RESULTS

•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.

Response of fecal coliform to Triclosan and Novobiocin in Pond Creek

0%10%20%30%40%50%60%70%

Tri (10) Nov (10) Nov (100) Nov (500)

Antibiotic Trt (ppm)

Per

cen

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Site 1

Site 2

Site 3

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

Membrane Filtration Method

•Filters (47 m) 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

•Colony Forming Units (cfu) were counted and recorded (Fig. 5)

ABSTRACTThird 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.

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).

•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.

•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.

CONCLUSION

INTRODUCTIONWidespread 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. 1 Aerial photograph of Third Creek near the UT Agricultural campus

Fig. 2. Collecting water samples on Pond Creek

P. Perrin, M. Pratten, A. Danovi, M. Garmon, B. Harris,

Fig.3. Idexx MPN plate Fig. 4. Filtering water samples

Fig. 5 Typical MF plate showing fecal coliform colonies

Fig.6. Replica plating

Sampling Site IDEXX M.F. IDEXX M.F.MPN (cells/100 mL) (CFU/100 mL) MPN (cells/100 mL) (CFU/100 mL)

1 2338 94 878 1572 2355 75 3206 2973 1596 210 3298 232

Third Creek Pond Creek

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

Fig. 7. Percentage of antibiotic resistant fecal coliforms in Third Creek.

Fig. 8. Percentage of antibiotic resistant fecal coliforms in Pond Creek.

Environmental and Soil Sciences, Biosystems Engineering and Environmental Science Dept.

Statistical comparison of the antibiotic resistance data averaged over the three sites revealed no significant difference (p0.05) 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.

Response of fecal coliform to Triclosan and Novobiocin in Third Creek

0%10%20%30%40%50%60%70%

Tri (10) Nov (10) Nov (100) Nov (500)

Antibiotic Trt (ppm)

Pe

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Site 1

Site 2

Site 3