**Methods and Techniques Used in Microbial Source Tracking Studies in
Missouri**

[Miya N. Barr]{.underline}

Missouri Water Science Center

U.S. Geological Survey

The Missouri Water Science Center has been conducting microbiology
source tracking (MST) studies since 1998. The methods and techniques
applied to the studies have changed over the years. Advances in
technology and the underlying science involved have aided scientists in
identifying sources of *Escherichia coli* (*E. coli*) in water bodies
across the State.

In the early stages of source tracking in Missouri, studies focused
mainly on determining if the source of *E. coli* was human or non-human.
Most data were obtained by performing ribotyping studies. A ribotype
study links a source of bacteria directly by using DNA. Because many
warm-blooded animals have unique and identifiable DNA ribopatterns, a
library of sources within a study area is one of the most important
tools in MST studies.

A study in the Blue River Basin near Kansas City, Missouri and Kansas,
beginning in 1998, tracked *E. coli* through ribotyping or genetic
fingerprinting, using a library of 900 combined isolates of dog, goose,
and human. Some variability existed in the results because only three
classes were used in classification; other classes, such as pig and
turkey, have similar banding patterns as the primary sources in the
study.

Beginning in 1999, a study in the Upper Shoal Creek Basin used
ribopattern analyses to determine human or non-human *E. coli* sources.
During 2000, a second-level test was performed with five source classes:
human, horse, cattle, turkey, and chicken. The library used in the basin
analysis contained less than 300 known sources. Most sources used for
cattle were obtained outside the study area, which caused uncertainty in
the data.

From 2001 to 2002, the Shoal Creek Basin analysis was changed to the
more technically advanced repeat element Polymerase Chain Reaction
(rep-PCR) to determine sources of *E. coli* using two classes: human and
non-human. As the rep-PCR technology advanced, a five-class comparison
was later performed using cattle, chicken, horse, human, and turkey.

Rep-PCR also was used in classifying bacteria sources in the Little Sac
River Basin from 1999 to 2001. A library of cattle, horse, and human
were used. The library was limited; however, the results were consistent
with the land uses in the study area.

An MST study was conducted on the Jacks Fork River from 1999 to 2004.
Ribotyping analyses were used during Phase II of the study to determine
the sources of the *E. coli*. Sewage, horse, and cattle were the three
classes that composed the basin library. An analysis of stream sediment
also was conducted, but was unsuccessful. During Phase II of the study,
a library containing sewage, horse, and cattle isolates from the Jacks
Fork Basin was developed, and rep-PCR was used in determining the three
classes. The increase of local sources within the library will aid in
the positive identification of *E. coli* sources in water samples from
the study area.

Currently (2005), a rep-PCR study is being conducted in the St. Louis,
Missouri and Illinois area. A library is being built, but difficulty
exists in determining the breakdown of classes among the non-human
sources, because only parts of three major river basins are contained in
the study area. Sources of the bacteria could come from upstream in each
basin outside the study area. The only "safe" way classification at this
time is human and non-human.

**Laboratory Support for Microbiological Monitoring Projects in the U.S.
Geological Survey**

> [Rebecca N. Bushon,]{.underline} Donna S. Francy, Amie M.G. Brady, and
> Donald M. Stoeckel
>
> U.S. Geological Survey
>
> Columbus, OH

The U.S. Geological Survey (USGS) Ohio District Microbiology Laboratory
(ODML) provides support for several projects by analyzing surface- and
ground-water samples for microorganisms of public health significance.
These projects range from routine monitoring to investigating processes
and factors that affect pathogens and indicator organisms in the
environment. For example, the ODML is partnering with the National Park
Service to identify a rapid method for the detection of fecal-indicator
bacteria so that park managers can supply daily information on the
safety of recreational waters. The ODML is also working with local
agencies to investigate the distribution and sources of *Escherichia
coli* in sediments and the use of statistical models to predict the
quality of recreational waters. A variety of microbiological
source-tracking techniques are currently being used by the ODML in
several settings, from studies of septic tank influences on ground water
to investigations of contaminant sources near Ohio beaches. The
occurrence and factors related to the presence of enteric viruses and
indicators in aquifers serving small public water supplies was recently
studied in a ground-water monitoring project. In another project,
stream-water samples were collected to investigate the occurrence of
waterborne pathogens and how they relate to concentrations of indicator
organisms.

