Microbial Ecology

, Volume 51, Issue 3, pp 267–276 | Cite as

Tetracyclines and Tetracycline Resistance in Agricultural Soils: Microcosm and Field Studies

  • Heike Schmitt
  • Krispin Stoob
  • Gerd Hamscher
  • Eric Smit
  • Willem Seinen


The influence of the use of antibiotics on the prevalence of resistance genes in the environment is still poorly understood. We studied the diversity of tetracycline and sulfonamide resistance genes as influenced by fertilization with pig manure in soil microcosms and at two field locations. Manure contained a high diversity of resistance genes, regardless of whether it stemmed from a farm operation with low or regular use of antibiotics. In the microcosm soils, the influence of fertilization with manure was clearly shown by an increase in the number of resistance genes in the soil after manuring. Spiking of the tetracycline compounds to the microcosms had only little additional impact on the diversity of resistance genes. Overall, the tetracycline resistance genes tet(T), tet(W), and tet(Z) were ubiquitous in soil and pig slurries, whereas tet(Y), tet(S), tet(C), tet(Q), and tet(H) were introduced to the microcosm soil by manuring. The diversity of tetracycline and sulfonamide [sul(1), sul(2), and sul(3)] resistance genes on a Swiss pasture was very high even before slurry amendment, although manure from intensive farming had not been applied in the previous years. The additional effect of manuring was small, with the tetracycline and sulfonamide resistance diversity staying at high levels for the complete growth season. At an agricultural field site in Germany, the diversity of tetracycline and sulfonamide resistance genes was considerably lower, possibly reflecting regional differences in gene diversity. This study shows that there is a considerable pool of resistance genes in soils. Although it is not possible to conclude whether this diversity is caused by the global spread of resistance genes after 50 years of tetracycline use or is due to the natural background in soil resistance genes, it highlights a role that environmental reservoirs might play in resistance gene capture.



We thank Joanne Chee-Sanford and Vincent Perreten for providing DNA extracts of the positive control strains; Remy Hillekens, Rogier Doornbos, and Karel Wernars for advice and technical assistance; Heinrich Höper (Geological Survey of Lower Saxony, Germany) for experimental setup and sampling of the German field study; and Jan-Dirk van Elsas for critically commenting on the progress of work. The Swiss field study was partly financed by the Swiss National Research Program NRP49 “Antibiotic Resistance” (4049-63282) and the Swiss Agency for the Environment, Forests and Landscape (SAEFL).


