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Effect of veterinary medicines and manure amendment on the abundance, diversity and transfer of bacterial antibiotic resistance genes in soil bacteria.

Prof. Dr. K. Smalla, Julius Kühn Institut Braunschweig

Summary

Large amounts of antibiotics are applied in veterinary medicine and reach agricultural fields by manure fertilization. In soil, the fate of these substances and their effects on the structure and function of bacterial communities and the development and spread of antibiotic resistance genes and mobile genetic elements were largely unknown. However, the respective knowledge is crucial for an assessment of risks associated with the application of antibiotics with manure to agricultural soils and potential effects on human health.
In this project we aimed at the assessment of effects of the veterinary medicines amoxicillin (AMX), difloxacin (DIF) and sulfadiazine (SDZ) amended via manure to soils on the abundance, diversity, and mobility of bacterial antibiotic resistance genes in soil bacteria, with special focus on the importance of the rhizosphere, the impact of repeated manure applications and the effects of varying soil moisture conditions. Therefore, manure spiked with antibiotics or collected from treated animals was once or repeatedly applied to agricultural soil in microcosm, mesocosm and field experiments and these treatments were compared to soil amended with manure free of antibiotics. The dissipation of the antibiotics in manure, bulk soil and rhizosphere was followed in cooperation with the A3 group and correlated to the abundance and transferability of resistance genes and mobile genetic elements, measured by quantitative real time PCR, Southern blot hybridization and exogenous plasmid isolation. We could demonstrate that manure itself is a reservoir of transferable antibiotic resistance plasmids of different incompatibility groups. In contrast to the diversity of plasmids and gene cassettes, a similar bacterial community structure and consistently high abundance of sulfonamide resistance genes was observed in field scale manures. The application of manure and antibiotics synergistically increased the abundance of antibiotic resistance genes in soil and their transferability. The repeated application of manure containing antibiotics even caused an accumulation of resistance genes, antibiotics and bacterial responders in bulk soil under controlled microcosm conditions. In the field, this accumulation was neither observed in bulk soil nor in the rhizosphere, which might be due to different reasons such as the lower concentrations of sulfadiazine but also organic matter in the manure from treated pigs, and an accelerated dissipation of SDZ in the rhizosphere. Varying soil moisture conditions were also tested as a possible reason for this discrepancy but seemed to have only a minor influence on the fate and effects of veterinary medicines applied with manure to soil. Mobile genetic elements which likely play a major role in horizontal spread of resistance genes between manure and soil bacteria were identified as IncP-1ε plasmids as well as the in this project firstly described LowGC-type plasmids. These plasmids showed a remarkable diversity of antibiotic resistance genes, the ability to efficiently transfer under soil conditions, and a correlation to antibiotic selective pressure which strongly suggest that these plasmids are important vectors for the spread of antibiotic resistance in the agro-ecosystem. Competition experiments in soil microcosms comparing the persistence of A. baylyi BD413 with or without plasmid pHHV216 in soil revealed a fitness advantage of the plasmid carrying strain when the soil was treated with SDZ spiked manure. The results obtained in this project provide significant progress in the understanding of the fate and effects of antibiotics applied with manure to soil and the associated risks for human health.

 

begutachtete Publikationen:

