, rolf kehr alfred wulf und kornelia smalla · heartwood-k3 polyethylene glass steel cf u/ 1 0 c m....

PS/SchönwälderBBABBA

Topic

Investigations on the hygienic properties of wood

Annett Schönwälder1, Rolf Kehr2 , Alfred Wulf2 und Kornelia Smalla1

Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, Germany

1Institute for Microbiology, Plant Virology and Biosafety2Institute for Plant Protection in Forests


Presentation

1. Interactions between bacteria and wood chips

2. Survival of bacteria on and in wooden boards

3. Preliminary test of wooden pallets in practice

4. Characterization of the antibacterial properties of wood


Experimental approach

Sample preparation(Inoculation of wood chips with Escherichia coli pIE639 or Enterococcus facium)

Incubation

Sampling

Recovery of bacteria by treatmentin a Stomacher-blender

Direct DNA extraction

Cultivation on nutrient agar

Determination of CFU

Purification/ PCR amplification

Agarose gel electrophoresis

Southern blot

Hybridization



Samples (wood chips) Stomacher treatment

crude DNA directly extracted from wood chipsInoculation


Interactions between bacteria and wood chips

Inoculation of different wood varieties and plastic with Escherichia coli pIE639

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+05

1,00E+06

1,00E+07

1,00E+08

1,00E+09

Pine

Spruce

Larch

Maple

Beech Oak

PoplarPolye

thylene

CFU

/g

0 h24 h48 h96 h216 h



L 0h 24h

48h

96h

216h

0h 24h

48h

96h

216h

L

Poplar Polyethylene

E.coli

a.)

b.)

L 0h 24h

48h

96h

216h

0h 24h

48h

96h

216h

0h 24h

48h

96h

216h

Pine SpruceLarch

E.coli

a.)

b.)

L

a) crude DNA of E.coli pIE639 directly extracted from wood chips on agarose gel

b) Southern blot after hybridization with sat3-probe specific for E.coli pIE639



Comparison of the survival of Escherichia coli pIE639 and Enterococcus faecium on different materials

1.00E+00

1.00E+02

1.00E+04

1.00E+06

1.00E+08

1.00E+10

0 50 100 150 200

time [h]

cfu/

g

polyethylen (E.coli) pine (E.coli) pine (E.faecium)

Comparison of the survival of Escherichia coli pIE639 on pine wood (right) andpolyethylen (left) after 24 h

Inoculum: 1*109 cfu/g, RT: 21°C, Relative atmospheric humidity: 55 %


Summary - Interactions between bacteria and wood chips

• Antibacterial effects were found, especially for Pinaceae.

• Recovery of the inoculated strain from pine sawdust was generally lower than that of the other wood varieties.

• Tested bacteria survived longest on plastic.

• On the tested wood Gram-positive bacteria survived longer than Gram-negative bacteria.

• Survival of bacteria on wood depended on wood variety, moisture content of the wood, temperature and humidity of the environment and on the characteristics and density of the inoculated strain.

• The molecular detection showed that the decrease of CFU numbers correlated with the decrease of DNA on wood.

• Some bacteria, especially Enterobacteriaceae, are more sensitive to wood ingredients than others .

• Wood ingredients from different wood types influence different bacterial species.


Survival of bacteria on and in wooden boards


Boards: pine, spruce, larch, poplar, beech, polyethylene

Bacteria: Escherichia coli pIE639Enterococcus faecium

Inoculation: The inoculum was deposited directlyon the board surface and spread with the pipette.The wooden boards were soaked in the inoculum for 15 min.

Sampling: agar contact platesmeasuring of wood shavings


Survival of bacteria on wooden boards

Recovery via agar contact plates, Initial inoculum size: 1x106 CFU/cm², RT: 21 °C

Survival of E. coli pIE639 on new wooden and plastic boards

0

50

100

150

200

250

300

350

400

Pine

Beech

Poplar

Spruce

Larch

Polyethylen

e

CFU/

10 c

m2 30 min

1 h2 h4 h24 h

Bacterial lawn > 350 CFU/cm²




Survival of E. coli pIE639 on new wooden and plastic boards before and after treatment K3

0

50

100

150

200

250

300

350

400

PinePine - K

3

Beech

Beech - K

3

PoplarPoplar

- K3

SpruceSpruce -

K3

LarchLarch - K

3Polyeth

ylene

CFU/

10 cm

2 30 min1 h2 h4 h24 h




Determination of CFU on the surface - measuring of shavings

0

1

2

3

4

5

6

7

8

Pine Pine - K3 Beech Poplar

log

CFU

/cm

2 30 min1h3 h5 h24 h

Initial inoculum size: 5 x 106 CFU/cm² E.coli pIE639, RT: 21 °C



Determination of CFU in the inherent structure (3 mm depth) - measuring of shavings

0

1

2

3

4

5

6

7

8

Pine Pine - K3 Beech Poplar

log

CFU

/cm

2 30 min1h3 h5 h24 h

Initial inoculum size: 5 x 106 CFU/cm² E.coli pIE639, RT: 21 °C



Multiple inoculation of pine wood boards - K3 with E. coli pIE 639

050

100150200250300350400

1.inocu

l.2.

inocul.3.

inocul.4.i

nocul.