The ODML supports these projects, as well as other microbiological
monitoring projects within the USGS nationally. The ODML analyzes for
fecal-indicator bacteria including total coliforms, fecal coliforms,
*Escherichia coli*, enterococci, and *Clostridium perfringens*.
Analytical methods are also available for viral indicators (coliphage)
and viral pathogens, such as enterovirus and hepatitis A virus, and two
protozoan pathogens, *Cryptosporidium* and *Giardia.* These
microorganisms and the methods used to detect them are described in the
poster.

**Rapid method for the determination of fecal-indicator bacteria
concentrations in recreational water**

[Rebecca N. Bushon]{.underline} and Amie M.G. Brady

U.S. Geological Survey

Columbus, OH

Standard plating methods to determine fecal-indicator bacteria
concentrations take 24 hours to obtain results―too long to adequately
assess water quality and take control measures. In a 3-year study
(2004--2006), the U.S. Geological Survey (USGS), in collaboration with
the National Park Service, is investigating the use of a rapid method
(\<1 hour) for the determination of fecal-indicator bacteria
concentrations in the Cuyahoga River within the Cuyahoga Valley National
Park, Ohio. The overall goal of this study is to determine whether the
rapid method yields accurate enough results that it can be used by park
managers in place of standard methods.

The immunomagnetic separation/adenosine triphosphate (IMS/ATP) rapid
method requires less than 1 hour from sample collection to availability
of results. In the original method, developed by researchers at the
University of Michigan, bacteria are first concentrated from the water
sample by serial filtration. Magnetic beads that are coated with
antibodies for either *Escherichia coli* (*E. coli*) or enterococci are
added to the concentrate. This mixture is then subjected to IMS, in
which the bacteria-antibody bead complex is separated from extraneous
materials in the sample by use of a magnet. The bacterial cells are then
ruptured and ATP is released and measured with a microluminometer.
Results are reported in relative light units (RLUs).

Daily samples, collected at three sites along the Cuyahoga River from
May through August 2004, were analyzed by use of the rapid and standard
methods for both *E. coli* and enterococci. Standard plating methods
included the use of modified mTEC media for the enumeration of *E. coli*
and mEI media for enterococci. Throughout the sampling period,
modifications were made to the original rapid method, including
elimination of the filtration step and the addition of sonication of the
water sample prior to IMS. The elimination of the filtration step
increased the method's performance and strengthened the correlations to
the standard methods. RLUs were significantly correlated to
concentrations found by use of standard methods for *E. coli* (r=0.704)
and enterococci (r=0.759). Further data analysis is needed before
determining whether the addition of sonication significantly increases
the correlations. Sampling is done bimonthly from October 2004--May 2005
and will be done daily during the recreational season of 2005
(June--September).

**Persistence and Growth Potential of *Escherichia coli* in Temperate
Forest Soils of a Great Lakes Coastal Watershed**

[M. N. Byappanahalli,]{.underline} D. A. Shively, and R. L. Whitman

Lake Michigan Ecological Research Station

U.S. Geological Survey

Porter, Indiana

The capabilities of *E. coli* to colonize, persist, and grow in forest
soils of a Lake Michigan coastal watershed were studied. *E. coli*
population recovery occurred within two weeks after hot water treatment
(88°C) of soil, and counts remained high ([\>]{.underline}1.0 X 10^2^ g
soil^-1^) for a year. Undisturbed, forest soils within six randomly
selected 0.5 m^2^ exclosure plots (covered by netting of 2.25 mm^2^ mesh
size) were monitored from April through October 2003 for *E. coli* in
order to describe the numerical and population characteristics of this
bacterium. *E. coli* occurred in 88% of the samples collected, with
overall mean counts (log MPN g dry soil^-1^) of 1.2 [+]{.underline} 0.1
SE, *n*=66. *E. coli* counts did not correlate with substrate moisture
content, air, or soil temperatures, suggesting that seasonality was not
a critical factor in population density control. *E. coli* distribution
within the exclosure was significantly higher than in immediate
surrounding area (*P* = 0.03, *n*= 60). *In vitro* studies at 35°C
showed that *E. coli* counts increased by 2 logs after 72 h in unamended
moist soil, and by 2-6 logs in 24 h in amended (bile salts, inorganic
fertilizer, or glucose) soils. These findings suggest that *E. coli* can
occur and persist for extended periods in undisturbed forest soils
independent of external inputs and season, and it may even grow in these
soils under certain conditions. Therefore, in assessing *E. coli*
sources within a stream, it is important to differentiate background
soil loadings from inputs derived from animal and human contamination.

