliform bacteria, 

including E. coli, and enterococci. 

Fecal coliform bacteria and Escherichia coli

Fecal coliform bacteria are a group of gram negative 

bacteria that ferment lactose at 44.5 degrees Celsius. They are 

usually present in high concentration in feces from humans 

and other warmblooded animals. Many fecal coliform bacteria are members of the Enterobacteriaceae family composed 

of many pathogens and nonpathogens, which are genetically 

similar and capable of exchanging genes. E. coli is a member 

of both the Enterobacteriaceae and fecal coliform bacteria 

groups. E. coli has been recommended by the U. S. Environmental Protection Agency (USEPA, 2000) as an indicator of 

sanitary water quality for surface waters. E. coli is part of the 

natural human intestinal flora, and most strains do not cause 

human illness. Certain pathogenic strains of E. coli, however, 

are capable of causing mild to serious human illnesses. 

Pathogenic E. coli (in particular the serotype E. coli

O157:H7) have been identified as the causative agents in 

several recreational waterborne-disease outbreaks (Lee and 

others, 2002). Most pathogenic E. coli belong to one of four 

major groups: enterhemorrhagic E. coli (EHEC), which 

include shiga toxin-producing E. coli (STEC); enterotoxigenic E. coli (ETEC); enteropathogenic E. coli (EPEC); and 

enteroinvasive E. coli (EIEC) (Nataro and Kaper, 1998). The 

most commonly seen pathogenic E. coli strains isolated in the 

United States belong to the EHEC/STEC group (Bopp and 

others, 2003). EHEC/STEC are defined by the severity of the 

disease they cause, and they commonly share similar genes 

responsible for their virulence. These genes include the intimin gene (eaeA), which is associated with close attachment of 

E. coli to intestinal cells, and the shiga toxin producing genes 

(stx1 and stx2) responsible for cell destruction. STEC strains 

usually have the eaeA gene and, in addition, carry one or both 

of the shiga toxin genes (stx1 and stx2). Recent findings suggest that strains of E. coli O157 with eaeA and stx2 are more 

virulent than those strains with only stx1 (Boerlin and others,

Map number USGS station number Station name County Watershed

1 04148035 Kearsley Creek at Mill Street at Ortonville, Mich. Oakland Flint

2 04143830 Shiawassee River at Holly, Mich. Oakland Shiawassee

3 04170000 Huron River at Milford, Mich. Oakland Huron

4 04170500 Huron River near New Hudson, Mich. Oakland Huron

5 04160800 Sashabaw Creek near Drayton Plains, Mich. Oakland Clinton

6 04160900 Clinton River near Drayton Plains, Mich. Oakland Clinton

7 04161000 Clinton River at Auburn Hills, Mich. Oakland Clinton

8 04161540 Paint Creek at Rochester, Mich. Oakland Clinton

9 04161580 Stony Creek near Romeo, Mich. Macomb Clinton

10 04161810 Clinton River at Yates, Mich. Oakland Clinton

11 04166000 River Rouge at Birmingham, Mich. Oakland Rouge

12 04166315 Upper River Rouge at Clarenceville, Mich. Oakland Rouge

13 04166100 River Rouge at Southfield, Mich. Oakland Rouge

14 04166200 Evans Ditch at Southfield, Mich. Oakland Rouge

Reference sites

04135500 Au Sable River near Grayling, Mich. Crawford Au Sable

04001000 Washington Creek at Windigo, Mich. (Isle Royale) Keweenaw Lake Superior

[USGS, U.S. Geological Survey]

Background 3
1999). More than 200 STEC serotypes have been isolated from 

persons with diarrhea and (or) hemolytic-uremic syndrome. 

In the United States, E. coli O157:H7 is the most frequently 

isolated STEC in clinical samples. However, increasingly, 

other non-O157 STEC strains are identified in outbreaks and 

sporadic illness (Bopp and others, 2003). STEC and EHEC 

strains have been found in fecal samples from humans with 

severe disease, farm animals, bird species (gulls, chickens, turkeys, and pigeons), and sewage from human and animal waste 

(Nataro and Kaper, 1998).

Isolating and identifying E. coli O157:H7 and other 

pathogenic organisms from environmental waters is often 

difficult because of the low expected concentrations of the 

pathogen and the high concentrations of other bacteria commonly found in environmental waters. Surface waters contain 

a wide range of environmental bacteria, many of which have 

not been identified. When water also contains fecal contamination, a very diverse set of bacteria, some similar to the target 

bacterium, may be present to interfere with assays. Most 

diagnostic techniques have been developed for clinical diagnosis of disease and for detection of contamination in food. 

These samples contain a limited and usually a known range of 

other bacteria species. As a result, when one uses techniques 

developed for food or clinical diagnosis, the limitations of the 

approach and the influence of environmental bacteria must be 

considered before interpreting the results. 

Commonly used methods for identifying STEC strains 

are based on the growth characteristics of STEC organisms 

on selective media, the presence of antigens specific to the 

strain, and the detection of genes responsible for virulence and 

pathogenicity. To isolate E. coli O157, cefixime and tellurite 

are added to sorbitol MacConkey growth medium (CT-SMAC) 

(Bopp and others, 2003). E. coli O157 ferments the sorbitol 

in this medium much more slowly than other E. coli, resulting in a colorless colony in comparison to pink colonies for 

those isolates that ferment sorbitol much more efficiently. The 

cefixime and tellurite are added to inhibit the growth of many 

fecal bacteria, decreasing the amount of background growth. 

However, there are still fecal coliform bacterial species other 

than E. coli O157 that can produce a similar non-sorbitol-fermenting CT-SMAC result. These bacteria include: Hafnia and 

Shigella (Zadik and others, 1993; Fujisaw and others, 2000).

Immunological assays are used to identify specific antigens present on E. coli O157. Commercial tests are available 

that use antibodies designed for the detection of E. coli O157 

(for example, the E. coli O157:H7 8 hour Reveal test; Neogen, 

Lansing, Mich.). This is a presence/absence test and is not 

quantifiable. This kit was designed for use in the food industry, and previous studies have shown other non-E. coli O157 

bacteria are capable of cross-reacting with the O157 antibodies 

used, specifically, Citrobacter freundii, Yersinia enterocolitica, 

Pseudomonas maltophilia, Brucella abortus, Escherichia 

hermannii, Hafnia alvei, Morganella morganii, and the Salmonella group N (Power and others, 2000). As a confirmatory 

test, molecular methods can be used to identify a gene specific 

for E. coli O157 (rfbO157). To identify samples that may

contain STEC, polymerase chain reaction (PCR) is used to 

detect genes commonly associated with STEC (eaeA, stx1, 

and stx2) that are responsible for virulence.

Enterococci

Enterococci are a group of gram-positive bacteria commonly associated with fecal material from mammals and 

birds. They are also found in the soil and associated with 

plant material. Although they are common members of the 

human bacterial flora, they are opportunistic pathogens that 

can cause disease particularly in elderly and immunocompromised patients (Aarestrup and others, 2002; Malani and others, 

2002). Enterococci have been identified as the second leading 

cause of nosocomial urinary tract and wound infections and 

the third leading cause of nosocomial bacteremia in the United 

States (Teixeria and Facklam, 2003). Enterococcus faecalis (E. 

faecalis) and Enterococcus faecium (E. faecium) account for 

more than 95 percent of clinical enterococci isolates (Teixeira 

and Facklam, 2003). The other enterococci species are less 

frequently identified in human infections. 

Overview of antibiotic resistance in bacteria 

Antibiotics are used in human and veterinary medicine, 

animal husbandry, and in the treatment of agricultural crops. 

Recent studies have shown the presence of antibiotics in 

waters across the United States, but in very low concentrations 

(Kolpin and others, 2002). Antibiotics and antibiotic-resistant 

bacteria may enter the environment in discharge from wastewater-treatment plants, pharmaceutical companies, or hospital 

waste; runoff from animal-feeding operations or land manure 

application; infiltration from agricultural application; or from 

landfill leachate. Antibiotic resistance is of increasing concern 

because of the emergence of multiple antibiotic resistant

pathogens, such as multiple-drug-resistant E. coli and Salmonella spp. (Walsh, 2003).

Intrinsic versus acquired resistance

Bacterial resistance to antibiotics can be either intrinsic

or acquired. Intrinsic antibiotic resistance is due to an inherent feature of the organism that prevents the antibiotic mode of 

action. This type of resistance is determined by chromosomal 

genes and is not due to the acquisition of new genes. For 

example, most fecal coliform bacteria are intrinsically resistant 

to vancomycin because their cellular structure consists of an 

outer membrane that prevents the vancomycin molecule from 

attacking its target (Rice and others, 2003). Intrinsic resistance is not transferable to other bacteria. Acquired antibiotic 

resistance may be transferred to other organisms. Resistance 

is acquired through a mutation in the chromosomal genes or 

through the acquisition of new genes responsible for antibiotic 

resistance. Genes responsible for acquired antibiotic resistance 

are often carried on genetic elements that can easily be trans4 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
ferred among bacteria (plasmids, transposons, and integrons; 

Roy, 1999). In many cases these genetic elements may carry 

several antibiotic resistance genes, thus transferring multiple 

antibiotic resistances to other organisms. The genetic elements 

can pick up new genes or lose genes as they transfer between 

organisms, and they are responsible for widespread antibiotic 

resistance in clinical isolates. Additionally, they have been 

frequently found in environmental bacterial isolates (Ash and 

others, 2002; Park and others, 2003; Roe and others, 2003). In 

some cases these genetic elements may also carry genes for 

resistance to metals or other toxic compounds (Ug and Ceylan, 

2003; Bass and others, 1999). Therefore, the transfer of these 

genetic elements under selective pressure of antibiotics, metals, or toxic chemicals may result in the transfer of several 

types of resistance genes. 

Without species identification, determining whether 

antibiotic resistance is acquired or intrinsic can be difficult. 

Several factors can indicate that the resistance is acquired. 

The most obvious is the presence of transferable genetic 

elements that are transferred among organisms. There are 

also antibiotic resistance patterns that are more indicative of 

acquired resistance. Both of these factors are described in 

more detail throughout this report. Antibiotic-resistant fecal 

coliform bacteria and enterococci in surface waters may pose a 

threat to human health because of the potential for transfer of 

antibiotic-resistance genes to pathogens.

Environmental versus clinical bacteria antibiotic 

resistance

Much of our knowledge of antibiotic resistance comes 

from the clinical setting in which antibiotic-resistant pathogens have made treatment very difficult. Only a small selection of antibiotics are actually used to treat human infections, 

and pathogens that are resistant to these select few antibiotics 

are very difficult to treat. Antibiotic-resistant bacteria have 

previously been isolated from surface waters (McArthur and 

Tuckfield, 2000; Ash and others, 2002; Roe and others, 2003; 

Yang and Carlson, 2003) but few studies have investigated the 

environmental occurrence of clinically significant antibiotic 

resistances such as resistance to second and third generation 

cephalosporins or vancomycin. In a study by Roe and others 

(2003), E. coli resistant to ceftriaxone, cephalothin, ampicillin, gentamicin, and streptomycin were isolated from the Rio 

Grande; of these, 32 percent were resistant to multiple antibiotics. In addition, integrons−genetic elements that can transfer 

antibiotic resistance genes to other organisms−were found 

in 13 percent of the E. coli isolates in that study. In another 

study by Ash and others (2002), antibiotic-resistant bacteria 

were isolated from 13 rivers across the United States at 22 

locations. All 22 sites sampled contained bacteria resistant to 

one or more cephalosporin antibiotics. Sixteen sites contained 

bacteria resistant to third-generation cephalosporins (ceftazidime and cefotaxime). In addition, 40 percent of the isolates 

resistant to ampicillin and at least one other tested antibiotic

harbored plasmids (genetic elements that may carry multiple 

antibiotic resistance genes). Most of the bacteria identified by 

Ash in this study were from the following genera: Acinetobacter, Alcalingenes, Citrobacter, Enterobacter, Pseudomonas, and Serratia. These findings suggest that bacteria resistant to antibiotics of clinical concern are present in surface 

waters; however, very little is known about the distribution 

of antibiotic-resistant bacteria in urban settings in the United 

States. Antibiotic-resistant fecal and nonfecal bacteria have 

been found in surface waters outside the United States. (GoñiUrriza and others, 2000; Reinthaler and others, 2003; Park and 

others, 2003). 

Because many organisms typically found in surface water 

have intrinsic antibiotic resistances (Farmer, 2003) or may not 

be pathogens, identification of the resistant bacteria is required 

to interpret the health significance. Pathogenic bacteria with 

resistance to an antibiotic used to treat the infection caused 

by that organism are an obvious concern. Less obvious is 

the significance of environmental (nonpathogenic) bacteria 

with antibiotic resistances. Of most concern is the ability of 

environmental bacteria to transfer antibiotic resistance genes 

to human pathogens. Therefore, identification of transferable 

genetic elements also is necessary. 

In a collaborative effort of numerous federal and state 

governmental agencies including the Food and Drug Administration (FDA), U.S. Department of Agriculture (USDA), 

and the Centers for Disease Control and Prevention (CDC), 

the National Antimicrobial Resistance Monitoring System 

(NARMS) has been established to track antibiotic-resistance patterns of enteric and foodborne pathogens (National 

Antimicrobial Resistance Monitoring System, 2005). Enteric 

pathogens including E. coli and Salmonella isolated from 

human and veterinary clinical samples, healthy farm animals, 

and raw meat from food animals are tested for resistance to 

various antibiotics including (but not limited to) ampicillin, 

cefoxitin, ceftiofur, ceftriaxone, cephalothin, gentamicin, 

streptomycin, and tetracycline. A select group of these results 

taken from CDC, (2003) and USDA (2004) is listed in table 2. 

