Reduce the Level of Salmonella Contamination by Control Its Multicellular Behavior in Food Processing Environment

Non-Technical Summary: Food contaminated with Salmonella causes an estimated 1.3 million people to become ill each year in the United States. Health experts estimate that the annual cost of all foodborne diseases in the United States is $5 to $6 billion in direct medical expenses and lost productivity. Infections with Salmonella alone account for $1 billion each year in direct and indirect medical cost. Salmonella has been long known to produce thin aggregative fimbriae. However, their relationships with another cell surface component, cellulose, and multicellular growth have not been studied until recently. Multicellularity offers a strategic advantage to a pathogen like Salmonella in comparison to free-living cells. It enhances cells' abilities to breakdown nutritional substrates and to combat environmental stresses. At the present time, thin aggregative fimbriae and cellulose - mediated Salmonella colonization on food and food-contact surfaces has not been appropriately addressed. We will evaluate quantify the amounts of thin aggregative fimbriae and cellulose produced by Salmonella, evaluate the role of the two cell surface components in attachment and biofilm formation by Salmonella on certain abiotic surfaces, and identify effective measures to degrade cellulose and thin aggregative fimbriae. <P> Approach: Isolates of Salmonella will be grown on Luria- Bertani (LB) no salt agar, supplemented with Congo red (40 ug/ml) and Coomassie brilliant blue G (20 ug/ml). The plates will be incubated at 28oC for 48-72 h. Colony morphologies will be examined visually as well as by light microscope in order to determine the expression of thin aggregative fimbriae by the Salmonella cells. Additionally, scanning electron micrographs will be taken in order to confirm the expression of the extracellular structure by the Salmonella cells. Cellulose production will be determined by growing the Salmonella cultures on LB no salt agar plates supplemented with 200 ug/ ml Calcofluor white stain. After incubation at 28oC for 48 h, fluorescent stain bound to the cellulose produced by Salmonella cells will be observed under UV light with a wavelength of 365-395 nm. Quantification of thin aggregative fimbriae expressed by Salmonella cells will be carried out using the procedures of Gophna et al. (2001) and Pawar and Chen (2005) with modifications. Quantification of cellulose produced by Salmonella cells will be carried out by a colorimetric method developed by Updegraff (1969) with modifications. All reagents used in the quantification of cellulose will be purchased from Fisher Scientific unless otherwise specified. Salmonella cultures with different surface properties will be grown in LB, LB plus 2% (w/v) glucose (LBG), LB no salt (LBNS), or LB no salt plus 2% (w/v) glucose (LBNSG). The cultures will be used for attachment (1-7 d at 28oC) and biofilm formation (7 d at 28oC) on abiotic surfaces made of polystyrene and glass, respectively. Twenty-four well polystyrene tissue culture plates and SepCap glass vials will be used as polystyrene and glass surface, respectively. Cells of Salmonella attached to and trapped in biofilm on the tested surfaces will be quantified using a crystal violet binding assay previously described by Sonak and Bhosle (1995) and Pawar and Chen (2005) with modifications. In the final phase of the project, enzymatic and chemical agents that can effectively degrade cellulose and thin aggregative fimbriae will be identified and evaluated.