**Modification of a numerical model to incorporate sediment flux of
fecal bacteria in a river**

[Tom Byl^1^]{.underline}, James Davis, III , Tiffany Hines, John Brew,
John Finke, and Lonnie Sharpe

^1^Dept. of Civil and Environmental Engineering, Tennessee State
University and

U.S. Geological Survey

Nashville, TN

Fecal pollution in surface waters is a serious water-quality problem. As
a result, scientists have developed a number of models in an attempt to
predict the fate and transport of fecal pollution in riverine systems.
Various models predict the rate of bacteria removal from the water
column based on density, settling rates, and water velocity. Such
models, however, do not consider survival and reproduction of bacteria
in sediments, or resuspension. Flume and stream experiments were
conducted to measure the survival, reproduction, and resuspension of
fecal bacteria in sediments. These results can be used to modify a
numerical model by incorporating survival of bacteria in bed sediments
and resuspension into the water column, in addition to other parameters
such as water velocity, initial bacteria concentration, and settling
rate. *E. coli* and *Klebsiella* were introduced into the
circulating-water flume at known concentrations and monitored as they
settled or remained suspended. Bacteria concentrations were measured in
the water column and the sediment along the flume to determine bacterial
fate and transport. The model accurately predicted bacteria settling
from the water column. The sediment fecal bacteria population declined
at an exponential rate over several weeks (experimental decay value =
-0.2735). This decomposition rate was coupled to the numerical model,
and additional tests were done in a small stream contaminated with fecal
coliform. The results of the numerical model and the field studies were
in good agreement for rates of settling (within 5 percent). Comparison
of the model-predicted bacteria resuspension and field-measured bacteria
resuspension were not in close agreement due to irregular resuspension
of bacteria-contaminated sediments. Additional work is needed to
understand factors that control resuspension of sediments, such as
organic content and other physical parameters.

**Enteric pathogen, fecal indicator and real-time environmental data at
inland beaches in Madison, Wisconsin: Monitoring and predictive
modeling.**

Robert J. Waschbusch, [Steven R. Corsi]{.underline}, Kirsti Sorsa, Jon
H. Standridge, and Tommye Schneider

U.S. Geological Survey

Middleton, WI

A comprehensive project was undertaken to study beach water quality at
three inland beaches in Madison, WI. Innovative monitoring tools were
used to determine microbial, physicochemical, and climatological
parameters and for detecting and communicating potential health risks to
beach users. A total of 436 samples were collected for fecal coliform,
*E. coli*, and enterococci, 231 samples were collected for F+ coliphage
quantitation and serotyping, 105 samples were collected for pathogen
analysis including *Salmonella, Cryptosporidium* and *Giardia,* and 152
samples were collected for *E. coli* 0157:H7*,*. Automated instruments
were used to collect water samples as well as real-time data on
rainfall, wind speed and direction, wave height, water temperature,
turbidity, dissolved oxygen, air temperature, and solar radiation.
Samples were collected on a fixed schedule and during event periods
(including rainfall events, high turbidity events, and high wave height
events). Pathogens were detected in 37% of samples collected for
pathogen analysis. Of the pathogens monitored, *Giardia* was present
most often followed by *Cryptosporidium* and then *Salmonella*. The
presence of *E. coli* O157:H7 was confirmed in only one sample.
Pathogens were present more often during event periods than in samples
collected on a fixed schedule. Additionally, fecal coliform and *E.
coli* concentrations were greater during event periods than in samples
collected on a fixed schedule. Results of F+ coliphage analysis
indicated that both DNA and RNA plaques were present throughout the
study period. Of the RNA plaques, all four of types I through IV were
present at some time during the study period. These results imply that
both human and nonhuman sources are likely to influence these beaches.
Statistical analysis was conducted to explore relations between
pathogens and microbial indicators, between pathogens and real-time
environmental parameters, and between microbial indicators and real-time
environmental parameters. Of these three analyses, a statistically
significant relation was found only between *E. coli* and real-time
environmental indicators. There was no significant relation between
pathogen occurrence and microbial indicators or between pathogen
occurrence and real-time environmental indicators. From this, a
statistical model was developed to predict exceedance of *E. coli*
thresholds. Important parameters for this model included antecedent
precipitation, the Universal Soil Loss Equation erosivity index,
specific conductance, water temperature, wave height, air temperature,
and wind speed and direction.

