RESULTS

1
Water Quality Assessment of Agricultural and
Urban Watersheds
ABSTRACT Third Creek in Knox County, Tennessee,
is listed as an impaired stream by the Tennessee
Department of Environment and Conservation. Its
impairments include sediment, habitat alterations
and pathogens due to compromised sewage lines and
seepage from nearby residential sources. Pond
Creek in Loudon, McMinn, and Monroe Counties,
Tennessee, is also chronically contaminated by
pathogens from livestock sources. Additionally,
the bacterial pathogens in these two streams are
exposed to various antibiotics and may have
developed certain levels of resistance. In urban
watersheds such as Third Creek, a commonly
detected antibacterial agent is triclosan, the
active ingredient in hand soaps, surface
disinfectants, mouthwashes and toothpaste. In
contrast, bacterial pathogens in agricultural
watersheds, such as Pond Creek, are often exposed
to antibiotics associated with animal production,
such as Novobiocin. Thus, the hypothesis of this
study was that the antibiotic resistance profiles
of bacterial pathogens in these two watersheds
would reflect sources of contamination. To
evaluate this hypothesis we enumerated total and
fecal coliforms and assessed antibiotic
resistance profiles using IDEXX technology and
membrane filtration.
P. Perrin, M. Pratten, A. Danovi, M. Garmon, B.
Harris,
Environmental and Soil Sciences, Biosystems
Engineering and Environmental Science Dept.
EXPERIMENTAL PROCEDURES

• IDEXX Colilert
• Used for detection of total coliforms in water
  (Fig. 3)
• Positive total coliform are indicated by yellow
• Positive E. coli indicated by flourescent yellow
• Provided an initial estimate of total coliform in
  the water samples

INTRODUCTION Widespread and indiscriminate use of
antibiotics can lead to the development of
antibiotic resistance in bacteria. As surface
bodies of water frequently become contaminated
with fecal coliforms of human and animal origins,
bacterial pollution is often used as a major
indicator of stream quality. Two streams were
studied. Third Creek was chosen due to extensive
pollution, primarily from urban/residential, and
its proximity to the campus (Fig. 1). Pond Creek
drains a small agricultural watershed in Loudon
County (Fig. 2). This area is characterized by
the presence of numerous farms and cattle
production. Thus, in contrast to Third Creek,
agricultural pollution affects this stream. This
study focused on bacteria resistance to two
commonly used antibiotics, Triclosan and
Novobiocin, based on different sources of
contamination of the two streams. Triclosan is an
antimicrobial agent widely used in residential
consumer products such as hand soaps, detergents,
toothpaste or surface disinfectants. Hence
bacterial contaminants in domestic wastewater are
likely to be exposed to significant levels of
Triclosan and consequently, may develop higher
levels of resistance than bacterial contaminants
from non-residential sources. Novobiocin is
widely used in animal production systems to
control bacterial pathogens and thus, we
anticipated that fecal contamination in Pond
Creek might be more resistant to this antibiotic.
Therefore, we decided to conduct a study to
determine if bacteria are becoming more resistant
when exposed to antibiotics, and to compare the
resistance of bacteria from both urban and
non-urban areas, when subjected to commonly used
antibiotics. Objectives Enumerate total fecal
coliforms in two streams with different sources
of contamination. Measure antibiotic resistance
levels of fecal coliforms to Triclosan and
Novobiocin in the two streams. Hypothesis
Coliform bacteria from Pond Creek are more
resistant to Novobiocin while coliform bacteria
in Third Creek are more resistant to Triclosan.
Fig.3. Idexx MPN plate
Fig. 4. Filtering water samples

• Membrane Filtration Method
• Filters (47 mm) were used in a filtration tower
  (Fig. 4)
• Creek samples (10 mL) were filtered through the
  columns
• Membrane filters were then transferred onto agar
  plates that contained differential and selective
  media for fecal coliforms and incubated at 44.5C
  for 24 hours
• Colony Forming Units (cfu) were counted and
  recorded (Fig. 5)

Fig. 5 Typical MF plate showing fecal coliform
colonies
Fig. 8. Percentage of antibiotic resistant fecal
coliforms in Pond Creek.

• Replica Plating
• Developed media that contained different types of
  antibiotics Novobiocin and Triclosan
• Novobiacin plates were made at 3 different
  concentrations (10, 100 500ppm)
• Using a stamping method, colonies were
  transferred from the control plates to antibiotic
  media and incubated for 24 hours prior to
  enumeration (Fig. 6).

• With increasing amounts of Novobiocin there was
  an obvious decrease in survival of colony forming
  units of bacteria (Fig. 8).
• Triclosan was more effective at inhibiting the
  growth of fecal coliforms but a surprisingly high
  percentage of Triclosan-resistant bacteria was
  observed at site 3.
• A relatively high percentage of
  Novobiocin-resistant fecal coliforms was observed
  at concentrations of 10 and 100 ppm but
  relatively few bacteria were resistant at 500 ppm.

Fig.6. Replica plating
RESULTS

• Fecal colifom abundance was greater in Pond than
  in Third Creek (Table 1).
• Total coliform levels exceeded contact standards
  at all 3 sites on Third Creek, and at 2 out of 3
  sites on Pond Creek.

Table 1. Total and fecal coliforms in Pond and
Third Creeks estimated by IDEXX MPN technology
and the M.F technique, respectively.
CONCLUSION

• Triclosan was more effective in inhibiting the
  growth of fecal coliforms than Novobiocin (Fig.
  7).
• Fecal coliform bacteria from site 3 on Third
  Creek were consistantly more resistant to
  Novobiocin at all concentrations when compared to
  sites 1 and 2.
• The percentage of Novobiocin resistant fecal
  coliforms decreased with increasing Novobiocin
  concentration.

Statistical comparison of the antibiotic
resistance data averaged over the three sites
revealed no significant difference (p?0.05)
between the percentage of Triclosan-resistant
fecal coliforms in Pond and Third Creeks. Thus
our hypothesis regarding contaminant source was
not supported with respect to Triclosan
resistance. However, for Novobiocin at the 10
and 100 ppm concentrations, percent resistant
coliforms in Pond Creek were significantly
greater than in Third Creek. Therefore our
hypothesis was supported by these data suggesting
that antibiotic resistance profile was related to
contaminant source (i.e. agricultural vs. urban).
Unequal variances prevented statistical
comparison of Novobiocin resistance at the 500
ppm concentration between the two streams.
Fig. 1 Aerial photograph of Third Creek near the
UT Agricultural campus
Fig. 7. Percentage of antibiotic resistant fecal
coliforms in Third Creek.
Fig. 2. Collecting water samples on Pond Creek