Structural Diversity of Soil
Microorganisms as a sensitive Indicator of
adverse Effects from
Pharmaceutical Antibiotics
Sören THIELE-BRUHN1, Rüdiger REICHEL1,
Ute HAMMESFAHR1, ANJA KOTZERKE2, LUCIA MICHELINI3
1Soil Science, Faculty of Geosciences, University of Trier, Germany
2Institute of Ecology, Berlin University of Technology, Germany
3Dept. of Agricultural Biotechnology, University of Padova, Agripolis, Italy
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 1
Input through contaminated manure
Direct input through grazing livestock
Input through medication in the field
Input through stable dust
Thiele-Bruhn 2
Introduction
UK France Netherlands Denmark Germany USA NZ Korea China Russia VMD, 2004 ANMV, 2003 FIDIN, 2004 DANMAP, 2005 BfT, 2012 MAF 1999 KFDA 2006
476 1,261 453 114 1,734 11,148 19 non-medical drugs
Consumption of antibiotics for livestock, t yr-1 (Kim et al. 2011, Schneidereit 2012, Sarmah et al. 2006)
Benbrook, 2002
Kenya 1995-99
13 1,278
Thiele-Bruhn S. (2003) Antibiotika. In: Blume et al. (eds.) Handbuch der Bodenkunde. Chap. 6.5.5, 1-19.
OH O O
OH
N(CH3)2OHCH
3
OHH H
OH
HH
OH
CONH2
S N
NH
NH
O
OMe
N
N N
O
F
H5C
2
COOH
O
OOH
OO
O
OH
OHO
OH
O
S
NNH
O O
N
S
OH
O
SN
N
NN
N
NH2
O
O
NH
OH
OH
OH
OOH
O
O
OH OH
NH
OHNH
NH2
NH2
NH2
NH
Tetracyclines: Oxytetracycline
Fluoroquinolones: Enrofloxacine
Benzimidazoles: Fenbendazole
ß-Lactams / Cephalosporines:
Cefotiam
Polyethers: Monensin
NH2
S
O
O
NH
N
N
Sulfonamides: Sulfadiazine
Aminoglycosides: Streptomycine OO
OH
OO
O
O
O O
N
OH
OH
Macrolides: Oleandomycine
NH
O
OH
O
N
O
O
NO
O
Polypeptides: Virginiamycine
Molecular structures of selected antibiotics
from often used structural classes
Introduction
Thiele-Bruhn 3
Concentrations of SDZ and major
transformation products in CaCl2 and
methanol extracts from soil (model fits = dashed lines).
(Förster et al. 2009. Env. Sci. Technol. 43, 1824-1830)
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 4
Fate of SDZ in manured soil
Time after application (d)
Luvisol + manure Cambisol + manure
0 50 100 150 200 0 50 100 150 200
SDZ concentration in soil
that was repeatedly treated
with contaminated manure. (Data: DFG FOR566, A. Focks)
Potential activity of ß-glucosidase
depending on sample treatment (liquid
manure and SDZ) and incubation time.
S0, S10, S100 = SDZ spiking C mg/kg. Hammesfahr et al. (2011) JPNSS 174, 614-623.
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 5
Effects of antibiotics on soil microbial functions
0 0.01 0.1 1 10 100 1,000 10,000 0
20
40
60
80
100
120
140 SDZ
Antibiotic Dose (µmol kg-1)
Fe(I
II)
red
ucti
on
(%
of
co
ntr
ol)
0 0.01 0.1 1 10 100 1,000 10,000 0
20
40
60
80
100
120 OTC SDT
0 0.01 0.1 1 10 100 1,000 10,000 0
20
40
60
80
100
120
Dose-related effects of antibiotics on microbial Fe(III)-reduction in Luvisol-Ah. Thiele-Bruhn (2005) Environ. Toxicol. Chem. 24:869–876
lter.kbs.msu.edu/.../ SEM_microbes_in_soil.jpg
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 6
o Mixed Communities with high Abundance of
Microorganisms.
o Structural Diversity of the Soil Microbial Community.
o Differently distributed and structured among Soil
Microcompartments: Rhizosphere, Aggregatosphere,
Drillosphere.
o Mixed Communities with high Abundance of
Microorganisms.
