Past project: Antibiotic resistance

Investigating the Spread of Antimicrobial Resistance near Animal Facilities: Mechanisms of Extracellular DNA Transport and Transfer

The use of antibiotics in animal production systems results in increased levels of antibiotic resistance in manure. During manure storage and through land application of treated manure the antibiotic resistance genes are transferred to soil, ground and surface waters, and crops, potentially creating an environmental reservoir of antibiotic resistance and increasing an already serious public health problem.
However, the study of this potential hazard has been difficult due to the widespread exchange of DNA among microorganisms and the limited techniques available for tracking specific resistance genes. We propose an interdisciplinary approach to identify physical and chemical factors in the lagoon and soil environments that control the transfer rate of extracellular DNA and therefore influence the spread of antibiotic resistance.
The resulting relationships will provide a basis for evaluating the public health risks associated with land application of manure and for identifying novel measures with the potential to control the spread of antibiotic resistance such as timing the land application of manure or choosing fields with appropriate soil and water chemistry to minimize transfer.

Bacteria obtain foreign genes by cell-to-cell transfer, viral transfer, and by the transfer of extracellular DNA. Some live bacteria release extracellular DNA actively, while others do so only upon cell lysis. It has been shown that soil microorganisms are more likely to acquire intibiotic-resistance genes through the uptake of extracellular plasmids than by direct cell-to-cell plasmid transfer (Demaneche et al., Appl. Environ. Microbiol., 2001). This process may be facilitated by soil particles to which the extracellular plasmids can adsorb after being released from a bacterial cell. Adsorbed plasmids gain protection from enzyme degradation while retaining their ability to transform competent organisms (e.g. Demaneche et al., Appl. Environ. Microbiol., 2001).

Extracellular DNA

Sources and fate of enxtracellular DNA in the Environment

We hypothesize that: (1) the persistence and mobility of extracellular DNA will depend on solution composition, including pH, ionic strength, and divalent cation concentration; and (2) the transformation of extracellular DNA will controlled primarily by physical and chemical parameters. These hypotheses will be tested for soil through the following objectives:
(1) Characterize the extent and mechanism of DNA adsorption to representative soil surfaces under a variety of environmental conditions using a quartz crystal microbalance with dissipation, a state of the art instrument for adsorption measurement;
(2) Identify relationships among environmental parameters, DNA adsorption, and the efficiency and rate of gene transfer for pure cultures of the soil bacterium Azotobacter vinelandii;
(3) Evaluate the frequency of gene transfer in soil samples with and without exposured to tetracycline.

The results of this study will provide a basis for both evaluating risk and identifying novel measures such as timing the land application of manure or choosing fields with appropriate soil and water chemistry to minimize the spread of resistance.

Extracellular DNA

Quartz crystal microbalance with dissipation (QCM-D) was used to study extracellular DNA adsorption and the conformation of the adsorbed DNA on silica and natural organic matter (NOM) surfaces. Gene transfer was assessed under the same conditions using natural transformation of chromosomal and plasmid DNA into soil bacteria
Azotobacter vinelandii to explore the relationships among environmental parameters, DNA adsorption, and the efficiency and rate of gene transfer for pure cultures. Experimental results indicate that solution ionic composition, such as calcium concentration and natural organic matter in groundwater, influence the amount of DNA that is adsorbed and protected from enzymatic degradation, but did not have a strong influence on natural transformation.
The presence of oxytetracycline in solution or in adsorbed form at environmentally-relevant concentrations also did not have a strong influence natural transformation of A. vinelandii. In addition, we found that oxytetracycline adsorbed to clay particles lost its ability to inhibit A. vinelandii growth. This work suggests that adsorbed DNA is fully bioavailable and could be an important reservoir for environmental gene transfer.