  1. 1.
    Agersø, Y, Jensen, LB, Givskov, M, Roberts, MC (2002) The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group. FEMS Microbiol Lett 214: 251–256CrossRefPubMedGoogle Scholar
  2. 2.
    Agersø, Y, Sengeløv, G, Jensen, LB (2004) Development of a rapid method for direct detection of tet(M) genes in soil from Danish farmland. Environ Int 30: 117–122CrossRefPubMedGoogle Scholar
  3. 3.
    American Academy of Microbiology (1999) Antimicrobial Resistance: An Ecological Perspective. American Society for Microbiology. Washington, DCGoogle Scholar
  4. 4.
    American Academy of Microbiology (2002) The Role of Antibiotics in Agriculture. American Society for Microbiology. Washington, DCGoogle Scholar
  5. 5.
    Aminov, RI, Chee-Sanford, JC, Garrigues, N, Teferedegne, B, Krapac, IJ, White, BA, Mackie, RI (2002) Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria. Appl Environ Microbiol 68: 1786–1793CrossRefPubMedGoogle Scholar
  6. 6.
    Aminov, RI, Garrigues-Jeanjean, N, Mackie, RI (2001) Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl Environ Microbiol 67: 22–32CrossRefPubMedGoogle Scholar
  7. 7.
    Andersen, JT, Schafer, T, Jorgensen, PL, Moller, S (2001) Using inactivated microbial biomass as fertilizer: the fate of antibiotic resistance genes in the environment. Res Microbiol 152: 823–833CrossRefPubMedGoogle Scholar
  8. 8.
    Andersen, SR, Sandaa, RA (1994) Distribution of tetracycline resistance determinants among gram-negative bacteria isolated from polluted and unpolluted marine sediments. Appl Environ Microbiol 60: 908–912PubMedGoogle Scholar
  9. 9.
    Brady, MS, White, N, Katz, SE (1993) Resistance development potential of antibiotic/antimicrobial residue levels designated as ‘safe levels.’ J Food Prot 56: 229–233Google Scholar
  10. 10.
    Bryan, A, Shapir, N, Sadowsky, MJ (2004) Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources. Appl Environ Microbiol 70: 2503–2507CrossRefPubMedGoogle Scholar
  11. 11.
    Chee-Sanford, JC, Aminov, RI, Krapac, IJ, Garrigues-Jeanjean, N, Mackie, RI (2001) Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl Environ Microbiol 67: 1494–1502CrossRefPubMedGoogle Scholar
  12. 12.
    Chopra, I, Roberts, M (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65: 232–260CrossRefPubMedGoogle Scholar
  13. 13.
    Chu, C, Chiu, CH, Wu, WY, Chu, CH, Liu, TP, Ou, JT (2001) Large drug resistance virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis. Antimicrob Agents Chemother 45: 2299–2303CrossRefPubMedGoogle Scholar
  14. 14.
    Clermont, D, Chesneau, O, De Cespedes, G, Horaud, T (1997) New tetracycline resistance determinants coding for ribosomal protection in streptococci and nucleotide sequence of tet(T) isolated from Streptococcus pyogenes A498. Antimicrob Agents Chemother 41: 112–116PubMedGoogle Scholar
  15. 15.
    De Liguoro, M, Cibin, V, Capolongo, F, Halling-Sorensen, B, Montesissa, C (2003) Use of oxytetracycline and tylosin in intensive calf farming: evaluation of transfer to manure and soil. Chemosphere 52: 203–212CrossRefPubMedGoogle Scholar
  16. 16.
    DePaola, A, Roberts, MC (1995) Class D and E tetracycline resistance determinants in Gram-negative bacteria from catfish ponds. Mol Cell Probes 9: 311–313CrossRefPubMedGoogle Scholar
  17. 17.
    Esiobu, N, Armenta, L, Ike, J (2002) Antibiotic resistance in soil and water environments. Int J Environ Health Res 12: 133–144CrossRefPubMedGoogle Scholar
  18. 18.
    FAO/OIE/WHO (2003) First Joint FAO/OIE/WHO Expert Workshop on Non-human Antimicrobial Usage and Antimicrobial Resistance: Scientific Assessment. FAO/OIE/WHO, GenevaGoogle Scholar
  19. 19.
    FEDESA (1999) Antibiotics for Animals. FEDESA. BrusselsGoogle Scholar
  20. 20.
    Garbeva, P, van Veen, JA, van Elsas, JD (2003) Predominant Bacillus spp. in agricultural soil under different management regimes detected via PCR-DGGE. Microb Ecol 45: 302–316CrossRefPubMedGoogle Scholar
  21. 21.
