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Cultivation-independent study of Cu-induced development of antibiotic resistance M.Sc. Jeanette Berg Supervisors: Drs Ole Nybroe, Kristian K. Brandt, Søren J. Sørensen, Lars H. Hansen. Control soil from the Hygum site Method: BrdU (Fig. 1) is a thymidine base analogue that can be taken up by bacteria and incorporated into their DNA. Department of Ecology Faculty of Life Sciences Universtiy of Copenhagen Introduction: Previous cultivation based studies have shown development of an increased frequency of antibiotic resistance after introduction of Cu to the soil. Here the incoporation of tritiated bromodeoxyuridine (BrdU) is tested as a cultivation independent measure for the effect of Cu exposure on the bacterial tolerance to Cu and antibiotics. Antibiotic resistance is a problem concerning public health. Cu is spread in agricultural soil with manure and sewage sludge. BrdU Here indigenous soil bacteria are shaked into a buffered suspension, challenged with a concentration array of antibiotics in triplicates in 2 ml vials and incubated with 100 nM BrdU (Fig. 2). Fig. 2 1 2 4 8 16 32 64 128 256 512 0 1 2 4 8 16 32 64 128 256 512 0 1 2 4 8 16 32 64 128 256 512 0 from the Hygum site Why? Fig. 3 Fig. 1 Thymidine 4000 ppm Cu soil Results: Fig. 3 above shows an increased tolerance to Cu in the 4000 ppm Cu contaminated soil - it takes about an order of magnitude higher [Cu] to inhibit the activity 50 % compared to the Control soil. See the ’s Over 99 % of indigenous soil bacteria are not able to grow on the classic agar plates, so cultivation independent test results are more representativ for the whole bacterial community. Below in Fig. 4 for the same soils a likely effect is seen for tolerance to tetracycline, less clear for nalidixic acid, where as no tolerance to olaquindox has developed in the 4000 ppm Cu contaminated soil. A rewiew of co-selection of antibiotic and metal resistance is written by Baker-Austin et al. in 2006. References: After 2 h the incubation is stopped and the DNA is precipitated by adding TCA, and the amount of BrdU incorporated into DNA is measured by scintillation counting as described in Bååth et al. 2001 for leucine and thymidine. Fig. 4 Technical assistance: S. Iversen and K. Hvidtfeldt Baker-Austin, C., Wright, M. S., Stepanauskas, R. and McArthur, J. V.(2006); Trends in Microbiology 14 (4); 176-182. Bååth, E., Pettersson, M. and Söderberg, K.H. (2001); Soil Biology and Biochemistry 33: 15711574. The project is affiliated with and supplements are partley finaced by CREAM