Structural Diversity of the Soil Microbial Community.
o Differently distributed and structured among Soil
Microcompartments: Rhizosphere, Aggregatosphere,
Drillosphere.
Microcosm/Pot exp. Mesocosm Field Experiment
---- Sandy Cambisol and Topsoil of a Luvisol from Loess -------------------
Zea mays L. (Cultivar RR39K13, Pioneer Hi-Bred)
Soil + pig slurry (control) ↓
Soil + pig slurry with SDZ /DIF ↓
1 / 10 / 100 mg SDZ 0.3 mg SDZ 1 mg SDZ (kg-1 soil)
pots à 0.004 m³ containers à 0.5 m³ field plots à 3 m²
1 x
1x
3x
sampling (d): ↓, 1, 4, 30, 60
-1, ↓0, 6, 13, 27, 41, 60
↓, 14, 48, ↓ 56, 132, ↓140, 252
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 7
Experimental Design
Lab. exp.
varied doses
flasks
Application↓: 1 x
Soil 1, 4, 32, 61, 125
------------ No plants
DFG FOR566
1
2
3
4
100 200 300 400 500 600Scan number
Inte
nsity
in a
rbitr
ary
units
x 1
0 6
C12
:0
C13
:0
C14
:0
i-C15
:0a-
C15
:0C
15:0
i-C16
:0ci
s-C
16:1
9C
16:0
i-C17
:0
C17
:0ci
s-C
17:0
cis-
C18
:19
trans
-C16
:19
C18
:0
C20
:0
C19
:0
cis-
C19
:0
Manure: SDZ
EUB338I
Manure: Control
EUB338I Manure: SDZ
DAPI
DGGE of 16S rRNA genes
GC-FID/GC-MS of PLFA
DNA sequencing
FISH and DAPI staining
DGGE of 16S rRNA genes (Heuer et al. 2002)
universal bacterial primers
group specific bacterial primers:
pseudomonas, α-proteobacteria, ß-proteobacteria,
actinomycetes, streptomycetes
Phospholipid fatty acids (PLFA)
(Zelles & Bai 1993) GC-FID, GC-MS various markers for
bacteria & fungi
Fluorescence in-situ hybridization (FISH)
and staining methods Markers: EUB338-I: active bacteria DAPI: DNA
Sequencing selected bands
Enzyme activities N-cycle C-cycle
Microbial biomass
Statistics ANOVA + HSD post- hoc (p < 0.05) Principal Response Curves (PRC) DA, CA and PCA
Experimental Design - Soil microbial community analysis
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 8
M48B1 1 0 0 0 1 0
M48B2 1 0 0 0 1 0
M48B3 1 0 0 1 0 0
M48B4 1 0 0 1 0 0
S48B1 0 1 0 1 0 0
S48B2 0 1 0 1 0 0
S48B3 0 1 0 1 0 0
S48B4 0 1 0 1 0 0
M48R1 0 1 0 1 0 0
1
2
3
4
100 200 300 400 500 600Scan number
Inte
nsity in
arb
itra
ry u
nits x
10 6
C1
2:0
C1
3:0
C1
4:0
i-C
15
:0a
-C1
5:0
C1
5:0
i-C
16
:0cis
-C16
:19
C1
6:0
i-C
17
:0
C1
7:0
cis
-C17
:0
cis
-C18
:19
tra
ns-C
16
:19
C1
8:0
C2
0:0
C1
9:0
cis
-C19
:0
Discriminant analysis of PLFA from soil
treated with manure and SDZ
(Hammesfahr et al. (2008) Soil Biol. Biochem. 40, 1583-1591)
Cambisol KS
DA 1 [62.6%]
-12 -6 0 6
day 1
day 32
day 4
day 1
day 4
day 32
DA
2 [2
2.8
%]
10
5
0
-5
-10
unfertilized soil
+ manure
+manure + 10 mg SDZ kg-1
+manure + 100 mg SDZ kg-1
Effect of SDZ in manured soil – lab. experiments – community structure
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 9
3-factorial ANOVA Factor PLFAtot
bac:fungi
gram–:gram+
stress
Soil 92.2 *** 59.3 *** 31.1 *** 8.1 **
Treatment 30.0 *** 56.6 *** 3.4 * 13.5 ***
Time 20.3 *** 6.2 ** 20.9 *** 2.6
Soil x Treatment 7.1 *** 5.4 ** 3.1 * 6.3 ***
Soil x Time 10.9 *** 11.2 *** 1.4 1.0
Treatment x Time 4.2 ** 3.6 ** 1.0 0.4
Soil x Treatment x Time 2.2 1.3 1.3 0.8
The universal tree of life Paul E.A. (2007) Soil Microbiology, Ecology,
and Biochemistry. 3rd ed.
