Structural Diversity of Soil

Structural Diversity of Soil

Microorganisms as a sensitive Indicator of adverse Effects from

Pharmaceutical Antibiotics

Sören T HIELE -B RUHN ¹ , Rüdiger R EICHEL ¹ ,

Ute H AMMESFAHR ¹ , A NJA K OTZERKE ² , L UCIA M ICHELINI ³

1

Soil Science, Faculty of Geosciences, University of Trier, Germany

2

Institute of Ecology, Berlin University of Technology, Germany

3

Dept. of Agricultural Biotechnology, University of Padova, Agripolis, Italy

Input through contaminated manure Direct input through grazing livestock

Input through medication in the field Input through stable dust

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

(Kim et al. 2011, Schneidereit 2012, Sarmah et al. 2006)

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)₂ CH₃ OH

O

H H H

OH

H H OH

CONH₂

S N

NH NH

O OMe

N

N N

O F

H₅C₂

COOH O

O O

H

O O

O

O H

O O H

OH O

S

N N

H

O O

N S

OH O

S

N N N N N

N H₂

O O NH O H O H

O H

O O H O

O O

H OH

NH OH NH

N H₂

N H₂

NH₂ NH

Tetracyclines: Oxytetracycline

Fluoroquinolones: Enrofloxacine Benzimidazoles: Fenbendazole ß-Lactams / Cephalosporines:

Cefotiam

Polyethers: Monensin

N H₂

S O

O NH

N N

Sulfonamides: Sulfadiazine

Aminoglycosides: Streptomycine

O H

O O

O O

O O

N OH OH

Macrolides: Oleandomycine

NH O

OH O

N O

O O N

O

Polypeptides: Virginiamycine

Molecular structures of selected antibiotics from often used structural classes

Introduction

Concentrations of SDZ and major transformation products in CaCl ₂ and

methanol extracts from soil

(model fits = dashed lines).

(Förster et al. 2009. Env. Sci. Technol. 43, 1824-1830)

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.

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(III) reduction (% of control)

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

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

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

Experimental Design

Lab. exp.

varied doses flasks

Application↓:

1 x

Soil 1, 4, 32, 61, 125

--- No plants

DFG FOR566

1 2 3 4

100 200 300 400 500 600

Scan number

Intensity in arbitrary units x 10 6 C12:0 C13:0 C14:0 i-C15:0 a-C15:0C15:0 i-C16:0 cis-C16:19C16:0 i-C17:0 C17:0cis-C17:0  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

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

100 200 300 400 500 600

Scan number

Intensity in arbitrary units x 10 6 C12:0 C13:0 C14:0 i-C15:0 a-C15:0 C15:0 i-C16:0 cis-C16:19C16:0 i-C17:0 C17:0cis-C17:0  cis-C18:19 trans-C16:19 C18:0 C20:0

C19:0

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

D A 2 [22. 8%]

10

5

0

-5

-10 unfertilized soil

+ manure

+manure + 10 mg SDZ kg

+manure + 100 mg SDZ kg

Effect of SDZ in manured soil – lab. experiments – community structure

3-factorial ANOVA

Factor PLFA_tot

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.

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

PLFAtot /Control

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

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

M MS M MS

day 29 day 57

*

*

*

*

*

*

-0.2 -0.1 0 0.1 0.2

N [mg g-1 TS 28d-1 ]

pot. Nitrification pot. Ammonification pot. N-Mineralization

0 0.5

1 1.5

2 2.5

3

N -M ineraliz at ion c ont rol

29 57

Incubation time [days]

* *

0 1 2 3 4 5

SI N c ont rol

*

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, 3

ed.)

Antibiotic effects on soil microbial functions

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

Abundances of AOB and AOA amoA genes in soil K in treatments.

Schauss et al. (2009) Environ. Microbiol. 11, 446–

456

Manure: Influence on SDZ effects in soil – pot experiments – functions

0 20 40 60 80

u re a se a ct ivity [ µg N g d m

2 h

]

0 20 40 80 0 20 40 80 0 20 40 80 manure [g kg dm

]

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

soil).

(Hammesfahr et al. (2008) J.

Plant Nutr. Soil Sci. 4, 614-623)

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

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

0 0

Transect 1 Transect 2

5 10 15 20 5 10 15

Earthworm burrow Bulk soil

Transect 2 Transect 1

DRIFT-derivedA:Bratio

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

DRIFT-derivedA:Bratio

0 Transect 3

mm mm

mm mm

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 Ps eud omonas Ca nonical coe ff ici ent (C

)

0,006 0,008 0,010 0,012 0,014 0,016 8

7 6

5 4

3 2

1 mm

DRIFT-derivedA:Bratio

0 25 50 75 100

0-5 mm > 10 mm

Residual SDZ [µg kg-1 ]

-Burrow soil-

-Bulk soil-

• Sampling of aggregates and rhizosphere soil.

Microcompartments: Aggregatosphere, Rhizosphere

Shell Core

• Ratio of extractable SDZ in rhizosphere and earthworm burrows vs.

bulk soil and macroaggregate shell vs. core. Reichel et al. (submitted)

Microcompartments: Enrichment of SDZ

Rhiz osph er e soil

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 (C

): decreasing with SDZ.

Rhizosphere: Effects on microbial biomass

Reichel et al. submitted.

0 10 20 30 40 50

0 10 20 30 40 50

PLF A

[nmo l g

]

Rhizosphere Bulk soil Rhizosphere Bulk soil

0 mg 1 10 0 1 10 0 mg 1 0 1

Microbial biomass (C

): 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

Reichel et al. submitted.

Drillosphere: Effects on community structure

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%]

PC2 [ 17. 8%]

Burrow soil Bulk soil

-1.0 1.5

-1. 0 1. 0

PC1 [46.9%]

PC 2 [18. 0%]

Bulk soil

Burrow soil

Aggregatosphere: Effects on microbial biomass and function

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

PLF A

[nmo l g

] Leucin -am in op eptid as e nmo l MUB (AMC) g

h

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

thiele@uni-trier.de

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