**Evaluation of *Bacteroides-Prevotella* Communities for Use in Fecal
Source Tracking Studies**

[Lisa. R. Fogarty]{.underline}, Diana L. Clough, Mary A. Voytek\
US Geological Survey-National Research Program

Reston, VA.

Current methods for detecting and for determining sources of fecal
contamination rely on cultivation of fecal coliforms, *E. coli*, and/or
enterococci. These groups of bacteria constitute a relatively small
percentage of the fecal flora, and cultivation methods may bias the
populations by selection, thus misrepresenting the source populations.
The *Bacteroides-Prevotella* group has recently been proposed as an
alternative. Each of these species represents a much larger proportion
of the fecal population than the fecal coliform and enterococci group.
They can also be detected by molecular methods and therefore could be
more sensitive indicators and source determinants of fecal
contamination. The variability in fecal bacterial communities within a
fecal sample, amongst fecal samples from individuals from the same
source species, or between different source species is poorly
understood. In this study, terminal restriction fragment length
polymorphism (T-RFLP) was used to evaluate differences in the
*Bacteroides-Prevotella* communities in fecal material from mammal and
avian source species and from waters contaminated with fecal material.
To determine spatial variation of bacterial populations, multiple
samples were collected from different geographical regions. DNA
extracted from the samples was amplified by PCR using published primers
for the *Bacteroides-Prevotella* group. T-RFLP fragments from different
enzymes were analyzed and compared using discriminant analysis to
develop criteria to distinguish source groups. This resulted in a set of
T-RFLP peaks that were more specific to particular fecal source groups.
In addition, markers previously identified using similar techniques were
evaluated with fecal samples collected for this study. Results of this
analysis showed that the cow specific marker accurately identified all
tested cow samples but the human marker failed to identify 80% of the
human samples tested. Results of this study not only indicate
differences in *Bacteroides-Prevotella* populations that occur between
source species but also suggest additional markers with potential to
identify sources of fecal contamination.

**Source-tracking tools for understanding fecal contamination and
predicting water quality at two Lake Erie Beaches**

[Donna S. Francy^1^]{.underline}, Rodney A. Sheets, and John Rhoades

^1^U.S. Geological Survey

Columbus, OH

Multiple lines of evidence and source-tracking methods were used to help
identify sources of fecal contamination at two Lake Erie
beaches---Edgewater, in Cleveland, Ohio; and Lakeview, in Lorain, Ohio.
In several field studies at Edgewater, investigators determined the
spatial distribution of *Escherichia coli* (*E. coli)* in nearshore
surveys and in lake-water samples collected within the bathing area.
Temporary shallow observation wells (piezometers) were installed at
Edgewater to determine the direction of ground-water flow and *E. coli*
concentrations in foreshore sands. At Lakeview, investigators tested the
use of multiple antibiotic resistance (MAR) patterns of *E. coli*
isolates as a source-tracking tool. Because humans are exposed to
antibiotics, the *E. coli* they harbor will be more resistant to
antibiotics than those *E. coli* found in the gastrointestinal tracts of
wild birds.

The results from nearshore surveys and lake-water sampling at Edgewater
indicate that fecal contamination is most likely of local origin.
Shallow ground-water flow directions were toward the lake during two
piezometer studies. The absence of *E. coli* in shallow ground water
\>50 ft inland and *E. coli* spikes at seemingly random locations
further indicates a local source; this local source may be a surface
source, such as bird excrement. The use of MAR patterns to distinguish
between contamination from humans and waterfowl at Lake Erie beaches
proved to be a promising method. In a small sampling at Lakeview, MAR
indexes were able to distinguish gull fecal samples from sewage samples,
and the MAR index for a bathing-water sample was similar to that of the
gull fecal samples.

