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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