Analysis by the CDC was done only for E. coli serotype O157:

H7 human clinical isolates, whereas data collected by the 

USDA for nonhuman samples are for E. coli in general. These 

isolates were not identified to the serotype level. The results of 

the NARMS studies provide a framework to compare E. coli

antibiotic-resistance profiles from this study to antibiotic-resistance profiles of E. coli from clinical and animal sources.

Clinically significant antibiotic resistance

Most of the antibiotics chosen for this study represent 

antibiotic resistances that are of concern for each respective 

bacterial group. Not all antibiotics are used to treat infections 

by all bacterial species. Therefore, this study focuses on two 

major antibiotic resistances that have become a major health 

concern for the two groups of bacteria included in this study: 

(1) extended-spectrum-β-lactamase-producing Enterobacteriaceae and (2) vancomycin-resistant Enterococcus. 

Background 5
Extended-spectrum-β-lactamase (ESBL)-

producing Enterobacteriaceae

Of increasing concern in the clinical setting are Enterobacteriaceae that produce extended-spectrum-β-lactamases 

(ESBL) (Rice, 2001; Diekema and others, 2004; McGowan 

and Tenover, 2004). ESBL-producing bacteria are typically 

resistant to all cephalosporins (except for cephamycins) and 

other β-lactam antibiotics such as the penicillins (ampicillin). 

ESBL-producing Enterobacteriaeceae are frequently resistant 

to other non-β-lactam antibiotics, such as the aminoglycocides 

and tetracylines, because plasmids that contain ESBL genes 

also carry other antibiotic-resistance genes, such as genes 

responsible for gentamicin, streptomycin, and/or tetracycline 

resistance (Nathisuwan and others, 2001). Being plasmidborne, these resistances can be transferred from one organism 

to another; such transfer has led to ESBL-producing pathogens 

including E. coli, Klebsiella, Pseudomonas, Enterobacter, and 

Salmonella species (Bradford, 2001; Pérez- Pérez and Hanson, 

2002; Philippon and others, 2002; Preston and other, 2003). 

Vancomycin-resistant enterococci (VRE)

In the United States, the number of reported vancomycin-resistant enterococci (VRE) from clinical samples has 

increased (Huycke and others, 1998; Cetinkaya and others, 

2000). Vancomycin is restricted to human medicine in the 

United States and the prevalence of VRE are rare outside the 

clinical setting (Cetinkaya and others, 2000); however, VRE 

have been found in hospital wastes (Harwood and others, 

2000). A few known species of enterococci are intrinsically 

resistant to vancomycin at levels less than 16 μg/mL (Kak

Antibiotic

CDC E. coli O157:H7 

human clinical 

isolates: 1996-2001a

(n=1,651)

USDA E. coli isolates-2002b

Diagnostic 

veterinary sources

(n=343)

Dairy cattle on farm

(n=1,389)

Fruit and vegetables

(n=736)

Environment

(n=130)

Ampicillin 2.0 53.90 21.0 3.5 80.0

Cefoxitin 0.4 20.10 0.4 1.0 0.0

Ceftriaxone 0.0 0.30 0.0 0.0 0.0

Cephalothin 1.4 28.30 4.0 1.9 2.3

Ceftiofur 1.0 13.40 0.6 0.7 0.0

Gentamicin 20.0 21.80 0.1 0.5 0.8

Streptomycin 3.0 28.50 4.0 4.9 0.8

Tetracycline 5.0 65.90 10.7 5.0 6.9

Table 2. Selected data from the National Antimicrobial Resistance Monitoring System (NARMS): Percentage of antibiotic-resistant E. coli

O157:H7 from human clinical samples and E. coli from diagnostic veterinary, dairy cattle, fruit and vegetables, and environmental samples.

[CDC, Centers for Disease Control; USDA, United States Department of Agriculture. E. coli; Escherichia coli; all data in percent]

and Chow, 2002). Higher levels of resistance to vancomycin 

(greater than 16 μg/mL) indicate acquired resistance, a result 

of vancomycin-resistance genes (vanA and vanB) carried on 

plasmids (Kak and Chow, 2002) that can be transferred to 

pathogenic Enterococcus or Staphylococcus species. 

Using antibiotic resistances to determine 

sources of fecal contamination

Recent studies have suggested using antibiotic-resistance 

patterns of fecal coliform bacteria and enterococci to determine sources of fecal contamination in surface waters (Wiggins and others, 1999; Harwood and others, 2000). This study 

was not designed to determine a source of fecal contamination; 

however, resistance patterns to antibiotics commonly used in 

particular practices may give some indication of the source of 

fecal contamination. Cephalosporin antibiotics are commonly 

used in human medicine but the first and second generation 

cephalosporin antibiotics are becoming more frequently used 

in veterinary medicine and livestock production. Ceftriaxone 

has remained primarily an antibiotic for human medicine. This 

premise is supported by Harwood and others (2000), in which 

they reported that fecal coliform bacteria resistant to the antibiotics cephalothin and ampicillin were most often associated 

with humans, whereas fecal coliform bacteria resistant to the 

antibiotics tetracycline and streptomycin were most commonly 

associated with animals. 

Enterococci resistant to low to intermediate levels of vancomycin have been reported to be more associated with animal 

feces than wastewater, most likely because of the greater 

prevalence of enterococci species with intrinsic resistance in 

animal feces (Harwood and others, 2000). Enterococci with 

a Data from CDC, 2003, National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS) 2001 annual report.

b Data from USDA, 2002 E. coli report, accessed January 2005 at http://www.arru.saa.ars.usda.gov/ECOLI/2002reports.htm. 

6 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
high levels of resistance to vancomycin, resulting from antibiotic-resistance genes carried on plasmids, are exceptionally 

rare in animal species in the United States (McDonald and 

others, 1997) where vancomycin has been restricted for human 

medicine.

Methods and Approach

To determine the sanitary water quality of the sampled 

streams, fecal coliform bacteria, E. coli, and enterococci were 

quantified, and resulting fecal coliform bacteria and enterococci isolates from those samples were tested for resistance to 

a selected group of antibiotics. Using molecular techniques, 

genetic elements responsible for antibiotic resistance were 

analyzed in E. coli and enterococci isolates. Three different 

methods were used to determine the presence of presumptive 

pathogenic E. coli for these sites. 

Sample collection

Water samples were collected August 5, 2003, as 1-L, 

grab samples from 14 streams in Oakland County, Mich. To 

determine whether results obtained in August were reproducible and to overcome difficulties with the methods applied in 

August, additional samples were collected September 8−9, 

2003, from these same sites. Reference samples were collected from Washington Creek (August 26, 2003) and the 

Au Sable River in Grayling, Mich. [August 4 (referred to as 

August sampling), and August 26 (referred to as the September sampling), 2003]. Station numbers and locations are listed 

in table 1. Samples collected in August were held on ice for 24 

hours before processing. Samples collected in September were 

processed immediately in the field for fecal indicator bacteria. 

Field parameters (water temperature, specific conductance, 

pH, dissolved oxygen, and stream discharge) and water-quality 

constituents were collected and measured at these same sites 

(Aichele and others, 2004).

Quantification of fecal indicator bacteria

Fecal coliform bacteria, E. coli, and enterococci were 

isolated from all samples by means of membrane filtration 

techniques (American Public Health Association 1998; U.S. 

Environmental Protection Agency, 2000). Sample volumes of 

100, 10, 1, and 0.1, mL and sterile buffered saline control were 

passed through individual sterile 0.45-μm-pore-size gridded 

cellulose nitrate membrane filters (Advantec MFS, Inc., Pleasanton, Calif.). Fecal coliform bacteria were identified on mFC 

agar LES medium (DIFCO Laboratories, Detroit, Mich.). For 

E. coli identification, membranes with 15−50 well-separated 

fecal coliform colonies were transferred to Nutrient Agar 

containing 4-methylumbelliferyl-β-D-glucuronide (NA-MUG; 

DIFCO). Colonies that fluoresced blue under UV light were 

identified presumptively as E. coli. Fecal coliform cultures 

on the 100-mL filter plate were preserved at -80°C in Luria 

Bertani broth and 20 percent glycerol and will be referred to 

hereafter as FC cultures. Enterococcus isolates were identified by means of membrane filtration on mEI agar as described 

by U.S. Environmental Protection Agency (2000). Colonies 

with blue halos were presumed enterococci. The mEI cultures 

were preserved at -80°C in tryptic soy broth and 20 percent 

glycerol and will be referred to as ENT cultures.

Determination of antibiotic resistance

For samples collected in August 2003 (after quantification), mFC filters that produced between 20 and 60 colonies 

were tested for clinically significant antibiotic resistance by 

means of replica-plating techniques (Carlton and Brown, 

1981). Antibiotic media were made by adding one of the 

following antibiotics to Mueller-Hinton (MH) agar in concentrations suggested by the National Committee for Clinical 

Laboratory Standards (NCCLS, 2002): cefoxitin (32 μg/mL), 

cephalothin (32 μg/mL), ceftriaxone (64 μg/mL), ampicillin 

(32 μg/mL), tetracycline (8 μg/mL), streptomycin (64 μg/mL), 

and gentamicin (16 μg/mL). These antibiotics were chosen to 

help define resistance that may be a threat to human health and 

also help to determine whether the sources of bacterial populations were different among the different sites. The colonies 

from the mFC plate, which included both fecal coliform and 

non-fecal-coliform colonies, were transferred on sterile velvet 

cloth to each of the MH-plus-antibiotic plates. Resistant colonies were not confirmed to be fecal coliform bacteria; rather, 

they were described as capable of growing at 44.5oC on mFC 

media and therefore might include non-fecal-coliform bacteria. Some plates were very overgrown, and colonies could not 

be differentiated. In these cases, results were reported as positive or negative resistance for that sample rather than a number 

of resistant colonies. Similarly, enterococci were transferred 

by replica-plating to MH agar with one of the following 

antibiotics in concentrations suggested by the NCCLS (2002): 

vancomycin (8 μg/mL), vancomycin (16 μg/mL), gentamicin 

(100 μg/mL), and streptomycin (1,000 μg/mL), and tetracycline (16 μg/mL). Again, these antibiotics represent clinically 

significant antibiotic resistances or resistances that may differ 

with sources of contamination. The plates were incubated 

for 18−24 hours at 37oC. Growth was recorded as a resistant 

colony. 

Because of the amount of overgrowth seen on the antibiotic plates from August, replica-plating was abandoned; 

instead, fecal coliform and enterococci colonies from the September sampling were picked individually and transferred with 

a sterile toothpick to each of the MH-plus-antibiotic plates 

(described above). Tetracycline was omitted for September 

enterococci isolates. For plates with 50 or more colonies, only 

50 representative colonies were picked. For plates with fewer 

than 50 colonies, all colonies were picked and transferred.

For antibiotic-resistance tests, control bacterial strains 

were tested alongside study samples as recommended by the 

NCCLS (2002). For fecal coliform bacteria analyses, E. coli

Methods and Approach 7
bile esculin agar (BEA), (3) growth in brain heart infusion 

broth containing 6.5 percent NaCl, (4) negative catalase 

reaction, (5) gram positive reaction, and (6) cell morphology. 

To better understand the threat to human health, presumptive 

enterococci were then identified to the species level by 

use of BioMérieux Strep 20 API test strips (BioMérieux; 

Hazelwood, Mo.) which test a series of biochemical reactions. 

ATCC control strain E. faecalis 29212 was used as a positive 

control tested alongside study isolates. Levels of resistance 

to vancomycin were determined for confirmed enterococci 

isolates that were vancomycin resistant at 16 μg/mL (four 

isolates from site 04148035 in September, six isolates 

from site 04161540 in September, and one isolate from site 

04166200 in August) by use of a microdilution technique 

established by the NCCLS. Again, E. faecalis 29212 (ATCC) 

was used a bacterial control for vancomycin tests. 

Determination of the presence of antibioticresistance genetic elements

By means of polymerase chain reaction (PCR), phenotypically confirmed E. coli were tested for the presence of 

integrons; protocols used were similar to those described by 

Lévesque and others, (1995). A 1-μL loop of overnight culture 

was suspended in 100 μL of phosphate-buffered saline, and 

1μL of this suspension was added to the PCR reaction. The 

PCR reaction consisted of a final concentration of 1X PCR 

buffer, 1.5 mM, MgCl2, 0.2 mM dNTPs, 0.2 μg/μL, bovine 

serum albumin (BSA), 0.2 μM of each primer (table 3), and 

1.25 units Taq polymerase (Promega, Madison, Wis.) in a final 

volume of 25 μL. A negative control was run with each reaction by omitting the addition of DNA to the reaction mix. 

DNA was amplified using a PerkinElmer GeneAmp PCR 

System 2400 Thermal Cycler with the conditions listed in 

table 3. The amplified PCR product was visualized on a 1.5-

percent agarose ethidium bromide stained gel under UV light. 