    Götz, A, Smalla, K (1997) Manure enhances plasmid mobilization and survival of Pseudomonas putida introduced into field soil. Appl Environ Microbiol 63: 1980–1986PubMedGoogle Scholar
  22. 22.
    Grape, M, Sundstrom, L, Kronvall, G (2003) Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources. J Antimicrob Chemother 52: 1022–1024CrossRefPubMedGoogle Scholar
  23. 23.
    Griffiths, RI, Whiteley, AS, O’Donnell, AG, Bailey, MJ (2003) Influence of depth and sampling time on bacterial community structure in an upland grassland soil. FEMS Microbiol Ecol 43: 35–43CrossRefPubMedGoogle Scholar
  24. 24.
    Halling-Sørensen, B, Nielsen, S, Lanzky, PF, Ingerslev, F, Lützhøft, HC, Jørgensen, SE (1998) Occurrence, fate and effects of pharmaceutical substances in the environment—a review. Chemosphere 36: 357–393CrossRefPubMedGoogle Scholar
  25. 25.
    Hamscher, G, Pawelzick, HT, Höper, H, Nau, H (2005) Different behaviour of tetracyclines and sulfonamides in sandy soils after repeated fertilization with liquid manure. Environ Toxicol Chem 24: 861–868CrossRefPubMedGoogle Scholar
  26. 26.
    Hamscher, G, Sczesny, S, Höper, H, Nau, H (2002) Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Anal Chem 74: 1509–1518CrossRefPubMedGoogle Scholar
  27. 27.
    Hansen, LM, Blanchard, PC, Hirsh, DC (1996) Distribution of tet(H) among Pasteurella isolates from the United States and Canada. Antimicrob Agents Chemother 40: 1558–1560PubMedGoogle Scholar
  28. 28.
    Heuer, H, Krögerrecklenfort, E, Wellington, EMH, Egan, S, van Elsas, JD, van Overbeek, L, Collard, J-M, Guillaume, G, Karagouni, AD, Nikolakopoulou, TL, Smalla, K (2002) Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42: 289–302CrossRefPubMedGoogle Scholar
  29. 29.
    Höper, H, Kues, H, Nau, H, Hamscher, G (2002) Eintrag und Verbleib von Tierarzneimittelwirkstoffen in Böden. Bodenschutz 4: 141–148Google Scholar
  30. 30.
    Hund-Rinke, K, Simon, M, Lukow, T (2004) Effects of tetracycline on the soil microflora: function, diversity, resistance. J Soils Sediments 4: 11–16CrossRefGoogle Scholar
  31. 31.
    Huysman, F, van Renterghem, B, Verstraete, W (1993) Antibiotic resistant sulphite-reducing clostridia in soil and groundwater as indicator of manuring practices. Water Air Soil Pollut 69: 243–255CrossRefGoogle Scholar
  32. 32.
    Jackson, CR, Fedorka-Cray, PJ, Barrett, JB, Ladely, SR (2004) Effects of tylosin use on erythromycin resistance in enterococci isolated from swine. Appl Environ Microbiol 70: 4205–4210CrossRefPubMedGoogle Scholar
  33. 33.
    Jensen, LB, Agerso, Y, Sengelov, G (2002) Presence of erm genes among macrolide-resistant Gram-positive bacteria isolated from Danish farm soil. Environ Int 28: 487–491CrossRefPubMedGoogle Scholar
  34. 34.
    Jensen, LB, Baloda, S, Boye, M, Aarestrup, FM (2001) Antimicrobial resistance among Pseudomonas spp. and the Bacillus cereus group isolated from Danish agricultural soil. Environ Int 26: 581–587CrossRefPubMedGoogle Scholar
  35. 35.
    Kay, P, Blackwell, PA, Boxall, AB (2004) Fate of veterinary antibiotics in a macroporous tile drained clay soil. Environ Toxicol Chem 23: 1136–1144CrossRefPubMedGoogle Scholar
  36. 36.
    Korthals, GW, Alexiev, AD, Lexmond, TM, Kammenga, JE, Bongers, T (1996) Long-term effects of copper and pH on the nematode community in an agroecosystem. Environ Toxicol Chem 15: 979–985CrossRefGoogle Scholar
  37. 37.
    Lanz, R, Kuhnert, P, Boerlin, P (2003) Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet Microbiol 91: 73–84CrossRefPubMedGoogle Scholar
  38. 38.
    Lee, C, Langlois, BE, Dawson, KA (1993) Detection of tetracycline resistance determinants in pig isolates from three herds with different histories of antimicrobial agent exposure. Appl Environ Microbiol 59: 1467–1472PubMedGoogle Scholar
  39. 39.
    Leng, Z, Riley, DE, Berger, RE, Krieger, JN, Roberts, MC (1997) Distribution and mobility of the tetracycline resistance determinant tetQ. J Antimicrob Chemother 40: 551–559CrossRefPubMedGoogle Scholar
  40. 40.
