Potential Impacts of Potential Impacts of Antibiotics in the Antibiotics in the
EnvironmentEnvironmentAmy Pruden
Assistant Professor, Civil Engineering, Colorado State
UniversityR1
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OverviewOverview
• Agricultural Antibiotics• Overview of potential impacts• Why study resistance genes?• Poudre River Study• Conclusions• Recommendations
Agricultural AntibioticsAgricultural Antibiotics
• More than ½ used in U.S. Animals• Subtherapeutic use promotes weight
gain.• Animal waste > 130 x human waste
– (United States Senate Committee on Agriculture, 1997)
• Antibiotics can be excreted unaltered.
• Animal Waste Treatment??
Antibiotics Antibiotics Used:Used:
• Tetracyclines– Chlortet., oxytet.,
tet. ..
• Sulfonamides• Macrolides
– Tylosin, erythromycin..
• Ionophores– Monensin..
-lactams Penicilillin
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Erythromycin (Ery)
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Roxithromycin (Rox)
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Tylosin (Tyl)
Tetracylcline (Tet)
Public ConcernPublic Concern
Potential ImpactsPotential Impacts
• Toxicity to Aquatic life (H. Ramsdell, CSU)– Planaria, flathead minnow, and Hyalella– Chlortetracycline, tylosin, sulfamethazine,
metronidizine, monensin and lyolocid showed toxicity
– Monensin strong toxicity and widespread use• LD50 = 5 ppm in water for minnows• LD50 = 20 ppm in sediment for Planaria• LD50 = 1 ppm in water for Hyalella
– Sublethal effects?
Potential ImpactsPotential Impacts
• Sub-lethal impacts: Endocrine disruptors– Micropollutants not
removed by wastewater treatment
– May cause hermaphroditism
– Effects on frogs– Fish in Chesapeake Bay
Sower et al., Env. Health Perspect. 2000
Potential ImpactsPotential Impacts
• Plant Uptake– Antibiotic uptake by plants from soil
fertilized with animal manure- Kumara et al. U. Minn.
• J Environ Qual (2005)
– Greenhouse studies: corn, green onion, & cabbage
– Uptake of chlortetracycline, but not tylosin• Low: 2 – 17 ng/g, but correlates with manure
concentration
– Implications for allergic individuals
Antibiotic Resistance Genes Antibiotic Resistance Genes (ARG)(ARG)
• Spread of ARG one of most urgent human health issues according to WHO
• Use of antibiotics selects for antibiotic resistant organisms– Shea, 2003; Fedorka-Cray et al., 2002; Smith
et al., 2002; Sørum and L’Abée-Lund, 2002; Teuber, 2001.
• Can be spread across microbial populations and in the environment– ARG as “pollutants”
Resistance Gene TransferResistance Gene Transfer
ASM News November, 2004ASM News November, 2004
Antibiotic Resistance GenesAntibiotic Resistance Genes
• If we can detect and quantify resistance genes, then we have an assay on the bioavailability/impact of the antibiotics.
Mechanisms of ResistanceMechanisms of Resistance
• Alteration of the antibiotic or target site– tetM tetS tetO tetW tetQ tetT tetBP
• Impaired uptake or enhanced efflux– tetA tetB tetC tetD tetE tetG tetH tetJ tetY tetZ
• Overproduce target so higher concentration of antibiotic needed– sul genes (PABA overproduction to make folic
acid)• Degrade antibiotic
-lactams• Resistance transfer:Resistance transfer:
– Plasmids can be exchanged within and Plasmids can be exchanged within and between species……between species……
MethodsMethods
• Plate counting:– R2A agar with antibiotics.
• Polymerase chain reaction (PCR) assays:– Presence/absence of a resistance gene
family.
• Quantitative real-time PCR (Q-PCR)– Quantify resistance gene families.
Goal: Indicator of Bioavailability/impact of AntibioticsGoal: Indicator of Bioavailability/impact of Antibiotics
Study Site: Poudre RiverStudy Site: Poudre River
Map of Study SitesMap of Study Sites
CFU at Sites: April 2004CFU at Sites: April 2004
site 1 site 2 site 3 site 4 site 5
CF
U P
er G
ram
of
Sed
imen
t
102
103
104
105
106
107
108
ChlortetracyclineOxytetracyclineMecolcyclineSulfamethoxazoleSulfamethazineErythromycinTylosinMonensinNo
Ab.