  1. Binh, CTT, Heuer, H, Gomes, NCM, Kotzerke, A, Fulle, M, Wilke, BM, Schloter, M, Smalla, K (2007): Short-term effects of amoxicillin on bacterial communities in manured soil. FEMS Microbiology Ecology 62, 290–302.
  2. Binh, CTT, Heuer, H, Kaupenjohann, M, Smalla, K (2008): Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. FEMS Microbiology Ecology 66, 25–37.
  3. Binh, CTT, Heuer, H, Kaupenjohann, M, Smalla, K (2009): Diverse aadA gene cassettes on class 1 integrons introduced into soil via spread manure. Research in Microbiology 160, 427–433.
  4. Ding, GC, Radl, V, Hai, B, Jechalke, S, Heuer, H, Smalla, K, Schloter, M (2014): Dynamics of soil bacterial communities in response to repeated application of manure containing sulfadiazine. PLoS ONE 9, e92958.
  5. Gaze, WH, Krone, SM, Larsson, DGJ, Li, X, Robinson, JA, Simonet, P, Smalla, K, Timinouni, M, Topp, E, Wellington, EM, Wright, GD, Zhu, YG (2013): Influence of humans on evolution and mobilization of environmental antibiotic resistome. Emerging Infectious Diseases 19, e120871.
  6. Heuer, H, Smalla, K (2007): Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months. Environmental Microbiology 9, 657–666.          
  7. Heuer, H, Focks, A, Lamshöft, M, Smalla, K, Matthies, M, Spiteller, M (2008): Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and manured soil. Soil Biology and Biochemistry 40, 1892–1900.
  8. Heuer, H, Kopmann, C, Binh, CTT, Top, EM, Smalla, K (2009): Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content. Environmental Microbiology 11, 937–949
  9. Heuer, H, Schmitt, H, Smalla, K (2011): Antibiotic resistance gene spread due to manure application on agricultural fields. Current Opinion in Microbiology 14, 236–243.
  10. Heuer H, Solehati Q, Zimmerling U, Kleineidam K, Schloter M, Müller T, Focks A, Thiele-Bruhn S, Smalla K (2011) Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine. Appl Environ Microbiol 77: 2527-2530.
  11. Heuer, H, Binh, CTT, Jechalke, S, Kopmann, C, Zimmerling, U, Krögerrecklenfort, E, Ledger, T, González, B, Top, E, Smalla, K (2012): IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes. Frontiers in Microbiology 3, Article 2.
  12. Heuer, H, Smalla K (2012): Plasmids foster diversification and adaptation of microbial populations in soil. FEMS Microbiology Reviews 36, 1083–1104.
  13. Jechalke, S, Kopmann, C, Rosendahl, I, Groeneweg, J, Weichelt, V, Krögerrecklenfort, E, Brandes, N, Nordwig, M, Ding, GC, Siemens, J, Heuer, H, Smalla, K (2013): Increased abundance and transferability of resistance genes after field application of manure from sulfadiazine-treated pigs. Applied and Environmental Microbiology 79, 1704–1711.
  14. Jechalke, S, Dealtry, S, Smalla K, Heuer H (2013). Quantification of IncP-1 plasmid prevalence in environmental samples. Applied and Environmental Microbiology 79, 1410–1413.
  15. Jechalke, S, Focks, A, Rosendahl, I, Groeneweg, J, Siemens, J, Heuer, H, Smalla, K (2014): Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs. FEMS Microbiology Ecology 87, 78-88.
  16. Jechalke, S, Kopmann, C, Richter, M, Moenickes, S, Heuer, H, Smalla, K (2013): Plasmid-mediated fitness advantage of Acinetobacter baylyi in sulfadiazine-polluted soil. FEMS Microbiology Letters 348, 127–132.
  17. Jechalke, S, Focks, A, Rosendahl, I, Groeneweg, J, Siemens, J, Heuer, H, Smalla, K (2014): Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs. FEMS Microbiology Ecology 87, 78-88.
  18. Jechalke, S., Heuer, H., Siemens, J., Amelung, W., Smalla, K. (2014): Fate and effects of veterinary antibiotics in soil. Trends in Microbiology  DOI: http://dx.doi.org/10.1016/j.tim.2014.05.005
  19. Kopmann, C, Jechalke, S, Rosendahl, I, Groeneweg, J, Krögerrecklenfort, E, Zimmerling, U, Weichelt, V, Siemens, J, Amelung, W, Heuer, H, Smalla, K (2013): Abundance and transferability of antibiotic resistance as related to the fate of sulfadiazine in maize rhizosphere and bulk soil. FEMS Microbiology Ecology 83, 125–134.

 

Weitere Publikationen:

  1. Jechalke, S., Heuer, H. Smalla, K (2013): Antibiotikaresistenzgene im Ackerboden. Biospektrum 19, 243-246.

 

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