25922 (American Type Culture Collection, ATCC) was tested 

for sensitivity to cephalothin, cefoxitin, ceftriaxone, gentamicin, streptomycin, and tetracycline. For enterococci analyses, 

E. faecalis 29212 (ATCC) was tested for sensitivity to vancomycin, gentamicin, streptomycin, and tetracycline. 

Determination of the presence of antibioticresistant E. coli

FC cultures which were successfully obtained from September samples were screened for isolates that would grow on 

MH medium with one of the cephalosporin antibiotics (cephalothin, cefoxitin, and ceftriaxone). Resistant isolates were then 

grown on mFC and further tested on NA-MUG to identify 

presumptive E. coli. Presumptive E. coli were confirmed by a 

negative oxidase reaction, a positive indol reaction, a positive 

o-nitrophenyl-β-D-galactopyranoside (ONPG) reaction, and 

a gram-negative morphology (Holt and others, 1994). ATCC 

control strain E. coli 25922 was used as a positive control for 

each reaction. Intermediate and high-level antibiotic resistance to cephalothin, cefoxitin, ceftriaxone, gentamicin, and 

ampicillin was determined for confirmed E. coli by means of 

microdilutions (National Committee for Clinical Laboratory 

Standards, 2002). Again, E. coli 25922 (ATCC) was used as a 

bacterial control for antibiotic resistance tests. 

Determination of the presence of vancomycinresistant enterococci

Because vancomycin-resistant enterococci pose a 

significant health threat, vancomycin-resistant enterococci 

were isolated by spread culturing from the ENT cultures (that 

originally contained presumptive vancomycin-resistant enterococci) on MH agar containing vancomycin (16 μg/ml). Colonies that grew on this medium were confirmed as enterococci 

by (1) growth on mEI medium, (2) bile esculin hydrolysis on

Gene Primers 5 ́-3 ́ Thermocycler conditions Reference

Integron F: GGC ATC CAA GCA FCA AG

R: AAG CAG ACT TGA CCT GA

5 min 94°C

35 cycles: 1 min 94°C

 1 min 95°C

 2 min 72°C

10 min at 72°C

Lévesque and other, 1995

vanA

vanB

F: GGG AAA ACG ACA ATT GC

R: GTA CAA TGC GGC CGT TA

F:ATG GGA AGC CGA TAG TC

R: GAT TTC GTT CCT CGA CC

3 min 96°C

40 cycles: 20 sec 95°C

 40 sec 55°C

 1.5 min 72°C

5 min 72°C

Dutka-Malen, 1995

Table 3. Primers and polymerase chain reaction conditions for integrons and vancomycin resistance genes.

[5 ́, five prime end; 3 ́, three prime end; F, forward primer; R, reverse primer; G, guanidine; C, cytosine; A, adenosine; T, tyrosine; min, minute; sec, second; 

°C, degrees Celsius]

8 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
A 100 base pair DNA size ladder was used to determine the 

size of DNA fragments. Gels were visualized with a foto/prep 

transilluminator (Fotodyne), imaged with a Kodak EDAS 290 

Zoom digital camera, and analyzed with the Kodak 1D-gel 

image analysis software.

The genes responsible for acquired high-level vancomycin resistance (vanA) and low- to intermediate-level resistance 

(vanB) were detected in identified enterococci isolates by 

means of protocols similar to those described by DutkaMalen and others (1995). A 1-μL loop of overnight culture 

was suspended in 50 μL of 0.05N NaOH. Cells were lysed 

by heating the suspension at 95°C for 10 minutes. Cellular 

debris was pelleted before 1μL of supernatant was added to 

the following PCR reaction: 1X PCR buffer, 2.5 mM MgCl2, 

0.2 mM dNTPs, 0.5 μg/μl BSA, 0.3 μM of each primer (table 

3), and 2.5 units Taq polymerase (Promega) in a final volume 

of 25 μL. DNA was amplified, and resulting PCR products 

were visualized and photographed as described above. Positive 

control strains also were tested along with a negative (nonDNA) control and study samples. Positive controls for these 

experiments included vancomycin-resistant E. faecium ATCC 

700221 containing the vanA gene, vancomycin-resistant E. 

faecalis ATCC 51299 containing the vanB gene, and vancomycin-susceptible E. faecalis ATCC 29212 lacking both vanA 

and vanB. 

Determination of the presence of pathogenic E. 

coli

Three different assays were used to detect the presence 

of potential pathogenic strains of E. coli. The first, a growth 

assay (GA), was used to determine whether E. coli O157 was 

present. The FC culture was serially diluted in sterile phosphate buffered saline solution and grown on sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC). 

CT-SMAC is commonly used in clinical settings to isolate and 

differentiate the E. coli O157 group (Zadik and others, 1993). 

Samples containing non-sorbitol-fermenting colonies (white 

or colorless) were recorded as presumptive E. coli O157 and 

will be hereafter referred to as GA positive. However, because 

of the background growth of sorbitol-fermenting pink colonies 

in some samples, white colonies may not have been detectable if they were in very low concentrations compared to pink 

colonies. In the second assay, Reveal O157:H7 8 hour E. coli

test kits (Neogen Inc., Lansing, Mich.) were used to detect E. 

coli O157 by immunological methods. This test was used to 

detect specific surface antigens that are expressed on E. coli

O157 cells (Bird and Hoerner, 2001; Power and others, 2000). 

Because this test was designed for detection of E. coli O157:

H7 in meat and had not previously been evaluated for surfacewater samples, it was used with slight modifications conducive to the samples to determine whether these antigens were 

present in the FC cultures. Briefly, from the FC culture, 100 

μL was added to 45 mL of the Reveal enrichment media. The 

sample was incubated at 42°C for 8 hours, and a portion of the 

enrichment was applied to a Reveal test device. Reveal positive 

results will hereafter be referred to as IA positive. Because of 

the amount of FC sample that could be used for this method 

and the ability to selectively enrich for the desired organisms, 

this resulted in our most sensitive detection of E. coli O157. 

Because certain non-E. coli O157 bacteria can cross-react 

with the antibodies used in this method, it is possible to obtain 

false-positive results. Therefore, samples were tested by means 

of a third assay to detect the E. coli O157-specific gene rfb0157. 

Owing to the constraints of this method, the amount of FC 

culture used for the rfb0157 analysis was often much less than 

that used for the IA test, resulting in a disagreement between 

immunological and gene-based methods reflective of detection-limit and test-sensitivity issues. 

In the third assay, PCR was used to identify the genes 

rfb0157, stx1, stx2, and eaeA. DNA was extracted from 1 to 

250 μL (dependent on number of fecal coliforms detected in 

the original water sample) of FC culture by use of the Qiagen 

DNeasy DNA extraction kit (Qiagen, Valencia, Calf.). The 

manufacturer’s instructions were followed for DNA extraction 

from bacterial cultures. DNA was collected into two 200-μL 

volumes of elution buffer and stored at -20°C until analysis. 

For the detection of the gene rfb0157, a 25-μL PCR 

reaction was set up with 5 μL of extracted DNA added to 

a final concentration of 1X PCR buffer, 5 mM MgCl2, 0.2 

mM dNTP’s, 0.1 μM primers (table 4), and 2.5 units of Taq 

polymerase (Promega) in buffer B. DNA was amplified, and 

resulting PCR products were visualized and photographed as 

described previously.

A multiplex PCR was used to detect the presence of 

the eaeA, stx1, and stx2 genes by methods similar to those 

described by Fagan and others (1999). Each 25-μL PCR 

reaction mixture consisted of 1μL of extracted DNA and 

a final concentration of 1X buffer II PCR reaction buffer, 

3 mM MgCl2, 0.2 mM dNTP’s, 0.1 μg/μL BSA, 0.05 μM 

of eaeA, stx2 and stx1 primers (table 4), 0.025 μM of the 

internal-control E. coli specific primers (EC; table 4), and 2.5 

units AmpliTaq Gold Polymerase. DNA was amplified, and 

resulting PCR products were visualized and photographed as 

described above. For both the rfb0157 and multiplex PCR, DNA 

extracted from a known strain of E. coli O157:H7 was added 

to one reaction as a positive control, and no DNA was added 

to another reaction for a negative control. The 16S rDNA gene 

specific for E. coli (EC, table 4) was included as a positivecontrol run with every sample. This control was an amplification of a gene specific for all strains of E. coli. To be sure the 

sample was amplifiable, this product must be visible. If it was 

not visible the sample was repeated. 

Results of Bacterial Analyses

Bacterial analysis of river water collected from 14 

locations in Oakland County in August and September 

2003 revealed high fecal indicator concentrations including 

exceedences of recreational water-quality standards throughout the county. Antibiotic-resistant fecal indicator bacteria 

Results of Bacterial Analyses 9
were isolated from all samples collected and tests indicative 

of pathogenic E. coli were positive in samples with high-level 

antibiotic resistances.

Fecal indicator bacteria enumeration

Fecal indicator bacteria (fecal coliforms, E. coli, and 

enterococci) were quantified in the samples from the 14 

Oakland County streams and two samples from reference 

sites collected in August and September 2003 (appendix A); 

results are shown in figure 2. E. coli concentrations ranged 

from 3 colony forming units (CFU) per 100 mL (August and 

September at site 04170500) to a high of 25,000 CFU/100 

mL (site 04166200 in August) and a high of 1,300 CFU/100 

mL (site 04166315) in September. The E. coli concentrations 

were compared to the Michigan water-quality standards for 

recreational waters (Michigan Water Quality Standard Rule 

62 of the Michigan Water Quality Standards: 130 E. coli/100 

mL of water as a 30-day geometric mean and 300 E. coli/100 

mL of water at any one sampling). Seven of the sites in August 

and 8 of the 14 sites in September exceeded the single sample 

criterion of 300 E. coli/100 mL of water (fig. 2). All seven 

of these samples in August and one in September had E. 

coli concentrations greater than 1,000 CFU/100 mL. E. coli

concentrations were greater in August than in September at 

nine of the sites and greater in September than August at just 

four sites. In August, the mean number of E. coli was 2,497 

CFU/100 mL with a median of 650 CFU/100 mL, compared 

to a mean of 375 CFU/100 mL in September with a median of 

315 CFU/100 mL. None of the three reference samples colGene Primers 5 ́-3 ́ Thermocycler conditions Reference

rfbO157 F: CGT GAT GAT GTT GAG TTG 

R: AGA TTG GGT TGG CAT TAC TG

5 min 94°C 

30 cycles: 1 min 94°C 

 1 min 53°C 

 1 min 72°C 

5 min 72°C 

Osek, 2003 

Maurer and others, 1999

Multiplex PCR

stx1 

stx2 

eaeA 

EC

F: ACA CTG GAT GAT CTC AGT GG 

R: CTG AAT CCC CCT CCA TTA TG 

F: CCA TGA CAA CGG ACA GCA GTT

R: CCT GTC AAC TGA GCA GCA CTT TG 

F: GTG GCG AAT ACT GGC GAG ACT 

R: CCC CAT TCT TTT TCA CCG TCG 

F: GGA AGA AGC TTG CTT CTT TGC TGA C 

R: AGC CCG GGG ATT TCA CAT CTG ACT TA

10 min 95°C 

35 cycles: 30 sec 95°C

 40 sec 56°C 

 1 min 72°C 

5 min 72°C

Gannon and others, 1992

Fagan and others, 1999

Sabat and others, 2000

Table 4. Primers and polymerase chain reaction (PCR) conditions for the rfb0157 gene, and multiplex PCR for the virulence genes.

[5 ́, five prime end; 3 ́, three prime end; F, forward primer; R, reverse primer; G, guanidine; C, cytosine; A, adenosine; T, tyrosine; min, minute; sec, second; 

°C, degrees Celsius]

lected (2 from site 04135500 and one from site 04001000) had 

concentrations that exceeded 300 E. coli/100 mL.

Enterococci and fecal coliform bacteria concentrations 

also were determined in August and September (figs. 3, 4, and 

appendix A). Although Michigan does not have water-quality 

standards for enterococci or fecal coliform bacteria, USEPA 

recommends that enterococci concentrations do not exceed 61 

CFU/100 mL water in a single sample for recreational waters 

(U.S. Environmental Protection Agency, 2000). In August, 

13 of 14 samples exceeded this recommendation, and 12 of 

14 samples exceeded the recommendation in September. In 

August, the mean number of enterococci was 464 CFU/100 

mL (median of 316 CFU/100 mL) and in September, 466 

CFU/100 mL (median of 370 CFU/100 mL). At seven sites, 

enterococci concentrations were greater in September than in 

August, in contrast to the E. coli concentrations which were 

more frequently greater in August. None of the reference 

samples exceeded the USEPA recommendations for enterococci. The highest concentration for a reference site was only 

14 enterococci/100 mL at site 04135500. Fecal coliforms are 

not currently a recommended indicator of water quality; however, prior to 1986, USEPA recommended that fecal coliforms 

not exceed 400 CFU/100 mL for a single sample for primary 

contact waters (U.S. Environmental Protection Agency, 

1976). This criterion was exceeded in 12 out of 14 Oakland 

County sites in August and 9 sites in September (fig. 1 and 

appendix A). No reference samples exceeded this criterion. 

In August fecal coliform bacteria concentrations ranged from 

6 CFU/100 mL at site 04170500 to 126,000 CFU/100 mL at 

site 04166200. In September, concentrations ranged from 3 

CFU/100 mL (site 04170500) to 3,300 CFU/100 mL (site 

10 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
0

250

500

750

1,000

1,250

1,500

1,750

2,000

04166000041661000416620004166315041608000416090004161000041615400416158004161810041438300414803504170000041705000413550004001000

USGS STATION NUMBER

C

OLO

N

Y FO

R

MIN

G

U

NIT

S PER 100

MILLILITER

W

ATER

S

A

MPLE

Aug Sept

25,000

Figure 2. Escherichia coli concentrations for Oakland County and reference samples collected in August and September 2003. Dotted 

line represents Michigan Water Quality Standard for single sample, limit of 300 colony forming units /100 mL for recreational waters. 

