Transcript Document

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
Fig.3. Idexx MPN plate
Response of fecal coliform to Triclosan and
Novobiocin in Pond Creek
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.
•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 44.5°C for 24 hours
Percent survival
Membrane Filtration Method
70%
60%
50%
40%
30%
20%
10%
0%
Site 1
Site 2
Site 3
Tri (10)
Nov (10)
Nov (100)
Nov (500)
Antibiotic Trt (ppm)
•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
•With increasing amounts of Novobiocin there was an obvious
•Novobiacin plates were made at 3 different concentrations (10, 100 & 500ppm)
Objectives:
Enumerate total fecal coliforms in two streams with different sources of
contamination.
decrease in survival of colony forming units of bacteria (Fig. 8).
•Using a stamping method, colonies were transferred from the control plates to antibiotic media
•Triclosan was more effective at inhibiting the growth of fecal
and incubated for 24 hours prior to enumeration (Fig. 6).
coliforms but a surprisingly high percentage of Triclosan-resistant
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. 4. Filtering water samples
RESULTS
Fig.6. Replica plating
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
Table 1. Total and fecal coliforms in Pond and Third Creeks estimated by
IDEXX MPN technology and the M.F technique, respectively.
•Fecal colifom abundance was greater in Pond than in
Third Creek (Table 1).
Sampling Site
•Total coliform levels exceeded contact standards at all 3
1
2
3
sites on Third Creek, and at 2 out of 3 sites on Pond
Third Creek
Pond Creek
IDEXX
M.F.
IDEXX
M.F.
MPN (cells/100 mL) (CFU/100 mL) MPN (cells/100 mL) (CFU/100 mL)
2338
94
878
157
2355
75
3206
297
1596
210
3298
232
relatively few bacteria were resistant at 500 ppm.
CONCLUSION
Creek.
•Triclosan was more effective in inhibiting the growth of fecal
Percent survival
Fig. 1 Aerial photograph of Third Creek near the UT
Agricultural campus
Response of fecal coliform to Triclosan and
Novobiocin in Third Creek
70%
60%
50%
40%
30%
20%
10%
0%
•Fecal coliform bacteria from site 3 on Third Creek were consistantly
Site 1
Site 2
Site 3
more resistant to Novobiocin at all concentrations when compared to
sites 1 and 2.
Tri (10)
Nov (10)
Nov (100)
Nov (500)
Antibiotic Trt (ppm)
Fig. 7. Percentage of antibiotic resistant fecal
coliforms in Third Creek.
Fig. 2. Collecting water samples on Pond Creek
coliforms than Novobiocin (Fig. 7).
•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.