The Great Lakes Beach Association: Communicating Beach Science and
Management Issues

[Sheridan K. Haack^1^]{.underline} and Richard L. Whitman

^1^US Geological Survey

Lansing, MI

During the Great Lakes Beach Conference, Chicago, IL, in 2000, a
consensus emerged that more communication and coordination was needed
among health, management and government recreational water quality
professionals. In the following year, the Great Lakes Beach Association
was founded to bring together beach managers, scientists, agencies,
officials and private groups to exchange information and improve
recreational water quality in the Great Lakes region. This is an
informal association, with no annual dues. The Association hosts an
e-mail listserve, a web page (www.great-lakes.net/glba), and an annual
meeting. There are currently over 300 members for the listserve. At the
most recent Annual Meeting in Parma, OH, there were over 100 attendees.
Each Great Lakes state and the regional beach coordinator is represented
on the board plus representatives of Canada, and of U. S. Federal,
county and municipal agencies. Partners include municipal treatment
facilities, health departments, planning commissions, federal
researches, park districts and private industry. The Great Lakes Beach
Association is the first organization of its type and a model for
communication between scientists, beach managers and regulatory
agencies. The history, structure and function of the Great Lakes Beach
Association, and the involvement of Federal, State and local entities,
will be discussed.

**Evaluation of real-time RT-PCR for detection of enterovirus and
hepatitis A virus in East Tennessee ground water**

[Trisha B. Johnson^1^]{.underline}, Larry D. McKay, and Alice C. Layton,
and G. Shay Fout

^1^University of Tennessee

This project involves development, validation testing and application of
a fast, efficient molecular method of measuring occurrence and
concentration of common human viral pathogens, enterovirus and hepatitis
A virus, in ground water samples using real-time reverse
transcription-polymerase chain reaction. Development of the real-time
RT-PCR assays for enterovirus and hepatitis A virus are complete and
they are being verified by comparison with cell culture assays and
traditional RT-PCR carried out at the U.S. EPA National Exposure
Research Laboratory in Cincinnati, OH. The application phase of the
project includes monitoring viral occurrence and concentration in ground
water samples from 8 community water supply wells and springs in karst
settings in east Tennessee. The wells/springs were chosen on the basis
of prior monitoring of fecal indicator bacteria and geochemical
parameters, as well as the presence or absence of likely input sources
of human sewage. The wells/springs used for sampling include four sites
that have a relatively high likelihood of containing human pathogens and
four that have a low likelihood of containing human pathogens. Sampling
for viruses took place between March and August 2004. These are expected
to be the first measurements of viral occurrence in ground water in the
state of Tennessee.

**Use of rep-PCR to detect off-site transport of fecal bacteria in
small, on-site septic systems through Ohio soils**

[Christopher Kephart]{.underline}

U.S. Geological Survey

Columbus, OH

Two studies were done to evaluate the effectiveness of fecal-indicator
organism removal at household sewage treatment systems (HSTS) in Ohio
soils. The working design parameter for septic systems in Ohio is that
fecal-indicator microorganisms are removed within the first two feet of
leach field. Although this assumption is based on bacterial travel
through unsaturated soil, in practice the design parameter is generally
applied to permitted leach-field-type septic systems. Deep perimeter
curtain drains are sometimes installed to lower the water table and
extend the zone of unsaturated soil.

An initial study, involved the sampling of piezometers that were
installed adjacent to HSTS at various horizontal spacings (5-23 feet)
from the leach line and at various vertical increments (4-8, 8-12, 12-16
feet) from land surface. Water samples from the piezometers were
analyzed for the presence of *Escherichia coli*, a fecal-indicator
bacterium. When possible, the curtain drain also was sampled. *E. coli*
subtypes were discriminated by rep-PCR genotyping. Subtypes detected in
sampling wells were compared with subtypes present in the septic tank
and in animal feces at the land surface to provide evidence of origin.
Congruence of genetic information from *E. coli* collected at the
primary source and test locations was observed at all three sites
tested. Specifically, *E. coli* rep-PCR fingerprints from subsurface
flow 4-8 feet deep and up to 5 feet laterally from the septic leach
field and effluent curtain drains located 10 feet from the leach lines
indicated transport of *E. coli* beyond the 2-foot treatment zone.