0

1,000

2,000

3,000

4,000

5,000

6,000

04166000041661000416620004166315041608000416090004161000041615400416158004161810041438300414803504170000041705000413550004001000

USGS STATION NUMBER

C

OLO

N

Y FO

R

MIN

G

U

NIT

S PER 100

MILLILITER

W

ATER

S

A

MPLE

Aug Sept

126,000

Figure 3. Fecal coliform concentrations for Oakland County and reference samples collected in August and September 2003. Dotted 

line 

units /100 mL for primary contact waters.

Results of Bacterial Analyses 11
04166100). In nine Oakland County samples in August and 

four in September, fecal coliform concentrations were greater 

than 1,000 CFU/100 mL. At all but one site, fecal coliform 

concentrations were greater in August than September. The 

median Oakland County fecal coliform number in August was 

2,200 CFU/100 mL and in September was 525 CFU/100 mL. 

In August, fecal coliform concentration for the tested reference site (04135500) was 320 CFU/100 mL. In September, 

fecal coliform bacteria concentrations were highest at site 

04001000 at 380 CFU/100 mL (fig. 2).

Samples collected in August were held beyond the 

recommended 6-hour holding time. In addition, stream water 

temperature was on average 3°C warmer in August. Both 

August and September samples were collected at low flow; 

however, streamflows were on average 5.3 ft3/s greater during 

the August sampling, which took place after a small rainstorm 

in the county. Field-measured characteristics corresponding to 

the September and August samplings are listed in appendix B.

Antibiotic resistances

Widespread antibiotic resistance was found among the 

fecal coliform and enterococci groups in both August and 

September. Multiple antibiotic resistant fecal coliform bacteria were found at all sites sampled. E. coli and enterococci 

resistant to antibiotics of clinical concern were recovered from 

several Oakland County sites. In addition, genetic elements 

that could serve as a vector for the transfer of antibiotic resistance also were found at several Oakland County sites.

Antibiotic-resistant fecal coliform bacteria

All 14 Oakland County samples contained fecal coliform isolates resistant to at least one of six tested antibiotics 

(ampicillin, cephalothin, cefoxitin, ceftriaxone, gentamicin, 

streptomycin) in both August and September 2003. Tetracycline was not included because results were not interpretable for all samples. Appendix C and D list the percentage 

or number of fecal coliform bacteria resistant to the tested 

antibiotics. Because of the methods used in August, antibiotic-resistant fecal coliform bacteria were difficult to identify. 

The percentage of fecal coliform bacteria resistant to each 

antibiotic could not be verified. In many cases, overgrowth of 

bacteria on the plate limited results to a finding of resistance 

to that particular antibiotic for that sample; quantifying the 

number of resistant isolates and verifying that the resistant 

isolates of fecal coliforms were precluded. In September, 

isolates were individually tested, and resistant fecal coliform 

bacteria were quantified. Every sample collected in September contained fecal coliform isolates resistant to multiple 

antibiotics (table 5). Out of a total of 518 fecal coliform 

isolates tested from Oakland County watersheds, only 30 of 

them did not show resistance to any of the 6 tested antibiotics, and only 149 were resistant to only 1 antibiotic. The 

0

150

300

450

600

750

900

1,050

1,200

1,350

1,500

1,650

04166000041661000416620004166315041608000416090004161000041615400416158004161810041438300414803504170000041705000413550004001000

USGS STATION NUMBER

C

OLO

N

Y FO

R

MIN

G

U

NIT

S PER 100

MILLILITER

W

ATER

S

A

MPLE

Aug Sept

Figure 4 . Enterococci concentrations for Oakland County and reference samples collected in August and September 2003. Dotted line 

repr 

waters. 

12 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
USGS 

station 

number

Total fecal 

coliform bacteria 

tested

Percentage of 

isolates sensitive 

to all antibiotics

Antibiotic resistances (percent)1

1 2 3 4 5a 6a

Rouge River watershed

04166000 50 44 40 10 6 0 0 0 

04166100 50 0 82 14 2 2 0 0 

04166200 8 0 0 50 38 13 0 0 

04166315 14 0 21 36 29 7 7a 0 

Clinton River watershed

04161580 36 0 0 0 3 25 50a 22a

04160900 45 0 42 36 18 4 0 0 

04161540 48 0 27 31 25 8 4a 4a

04161810 50 2 10 20 40 20 8a 0 

04161000 50 0 16 28 56 0 0 0 

04160800 50 0 0 30 36 20 14a 0 

Other Oakland County watersheds

04148035 50 6 32 38 18 6 0 0 

04143830 29 0 3 7 69 17 3a 0 

04170500 5 0 0 100 0 0 0 0 

04170000 33 6 30 15 36 12 0 0 

Reference sites

04001000 30 0 37 33 27 0 3a 0 

04135500 5 40 40 20 0 0 0 0 

1 Percentage of isolates resistant to 1, 2, 3, 4, 5, or 6, different antibiotics.

a Bold samples represent isolates in which multiple antibiotic resistances may be a result of an acquired resistance.

Table 5. Percentage of fecal coliform bacteria resistant to multiple antibiotics in September 2003.

remaining 339 fecal coliform isolates were resistant to 2 or 

more antibiotics.

At Stony Creek near Romeo (04161580), 22 percent of 

the fecal coliform isolates were resistant to all six antibiotics. 

Similarly, 4 percent of the fecal coliform isolates from Paint 

Creek at Rochester (04161540) also were resistant to all 6 

antibiotics. Both sites are within the Clinton River watershed. 

In addition, 50 percent and 4 percent of the fecal coliforms 

from these two sites (04161580 and 04161540, respectively) 

were resistant to five of the tested antibiotics. Two other Clinton River watershed samples also contained fecal coliform isolates resistant to 5 of the 6 tested antibiotics: Sashabaw River 

near Drayton Plains (04160800), 14 percent and Clinton River 

at Yates (04161810), 8 percent. Two other Oakland County 

sites also had fecal coliform isolates that were resistant to 5 

of the 6 antibiotics: Shiawassee River at Holly (04143830), 3 

percent; and Upper River Rouge at Clarenceville (04166315), 

7 percent. Neither the reference sample from Washington 

Creek (04001000) nor that from Au Sable River at Grayling 

(04135500) contained any fecal coliform isolates that were 

resistant to all six tested antibiotics. However, at site 

04001000, 3 percent of the fecal coliform isolates were resistant to five antibiotics. 

Fecal coliform bacteria resistant to cephalosporins were 

found in all samples collected from Oakland County (table 

6). In the Clinton River watershed, fecal coliform bacteria 

with antibiotic-resistance profiles indicative of ESBLs were 

found at five out of six sites (04161580, 04161540, 04160800, 

04161810, and 04160900; fig. 5). These isolates were resistant 

to cephalothin, ceftriaxone, and ampicillin and were susceptible to cefoxitin. In addition, isolates from four of these sites 

(04161580, 04161540, 04161810, and 04160800) not only 

had this ESBL profile but also were resistant to other non-βlactam antibiotics tested. Of most interest were isolates from 

sites 04161580, 04161540, and 04161810 in which 2, 1, and 1 

isolate, respectively, were resistant to cephalothin, ceftriaxone, 

ampicillin, tetracycline, gentamicin, and streptomycin. This is 

a strong indication that this resistance may be due to a plasmid-carrying multiple antibiotic-resistance genes. In contrast, 

only one of the four River Rouge sites had isolates resistant to 

Results of Bacterial Analyses 13
cephalothin, ceftriaxone, and ampicillin with a susceptibility 

to cefoxitin (04166100; fig. 5). Sites 04166000 and 04166315 

in this same watershed had isolates resistant to cephalothin and 

cefoxitin but susceptible to ampicillin. The remaining River 

Rouge watershed sample did not have any isolate resistant to 

cephalothin and ceftriaxone. Other sites sampled in Oakland 

County also resulted in fecal coliform isolates with an ESBL 

profile, but none of these were resistant to the non-β-lactam 

antibiotics (table 6 and fig. 5).

The fecal coliform isolates in this study were not identified to the genus or species level and were not tested for antibiotic-resistance genes; intrinsic resistance, therefore, cannot 

be ruled out. However, many of fecal coliform bacteria in this 

study showed resistance patterns indicative of acquired resistance, such as multiple resistances to antibiotics in different 

groups and resistance patterns indicative of ESBL production. 

Two Clinton River watershed sites, 04161580 and 04161540, 

had isolates resistant to all tested antibiotics and isolates with 

ESBL patterns of resistance. By use of molecular techniques, 

resistant organisms could be identified and genes responsible 

for resistance could be detected; such an analysis would yield 

more information as to the significance of these results. 

Antibiotic-resistant E. coli

From the FC cultures, presumptive E. coli (isolates that 

grew on mFC at 44.5°C and fluoresced under UV light on 

nutrient agar with MUG) that were resistant to one of the 

cephalosporin antibiotics were isolated from seven Oakland 

County sites (04166000, 04160900, 04148035, 04161540, 

04160800, 04161580, and 04161810). By means of a set of 

additional biochemical tests, isolates phenotypically confirmed 

to be E. coli were obtained from only three sites (04166000, 

04160900, and 04161540). The remainder were determined 

USGS 

station 

number

Total fecal 

coliform 

isolates 

tested

CEP 

only

FOX 

only

AXO 

only

CEP + 

FOX

CEP + 

AXOa

FOX + 

AXO

CEP + 

FOX + 

AXO

No cephalosporin 

resistance

River Rouge watershed

04166000 50 2 0 1 3 1a 0 0 7

04166100 50 46 0 0 2 2a 0 0 0

04166200 8 0 1 1 2 0 1 3 0

04166315 14 4 0 0 6 2a 0 2 0

Clinton River watershed

04160800 50 22 0 0 14 6a 0 8 0

04160900 45 21 2 0 15 4a 0 3 0

04161000 50 17 0 0 32 0 0 0 1

04161540 48 18 0 0 4 16a 0 10 0

04161580 36 0 0 0 0 5a 0 31 0

04161810 50 14 1 0 23 3a 0 4 5

Other Oakland County watersheds

04143830 29 5 0 0 23 0 0 1 0

04148035 50 30 1 1 9 1a 0 2 6

04170000 33 7 0 0 15 2a 0 0 9

04170500 3 0 0 0 0 0 3 0 0

Reference sites

04001000 30 21 0 0 5 1a 0 2 1

04135500 5 2 1 0 0 0 0 0 2

Total 551 209 6 3 153 43 4 66 31

a Pattern indicative of extended-spectrum-β-lactamase (ESBL)-producing fecal coliform bacteria.

Table 6. Resistances of individual fecal coliform bacteria to cephalosporin antibiotics, September 2003.

[Bold samples represent resistance of clinical significance; CEP, cephalothin; FOX, cefoxitin; AXO, ceftriaxone] 

14 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Figure 5. Oakland County, Mich., sites that contain fecal coliform bacteria with antibiotic resistance profiles indicative of extendedspectrum-β-lactamases (ESBLs) [cephalothin and ceftriaxone; cephalothin, ceftriaxone, and ampicillin; cephalothin, ceftriaxone, 

ampicillin, and one or more non-β-lactam antibiotics (tetracycline, streptomycin, and/or gentamicin)].

to be false positives on the basis of a negative indol reaction. 

Other studies have reported several bacterial genera that could 

result in a false positive for β-glucuronidase reaction including 

Klebsiella, Enterobacter, Shigella, and Salmonella (Sarhan 

and Foster, 1991; McDaniels and others, 1996; Geissler and 

others, 2000). All of these genera contain species that are 

potential pathogens of human health concern. More investigation is necessary to determine the identity of these non-E. coli

isolates from Oakland County. 

Phenotypically confirmed E. coli resistant to one of three 

tested cephalosporin antibiotics were tested for intermediatelevel (I) and high-level (H) resistance to cephalothin (I=16 

μg/mL, H=32 μg/mL), cefoxitin (I=16 μg/mL, H=32 μg/mL), 

ceftriaxone (I=16 μg/mL, H=64 μg/mL), gentamicin (I= 8 

μg/mL, H=16 μg/mL), and ampicillin (I=16 μg/mL, H=32 μg/

mL). Results are listed in table 7. One E. coli isolate obtained 

from site 04166000 showed a high level of resistance to cephalothin, cefoxitin, and ceftriaxone and intermediate resistance 

to gentamicin. Other E. coli isolates from this site also showed 

high-level resistance to cephalothin. E. coli isolates with highlevel resistance to both cephalothin and ampicillin were also

recovered from site 04160900. E. coli were recovered from 

site 04161540 with high-level of resistance to cephalothin. 

Integrons were detected in E. coli isolated from sites 

04160900 and 04166000 (table 7). Integrons are transferable among similar organisms. Further studies are needed to 

determine whether the antibiotic resistances seen in the E. coli

isolates in this study are encoded by genes on the integrons 

that are present. Other genes responsible for resistance to the 

cephalosporin antibiotics and ampicillin were not evaluated 

in this study; however, these genes should not be overlooked 

as additional possibilities to explain the resistance patterns 

detected. 

