Transcript SchaferSpr09

Antibiotic Resistant in Commercial Septic Discharge
Jacob Schafer, Jay Nielsen, Michael Servi and Matt Haak
Faculty Sponsors: Sasha Showsh, Crispin Pierce, and Eli Gottfried
Introduction
Fig.2 Soil Sampling Site
Fig. 1 Land spreading of septic tank effluent.
 Antibiotic resistance is evident when a drug
http://www.limemaster.com/Land_Spreading.html
can no longer inhibit the growth of the target
bacteria.
 According to the National Nosocomial
Control
Infections Surveillance (NNIS) System data on
intensive care units (ICUs) in the U.S, 28.5%
of enterococci associated with nosocomial
infections in ICU patients in 2003 were
resistant to vancomycin and resistance rates
of Pseudomonas aeruginosa were 31.9% (to
ceftazidime)
Treated
 More than 80 pharmaceuticals and drug
metabolites, have been measured in μg/llevels in sewage samples and downstream
surface waters. (4)
 Recent research has shown certain bacteria
Fig. 3 Petri plates with sequential dilutions.
may survive on a diet of the antibiotic
Vancomycin. (5)
http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displa
yarticle&art_id=273&theme=printer
• In this small study, treatment of
an agricultural field with septic
system effluent did not
significantly change the fraction
of bacteria that were resistant to
five antibiotics (Kanamycin,
Tetracycline, Eosine Methylene
Blue, Erythromycin and
Ampicillin).
Materials and methods
1) Twenty-Seven soil samples (19 control and
8 treated) were collected from a crop field
near Eau Claire treated with septic system
effluent (Figs. 1 and 2). These samples
were mixed with water and plated on
Colombian Blood Agar and five antibiotics
(Kanamycin, Eosine Methylene Blue,
Tetracycline, Erythromycin and
Ampicillin, Fig. 3).
2) Bacterial antibiotic resistance generated
from a large retail store and a small chain
restaurant was measured. Several samples
of effluent from the respective septic
systems were analyzed for bacterial
resistance to ampicillin, kanamycin,
tetracycline, and erythromycin.
Methods to conduct the experiment…
Results
•Although the fraction of antibiotic-resistant colonies tended to be higher for control vs. treated
samples, this apparent difference did not reach a significance of p<0.05 (one-tailed t-test, equal
variance, Fig. 4).
•The restaurant contained higher levels of resistance to ampicillin, kanamycin, and tetracycline,
with an average of 51.79%, 27.54%, and 30.78% of microbial growth resistant to the respective
antibiotics. The store generated an average of 17.50%, 17.87%, and 5.97% resistance to
ampicillin, kanamycin, and tetracycline. Erythromycin resistance was greater in effluent
discharged from the store at 7.00% than it was in the restaurant at 2.87%.
Fig. 4 Colony growth and fraction of antibiotic resistance in control and treated (septic effluentapplied) samples for five antibiotics.
Columbia Blood
Agar
 Mix 10 grams of soil sample (or liquid effluent)
in Erlenmeyer flask containing 50ml of water.
Stir for 1 minute and let rest for another 1
minute
 Make three dilutions aseptically transferring
500µml of solution into a test tube
 Transfer 100µml of each diluted solution to a
CBA plate or a CBA plate containing a
particular antibiotic
Kanamycin
4500000
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1/amount plated * number of colonies
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Colony
Count Test
Colony Count Control
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KanamycinControl
Erythromycin
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KanamycinTest
Ampicillin
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TetracyclineControl
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 Surrounding non-treated area was used as
control
 27 samples collected and store on ice
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ErythromycinControl
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Erythromycin-test
1.Paterson, David . "Update on Antibiotic
Resistance in Hospitals." The Prevalence of
Antibiotic Resistance in the Hospital Setting.
2006. 20 Apr 2007
<http://www.medscape.com/editorial/cmeto
go/5541>.
0
AmpicillinControl
0
Ampicillintest
2. Fridkin, Scott K. et al. "Temporal
Changes in Prevalence of Antimicrobial
Resistance in 23 U.S. Hospitals." Emerging
Infectious Diseases Vol. 8, No. 7,(2002): 697701.
3. Lipsitch, Marc. "The epidemiology of
antibiotic resistance in hospitals:Paradoxes
and prescriptions." PNAS vol.97(2000): 1938-
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BIBLIOGRAPHY
1943.
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Methods to collect the soil samples…
 Samples taken seven inches below surface
from area near Eau Claire treated with septic
effluent
TetracyclineTest
Eosine Methylene
Blue
70
70
• Measurements from the chain
restaurant and large store are
significant because they create a
baseline percentage of normal or
expected antibiotic resistance
discharged from commercial
sites.
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 Store at 37°C for 24hrs
 Use CFU/ml = Total Dilution Factor *
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 Observe and count colonies.
Tetracycline
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Conclusions
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Eosin Methylene
Blue-Control
Eosin Methylene
Blue-Test
4. Herberer, Thomas. "Toxicology Letters:
Occurrence, Fate, and Removal of
Pharmaceutical Residues in the Aquatic
Environment: a Review of Recent Research
Data." ScienceDirect. 15 Mar. 2002. Toxicology
Letters. 7 Apr. 2008
5. "Antibiotic-eating germ alarms doctors Enterococcus faecium bacterium survives on
diet of the antibiotic vancomycin - Biomedicine Brief Article". Science News. Dec 21, 1996.