1. F A C U L T Y O F L I F E S C I E N C E S U N I V E R S I T Y O F C O P E N H A G E N Growth Conditions Influence Initial Adhesion and Cell Surface Characteristics of Listeria monocytogenes Anne Skovager*, Marianne H. Larsen**, Nils Arneborg* and Hanne Ingmer** * Department of Food Science, Food Microbiology, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark ** Department of Veterinary Disease Biology, Section for Microbiology, Faculty of Life sciences, University of Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, Denmark Introduction In the present work, it is hypothesized that changes in nutrient composition influence on the initial adhesion of Listeria monocytogenes. The cell characteristics may change as a consequence of different nutrients present. Aim The aim is to examine if different nutrients (carbohydrates and amino acids) alter the initial adhesion of L. monocytogenes to fine polished stainless steel, and if so to determine altered surface characteristics. Conclusion It can be concluded that specific growth conditions (nutrients) have a significant influence on the initial adhesion rate (IAR) of L. monocytogenes on fine polished stainless steel and on the cell surface protein composition. Introduction In the present work, it is hypothesized that changes in nutrient composition influence on the initial adhesion of Listeria monocytogenes. The cell characteristics may change as a consequence of different nutrients present. Aim The aim is to examine if different nutrients (carbohydrates and amino acids) alter the initial adhesion of L. monocytogenes to fine polished stainless steel, and if so to determine altered surface characteristics. Conclusion It can be concluded that specific growth conditions (nutrients) have a significant influence on the initial adhesion rate (IAR) of L. monocytogenes on fine polished stainless steel and on the cell surface protein composition. Results and Discussion  Different nutrients had an influence on IAR (Fig.1A).  2.5 g/L mannose and 2.5 g/L glucose +100 mM L-leucine resulted in the lowest and highest IAR, respectively (P<0.05).  No correlation was found between cell size, hydrophobicity, electron donating and accepting properties and IAR.  The different growth conditions resulted in altered fatty acid composition, however it could not be correlated to IAR. Results and Discussion  Different nutrients had an influence on IAR (Fig.1A).  2.5 g/L mannose and 2.5 g/L glucose +100 mM L-leucine resulted in the lowest and highest IAR, respectively (P<0.05).  No correlation was found between cell size, hydrophobicity, electron donating and accepting properties and IAR.  The different growth conditions resulted in altered fatty acid composition, however it could not be correlated to IAR. Figure 1. A) Initial adhesion of L. monocytogenes cells, grown with different nutrients (cells/cm 2 vs. time). B) SDS-PAGE protein gel of cell surface proteins (3 growth conditions).  Expression of cell surface proteins was dependent on the growth media composition, notably in the presence of 2.5 g/L mannose; 2.5 g/L glucose; and 2.5 g/L glucose +100 mM L-leucine (Fig. 1B).  Proteins with altered expression will be identified in the future.  Expression of cell surface proteins was dependent on the growth media composition, notably in the presence of 2.5 g/L mannose; 2.5 g/L glucose; and 2.5 g/L glucose +100 mM L-leucine (Fig. 1B).  Proteins with altered expression will be identified in the future. Examine effect of: Examine effect of:  Nutrients: Glucose, mannose, fructose, L-leucine, L-isoleucine, L-valine On:  Initial adhesion under liquid flow to stainless steel  Hydrophobicity and electron accepting/ donating properties (Microbial Adhesion To Solvents, MATS) donating properties (Microbial Adhesion To Solvents, MATS)  Cell size  Fatty acid composition (GLC – of whole cells)  Cell surface proteins (SDS-PAGE) Examine effect of: Examine effect of:  Nutrients: Glucose, mannose, fructose, L-leucine, L-isoleucine, L-valine On:  Initial adhesion under liquid flow to stainless steel  Hydrophobicity and electron accepting/ donating properties (Microbial Adhesion To Solvents, MATS) donating properties (Microbial Adhesion To Solvents, MATS)  Cell size  Fatty acid composition (GLC – of whole cells)  Cell surface proteins (SDS-PAGE) Methods  Strain / growth condition Listeria monocytogenes EGD/pNF8 TSB + additional nutrients (22 h at 37°C)  Flow perfusion experiments Fluorescence microscopy and a flow perfusion system (shear stress: 0.10 Pa) was used for determination of IAR under liquid flow (cells/cm 2 *min) (Fig.2). Methods  Strain / growth condition Listeria monocytogenes EGD/pNF8 TSB + additional nutrients (22 h at 37°C)  Flow perfusion experiments Fluorescence microscopy and a flow perfusion system (shear stress: 0.10 Pa) was used for determination of IAR under liquid flow (cells/cm 2 *min) (Fig.2). Cell suspension Flow equalizing system Waste Pumps Digital camera Fluorescence microscope Flow perfusion chamber Figure 2. Experimental setup: The flow chambers are glued on fine polished stainless steel and placed under a fluorescence microscope, connected with a digital camera. The flow chamber is via tubes connected to two pumps. Between the inlet of the perfusion chamber and the inlet pump a flow equalizing system is installed. Sponsor: The Danish Research Council for Technology and Production Sciences; Contact: Anne Skovager, annesj@life.ku.dk A B ▲ ● ♦ Time (min) Cells/cm 2