Structural Diversity of Soil Microorganisms as a sensitive ... · Structural Diversity of Soil Microorganisms as a sensitive Indicator of adverse Effects from Pharmaceutical Antibiotics

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)


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(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


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


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


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


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.


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


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.)


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

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)


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

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


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


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

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


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


40 d / SDZ / no manure

Field experiment




Drillosphere: Effects on community structure





Field experiment


• 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



Laboratory experiment - Structure


Field experiment - Function


• 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.


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

Documents

Structural Diversity of Soil Microorganisms as a sensitive ... · Structural Diversity of Soil Microorganisms as a sensitive Indicator of adverse Effects from Pharmaceutical Antibiotics