A subsequent study further evaluated the occurrence of potentially
pathogenic wastewater in curtain drains at traditional leach-line and
evapotranspiration (ETA) septic systems in Medina County, Ohio. Samples
were collected from the septic tank and curtain drain at 10 sites for
*E. coli* characterization using rep-PCR, just as in the previously
mentioned study. Congruence of genetic information supported the
hypothesis that individual septic systems were the source of *E. coli*
detected in some curtain drain effluents.

**Fixed and Random Factors Affecting *E. coli* Results at Southern Lake
Michigan Beaches**

[Meredith B. Nevers]{.underline} and Richard L. Whitman

Lake Michigan Ecological Research Station

U.S. Geological Survey

Porter, Indiana

The southern Lake Michigan shoreline beaches are recreationally and
economically important to the region, so managers depend on the
reliability of their *E. coli* monitoring program to ensure healthy
recreational opportunities. Studies undertaken at Indiana and Illinois
beaches have revealed numerous random and fixed factors that influence
the outcome of *E. coli* results taken as part of routine beach
monitoring. Abundant *E. coli* in the nearshore sand, often as high as 4
log/100 ml at 63^rd^ Street Beach in Chicago, strongly influenced its
counts in the beach water. In addition to the sand acting as a source of
*E. coli* to the water, amassed *Cladophora* algae and shoreline birds
may influence *E. coli* counts in water. Location of a beach within the
broader region may also affect overall *E. coli* counts. Fixed factors
including time of sampling also directly influenced *E. coli* results,
with lower *E. coli* densities in afternoon samples than in morning
samples (p\<0.01) as a result of inactivation by sunlight. Also, the
number and location of replicate samples greatly influences the outcome
of *E. coli* monitoring; at the subject Chicago beach, replicates
collected across the entire 500-m beach were far more variable than
replicates collected intensively at one fixed sampling site. Ongoing
research along southern Lake Michigan beaches focuses on developing and
calibrating predictive models as an alternative to current techniques in
order to minimize error and increase reliability.

Widespread Occurrence and Growth of E. coli and Enterococci in Green
Algae (Cladophora spp.) on Beach Sands and in Swimming Waters of Lake
Michigan

Whitman, R. L., [D. Shively]{.underline}, H. Pawlik, and M. N.
Byappanahalli

Lake Michigan Ecological Research Station

U.S. Geological Survey

Porter, Indiana

Each summer the nuisance green algae, *Cladophora* spp. (mostly *C.
glomerata*) amasses along some beaches of Lake Michigan, creating near
shore anoxia and unsightly, malodorous mats that can attract problem
animals and detract from visitor enjoyment. The relationship between
fecal indicator bacteria and these algae remains essentially unstudied.
In this investigation, we sought to describe the local and regional
density of *E. coli* and enterococci in *Cladophora* spp. along four
states bordering Lake Michigan. Having established that the occurrence
of *E. coli* and enterococci populations within *Cladophora* spp. was
dense (median 10^4^ and 10^2^ CFU/g d.w., respectively), ubiquitous (up
to 95% occurrence), interrelated (p\<0.0001), and apparently independent
of point sources, we explored the growth potential of these bacteria
within the algae *in situ* and *in vitro*. *Cladophora* spp.-borne *E.
coli* increased in numbers as much as 65% when the algal mats were
incubated on a sand bed in full sunlight for 6 hours in August; growth
of *E. coli* was directly related to mat thickness (p=0.035). Indigenous
*E. coli* and enterococci grew exponentially during the first 18 hr at
35°C in a *Cladophora* spp. elutriant obtained after steeping the algae
in lake water for 48 hr at 4 °C. *Cladophora* spp*.*, even in small
quantities, stimulated impressive *in vitro* growth of *E. coli* in
sterile sand. Thus, *C. glomerata* may be an important reservoir and
source of indicator bacteria for beaches amassing these algal mats along
their shores. The study raises issues concerning bacteria source
paradigms, application of these bacteria as fecal indicators, and the
related health implication of these algae for freshwater recreational
beaches.