CFU at Sites: February 2005CFU at Sites: February 2005
site 1 site 2 site 3 site 4 site 5
CF
U P
er G
ram
of
Sed
imen
t
102
103
104
105
106
107
108
ChlortetracyclineOxytetracyclineMecolcyclineSulfamethoxazoleSulfamethazineErythromycinTylosinMonensinNo Ab.
Pitfalls of Culture-Based Pitfalls of Culture-Based MethodsMethods
• 99% of environmental organisms cannot be cultured on standard media (Amann et al., Pace et al.).
• 16S rRNA gene as a target for detecting microorganisms in environmental samples (Woese et al.).
• Targeting of functional genes….
Molecular Biology ApproachMolecular Biology Approach
• Polymerase Chain Reaction (PCR)– Exponentially amplify target genes using
primers specific to the target.– Low detection limit.– Provides a means of presence/absence
detection.
PhylogenetiPhylogenetics of cs of SulSul GenesGenes
sul I
sul III
sul A
sul BC
sul Bcr
sul D
sul II
New New SulSul Primers Primers
Specificity verified by cloning and sequencing the inserts.Specificity verified by cloning and sequencing the inserts.
Detection of PCR ProductDetection of PCR Product
PCR Presence / Absence PCR Presence / Absence AssayAssay
Gene ID
April 2004 high-flow spring
February 2005 low-flow winter
+ control
Site 1
Site2
Site3
Site4
Site5
Site1
Site2
Site3
Site4
Site5
tetB(P)
- - - - + - - - - - +
tet(O) + - + + + + + + + + +
tet(S) - - - - + - - - - - +
tet(T) - - + + + - - - - - +
tet(W) - + + + + + + + + + +
sul(I) + + + + + + + + + + +
sul(II) - - + + + - - + + + +
sul(III)
- - + + + - - - - - +
sul(A) - - + - + - - - - - +
Real-time PCRReal-time PCR
Number of Cycles
Flu
ore
scen
ce
0 5 10 15 20 25 30 35 40 45 50
Sul ISul I Gene Calibration Gene Calibration
Threshold Cycle (CT) Value
0 10 20 30 40 50
Lo
g C
op
y o
f s
ul I
Gen
es
pe
r R
eac
tio
n
0
2
4
6
8
10
12y = -0.22 x + 12.13
r2 = 0.9978
April, 2004: Spring High-April, 2004: Spring High-FlowFlow
site 1 site 2 site 3 site 4 site 5Co
py
of
AR
G /
Co
py
of
16S
Ge
ne
s
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2sul(I)sul(II)tet(W)tet(O)
Feb, 2005: Winter Low-FlowFeb, 2005: Winter Low-Flow
site 1 site 2 site 3 site 4 site 5
Co
py
of
AR
G /
Co
py
of
16
S g
en
es
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2sul(I)sul(II)tet(W)tet(O)
Aug, 2005: Summer Low-Aug, 2005: Summer Low-FlowFlow
site 1 site 2 site 3 site 4 site 5
Co
py
of
AR
G /
Co
py
of
16
S g
en
es
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2sul(I)sul(II)tet(W)tet(O)
ConclusionsConclusions
• Resistance genes in Poudre sediments– correlate with human and agricultural
activity
• No direct correlation with antibiotics– High sulfonamide resistance compared to
tet resistance– Fate of antibiotics vs fate of genes?
• High-flow versus low-flow?– Implications for transport?
RecommendationsRecommendations
• Need further studies into the origin of the antibiotic resistance genes and their fate– Human vs agricultural– Do genes persist longer than antibiotics?
• Investigate and apply treatment strategies for mitigating risk.
Composting Field StudyComposting Field Study
““Biodegradation” of ARGBiodegradation” of ARG
Students!!!Students!!!
Thank You!!Thank You!!• Thank you to USDA NRI and to the
CSU Agricultural Research Station for supporting this research!!
• Ken Carlson & Sung-chul Kim• Jessica Davis & Kathy Doesken
•Questions??Questions??