Antibiotic-resistant enterococci

Enterococci isolates from both August and September were tested for resistance to a low level (8 μg/mL) and 

intermediate level (16 μg/mL) of vancomycin. Low-level 

vancomycin resistance was found in enterococci from 8 of the 

15 samples collected in August and 13 of the 15 samples with 

enterococci in September (table 8). Some enterococci species

83 °45 ’ 83 °30 ’ 83 °15 ’ 83°

42 °45 ’

42 °30 ’ H uron R iv er

R iv e r

S hiawassee

Kearsley

C reek

Uppe r R iver R ouge

E vans Ditch

R iver R ouge

Stony

C reek

Paint C reek

S ash abaw C reek C linto n

R iver

C linton R iver

0 5 10

0 5 10

MILES

KILOMETERS

River

Rouge

watershed

Shiawassee

River

watershed

Clinton River

watershed

Flint River

watershed

Huron

River

watershed

EXPLANATION

Analytical resultspositive results shades,

negative results left blank

Cephalothin and ceftriaxone

Cephalothin, ceftriaxone,

and ampicillin

Cephalothin, ceftriaxone, ampicillin,

and other non-β-lactam antibiotic

1

2

3

4

5

6 7

8

9

10

11

12

13 14

Map

Number

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Site

Number

04148035

04143830

04170000

04170500

04160800

04160900

04161000

04161540

04161580

04161810

04166000

04166315

04166100

04166200

Map

Number

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Site

Number

04148035

04143830

04170000

04170500

04160800

04160900

04161000

04161540

04161580

04161810

04166000

04166315

04166100

04166200

Results of Bacterial Analyses 15
have an intrinsic resistance to low levels of vancomycin, but 

these species are usually not resistant to higher levels. Intermediate to high levels of vancomycin resistance are often a result 

of a resistance gene that is carried on a plasmid. Enterococci 

resistant to intermediate levels of vancomycin were found at 

one site in August (04166200) and at five sites in September 

(04170500, 04161540, 04148035, 04143830, and 04160800; 

table 8). For all but site 04170500, enterococci concentrations 

were at least 300 CFU/100 mL. Not all samples with concentration greater than 300 CFU/100 mL contained vancomycinresistant colonies. 

In addition to vancomycin, all August and September 

enterococci isolates were tested for their resistance to gentamicin and streptomycin. Gentamicin-resistant enterococci were 

isolated from 10 Oakland County sites in August and 3 sites in 

September (table 8). Only one site contained gentamicin-resistant enterococci on both dates (site 04160800). Streptomycinresistant enterococci were isolated from 8 Oakland County 

sites in August and 5 sites in September; samples from only 

three of those sites contained streptomycin-resistant enterococci on both dates (table 8). Neither gentamicin nor streptomycin-resistant enterococci were found at the reference sites.

USGS station number Isolate number AMP CEP FOX AXO GEN Integron

04166000 1 S R R R I -

2 I R S S S +

3 S R S S S -

4 I I S S S -

5 S I S S S -

04160900 1 R R S S S +

2 R R S S S -

3 S R S S S -

4 R I S S I -

5 S I S S S -

04161540 1 S R I S S -

2 S R S S S -

3 S I S S S -

S1 < 8 μg/mL < 8 μg/mL < 8 μg/mL < 8 μg/mL < 4 μg/mL

I

1 16 μg/mL 16 μg/mL 16 μg/mL 16-32 μg/mL 8 μg/mL

R1 > 32 μg/mL > 32 μg/mL > 32 μg/mL > 64 μg/mL > 16 μg/mL

Table 7. Escherichia coli antibiotic-resistance profiles and integron-detection results.

[AMP, ampicillin; CEP, cephalothin; FOX, cefoxitin; AXO, ceftriaxone; GEN, gentamicin; S, susceptible; I, intermediate level resistance; H, high level resistance, μg/mL, microgram per milliliter; +, positive result; -, negative result, >, greater than or equal to; <, less than or equal to]

1 Resistance levels based on NCCLS minimum inhibitory concentration (MIC) interpretive standard for Enterobacteriaceae (National Committee for Clinical 

Laboratory Standards, 2002).

Three enterococci isolates resistant to high levels of vancomycin (> 64 μg/mL) were isolated from the ENT cultures 

from the August Evans Ditch in Southfield (04166200) and 

September Kearsley Creek at Ortonville (04148035) samples. 

The Evans Ditch isolate and two of the Kearsley Creek enterococci isolates were resistant to 128 μg/mL vancomycin. Api 

Strep20 identification strips identified these isolates as E. faecium. By use of PCR, the vanA gene, which is responsible for 

acquired high-level vancomycin resistance (> 128 μg/mL), was 

detected in the isolate from Evans Ditch and in one of the isolates from Kearsley Creek. E. faecium is the most frequently 

isolated vancomycin-resistant Enterococcus in clinical settings 

(Kak and Chow, 2002). The vanA gene is usually carried on 

plasmids, which are easily transferred among similar organisms. The transfer of this resistance is possible with the presence of the vanA gene. Other isolates resistant to intermediate 

levels of vancomycin also were isolated from these two sites 

and from Paint Creek at Rochester (04161540); these isolates 

were identified as E. faecium and E. faecalis. Both of these 

enterococci species have potential to cause disease in humans. 

Vancomycin-resistant isolates would make treating such infection extremely difficult. 

16 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
August 2003a September 2003b

USGS station 

number

Total 

enterococci isolates

VAN 8 

μg/mL

VAN 16 

μg/mL

STR 2000 

μg/mL

GEN 100 

μg/mL

Total 

enterococci 

isolates

VAN 8 

μg/mL

VAN 16 

μg/mL

STR 2000 

μg/mL

GEN 100 

μg/mL

River Rouge watershed

04166000 844 6 0 0 0 42 3 0 0 0

04166100 596 0 0 8 0 50 0 0 0 2

04166200 >1,000 6 2 2 2 35 0 0 0 0

04166315 1,096 0 0 40 92 50 3 0 1 0

Clinton River watershed

04160800 96 0 0 16 38 30 10 2 0 1

04160900 906 2 0 0 2 47 4 0 2 0

04161000 100 2 0 2 14 39 1 0 3 0

04161540 >1,000 0 0 14 2 35 7 3 2 0

04161580 118 4 0 0 8 50 1 0 1 0

04161810 1,032 0 0 2 36 50 4 0 0 0

Other Oakland County watersheds

04143830 206 0 0 0 26 39 6 3 0 0

04148035 416 0 0 0 0 50 11 4 0 2

04170000 70 2 0 0 0 35 2 0 0 0

04170500 2 4 0 2 2 13 5 2 0 0

Reference sites

04001000 NA NA NA NA NA 0 NA NA NA NA

04135500 5 4 0 0 0 14 1 0 0 0

Table 8. Percentage of enterococci isolates resistant to tested antibiotics.

[VAN, vancomycin; STR, streptomycin; GEN, gentamicin; NA, not available; >, greater than]

aAugust antibiotic resistance test included all mEI isolates which may include non-enterococci.

bSeptember antibiotic resistance test was on individual mEI positive isolates, therefore, tested isolates are all presumptive enterococci.

Potential sources of resistance

In Oakland County, cephalothin resistance was seen in a 

large percentage of isolates from nearly all sites in September 

(appendixes C and D). The exceptions were the River Rouge 

site at Birmingham (04166000) and the Huron River site at 

Milford (04170500), where only 10 percent of the isolates 

were resistant to cephalothin at 04166000 and none of the isolates at site 04170500. At site 04170500, there were only three 

fecal coliform bacteria per 100 mL water to test, and all three 

were resistant to ceftriaxone and cefoxitin. Fecal coliform 

bacteria resistant to ampicillin were also found at every site in 

September; however, the percentage of the isolates resistant 

to ampicillin varied with watershed. Fecal coliform bacteria 

resistant to ceftriaxone were found throughout the county 

(appendixes C and D). Only at the Clinton River at Auburn 

Hills (04161000) were ceftriaxone-resistant fecal coliform 

bacteria absent. This site did have high levels of cephalothin-, 

cefoxitin-, and ampicillin-resistant fecal coliform bacteria. In 

contrast, the percentages of fecal coliform bacteria resistant to 

tetracycline, streptomycin, and gentamicin were lower than the 

percentages at the other Oakland County sites (appendixes C 

and D). 

The two reference sites included in this study are areas 

with less human presence and activity then in Oakland 

County. The first is a site on the Au Sable River in Grayling 

(04135500) and the second, Washington Creek, is on Isle 

Royale (04001000). Only 5 CFU/100 mL fecal coliform 

bacteria were present in the September Au Sable River sample 

to test for antibiotic resistance. These fecal coliform bacteria 

were all susceptible to ceftriaxone, ampicillin, and gentamicin; 

60 percent, 40 percent, 20 percent, and 20 percent of the 

Results of Bacterial Analyses 17
isolates were resistant to tetracycline, cephalothin, streptomycin, and cefoxitin, respectively. Antibiotic-resistance patterns 

in September at Washington Creek were similar to those for 

many of the samples collected in Oakland County (appendixes 

C and D); 97 percent of the Washington Creek fecal coliform 

isolates were resistant to cephalothin. Resistance for the other 

tested antibiotics ranged from 20 to 27 percent. 

For the enterococci isolates, resistance to intermediate 

levels of vancomycin (16 μg/mL) was found at only one site 

in August, the Evans Ditch at Southfield site (04166200), 

and at five Oakland County sites in September. These sites 

include two from the Clinton River watershed (04160800 

and 04161540), the Shiawassee River site (04143830), the 

Flint River watershed site (04148035), and the Huron River 

(04170500). No sample from the River Rouge watershed in 

September contained enterococci resistant to intermediate 

levels of vancomycin. Streptomycin-resistant enterococci 

were found at four out of the six Clinton River watershed sites 

(04160900, 04161000, 04161540, and 04161580), but at only 

one of the River Rouge watershed sites (04166315). The gene 

responsible for high-level vancomycin resistance commonly 

found on plasmids was found in E. faecium isolated from two 

Oakland County sites, Kearsley Creek (04148035) in September and at Evans Ditch (04166200) in August.

Although the source of the fecal contamination cannot 

be determined from these data, there is evidence that the fecal 

indicator bacteria communities are different among the various 

watersheds. The Huron River watershed samples had much 

less indication of fecal contamination than the other Oakland County sites. All fecal indicator bacteria concentrations 

were much smaller for Huron River samples than for samples 

from other watersheds (in most cases meeting water-quality 

standards) and in some cases lower than those at the reference 

sites (figs. 2-4). The Clinton River watershed samples tended 

to have more multiply-resistant isolates. For example, four of 

the six sites contained fecal coliform isolates resistant to five 

or six antibiotics, whereas only one River Rouge watershed 

sample had fecal coliform isolates resistant to many antibiotics 

(table 5). In addition, five of the Clinton River watershed samples had fecal coliform isolates with resistances to cephalothin, ceftriaxone, and ampicillin, in contrast to just one sample 

from the River Rouge watershed and one from the Huron 

River watershed (fig. 5). Finally, enterococci with intermediate- to high-level vancomycin resistance were not frequently 

detected in either the Clinton or River Rouge watersheds 

(two sites in the Clinton and one in the Rouge), but they were 

detected in all but one of the other Oakland County samples 

(table 8). The differences seen in both patterns of antibiotic 

resistance and concentrations of fecal indicator bacteria suggest the communities of fecal indicator bacteria are different 

between the major watersheds in the County. 

Water-chemistry data for September 2003 also suggest sources may differ between samples and watersheds. 

Sodium, chloride, calcium, and phosphorus were all higher 

in the Rouge River watershed samples in September than in 

any other sample (Aichele and other, 2004). “Wastewater 

constituents” (a suites of analytes including pharmaceuticals, 

hormones, and household chemical products) were detected at 

all Oakland County sites (Aichele and others, 2004). Certain 

groups of these constituents were more commonly found in 

particular watersheds. For example, cholesterol and prometon 

(herbicide) were detected in three of the four River Rouge 

watershed samples, but were not detected in any other sample 

during this time period. The detergent-degradation compound, 

4-tert-octylphenol was detected in five of the six Clinton River 

watershed samples and in only one River Rouge and one 

Huron River watershed sample. 

Pathogenic bacteria

Based on previous studies (Boerlin and others, 1999), E. 

coli O157 that possess the eaeA and stx2 genes pose a greater 

human health threat than other serotypes with other gene combinations. Multiple lines of evidence were used to identify E. 

coli O157 in this study, as previously mentioned. E. coli O157 

is indicated by positive GA, IA, and/or rfb0157 results. Results 

of these analyses are shown in figure 6 and listed in table 9.

Indications of potentially pathogenic E. coli were 

detected throughout Oakland County watersheds. Positive IA 

and rfb0157 results were obtained for three sites: 04161000, 

04161810, and 04166000 in August and 04166000 again in 

September. Only two of these sites (04161000 and 04161810) 

were GA positive. Three other sites (04143830 and 04166200 

in August, and 04166315 in September) were GA and IA positive but rfb0157 negative. No Oakland County sites were rfb0157

positive and IA negative, but seven sites were GA positive and 

IA negative, all of which were from the September sampling 

(fig. 6 and table 9). Further evaluation would be necessary to 

positively identify these samples as carrying E. coli O157. 

However, the multiple lines of evidence used in this study 

do indicate the potential for these organisms to be present in 

waters within Oakland County. In contrast, the three samples 

from the reference sites were all IA and rfb0157 negative; one 

sample was GA positive. 

18 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
USGS 

station 

number

August 2003 September 2003

PCR amplified genes PCR amplified genes

IA

GA 

(CFU/μL 

mFC 

stock) eaeA stx1 stx2 rfb0157 IA

GA 

(CFU/μL 

mFC 

stock) eaeA stx1 stx2 rfb0157

Rouge River watershed

04166000 + 0 + - - + + 0 + - + +

04166100 - 0 + - + - - 0 + - - -

04166200 + 100 + - + - - 1,500 + - + -

04166315 - 0 + - + - + 8 - - - -

Clinton River watershed

04160800 - 0 + - - - - 300 + - - -

04160900 - 0 + - + - - 0 + - + -

04161000 + 10,000 + - - + - 200 + - - -

04161540 - 0 + - - - - 1 + - + -

04161580 - 0 + + - - - 0 + - + -

04161810 + 100 + - - + - 7 + - + -

Other Oakland County watersheds

04170000 - 0 + - - - NT NT NT NT NT NT

04170500 - 0 - - - - NT NT NT NT NT NT

04143830 + 100 + - + - - 6,000 + - + -

04148035 + 0 + - - - - 8,000 + - - -

Reference sites

04135500 - 1 + + - - - 0 + - - -

04001000 NT NT NT NT NT NT - 0 + - - -

Table 9. Results of pathogenic Escherichia coli tests.