5.inocu

l.6.i

nocul.

7. inocul.

8.inocu

l.9.i

nocul.

10.in

ocul.

CFU

/ 10

cm² 0,5 h

1h2 h4 h24 h 50

100150200250300350400

1

CFU

/10

cm²

Multiple inoculation of plastic boards with E. coli pIE 639

0

.inocul.

2. inocul.

3. inocul.

4.inocul.

5.inocul.

6.inocul.

7. inocul.

8.inocul.

9.inocul.

10.inocul.

0,5 h1h2 h4 h24 h

Recovery via agar contact plates, Initial inoculum size: 1 x 106 CFU/cm²,Inoculation each 3 – 5 days, RT: 21 °C




S urvival o f E. c o li pIE639 o n ne w and us e d wo o de n bo ards

0

50

100

150

200

250

300

350

400

2 4 24 48 120

Tim e a f te r inc uba t io n [h]

1999199619941987

Bacteria l lawn > 350 CFU/cm²



Pallets in use

0

50

100

150

200

250

300

350

400

Pine woodpallet (top)

Pine woodpallet (side)

Pine woodpallet

(below)

Pine woodpallet (ref)

Plasticpallet (top)

Plasticpallet

(below)

Plasticpallet (side)

Plasticpallet (ref)

CFU

/10

cm² 3 h

24 h120 h



Summary – Survival of bacteria on and in wooden boards

• The antibacterial effect of pine could be confirmed.

• The antibacterial effect of pine was stable during a number of contaminations.

• Bacterial survival and hygienic properties of wood depended on a number of factors such as wood species, the initial inoculum size and the characteristic of the inoculated strain.

• There was a significant difference in bacterial recoveries from pine wood and plastic. On pine wood the bacteria had the lowest survival compared with other woods (spruce, poplar, beech) and plastic.

• On the tested wood Gram-positive bacteria survived longer than Gram-negative bacteria.

• The antibacterial effect and the effectiveness of the germ reduction was comparable on new and used pine wood boards.

• The decrease of CFU on the surface of pine wood boards corraleted with the decrease of CFU in theinner structure of pine wood.


Characterization of the antibacterial properties of wood

Reasons for the antibacterial effect may be:

1. That the porous and hygroscopic wood take the moisture away from the bacteria, which they need to survive (desiccation). - physical reason

2. Wood ingredients (polyphenols or tannins) cause the antibacterial effect. - chemical reason


Mechanisms of tannin toxicity

1. Astringency: enzyme inhibition and substrate deprivation

Field und Lettinga, 1992



2. Action on membranes

Scalbert, 1991



2. Metal ions deprivation

Insoluble tannin-ferric complexesScalbert, 1991


Microbial defences against tannins

1. Secretion of tannin-binding polymers

2. Tannin-resistant enzymes

3. Tannin oxidation

4. Siderophores

5. Tannin biodegradation


Conclusions

Comparison of different materials after inoculation with E.coli pIE639 (1 x 106 cfu/cm2) - agar contact plates

050

100150200250300350400

Pine-heartwood-K3

Polyethylene Glass Steel

CFU

/10

cm2

0,5 h1 h2 h4 h24 h48 h120 h216 h

• Different wood varieties have different hygienic characteristics!

• Pine wood – a natural raw material with antibacterial properties.

• Pine wood posses clear hygienic advantages opposite other woods and plastic.


Acknowledgments

Heinrich Wilms Company Gustav Wilms, Bad Essen-Barkhausen

Kornelia Smalla Federal Biological Research Centre for Agriculture and Forestry, Institute for Microbiology, Plant Virology and Biosafety, Braunschweig

Alfred Wulf Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Forests, Braunschweig

Rolf Kehr Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Forests, Braunschweig

Helmut Steinkamp German Institute for Food Technology, Quakenbrück

, rolf kehr alfred wulf und kornelia smalla · heartwood-k3 polyethylene glass steel cf u/ 1 0 c m....

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