Investigations on the hygienic properties of wood

Transcription

1 Topic Investigations on the hygienic properties of wood Annett Schönwälder 1, Rolf Kehr 2, Alfred Wulf 2 und Kornelia Smalla 1 Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, Germany 1 Institute for Microbiology, Plant Virology and Biosafety 2 Institute for Plant Protection in Forests

2 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

3 Experimental approach Sample preparation (Inoculation of wood chips with Escherichia coli pie639 or Enterococcus facium) Incubation Sampling Recovery of bacteria by treatment in a Stomacher-blender Cultivation on nutrient agar Determination of CFU Direct DNA extraction Purification/ PCR amplification Agarose gel electrophoresis Southern blot Hybridization

4 Experimental approach Samples (wood chips) Stomacher treatment Inoculation crude DNA directly extracted from wood chips

5 Interactions between bacteria and wood chips Inoculation of different wood varieties and plastic with Escherichia coli pie639 CFU/g 1,00E+09 1,00E+08 1,00E+07 1,00E+06 1,00E+05 1,00E+04 1,00E+03 1,00E+02 1,00E+01 1,00E+00 0 h 24 h 48 h 96 h 216 h Pine Spruce Larch Maple Beech Oak Poplar Polyethylene

6 Interactions between bacteria and wood chips Pine Larch Spruce Poplar Polyethylene L 0h 24h 48h 96h 216h 0h 24h 48h 96h 216h 0h 24h 48h 96h 216h L L 0h 24h 48h 96h 216h 0h 24h 48h 96h 216h L E.coli E.coli a.) a.) b.) b.) 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

7 Interactions between bacteria and wood chips Comparison of the survival of Escherichia coli pie639 and Enterococcus faecium on different materials 1.00E E+08 polyethylen (E.coli) pine (E.coli) pine (E.faecium) cfu/g 1.00E E E E time [h] Inoculum: 1*10 9 cfu/g, RT: 21 C, Relative atmospheric humidity: 55 % Comparison of the survival of Escherichia coli pie639 on pine wood (right) and polyethylen (left) after 24 h

8 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.

9 Survival of bacteria on and in wooden boards Experimental approach Boards: pine, spruce, larch, poplar, beech, polyethylene Bacteria: Escherichia coli pie639 Enterococcus faecium Inoculation: The inoculum was deposited directly on the board surface and spread with the pipette. The wooden boards were soaked in the inoculum for 15 min. Sampling: agar contact plates measuring of wood shavings

10 Survival of bacteria on wooden boards Survival of E. coli pie639 on new wooden and plastic boards Bacterial lawn > 350 CFU/cm² 300 CFU/10 cm min 1 h 2 h 4 h 24 h 50 0 Pine Beech Poplar Spruce Larch Polyethylene Recovery via agar contact plates, Initial inoculum size: 1x10 6 CFU/cm², RT: 21 C

11 Survival of bacteria on wooden boards Survival of E. coli pie639 on new wooden and plastic boards before and after treatment K Bacterial lawn > 350 CFU/cm² 300 CFU/10 cm min 1 h 2 h 4 h 24 h Pine Pine - K3 Beech Beech - K3 Poplar Poplar - K3 Spruce Spruce - K3 Larch Larch - K3 Polyethylene Recovery via agar contact plates, Initial inoculum size: 1x10 6 CFU/cm², RT: 21 C

12 Survival of bacteria on and in wooden boards Determination of CFU on the surface - measuring of shavings 8 7 log CFU/cm min 1h 3 h 5 h 24 h 0 Pine Pine - K3 Beech Poplar Initial inoculum size: 5 x 10 6 CFU/cm² E.coli pie639, RT: 21 C

13 Survival of bacteria on and in wooden boards Determination of CFU in the inherent structure (3 mm depth) - measuring of shavings 8 7 log CFU/cm min 1h 3 h 5 h 24 h 0 Pine Pine - K3 Beech Poplar Initial inoculum size: 5 x 10 6 CFU/cm² E.coli pie639, RT: 21 C

14 Survival of bacteria on wooden boards Multiple inoculation of pine wood boards - K3 with E. coli pie 639 Multiple inoculation of plastic boards with E. coli pie ,5 h h h h h ,5 h 1h 2 h 4 h 1.inocul. 2. inocul. 3. inocul. 4.inocul. 5.inocul. 6.inocul. 7. inocul. 8.inocul. 9.inocul inocul. CFU/ 10 cm² CFU/10 cm² 24 h.inocul. 2. inocul. 3. inocul. 4.ino cul. 5.inocul. 6.inocul. 7. ino cul. 8.inocul. 9.inocul. 10.ino cul. Recovery via agar contact plates, Initial inoculum size: 1 x 10 6 CFU/cm², Inoculation each 3 5 days, RT: 21 C

15 Survival of bacteria on wooden boards Survival of E. co li pie639 on new and used wooden boards Bacterial lawn > 350 CFU/cm² Time after incubatio n [h] Recovery via agar contact plates, Initial inoculum size: 1x10 8 CFU/cm², RT: 21 C

16 Survival of bacteria on wooden boards Pallets in use Bacterial lawn > 350 CFU/cm² 300 CFU/10 cm² h 24 h 120 h Pine wood pallet (top) Pine wood pallet (side) Pine wood pallet (below) Pine wood pallet (ref) Plastic pallet (top) Plastic pallet (below) Plastic pallet (side) Plastic pallet (ref)

17 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 the inner structure of pine wood.

18 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

19 Mechanisms of tannin toxicity 1. Astringency: enzyme inhibition and substrate deprivation Field und Lettinga, 1992

20 Mechanisms of tannin toxicity 2. Action on membranes Scalbert, 1991

21 Mechanisms of tannin toxicity 2. Metal ions deprivation Scalbert, 1991 Insoluble tannin-ferric complexes

22 Microbial defences against tannins 1. Secretion of tannin-binding polymers 2. Tannin-resistant enzymes 3. Tannin oxidation 4. Siderophores 5. Tannin biodegradation

23 Conclusions Comparison of different materials after inoculation with E.coli pie639 (1 x 10 6 cfu/cm 2 ) - agar contact plates CFU/10 cm Pineheartwood-K3 Polyethylene Glass Steel 0,5 h 1 h 2 h 4 h 24 h 48 h 120 h 216 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.

24 Acknowledgments Heinrich Wilms Kornelia Smalla Alfred Wulf Rolf Kehr Company Gustav Wilms, Bad Essen-Barkhausen Federal Biological Research Centre for Agriculture and Forestry, Institute for Microbiology, Plant Virology and Biosafety, Braunschweig Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Forests, Braunschweig Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Forests, Braunschweig Helmut Steinkamp German Institute for Food Technology, Quakenbrück