[IA, immunological assay; GA, growth-based assay; CFU, colony forming units; mFC, fecal coliform media; μL, microliters; PCR, polymerase chain reaction; +, positive result; -, negative result; NT, not tested]

Three genes commonly associated with STEC were 

analyzed for this study: eaeA, stx1, and stx2. As previously 

mentioned, the combination of eaeA and stx2 genes poses the 

greatest threat to human health. This gene combination was 

detected in FC cultures from five Oakland County sites in 

August and seven sites in September (fig. 6 and table 9). Of 

these, only three sites had FC cultures that were also IA positive (sites 04166200 and 04143830 in August and 04166000 

in September) and two that were also GA positive (sites 

04166200 and 04143830 in August). In September, the FC 

culture from site 04166000 was IA and rfb0157 positive and 

had both the eaeA and stx2 genes. No FC culture was positive for all three indicators of E. coli O157 as well as the eaeA 

and stx2 genes. Only the FC culture from site 04161580 was 

positive for the stx1 gene (fig. 6 and table 9). The eaeA gene 

was present in all three of the FC cultures from the reference 

samples, but stx2 was absent from all three samples. The stx1 

gene was detected in one reference samples (table 9). Representative gel images resulting from the PCR for the toxin 

genes, eaeA, stx1, and stx2 are shown in figs. 7 and 8. Results 

of the toxin gene PCR are listed in table 9.

Results of Bacterial Analyses 19
83 °45 ’ 83 °30 ’ 83 °15 ’ 83°

42 °45 ’

42 °30 ’ H uron R iv er

R iv e r

S hiawassee

Kearsley

C reek

Uppe r R iver R ouge

E vans Ditch

R iver R ouge

Stony

C reek

Paint C reek

S ash abaw C reek

C linto n

R iver

C linton R iver

River

Rouge

watershed

Shiawassee

River

watershed

Clinton River

watershed

Flint River

watershed

EXPLANATION

Analytical resultspositive results shades,

negative results left blank

1

2

3

4

5

6 7

8

9

10

11

12

13 14

Map

Number

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Site

Number

04148035

04143830

04170000

04170500

04160800

04160900

04161000

04161540

04161580

04161810

04166000

04166315

04166100

04166200

Map

Number

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Site

Number

04148035

04143830

04170000

04170500

04160800

04160900

04161000

04161540

04161580

04161810

04166000

04166315

04166100

04166200

A S

A S

A

A

A S

A S

A S

A S

A S

A S

A S

A S

A S A S

GA

IA

rfbO157

stx1

stx2

A (August)

S (September)

0 5 10

0 5 10

MILES

KILOMETERS

Huron

River

watershed

Figure 6. Oakland County, Mich., sites that contain positive results for the following: IA, GA and rfb0157 for the detection of E. coli O157; 

and genes (eaeA, stx1, and stx2) responsible for pathogenicity.

041705000417000004161000041608000414383004161580041480350416600004166315041661000416154004160900041618100416620004135500

control

Blank

E. Coli O157:H7

eaeA

stx2

stx1

E. coli specific gene

Figure 7. Polymerase chain reaction detection of toxin genes (eaeA, stx1, and stx2) and an Escherichia coli (E. coli) specific gene, in 

samples collected August 2003 in Oakland County watersheds, Mich. 

20 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
control

Blank

E. Coli O157:H7

eaeA

stx2

stx1

E. coli specific gene

0416610004160800041610000414383004161810041662000400100004161580041480350416631504135500041609000416154004166000

Figure 8. Polymerase chain reaction detection of toxin genes (eaeA, stx1, and stx2) and an Escherichia coli (E. coli) specific gene, in 

samples collected September 2003 in Oakland County watersheds, Mich. 

Summary and Conclusions 

To better understand the microbial water quality of 

streams in Oakland County, Mich., the USGS in cooperation with Oakland County Department of Human Services 

conducted a preliminary study of streams in the County to 

determine if antibiotic resistances of clinical significance 

occurred in fecal indicator bacteria and if potentially pathogenic E. coli could be detected. Results of this study indicate 

several exceedences in fecal indicator concentrations, antibiotic resistances of clinical concern, indications of acquired 

antibiotic resistances, and evidence of potentially pathogenic 

E. coli from surface-water samples. Samples for this study 

were initially collected in August 2003 from 14 streams in 

Oakland County. Interpretations of the results from the August 

sampling are limited because of difficulties in sampling and 

processing of these samples. In particular, samples collected 

in August were held beyond the 8-hour recommended holding 

time and overgrowth of antibiotic-resistant bacteria from the 

fecal coliform bacteria plates prevented accurate quantification 

of resistant fecal coliform bacteria. To address these issues, 

samples were collected again in September from these same 

sites in Oakland County; these samples were processed within 

the recommended holding time, and a change in the technique 

testing antibiotic resistances allowed for accurate quantification of antibiotic-resistant fecal coliform bacteria. In addition, 

reference samples were also collected from two sites outside 

of Oakland County representing significantly different land 

uses. 

E. coli and enterococci concentrations exceeded standards for recreational waters at several sites in August and 

September 2003. E. coli exceeded the Michigan single-sample 

standard for water quality at 7 of 14 sites in August and 8 of 

14 sites in September. Enterococci concentrations exceeded 

USEPA standards for recreational waters at 13 of 14 sites in 

August and 12 of 14 sites in September. None of the reference 

samples collected exceeded the recommend E. coli standards.

Fecal coliform bacteria and E. coli with resistance to 

ampicillin, cephalothin, and ceftriaxone and sensitivity to 

cefoxitin may carry extended-spectrum-β-lactamases (ESBLs). 

This is significant because ESBL-producing E. coli and other 

Enterobacteriaceae have been cited as a critical resistance in 

the healthcare setting. In most cases this type of resistance 

is acquired and transferable. This pattern was found in fecal 

coliform bacteria isolated from the following sites: Sashabaw 

Creek near Drayton Plains (04160800), Clinton River near 

Drayton Plains (04160900) and at Yates (04161810), Paint 

Creek at Rochester (04161540), Stony Creek near Romeo 

(04161580), River Rouge at Southfield (04166100), Kearsley 

Creek at Ortonville (04148035), and Huron River at Milford 

(04170000). This pattern was not found in the reference 

samples. 

The vanA gene in enterococci and class I integrons in 

E. coli indicate an acquired resistance with the ability to 

transfer this resistance to similar organisms. The vanA gene 

was detected in enterococci from Evans Ditch at Southfield 

(04166200) in August and Kearsley Creek at Ortonville 

(04148035) in September. Isolates with the vanA gene were 

identified as E. faecium. One type of acquired resistance found 

in the Enterobacteriaceae family is due to the acquisition of 

an integron. Integrons were detected in the E. coli isolates 

recovered from September FC cultures from River Rouge at 

Birmingham

Summary and Conclusions 21
(04166000) and Clinton River near Drayton Plains 

(04160900). Integrons have the capability of carry multipleantibiotic-resistance genes that could easily be transferred to 

other Enterobacteriaece organisms. These transferable genes 

(vanA and integrons) were not found in the reference samples. 

Multiple antibiotic resistances are often a result of transferable genetic elements. Multiple-antibiotic resistant fecal 

coliforms were isolated from every September sample. Only 

a selected number of E. coli isolates recovered from the FC 

culture were analyzed for the presence of integrons. However, 

fecal coliforms also may carry this element or other transferable elements that were not analyzed for in this study. In samples with isolates resistant to five or more antibiotics, the likelihood that this resistance is a result of a transferable genetic 

element is higher. There were six sites in Oakland County 

where fecal coliform isolates were resistant to five or six 

antibiotics: Upper River Rouge at Clarenceville (04166315), 

Stony Creek near Romeo (04161580), Paint Creek at Rochester (04161540), Clinton River at Yates (04161810), Sashabaw 

Creek near Drayton Plains (041610800), and Shiawassee River 

at Holly (04143830). The Washington Creek reference sample 

also had fecal coliform isolates resistant to five antibiotics. 

Most of the fecal coliform isolates tested in this study were 

presumed to be fecal coliform bacteria and were not further 

identified; therefore, the clinical significance of the antibiotic 

resistance cannot be determined. However, because multiple 

antibiotic resistances are an indication of acquired resistance it 

is possible that this resistance could be transferred to pathogens. 

Presumptive E. coli O157 was identified by three different methods. Sites in which all three methods (IA, GA, rfb0157

assay) identified potential E. coli O157 include 04161000 and 

04161810 in August. IA, the most sensitive assay, identified 

four other sites in August (04166000, 04166200, 04143830, 

and 04148035) and two other sites in September (04166000 

and 04166315) with potential for E. coli O157. Both the IA 

and rfb0157 assay were negative for all three reference samples. 

Only the reference site Au Sable River at Grayling (04135500) 

was positive for the GA. 

Virulence genes eaeA and stx2 have been reported to 

be associated with human disease. This gene combination 

was found in samples from the following sites: 04166000, 

04166200, 04160900, and 04143830 in August and September; 04166100 and 04166315 in August; and 04161540, 

044161580, and 04161810 in September. The eaeA and stx2 

gene combination was not found in any of the three tested 

reference samples. 

One site of interest was the River Rouge at Birmingham 

site (04166000). Pathogenic E. coli tests revealed a high likelihood that E. coli O157 was present. This water sample also 

contained the eaeA and stx2 genes. At this site, fecal coliform 

bacteria were also isolated that were resistant to cephalothin 

and ceftriaxone, and presumptive E. coli was also recovered 

that was resistant to cepahalothin, ceftriaxone, and cefoxitin, 

with intermediate resistance to gentamicin. In addition, E. coli

carrying an integron was detected at this particular site. The 

combination of potential pathogens, antibiotic resistances, and 

transferable genetics is of clinical significance. 

Very little data are available on the prevalence of clinically significant antibiotic-resistant bacteria and pathogenic E. 

coli in environmental waters. This study included two reference sites with much less urbanization than those in Oakland 

County. Antibiotic resistance was found at both of these sites; 

however, the pattern of resistance was much different. Further 

studies would be needed to determine whether the results 

seen in Oakland County are representative of what is typically 

seen in the environment or whether these results are typical of 

more urban/suburban areas. In addition, this study was unable 

to determine whether particular environmental factors (such 

as temperature and rainfall patterns, wildlife, and so forth) 

would influence study results. It may be possible to use DNA 

sequence information to determine the variants of stx2 genes 

found at these sites. Many studies have suggested that particular stx2 variants are more commonly associated with certain 

non-human host animals and that only some of the variants 

have been associated with human disease. 

Suggestions for Future Studies

Results of this study indicate that clinically significant 

antibiotic resistances and pathogenic bacteria are present in the 

waters of Oakland County. However, indications of multiple 

antibiotic resistances and of pathogenic E. coli were found 

in reference samples collected outside of Oakland County. 

Because very few reference samples were collected for this 

study, additional reference samples would need to be collected from other sites to better understand how the waters in 

Oakland County compare to other waters. Studies identifying 

sources of fecal contamination, seasonality of bacteria concentrations, changes in flow, and rainfall patterns, would help in 

understanding the extent of the problems that do occur. 

The threat that fecal indicator bacteria resistant to antibiotics poses to human health is not clearly known. However, the 

presence of transferable genetic elements in the environment 

does present a possibility that clinically-significant antibiotic 

resistance could be transferred to pathogens. Further studies 

investigating the occurrence of transferable antibiotic-resistance elements and their relation to other constituents such as 

antibiotics, metal, detergents, or other waste products would 

improve our understanding of the role the environment has on 

antibiotic resistances. In addition, an evaluation of particular 

acquired antibiotic-resistance genes, such as vanA, could 

determine how closely genes found in the environment are 

related to those of concern in the clinical setting. 

This study also has indicated potentially pathogenic E. 

coli are present in streams throughout Oakland County. The 

extent of the problem and threat to human health is unknown. 

Further studies addressing the occurrence of pathogenic E. 

coli markers (temporally and with streamflow changes) would 

improve our understanding. 

22 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Acknowledgments

We would like to thank the Oakland County Department 

of Human Services and Dr. Thomas Gordon, Director, for 

cooperation. Special thanks to Kathy Forzley and Barb Weberman with the Oakland County Human Health Division for 

their help and review of this report. 

References

Aarestrup, F.M., Butaye, P., and Witte, W., 2002, Nonhuman 

reservoirs of enterococci, in The Enterococci−Pathogenesis, 

Molecular Biology, and Antibiotic Resistance, Gilmore, 

M.S., ed.: American Society of Microbiology Press, Washington, D.C., p. 55−100. 

Aichele, S.S., Crowley, S.L., Taricska, C.K., and Stopar, J., 

2004, Water Resources Data, Oakland County, Michigan, 

2001−2004: U.S. Geological Survey Open File Report 

2004-1417, 75 p.

American Public Health Association, 1998, Standard Methods 

for the Examination of Water and Wastewater, 20th ed.: 

Washington D. C., American Public Health Association, p. 