    Mathew, AG, Saxton, AM, Upchurch, WG, Chattin, SE (1999) Multiple antibiotic resistance patterns of Escherichia coli isolates from swine farms. Appl Environ Microbiol 65: 2770–2772PubMedGoogle Scholar
  41. 41.
    Onan, LJ, LaPara, TM (2003) Tylosin-resistant bacteria cultivated from agricultural soil. FEMS Microbiol Lett 220: 15–20CrossRefPubMedGoogle Scholar
  42. 42.
    Perreten, V, Boerlin, P (2003) A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob Agents Chemother 47: 1169–1172CrossRefPubMedGoogle Scholar
  43. 43.
    Rajic, A, McFall, ME, Deckert, AE, Reid-Smith, R, Manninen, K, Poppe, C, Dewey, CE, McEwen, SA (2004) Antimicrobial resistance of Salmonella isolated from finishing swine and the environment of 60 Alberta swine farms. Vet Microbiol 104: 189–196CrossRefPubMedGoogle Scholar
  44. 44.
    Riesenfeld, CS, Goodman, RM, Handelsman, J (2004) Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Environ Microbiol 6: 981–989CrossRefPubMedGoogle Scholar
  45. 45.
    Roberts, MC (2003) Acquired tetracycline and/or macrolide–lincosamides–streptogramin resistance in anaerobes. Anaerobe 9: 63–69CrossRefPubMedGoogle Scholar
  46. 46.
    Rooklidge, SJ (2004) Environmental antimicrobial contamination from terraccumulation and diffuse pollution pathways. Sci Total Environ 325: 1–13CrossRefPubMedGoogle Scholar
  47. 47.
    Sczesny, S, Nau, H, Hamscher, G (2003) Residue analysis of tetracyclines and their metabolites in eggs and in the environment by HPLC coupled with a microbiological assay and tandem mass spectrometry. J Agric Food Chem 51: 697–703CrossRefPubMedGoogle Scholar
  48. 48.
    Sengeløv, G, Agersø, Y, Halling-Sørensen, B, Baloda, SB, Andersen, J, Jensen, LB (2003) Bacterial antibiotic resistance levels in Danish farmland as a results of treatment with pig manure slurry. Environ Int 28: 587–595CrossRefPubMedGoogle Scholar
  49. 49.
    Smit, E, Leeflang, P, Gommans, S, van den Broek, J, van Mil, S, Wernars, K (2001) Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods. Appl Environ Microbiol 67: 2284–2291CrossRefPubMedGoogle Scholar
  50. 50.
    Smith, HW (1975) Persistence of tetracycline resistance in pig E. coli. Nature 258: 628–630CrossRefPubMedGoogle Scholar
  51. 51.
    Stanton, T, McDowall, JS, Rasmussen, MA (2004) Diverse tetracycline resistance genotypes of Megasphaera elsdenii strains selectively cultured from swine feces. Appl Environ Microbiol 70: 3754–3757CrossRefPubMedGoogle Scholar
  52. 52.
    Stanton, TB, Humphrey, SB (2003) Isolation of tetracycline-resistant Megasphaera elsdenii strains with novel mosaic gene combinations of tet(O) and tet(W) from swine. Appl Environ Microbiol 69: 3874–3882CrossRefPubMedGoogle Scholar
  53. 53.
    Tauch, A, Puhler, A, Kalinowski, J, Thierbach, G (2000) TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems. Plasmid 44: 285–291CrossRefPubMedGoogle Scholar
  54. 54.
    Ungemach, FR (2000) Figures on quantities of antibacterials used for different purposes in the EU-countries and interpretation. Acta Vet Scand 93: 89–98Google Scholar
  55. 55.
    van Overbeek, LS, Wellington, EMH, Egan, S, Smalla, K, Heuer, H, Collard, J-M, Guillaume, G, Karagouni, AD, Nikolakopoulou, TL, van Elsas, JD (2002) Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. FEMS Microbiol Ecol 42: 277–288PubMedGoogle Scholar
  56. 56.
    WHO (2001) Global Strategy for Containment of Antimicrobial Resistance. Report No. WHO/CDS/CSR/DRS/2001.2a. World Health Organisation, GenevaGoogle Scholar
  57. 57.
    Witte, W (1998) Medical consequences of antibiotic use in agriculture. Science 279: 996–997CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Heike Schmitt
    • 1
  • Krispin Stoob
    • 2
  • Gerd Hamscher
    • 3
  • Eric Smit
    • 4
  • Willem Seinen
    • 1
  1. 1.Institute for Risk Assessment Sciences (IRAS)Utrecht UniversityUtrechtThe Netherlands
  2. 2.EAWAGDübendorfSwitzerland
  3. 3.Department of Food ToxicologyUniversity of Veterinary Medicine Hannover, FoundationHannoverGermany
  4. 4.RIVMBilthovenThe Netherlands

Personalised recommendations