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 10
Antibiotic effects on soil microbial community structure
• Shift from bacteria to fungi
• Shift from bacteria to archaea
• Effects on pseudomonads and
ß-proteobacteria
• Shifts from Gram- to Gram+ bacteria
Luvisol + manure
0 50 100 150 200
Time after application (d) 0 50 100 150 200
Long-term effects on microorganisms
Total-PLFA from Luvisol-Ah Merzenhausen
treated with manure and SDZ
(Hammesfahr 2011, PhD thesis)
Control
+ 8.6 mg SDZ
+manure
0.2
0.7
1.2
1.7
2.2
PLF
A to
t /C
ontr
ol
0 25 50 75 100 125
Incubation time [days]
M/U
M/S
M/M
M/MS
a
a,b
a,b b
a
a
b b
M/U
M/S
M/M
M/MS
a
a,b
a,b b
a
a
b b
Effect of SDZ in manured soil – lab. experiments – community structure
+manure + 8.6 mg SDZ
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 11
Factor pot. Nitrification pot. Ammonification pot. N-Mineralization N-Mineral. control-1 SIN control-1
+/- SDZ 120*** 2162*** 9.4** 111*** 15.6***
Storage 0.3 9.9** 0.0 98.6*** 9.5**
Time 3.0 1.4 3.6 127** 1.4
Storage x +/-SDZ 0.8 104*** 6.6* 39.4*** 1.4
+/- SDZ x Time 3.0 27.3*** 0.7 4.9* 1.4
Storage x Time 0.0 16.5*** 0.4 16.9*** 4.0
Storage x +/-SDZ x Time 7.3* 12.1** 4.3* 8.2** 14.7***
M MS M MS
day 29 day 57
* *
*
*
*
*
-0.2
-0.1
0
0.1
0.2
N [m
g g
-1 T
S
28
d -1
] pot. Nitrification pot. Ammonification pot. N-Mineralization
0
0.5
1
1.5
2
2.5
3
N-M
ine
raliz
atio
n c
on
tro
l-1
29 57 Incubation time [days]
* *
0
1
2
3
4
5
SIN
co
ntro
l -1
*
M N-min MS N-min M SIN MS SIN
Effect of SDZ in manured soil – lab. experiments – functions
(Hammesfahr et al. (2011) Europ. J. Soil Biol. 47, 61-68)
Major elements of the terrestrial nitrogen cycle (from Paul, E.A. (2007) Soil Microbiology, Ecology, and Biochemistry, Elsevier – AP, 3rd ed.)
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 13
Antibiotic effects on soil microbial functions
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 14
Effect of SDZ in manured soil – lab. experiments – functions
(Hammesfahr et al. (2011) J. Plant Nutr. Soil Sci. 4, 614-623)
Effects of SDZ on different microbial endpoints in a sandy Cambisol
Proportion of AOA and AOB in total
simulated nitrate production.
SDZ0 SDZ10 SDZ100
Effect of SDZ in manured soil – lab. experiments
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 15
Abundances of AOB and
AOA amoA genes in soil K
in treatments.
Schauss et al. (2009) Environ. Microbiol. 11, 446–
456
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 16
Manure: Influence on SDZ effects in soil – pot experiments – functions
0
20
40
60
80
ure
ase a
ctivity [
µg N
g d
m-1
2h
-1]
0 20 40 80 0 20 40 80 0 20 40 80
manure [g kg dm-1]
day 1
day 8
day 32
0 µg SDZ 10 µg SDZ 100 µg SDZ
Manure and SDZ interact in their effects on microorganisms
Potential activities of
urease depending on
the sample treatment (SDZ spiking concentration
in mg kg-1 soil).
