Potential impacts of antibiotics in the environment
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Potential Impacts of Antibiotics in the Environment. Amy Pruden Assistant Professor, Civil Engineering, Colorado State University. Overview. Agricultural Antibiotics Overview of potential impacts Why study resistance genes? Poudre River Study Conclusions Recommendations.

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Potential Impacts of Antibiotics in the Environment

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Potential impacts of antibiotics in the environment

Potential Impacts of Antibiotics in the Environment

Amy Pruden

Assistant Professor, Civil Engineering, Colorado State University



  • Agricultural Antibiotics

  • Overview of potential impacts

  • Why study resistance genes?

  • Poudre River Study

  • Conclusions

  • Recommendations

Agricultural antibiotics

Agricultural 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??

Antibiotic pathways

Antibiotic Pathways

Modified from www.usda.gov

Antibiotics used

Antibiotics Used:

  • Tetracyclines

    • Chlortet., oxytet., tet. ..

  • Sulfonamides

  • Macrolides

    • Tylosin, erythromycin..

  • Ionophores

    • Monensin..

  • b-lactams

    • Penicilillin

Tetracylcline (Tet)

Public concern

Public Concern

Potential impacts

Potential 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 impacts1

Potential 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 impacts2

Potential 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 arg

Antibiotic Resistance Genes (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 transfer

Resistance Gene Transfer

ASM News November, 2004

Antibiotic resistance genes

Antibiotic Resistance Genes

  • If we can detect and quantify resistance genes, then we have an assay on the bioavailability/impact of the antibiotics.

Mechanisms of resistance

Mechanisms 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


  • Resistance transfer:

    • Plasmids can be exchanged within and between species……



  • 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 Antibiotics

Study site poudre river

Study Site: Poudre River

Map of study sites

Map of Study Sites

Cfu at sites april 2004

CFU at Sites: April 2004

Cfu at sites february 2005

CFU at Sites: February 2005

Pitfalls of culture based methods

Pitfalls of Culture-Based Methods

  • 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 approach

Molecular 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.

Phylogenetics of sul genes

Phylogenetics of Sul Genes

sul D

sul BC

sul Bcr

sul A

sul III

sul I

sul II

New sul primers

New Sul Primers

Specificity verified by cloning and sequencing the inserts.

Detection of pcr product

Detection of PCR Product

Pcr presence absence assay

PCR Presence / Absence Assay

Real time pcr

Real-time PCR


0 5 10 15 20 25 30 35 40 45 50

Number of Cycles

Sul i gene calibration

Sul I Gene Calibration

April 2004 spring high flow

April, 2004: Spring High-Flow

Feb 2005 winter low flow

Feb, 2005: Winter Low-Flow

Aug 2005 summer low flow

Aug, 2005: Summer Low-Flow



  • 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?



  • 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 study

Composting Field Study

Biodegradation of arg

“Biodegradation” of ARG



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??

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