**Comparison of Seven Protocols to Identify Fecal Contamination Sources
using *Escherichia coli***

[Donald M. Stoeckel^1^]{.underline}, Melvin V. Mathes, Kenneth E. Hyer,
Charles Hagedorn, Howard Kator, Jerzy Lukasik, Tara L. O'Brien, Terry W.
Fenger, Mansour Samadpour, Kriston M. Strickler, and Bruce A. Wiggins

^1^U.S. Geological Survey

Columbus, Ohio

Microbial source tracking (MST) uses various approaches to classify
fecal-indicator microorganisms to source hosts. Reproducibility,
accuracy, and robustness of 7 phenotypic and genotypic MST protocols
were evaluated by use of *Escherichia coli* from an 8-host library of
known-source isolates and a separate, blinded challenge library. In
reproducibility tests, measuring each protocol's ability to reclassify
blinded replicates, only one (pulsed-field gel electrophoresis; PFGE)
correctly classified all test replicates to host species; three
protocols classified 48-62% correctly, and the remaining three
classified fewer than 25% correctly. In accuracy tests, measuring each
protocol's ability to correctly classify new isolates, ribotyping with
*Eco*R1 and *Pvu*II approached 100% correct classification but only 6%
of isolates were classified; four of the other six protocols (antibiotic
resistance analysis, PFGE, and two repetitive-element PCR protocols)
achieved better than random accuracy rates when 30-100% of challenge
isolates were classified. In robustness tests, measuring each protocol's
ability to recognize isolates from non-library hosts, three protocols
correctly classified 33-100% of isolates as "unknown origin," whereas
four protocols classified all isolates to a source category. A relevance
test, summarizing interpretations for a hypothetical water sample
containing 30 challenge isolates, indicated that false-positive
classifications would hinder interpretations for most protocols. Study
results indicate that more representation in known-source libraries and
better classification accuracy would be needed before field application.
Thorough reliability assessment of classification results is crucial
before and during application of MST protocols.

**The Role of Suspended Sediment in *E. coli* Transport**

[Pamela Struffolino]{.underline}^1^, Von Sigler, Daryl Dwyer

^1^Department of Earth, Ecological and Environmental Science

University of Toledo,

Toledo, Ohio

Although the survival of *Escherichia coli* (E*. coli*) in bottom
sediments have been the focus of recent publications, the role of
suspended sediments in facilitating fecal bacteria transport and
elevated densities are unknown. To determine the impact of suspended
sediments on *E coli* densities, sediment collectors were designed to
trap suspended sediment from the water column. The collectors consist of
five sets of vertical cylinders attached to a stable base that were
deployed throughout Maumee Bay, Oregon Ohio, at the mouths of possible
inputs of *E. coli* into the bay. At approximately two-week intervals,
trapped sediments, bottom sediments, and water were collected and
analyzed for *E. coli* densities. *E. coli* densities in trapped
sediments were significantly higher than those of bottom sediments and
water (p \<0.01), indicating the potential for sediment related *E.
coli* transport and accumulation. To investigate the potential for
sediment mediated *E. coli* transport, the *E. coli* population
structure in trapped- and bottom sediments from three trap locations was
compared following DNA extraction and denaturing gradient gel
electrophoresis (DGGE). The results showed that populations in trapped
sediment were structurally distinct from those of the bottom sediment,
confirming sediment-mediated transport of discrete *E. coli* populations
throughout the Bay. Overall, our results suggest that while suspended
sediments harbor differing densities of *E. coli* compared to bottom
sediments and water, the populations associated with these sediments are
composed of novel *E. coli* populations that are likely transported
continuously throughout the Bay.

**Solar and Temporal Effects on *Escherichia coli* Concentration at a
Great Lakes Swimming Beach**

[Richard L. Whitman,]{.underline} Meredith B. Nevers, Ginger C. Korinek,
and Muruleedhara N. Byappanahalli