(Hammesfahr et al. (2008) J.
Plant Nutr. Soil Sci. 4, 614-623)
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 17
Manure: Effects of medication on slurry composition
-5
-4
-3
-2
-1
0
1
2
3
4
-10 -8 -6 -4 -2 0 2 4
DA
2 (
31 %
)
DA 1 (57 %)
Control slurry from pigs without medication
Slurry from DIF-medicated pigs
Slurry from SDZ-medicated pigs
Pig slurries (PyFIMS)
• Antibiotic medication affects digestive tract system and
molecular composition of excreta/slurry.
Discriminant analysis of slurry composition determined by pyrolysis-field ionization
mass spectrometry (Py-FIMS) obtained from medicated and control pigs. Reichel et al. (2013) Soil Biology Biochemistry 62, 82-91
• Change of excreted and survival of manure-borne,
possibly antibiotic resistant microorganisms in soil.
DGGE band
Genus (otu) and most related bacterial sequence(s)#
% Identity (bases of match)#
Accession no.#
Source and notes
3f Pseudomonas (otu_3227) Pseudomonas pseudoalcaligenes str. W-20 …
100.00 (435) 100.00 (435) …
EU187489.1 EU395787.1 …
Pseudomonas strains with special degradation potentials
3g Pseudomonas (otu_3227) Pseudomonas sp. BBTR25 Pseudomonas sp. str. 91S1 Pseudomonas sp. str. HY-14 Pseudomonas sp. str. d130 (unclassified)
97.70 (435) 97.70 (435) 97.70 (435) 97.25 (406)
DQ337603.1 EU370417.1 EU620679.2 FJ950669.1
Swine effluent amended soil Pig manure - Treated oxytetracycline production wastewater
3h Pseudomonas (otu_3227) Pseudomonas sp. str. SKU Pseudomonas sp. BBTR25 Pseudomonas sp. str. 91S1 Pseudomonas sp. str. 98S1 Pseudomonas sp. str. HY-14
97.47 (413) 97.47 (413) 97.47 (413) 97.47 (413) 97.47 (413)
AY954288.1 DQ337603.1 EU370417.1 EU370416.1 EU620679.2
- Swine effluent amended soil Pig manure Pig manure -
a Classification and identification according to the BlastN analysis of the Greengenes database
Tab. Sequencing results of excised DGGE bands and most closely related bacterial sequences
Pseudomonas 16S rRNA gene DGGE
from mesocosm experiments; 13 d
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 18
Manure: Effects on community structure – 16S rRNA gene sequencing
• Specific properties of earthworm burrows and sampling.
Microcompartments: Drillosphere
0.005
0.010
0.015
0.020
00
Transect 1 Transect 2
5 10 15 20 5 10 15
Earthworm burrow
Bulk soil
Transect 2
Transect 1
DR
IFT
-de
rive
dA
:Bra
tio
Transect 4
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0 5 10 15 20 25 30 35 5 10 15 20 25 30
DR
IFT
-de
rive
dA
:Bra
tio
0
Transect 3
mmmm
mmmm
Transect 4
Transect 3
10 mm
10 mm
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 19
0.70
0.50
0.55
0.60
0.65
Lining 0-5 5-10 > 10 mm
Pse
ud
om
on
as
Can
on
ical
co
eff
icie
nt
(Cd
t)0,006
0,008
0,010
0,012
0,014
0,016
87654321 mm
DR
IFT
-de
riv
ed
A:B
rati
o
0255075
100
0-5 mm > 10 mm
Re
sid
ua
l S
DZ
[µg
kg
-1] -Burrow soil-
-Bulk soil-
• Sampling of aggregates and rhizosphere soil.