Transformation Frequency

Natural transformation of A. vinelandii. Transformation experiments were conducted with dissolved DNA in the transformation buffer (A) and with dissolved DNA (B and C), DNA adsorbed to silica surface (D and E), and DNA adsorbed to NOM surface (F to J) in solutions of no salt (solid triangle), Na+ (solid square), and Ca2+ (open circle) at pH 7.2. Where multiple letters are listed in the same category, for example, B and C, they represent replicate experiments performed with different batches of competent cells. The transformation frequency was calculated as the ratio of the number of Nif+ transformants to the number of viable cells. Each data point was calculated from at least three replicates, and independent experiments are shown separately. The DNA concentration was 0.2 ug/ml, except for J, which contained 0.025 ug DNA/ml.Figure from Lu et al., 2010.

We further investigated the mechanism of oxytetracycline (OTC) adsorption to a silty clay loam soil using sorption isotherm experiments, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) spectroscopy. Sorption data fit well to a cation-exchange capacity sorption model. Spectroscopic data indicate that the interactions between oxytetracycline and silty clay loam soil were primarily through electrostatic interactions between the protonated dimethylamino group of oxytetracycline and the negatively charged moieties on the surface of the soil. Based on XRD results, OTC adsorption appeared to inhibit the ethylene glycol solvation of the expandable clay minerals suggesting OTC had diffused into the clay interlayer space. The presence of adsorbed OTC did not significantly affect the transformation frequency of the soil bacterium Azotobacter vinelandii with plasmid DNA. Growth was inhibited by adsorbed OTC, although a greater mass of adsorbed OTC were required to achieve the same degree of inhibition as the system of dissolved OTC alone. These results suggest that the interactions of tetracyclines at soil/water interface will affect the growth of sensitive microorganisms in soil microbial communities.


  1. Goetsch*, H.E.; Mylon, S.E.; Butler, S; Zilles, J.L.; Nguyen, T.H., Oxytetracycline interactions at the soil/water interface: the effects of environmental surfaces on natural transformation and growth inhibition of Azotobacter vinelandii, Environmental Chemistry and Toxicology, 2012, Vol. 31, p. 2217-2224, full text.
  2. Lu*, N., Mylon, S.E., Kong, R., Bhargava,R., Zilles, J.L., Nguyen, T.H., Interactions between dissolved natural organic matter and adsorbed DNA and their effect on natural transformation of Azotobacter vinelandii, Science of the Total Environment, 2012, Vol. 426, p. 430–435, full text.
  3. Lu*, N., Zilles, J.L., and Nguyen, T.H. , “Adsorption of extracellular chromosomal DNA and its effects on natural transformation of Azotobacter vinelandii, Applied and Environmental Microbiology, 2010, Vol. 76, p. 4179-4184, full text.
  4. Nguyen, T.H., Chen K.L. and and Elimelech M., Adsorption Kinetics and Reversibility of Linear Plasmid DNA on Silica Surfaces: Influence of Alkaline Earth and Transition Metal Ions, Biomacromolecules, 2010, Vol. 11, p. 1225-1230,full text.
  5. Nguyen, T.H. and Chen K.L., Role of Divalent Cations in Plasmid DNA Adsorption to a Silica Surface Coated with Natural Organic Matter, Environmental Science and Technology, 2007, Vol. 41, p. 5370-5375, full text.
  6. Nguyen, T.H. and Elimelech M., Adsorption of Plasmid DNA to a Natural Organic Matter Coated Silica Surface: Kinetics, Conformation, and Reversibility, Langmuir, 2007, Vol. 23, p. 3273-3279, full text.
  7. Nguyen, T.H. and Elimelech M., Plasmid DNA Adsorption on Silica: Kinetics and Conformational Changes in Mono and Divalent Salts”, Biomacromolecules, 2007, Vol. 8, p. 24-32, full text.


Nanxi Lv

(graduate student, MS in 2010)

Heather Goetsch

(graduate student, MS in 2011)

Dr. Julie Zilles, Department of Civil and Environmental Engineering, University of Illinois.
USDA Water and Watershed Program, University of Illinois.

Last update: Nov. 20, 2012