9−1 to 9−76.

Ash, R.J., Mauck, B., and Morgan, M., 2002, Antibiotic 

resistance of gram-negative bacteria in rivers, United States: 

Emerging Infectious Diseases, v. 8, p. 713−716. 

Bass, L., Liebert, C.A., Lee, M.D., Summers, A.O., White, 

D.G., Thayer, S.G., and Maurer, J.J., 1999, Incidence and 

characterization of integrons, genetic elements mediating 

multiple-drug resistance, in avian Escherichia coli: Antimicrobial Agents and Chemotherapy, v. 43, p. 2925−2929.

Bird, C.B., and Hoerner, R.J., 2001, Reveal 8-hour test 

system for detection of Escherichia coli O157:H7 in 

raw ground beef, raw beef cubes, and iceberg lettuce 

rinse−Collaborative study: Journal of the Association of 

Analytical Communities International, v. 84, p. 720−735.

Boerlin, P., McEwen, S.A., Boerlin-Petzold, F., Wilson, J.B., 

Johnson, R.P., and Gyles, C.L., 1999, Associations between 

virulence factors of shiga toxin-producing Escherichia coli

and disease in humans: Journal of Clinical Microbiology, v. 

37, p. 497−503.

Bopp, D.J., Sauders, B.D., Waring, A.L., Ackelsberg, J., 

Dumas, N., Brau-Howland, E., Dziewulski, D., Wallace, 

B.J., Kelly, M., Halse, T., Musser, K.A., Smith, P.F., Morse, 

D.L., and Limberger, R.J., 2003, Detection, isolation, and 

molecular subtyping of Escherichia coli 0157:H7 and Campylobacter jejuni associated with a large waterborne outbreak: Journal of Clinical Microbiology, v. 37, p. 174−180.

Bradford, P.A., 2001, Extended-spectrum β-lactamases in the 

21st century: Characterization, epidemiology, and detection 

of this important resistance threat: Clinical Microbiology 

Reviews, v. 14, p. 933−951.

Carlton, B.C., and Brown, B.J., 1981, Gene Mutation, in 

Nester, E.W. ed., Manual of Methods for General Bacteriology: Washington, D.C., American Society for Microbiology, 

p. 222-−242.

Centers for Disease Control, 2003, National antimicrobial 

resistance monitoring system for enteric bacteria (NARMS) 

− 2001 Annual report: Atlanta, Ga., U.S. Department of 

Health and Human Services, p. 9−14.

Cetinkaya, Y., Falk, P., and Mayhall, C.G., 2000, Vancomycinresistant enterococci: Clinical Microbiology Reviews, v. 13, 

p. 686−707.

Diekema, D.J., BootsMiller, B.J., Vaughn, T.E., Woolson, 

R.F., Yankey, J.W., Ernst, E.J., Flach, S.D., Ward, M.M., 

Franciscus, C.L., Pfaller, M.A., Doebbeling, B.N., 2004, 

Antimicrobial resistance trends and outbreak frequency in 

United States hospitals: Clinical Infectious Disease, v. 78, p. 

78−85.

Dutka-Malen, S., Evers, S., and Courvalin, P., 1995, Detection 

of glycopeptide resistance genotypes and identification to 

the species level of clinically relevant enterococci by PCR: 

Journal of Clinical Microbiology, v. 33, p. 24−27.

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

stx2), intimin (eaeA), and Enterhemmorrahagic Escherichia 

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

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

Farmer, J.J., 2003, Enterobacteriaceae−Introduction and 

Identification, in Murray, P.R., Baron, E.J., Jorgensen, 

J.H., Pfaller, M.A., Yolken, R.H. (eds.), Manual of Clinical 

Microbiology, 8th ed.: Washington, D.C., ASM Press, p. 

636−653.

Fujisawa, T., Sata, S., Aikawa, K., Takahashi, T., Yamai, S., 

and Shimada, T., 2000, Modification of sorbitol MacConkey 

medium containing cefixime and tellurite for isolation of 

Escherichia coli O157:H7 from radish sprouts: Applied and 

Environmental Microbiology, v. 66, p. 3117−3118.

Gannon, V.P., D’Souza, S., Graham, T., and King, R.K., 1992, 

Rapid and sensitive method for detection of Shiga-liketoxin-producing Escherichia coli in ground beef using the 

polymerase chain reaction: Applied and Environmental 

Microbiology, v. 58, p. 3809−3815.

Geissler, K., Manafi, M., and Alonso, J.L., 2000, Quantitative 

determination of total coliforms and Escherichia coli in 

marine waters with chromogenic fluorogenic media: Journal 

of Applied Microbiology, v. 88, p. 280−285

Acknowledgments 23
Goñi-Urriza, M., Capdepuy, M., Arpin, C., Raymond, N., Caumette, P., and Quentin, C., 2000, Impact of an urban effluent 

resistance of riverine Enterobacteriaceae and Aeromonas

spp.: Applied and Environmental Microbiology, v. 66, p. 

125−132.

Harrison, K.G. (ed.), 2003, State of Michigan’s Environmental 

2003−Second Biennial Report, December 2003. Special 

Projects: Lansing. Mich., Michigan Department of Environmental Quality, p. 60−61.

Harwood, V.J., Whitlock, J., and Withington, V., 2000, Classification of antibiotic resistance patterns of indicator bacteria 

by discriminate analysis−use in predicting the source of 

fecal contamination in subtropical waters: Applied and 

Environmental Microbiology, v. 66, p. 3698−3704.

Harwood, V.J., Brownell, M., Perusek W., and Whitlock, 

J.E., 2001, Vancomycin-resistant Enterococcus spp. 

isolated from wastewater and chicken feces in the United 

States: Applied and Environmental Microbiology, v. 67, p. 

4930−4933. 

Holt, J.G., Kreig, N.R., Sneath, P.H., Staley, J.T., and Williams, S.T., 2004, Group 5 Facultatively anaerobic gramnegative rods, in Bergey’s Manual of Determinative Bacteriology ninth edition, Baltimore, MD, Williams and Wilkins, 

p. 175−290.

Huycke, M.M., Sahm, D.F., Gilmore, M.S., 1998, Multipledrug resistant enterococci−The nature of the problem and an 

agenda for the future: Emerging Infectious Disease, online 

accessed January 2005 at http://www.cdc.gov/ncidod/EID/

vol4no2/huycke.htm.

Kak, V., and Chow, J.W., 2002, Acquired antibiotic resistances 

in enterococci, in The Enterococci: Pathogenesis, Molecular 

Biology, and Antibiotic Resistance, Gilmore, M.S (ed.): 

Washington, D.C., American Society of Microbiology 

Press, p. 355−384.

Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., 

Zaugg, S.D., Barber, L.B., and Buxton, H.T., 2002, Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999−2000−A national reconnaissance: Environmental Science & Technology, v. 36, p. 

1202−1211.

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

R.L., 2002, Surveillance for waterborne-disease outbreaks − 

United States, 1999−2000: Morbidity and Mortality Weekly 

Report Surveillance Studies, 51(SS08); p. 1−28.

Lévesque, C, Piche, L, Larose, C, Roy, P.H., 1995, PCR mapping of integrons reveals several novel combinations of 

resistance genes: Antimicrobial Agents and Chemotherapy, 

v. 39, p. 185−191.

Malani, P.N., Kauffman, C.A., and Zervos, M.J., 2002, 

Enterococcal disease, epidemiology, and treatment, in The 

Enterococci: Pathogenesis, Molecular Biology, and Antibiotic Resistance, Gilmore M.S., (ed.), Washington, D.C., 

American Society of Microbiology Press, p. 385−408.

Maurer, J.J., Schmidt, D., Petrosko, P., Sanchez, S., Bolton, L., 

and Lee, M.D., 1999, Development of primers to O-antigen 

biosynthesis genes for specific detection of Escherichia coli

O157 by PCR: Applied and Environmental Microbiology, v. 

65, p. 2954−2960.

McArthur, J.V., and Tuckfield, R.C., 2000, Spatial patterns 

in antibiotic resistance among stream bacteria−effects of 

industrial pollution: Applied and Environmental Microbiology, v. 66, p. 3722−3726.

McDaniels, A.E., Rice, E.W., Reyes, A.L., Johnson, C.H., 

Hugland, R.A., and Stelma, G.N., 1996, Confirmational 

identification of Escherichia coli, a comparison of genotype 

and phenotype assays for glutamate decarboxylase and ß-Dglucuronidase: Applied and Environmental Microbiology, v. 

62, p. 3350−3354.

McDonald, L.C., Kuehnert, M.J., Tenover, F.C., and Jarvis, 

W.R., 1997, Vancomycin-resistant enterococci outside 

the health-care setting−prevalence, sources, and public 

health implications: Emerging Infectious Disease, v. 3, p. 

311−317.

McGowan, J.E, .Jr., and Tenover, F.C., 2004, Confronting 

bacterial resistance in healthcare settings−A crucial role for 

the microbiologist: Nature Reviews Microbiology, v. 2, p. 

251−258.

Nataro, J.P., and Kaper, J.B., 1998, Diarrheagenic Escherichia 

coli: Clinical Microbiological Reviews, v. 11, p. 142−201.

Nathisuwan, S., Burgess, D.S., and Lewis, J.S., 2001, 

Extended-spectrum-beta-lactamases−epidemiology, detection, and treatment: Pharmacotherapy, v. 21, p. 920−928.

National Antimicrobial Resistance Monitoring System Working Group, 2001 annual report online accessed January 

2005 at http://www.cdc.gov/narms/annual/2001/annual_

01.htm

National Antimicrobial Resistance Monitoring System Working Group, 2005, Accessed January 2005 at http://www.fda.

gov/cvm/index/narms/narms_pg.html

National Committee for Clinical Laboratory Standards, 2002, 

Performance standards for antimicrobial susceptibility testing; twelfth informational supplement, M-100-S12: v. 22, 

no. 1.

Osek, J., 2003, Development of a multiplex PCR approach for 

the identification of Shiga toxin-producing Escherichia coli

strains and their major virulence factor genes: Journal of 

Applied Microbiology, v. 95, p. 1217−1225.

24 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Park, J.C., Lee, J.C., Oh, J.Y, Jeong, Y.W., Cho, J.W., Joo, 

H.S., Lee, W.K., and Lee, W.B., 2003, Antibiotic selective 

pressure for the maintenance of antibiotic resistant genes 

in coliform bacteria isolated from the aquatic environment: 

Water Science and Technology, v. 47, p. 249−253.

Pérez- Pérez, F.J., and Hanson, N.D., 2002, Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates 

by using multiplex PCR: Journal of Clinical Microbiology, 

v. 40, p. 2153−2162.

Philippon, A., Guillaume, A., and Jacoby, G.A., 2002, 

Minireview−Plasmid-determined AmpC-type β-lactamases: 

Antimicrobial Agents and Chemotherapy, v. 46, p. 1−11.

Power, C.A., Johnson, R.P., McEwen, S.A., McNab, W.B., 

Griffiths, M.W., Usborne, W.R., and De Grandis, S.A., 

2000, Evaluation of the Reveal and Safepath rapid Escherichia coli O157 detection tests for use on bovine feces and 

carcasses: Journal of Food Protection, v. 63, p. 860−866.

Preston, K.E., Graffunder, E.M., Evans, A.M., and Venezia, 

R.A., 2003, Survey of plasmid-associated genetic markers in Enterobacteriaceae with reduced susceptibilities to 

cephalosporins: Antimicrobial Agents and Chemotherapy, v. 

47, p. 2179−2185.

Reinthaler, F.F., Posch, J., Feierl, G., Wust, G., Haas, D., 

Ruckenbauer, G., Mascher, F., and Marth, E. 2003, Antibiotic resistance of E. coli in sewage and sludge: Water 

Research, v. 37, p. 1685−1690.

Rice, L.B., 2001, Evolution and clinical importance of 

extended-spectrum-beta-lactamases: Chest, v. 119 (2 

Suppl), p. 391S−396S.

Rice, L.B., Sahm, D., and Bonomo, R.A, 2003, Mechanisms 

of resistance to antibacterial agents. in Murray, P.R., Baron, 

E.J., Jorgensen, J.H., . Pfaller, M.A, and Yolken, R.H. (eds.), 

Manual of Clinical Microbiology, 8th ed. Washington, D.C , 

ASM Press, p. 2098−2107.

Roe, M.T., Vega, E., and Pillai, S.D., 2003, Antimicrobial 

resistance markers of class 1 and class 2 integron-bearing Escherichia coli from irrigation water and sediments: 

Emerging Infectious Disease, v. 9, p. 822-826.

Rose, J.B., and Grimes, D.J., 2001, Reevaluation of microbial water quality−powerful new tools for detection and 

risk assessment: Washington, D.C., American Academy of 

Microbiology. p. 1−18.

Roy, P.H., 1999, Horizontal transfer of genes in bacteria: 

Microbiology Today, v. 26, p. 168−170.

Sabat, G., Rose, P., Hickey, W.J., and Harkin, J.M., Selective 

and sensitive method for PCR amplification of Escherichia 

coli 16S rRNA genes in soil: Applied and Environmental 

Microbiology, v. 66, p. 844−849.

Sarhan, H.R., and Foster, H.A., 1991, A rapid fluorogenic 

method for the detection of Escherichia coli by the production of beta-glucuronidase: Journal of Applied Bacteriology, 

v. 70, p. 394−400.

Taylor, D.E., 1999, Bacterial tellurite resistance: Trends in 

Microbiology, v. 7, p. 111−115.