Lake Michigan Ecological Research Station

U.S. Geological Survey

Porter, Indiana

Studies on solar inactivation of *E. coli* in freshwater and *in situ*
have been limited. At 63^rd^ Street Beach, Chicago, this study was
undertaken to analyze the factors, particularly insolation, influencing
the daily periodicity of culturable *E. coli*. Water samples for *E.
coli* analysis were collected twice daily between April and September
2000 three times a week along five transects in two depths of water.
Hydrometerological conditions were continuously logged: UV radiation,
total insolation, wind speed and direction, wave height, and lake stage.
On ten days, transects were sampled hourly from 0700 to 1500 h. *In
situ* mesocosms were also used to examine the effect of sunlight on *E.
coli* inactivation. During sunny days, *E. coli* decreased exponentially
with day length and exposure to insolation, but on cloudy days, the
decrease was diminished and less dependent on insolation; decay rate was
influenced by initial concentration. *In situ* experiments confirmed
that insolation primarily inactivated *E. coli*; UV radiation only
marginally affected concentration. The relationship between insolation
and *E. coli* is complicated by relative lake stage, wave height, and
turbidity. There was no evidence of *in situ* multiplication or
resuscitation, but continuous importation and nighttime replenishment of
*E. coli* was evident. These findings have implications for management
strategies of non-tidal waters and the use of *E. coli* as an indicator
organism.

Monitoring Water-Quality Effects of Septic Tank Effluent for Two Streams
in Duval County, Florida

[Shaun M. Wicklein]{.underline}

U.S. Geological Survey

Altamonte Springs, FL

The U.S. Geological Survey undertook a study (1999-2004) to monitor the
effects of septic tanks and sanitary sewer system installation and
subsequent septic tank removal for two tributaries to the St. Johns
River in Jacksonville, Florida. In order to provide data to evaluate the
effects of future remedial activities in the watersheds of selected
tributaries, surface-water samples were analyzed for wastewater
compounds, fecal coliform concentrations, and source of fecal coliform
bacteria populations. These data were used to document septic tank
influences on the water quality of selected tributaries (Wicklein,
2003).

Organic wastewater compounds detected above the minimum
laboratory-reporting limit included detergents, antioxidants and flame
retardants, manufactured polycarbonate resins, industrial solvents, and
mosquito repellant. The most commonly detected compound was
para-nonylphenol, which is a detergent metabolite. Other compounds
detected were N,N-diethyl-meta-toluamide (DEET) and bisphenol-a. Most of
the compounds detected are commonly associated with grey water
discharges to septic tanks in the study areas; therefore, their presence
indicates that study basins are influenced by wastewater from septic
tanks.

For 63 percent of the samples, fecal coliform bacteria concentrations
exceeded the State of Florida fecal coliform bacteria standard for Class
III surface waters of 800 colonies per 100 milliliters of water on any 1
day. Concentrations of fecal coliform bacteria generally increased from
upstream to downstream. Septic tank density also effected observed
concentrations of fecal coliform bacteria, as indicated by analysis of
sample populations for each of the two study basins.

The antibiotic resistance analysis used for this study is based on the
assumption that fecal coliform bacteria from human sources have a
different resistance to selected antibiotics than those from different
animal sources (Simpson, 2002). Water samples were analyzed by the
Department of Biology, University of South Florida, Tampa, Florida,
using materials, methods, and statistical analysis described by Harwood
and others (2000). Significant sources of fecal coliform bacteria
included wild animals, dogs, and humans. A majority of the fecal
coliform bacteria were classified to be from human sources.

Detections of fecal coliform bacteria isolates at one sampling site
indicated a change in known source classification from human to a mix of
human and wild animal and, later, dog and wild animal over the study
period. The change in classification of known sources likely occurred
due to drainage changes made in the stream and other parts of the basin
during construction and connection of residences to the sanitary sewer
system following its completion. Results of the fecal coliform bacteria
antibiotic resistance analysis indicate that movement of septic tank
effluent through the soil matrix, and discharging to the stream,
contributes to fecal coliform bacteria populations observed in the
stream water.

# Literature Cited

Harwood, Valerie J., Whitlock, John, and Withington, Victoria, 2000,
Classification of antibiotic resistance patterns of indicator bacteria
by discriminant analysis---Use in predicting the source of fecal
contamination in subtropical waters: Applied and Environmental
Microbiology, v. 66, no. 9, p. 3698-3704.

Simpson, J.M., Santo Domingo, J.W., Reasoner, D.J., 2002, Microbial
source tracking: State of the science---Environmental Science and
Technology, v. 36, no. 24, p. 5279-5288.

Wicklein, S. M., 2003, Evaluation of water quality for two St. Johns
River tributaries receiving septic tank effluent, Duval County, Florida:
U. S. Geological Survey Water-Resources Investigations Report 03-4299,
28 p.