Microcompartments: Aggregatosphere, Rhizosphere
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 20
Shell Core
• Ratio of extractable SDZ in rhizosphere and earthworm burrows vs.
bulk soil and macroaggregate shell vs. core. Reichel et al. (submitted)
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 21
Microcompartments: Enrichment of SDZ
Rh
izo
sp
he
re s
oil
Earthworm
burrow
Soil macro-
aggregates
Distribution of residual SDZ [M:X]
-1
-1
*
*
*
* significant at p < 0.05
• Total PLFA in rhizosphere and bulk soil.
Microbial biomass (Cmic): decreasing with SDZ.
Rhizosphere: Effects on microbial biomass
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 22
Reichel et al. submitted.
0
10
20
30
40
50
0
10
20
30
40
50
PL
FA
tot [n
mo
l g
-1]
Rhizosphere Rhizosphere Bulk soil Bulk soil
0 mg 1 10 0 1 10 0 mg 1 0 1
Microbial biomass (Cmic): decreasing with SDZ
c c
ab ab a b
a b
c d
Laboratory experiment
63 d / SDZ / spiked manure
Field experiment
132 d / SDZ / medication
• Principal component analysis of Pseudomonas 16S rRNA gene DGGE
fingerprints from rhizosphere and bulk soil.
Rhizosphere: Effects on community structure
Laboratory experiment
40 d / SDZ / no manure
Field experiment
132 d / SDZ / medication
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 23
Reichel et al. submitted.
Drillosphere: Effects on community structure
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 24
Reichel et al. submitted.
Laboratory experiment
14 d / SDZ / spiked manure
Field experiment
252 d / SDZ / medication
• Principal component analysis of Pseudomonas 16S rRNA gene DGGE
fingerprints from earthworm burrows and bulk soil.
-1.0 1.0
-0.6
0
.8
PC1 [60.2%]
PC
2 [
17
.8%
]
Burrow soil
Bulk soil
-1.0 1.5
-1.0
1
.0
PC1 [46.9%]
PC
2 [1
8.0
%]
Bulk soil
Burrow soil
Aggregatosphere: Effects on microbial biomass and function
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 25
Reichel et al. submitted.
Laboratory experiment - Structure
14 d / SDZ / spiked manure
Field experiment - Function
252 d / SDZ / medication
• Total PLFA and enzyme activity, respectively, in soil macroaggregate
shell and core soil.
0
10
20
30
40
50
60
a a
b
c
…Shift to fungi in shells of SDZ treated aggregates.
Core Shell
Control SDZ Control SDZ
PL
FA
tot [n
mol g
-1]
Leu
cin
-am
ino
pep
tid
ase
nm
ol M
UB
(A
MC
) g
-1 h
-1
Core Shell
Control SDZ Control SDZ 0
500
1000
1500
2000
2500 a b b b
• Antibiotics reach the soil environment via excrements
in considerable amounts.
• Antibiotic medication alters the molecular and microbial
composition of excreta.
• Mid- to long-term effects of antibiotics and manure interact and
manure borne microorganisms may survive in soil on a mid-term.
• Effects on functional and structural diversity of soil microorganisms.
• Tests on biodiversity are more sensitive than single endpoint tests.
• Structural community shifts may be accompanied by functional
redundancy. Community structure is a more sensitive parameter.
• Long-term effects occur apparent concentration independence.
• Accumulation and effects are different in soil microcompartments.
Thiele-Bruhn, UBA Workshop, Dessau 18-19 June 2013 26
Summary & Conclusions
● to our colleagues, especially from Research Unit FOR 566,
● for funding,
● for your attention.
Soil Science
FB VI Geography/Geosciences, Universität Trier [email protected]
Thanks
Amelung W., Rosendahl I. INRES-Soil Science and Soil Ecology, University of Bonn
Groeneweg J. ICG-4, FZ Jülich
Lamshöft M., Spiteller M. INFU, TU Dortmund
Smalla K., H. Heuer H. JKI, Brunswik
Schloter M., Kleineidam K. German Research Center for Environmental Health, Neuherberg
Wilke B.-M., Kotzerke A., Kindler R. Berlin University of Technology, Institute of Ecology, Berlin
Peeters E., Focks A. Aquatic Ecology & Water Quality Management, Wageningen UR