Teixeira, L.M. and Facklam, R.R., 2003, Enterococcus, in 

Murray, P.R., Baron, E.J., Jorgensen, J.H., Pfaller, M.A, and 

Yolken, R.H. (eds.), Manual of Clinical Microbiology, 8th 

ed. Washington, D.C , ASM Press, p. 422−433.

Ug, A., and Ceylan, Ö., 2003, Occurrence of resistance to 

antibiotics, metals, and plasmids in clinical strains of 

Staphylococcus spp.: Archives of Medical Research, v. 34, 

p. 130−136.

U.S. Department of Agriculture, 2004, Accessed January, 

2005 at http://www.arru.saa.ars.usda.gov/

U.S. Environmental Protection Agency, 1976, Quality criteria 

for water: Washington, D.C., U.S. Environmental Protection Agency, Office of Research and Development, EPA 

440/9−76/023, p. 537

U.S. Environmental Protection Agency, 1986, Ambient water 

quality criteria for bacteria—1986: Washington, D.C., U.S 

Environmental Protection Agency, Office of Research and 

Development, EPA 440/5−84−-002, p. 118.

U. S. Environmental Protection Agency, 2000, Improved 

enumeration methods for the recreational water quality 

indicators−Enterococci and Escherichia coli: Washington 

D.C., U.S. Environmental Protection Agency, Office of Science and Technology: EPA 821−R−97−004, p. 1−48.

Walsh, C., 2003, Antibiotics actions, origins, resistance: 

Washington, D. C., American Society of Microbiology 

Press, p. 285−295.

Wiggins, B.A., Andrews, R.W., Conway, R.A., Corr, C.L., 

Dobratz, E.J., Dougherty, D.P., Eppard, J.R., Knupp, S.R., 

Limjoco, M.C., Mettenburg, J.M., Rinehardt, Sonsino, J., 

Toorijos, R.L., and Zimmerman, M.E., 1999, Use of antibiotic resistance analysis to identify nonpoint sources of fecal 

pollution: Applied and Environmental Microbiology, v. 65, 

p. 3483−3486.

Yang, S., and Carlson, K., 2003, Evolution of antibiotic occurrence in rivers through pristine, urban, agricultural landscapes: Water Research, v. 37, p. 4645−4656.

Zadik, P.M., Chapman, P.A., and Siddons, C.A., 1993, Use 

of tellurite for the selection of verocytotoxigenic Escherichia coli O157: Journal of Medical Microbiology, v. 9, p. 

155−158.

References 25
Glossary

Acquired antibiotic resistance − A resistance to an antibiotic 

as a result of a genetic mutation or acquisition of a gene(s) 

that causes resistance in an otherwise sensitive organism. 

Clinically significant antibiotic resistances − Antibiotic 

resistances that include pathogens resistant to those antibiotics used to treat an infection or genes responsible for 

resistance that can be transferred to pathogens.

ENT cultures − Preserved stock of all isolates capable of 

growing on mEI media at 41.5°C from 100 mL of filtered 

water.

FC cultures − Preserved stock of all isolates capable of growing on mFC media at 44.5°C from 100 mL of filtered water.

GA − Assayed based on growth on sorbitol MacConkey 

medium containing cefixime and tellurite (CT-SMAC).

IA − Immunological assay based on the presence of an antigen 

specific to the E. coli O157group.

Integron − Transferrable genetic elements that may carry 

multiple genes including genes responsible for antibiotic 

resistance.

Intrinsic antibiotic resistance − An inherent resistance not 

caused by exposure to the antibiotic or acquired genes; 

rather, it is caused by the biology of the organism. Intrinsic 

resistance is not transferable to other bacteria.

Isolates − Individual colony forming unit.

Multiple-antibiotic-resistant bacteria − Bacteria resistant to 

two or more antibiotics.

Nosocomial − Infections acquired while in the hospital.

Pathogenic bacteria − Bacteria capable of causing disease in 

humans or animals.

Serotype − A serological characterization of a microorganism at the species level based on the organism’s antigenic 

properties.

Transferable genetic elements − Plasmids, transposons, integrons, or genes that may carry genetic sequences responsible for antibiotic resistance and that can be transferred to 

other organisms. 

26 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Appendix A. 

Fecal indicator bacteria concentrations in river-water samples collected from Oakland County, Mich., in August and September 

2003.

Appendix A 27
USGS 

Station 

Number

Fecal coliforms CFU/100 

mL (P31616)

E. coli

CFU/100 mL

(P50278) 

Percentage of fecal 

coliforms that are 

E.coli

Enterococci

CFU/100 mL

(P90909)

Aug Sept Aug Sept Aug Sept Aug Sept

River Rouge watershed

04166000 2,000 D 910 D 1,000 D 910 D 50 100 852 D 420 D

04166100 5,400 D 3,300 D 1,500 D 350 D 28 11 596 D 1,380 D

04166200 126,000 D 80 25,000 D 50 20 63 1,000 D 350 D

04166315 5,400 D 1,400 D 1,500 D 1,300 D 28 93 1,120 D 1,600 D

Clinton River watershed

04160800 430 D 1,040 D 120 D 220 D 28 21 104 D 300 D

04160900 3,700 D 450 D 1,700 D 430 D 46 96 900 D 470 D

04161000 1,620 D 1,030 D 110 D 320 D 7 31 102 D 400 D

04161540 3,500 D 480 D 1,500 D 470 D 43 98 >1,000 D 350 D

04161580 990 D 360 D 270 D 260 D 27 72 126 D 95 D

04161810 3,400 D 570 D 1,800 D 310 D 53 54 1,060 D 69 D

Other Oakland County watersheds

04143830 980 D 290 D 140 D 100 D 14 34 210 D 390 D

04148035 2,400 D 600 D 300 D 500 D 13 83 422 D 650 D

04170000 190 D 33 20 23 11 70 70 35

04170500 6 D 3 3 3 50 100 2 13

Reference sites

04135500 320 D 36 290 D 34 91 94 5 14

04001000 NT 380 D NT 260 D NT 68 NT 0

Appendix A. Fecal indicator bacteria concentrations in river-water samples collected from Oakland County, Mich., in August and 

September 2003.

[Aug, August; Sept, September; E. coli, Escherichia coli; CFU, colony forming units; mL, milliliter; NT, not tested; D, quantification based on dilution]

28 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Appendix B. 

Field measurements at sites in Oakland County, Mich., in August and September 2003.

Appendix B 29
USGS station 

number Date sampled pH

Water 

temperature 

(°C)

Specific 

conductance 

(μS/cm)

Dissolved 

oxygen (mg/L) Discharge (ft3/s)

04143830 8/5/2003 8.06 25.79 636 7.38 9.7

04148035 8/5/2003 8.07 20.39 868 8.40 6.1

04160800 8/5/2003 7.99 23.46 707 6.60 2.2

04160900 8/5/2003 7.58 22.47 933 6.67 3.0

04161000 8/5/2003 7.73 22.32 1,164 9.01 21.0

04161540 8/5/2003 8.01 19.96 710 8.22 12.0

04161580 8/5/2003 8.34 23.46 700 8.50 3.2

04161810 8/5/2003 8.19 22.04 913 9.06 62.8

04166000 8/5/2003 8.08 22.50 1,230 7.33 9.8

04166100 8/5/2003 7.85 20.38 1,254 6.81 17.0

04166200 8/5/2003 7.67 20.65 743 4.73 3.2

04166315 8/5/2003 7.98 20.08 1,378 7.08 5.9

04170000 8/5/2003 7.66 21.86 866 6.10 31.0

04170500 8/5/2003 8.03 24.50 779 7.17 33.0

04143830 9/9/2003 7.58 19.55 611 6.17 4.5

04148035 9/9/2003 7.99 14.76 878 8.45 4.2

04160800 9/9/2003 8.13 18.52 751 8.33 0.6

04160900 9/8/2003 7.55 21.30 918 5.60 4.2

04161000 9/8/2003 7.90 21.85 1,038 9.65 21.0

04161540 9/9/2003 8.25 16.42 872 10.36 ND

04161580 9/9/2003 8.28 19.78 643 9.56 1.70

04161810 9/9/2003 8.49 19.33 1,016 10.28 33.5

04166000 9/8/2003 8.30 20.95 1,211 8.46 4.2

04166100 9/8/2003 8.01 17.51 1,394 8.20 11.0

04166200 9/8/2003 7.91 18.05 3,040 6.60 1.40

04166315 9/8/2003 8.18 17.57 1,451 9.02 3.7

04170000 9/8/2003 7.66 19.57 875 6.51 27.0

04170500 9/8/2003 8.01 22.55 774 8.49 22.0

Appendix B: Field measurements at sites in Oakland County, Mich., in August and September 2003.

[°C, degrees Celsius; μS/cm, microsiemens per centimeter; mg/L, milligrams per liter; ft3/s, cubic feet per second; ND, not determined]

30 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Appendix C. 

N 

collected in Oakland County, Mich.

Appendix C 31
Fecal coliform antibiotic resistance (CFU/100 mL)1 Enterococci antibiotic resistance (CFU/100 mL)

USGS 

station 

numberTotal fecal coliform isolatescefoxitin1 32 μg/mLceftriaxone 64 μg/mLcephalothin1 32 μg/mLampicillin1 32 μg/mLtetracycline 8 μg/mLgentamicin 16 μg/mLstreptomycin 64 μg/mLTotal enterococci isolates vancomycin 8 μg/mLvancomycin 16 μg/mLstreptomycin 2000 μg/mLgentamicin 100 μg/mLtetracycline 16 μg/mL

Rouge River watershed

04166200 25,000 + 1,000 + + + + 5,000 >1,000 6 2 2 2 104

04166000 1,000 + 300 + + 100 0 0 844 6 0 0 0 64

04166315 1,500 + 800 + + 80 900 100 1,096 0 0 40 92 146

04166100 1,500 + 200 + + 700 400 100 596 0 0 8 0 54

Clinton River watershed

04161580 270 + 90 + + 190 110 0 118 4 0 0 8 34

04160900 1,700 + 1,600 + + + + 0 906 2 0 0 2 68

04161540 1,500 + 600 + + 400 600 0 >1,000 0 0 14 2 90

04161810 1,800 + 1,700 + + 600 1,600 400 1,032 0 0 2 36 56

04161000 110 + 40 + + + + 90 100 2 0 2 14 6

04160800 120 + 80 + + + + + 96 0 0 16 38 58

Other Oakland County watersheds

04148035 300 + 20 + + 10 10 0 416 0 0 0 0 56

04143830 140 + 90 + + 190 110 0 206 0 0 0 26 2

04170500 3 + 2 + + 2 0 0 2 4 0 2 2 2

04170000 20 + 20 + + 160 460 0 70 2 0 0 0 4

Reference sites

04135500 290 + 160 + + 70 60 0 5 4 0 0 0 0

04001000 NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Appendix C. Number of fecal coliform and enterococci isolates resistant to tested antibiotics that were isolated in August 2003 from 

samples collected in Oakland County, Mich.

[CFU, colony forming unit; mL, milliliter; μg, microgram; +, positive result; >, greater than; NA, not available]

1 + Indicates plates were overgrown and bacteria colonies could not be counted.

32 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Appendix D. 

samples collected in Oakland County, Mich.

Appendix D 33
Percent fecal coliform antibiotic resistance Percent enterococci antibiotic resistance

USGS 

station 

numberTotal. fecal coliform isolatescefoxitin1 32 μg/mLceftriaxone 64 μg/mLcephalothin1 32 μg/mLampicillin1 32 μg/mLtetracycline 8 μg/mLgentamicin 16 μg/mLstreptomycin 64 μg/mLTotal enterococci isolates vancomycin 8 μg/mLvancomycin 16 μg/mLstreptomycin 2000 μg/mLgentamicin 100 μg/mL

Rouge River watershed

04166000 50 6 4 10 50 NA 0 2 42 7 0 0 0

04166100 50 4 4 100 12 NA 2 2 50 0 0 0 4

04166200 8 88 63 63 38 0 0 13 35 0 0 0 0

04166315 14 57 29 100 33 NA 0 50 50 6 0 2 0

Clinton River watershed

04160800 50 46 30 100 1 44 30 12 30 33 7 0 3

04160900 45 44 16 98 16 56 4 11 47 9 0 4 0

04161000 50 64 0 98 100 NA 0 0 39 3 0 8 0

04161540 48 29 54 100 2596 15 23 13 35 20 9 6 0

04161580 36 86 100 97 100 72 39 69 50 2 0 2 0

04161810 50 56 14 88 76 24 32 6 50 8 0 0 0

Other Oakland County watersheds

04143830 29 83 3 100 97 21 17 10 39 15 8 0 0

04148035 50 24 8 84 56 NA 12 2 50 22 8 0 4

04170000 33 45 6 73 73 0 0 24 35 6 0 0 0

04170500 3 100 100 0 0 0 0 0 13 38 15 0 0

Reference sites

04001000 30 23 0 97 23 27 20 27 0 NA NA NA NA

04135500 5 20 0 40 0 60 0 20 14 7 0 0 0

Appendix D. Percentage of fecal coliform and enterococci isolates resistant to tested antibiotics that were isolated in September 2003 

from samples collected in Oakland County, Mich.

[CFU, colony forming unit; μg, microgram; mL, milliliter; NA, not available]

34 Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic Escherichia coli
Lisa R. Fogarty, Joseph W. Duris, and Stephen S. Aichele—Preliminary Survey of Antibiotic-Resistant Fecal Indicator Bacteria and Pathogenic

Escherichia coli from River-Water Samples Collected in Oakland County, Mich., 2003—Scientific Investigations Report 2005-5058

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