Salmonella Surface Characteristics and Adhesion Rates Samantha Begnoche, Olgun Zorlu, Dr. Sharon Walker Department of Chemical and Environmental Engineering, University of California – Riverside, Riverside, CA Acknowledgments: I would like to thank the National Science Foundation for the research opportunities offered to undergraduates. Thanks to Gexin Chen, Indranil Chowdury, Amy Gong, Berat Haznedaroglu, Ian Marcus, Brian Perez, and Chad Thomsen for all of their help and Jun Wang for coordinating the program. From 1988 to 1995, the number of reported cases of salmonellosis varied between 40,000 and 50,000 each year, excluding cases of typhoid fever, which has a fatality rate of 10% (compared to about 1% for most forms of salmonellosis) (1). Research into Salmonella outbreaks has been mostly limited to the genotypic nature of the cells, and not emphasizing phenotypic (physical and chemical) features of the bacteria. The Salmonella strains utilized in this research include: SA 5983 ( Typhimurium ), SGSC 4910 ( Newport ), and SGSC 2377 ( Enteritidis ). These three strains were selected as they have differing motility. The first step and focus here is characterization of the strains of bacteria. The following step is the determination of dynamic attachment of these three strains on abiotic and biotic surfaces by using parallel plate flow chamber. The objective of this study was to fully characterize these three strains of Salmonella to lead to deeper understanding of the surface chemistry. After characterization adhesion rates of Salmonella on food and engineered surfaces will be investigated utilizing a parallel plate flow chamber. Introduction Methods Results One strain at a time was cultured, harvested, and washed in KCl solution prior to completing one or more of the following: Viability assessment (live/dead) 1 mL of stock bacteria is mixed with 4 mL blank. 1 mL from this mix is added to 3 μL of dye. Vortex and wait 15 minutes. Live (green) and dead (red) bacteria can be counted. Size measurements The length and breadth are measured using images taken from a phase contrast microscope. From this the average effective spherical radii are calculated. Hydrophobicity measurement Using the microbial adhesion to hydrocarbons (MATH) test with n-dodecane, the percentage of cells that choose the hydrocarbon versus the electrolyte condition can be measured. Cells less than or equal to approximately 40% are considered hydrophilic. Conclusion and Future Work Zeta Potential/Electrophoretic mobility measurement Using ZetaPALS the electrophoretic mobility and zeta potential are calculated from a solution diluted to an optical density between.200 and.225. Determination of adhesion rate in Parallel Plate flow chamber: Images are taken of bacteria flowing through the plate at 20 second intervals for a set amount of time. The number of cells depositing on the surface is counted and plotted as a function of time. The adhesion rate (aka mass transfer rate) is calculated from the slope of this plot (2). SIZE MEASUREMENT SA 5983: ± µm SGSC 4910: ± µm SGSC 2377: ± µm References: 1. FDA Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. 2. Chen et al Langmuir 25 (3), Characterization has shown diversity in surface features and chemistry between Salmonella strains. From the data collected thus far it is difficult to draw any inferences on the contribution of motility on adhesion trends. Work is ongoing. Continuing research will include running these strains through 96 well plate adhesion assays. Wells will be coated to mimic the surface layer of foods, for example lettuce and spinach, instead of simply the plastic surface. How modifying the surface chemistry impacts extent of adhesion, provides insight into how these potentially pathogenic strains may adhere to foods and other sanitary surfaces. Ultimate goal is to identify properties of surfaces that inhibit adhesion and lead to a solution to foodborne outbreaks. PARALLEL PLATE FLOW CHAMBER SA 5983 in a 1mM KCl solution exhibits no attachment in a parallel plate system at 1.5 and 2 mL/min. It is expected that increasing ionic strength increases the amount of deposition of Salmonella strain. Furthermore, deposition rate is expected to increase at lower flow rates. HYDROPHOBICITY VIABILITY SA mM KCl: ± % 10 mM KCl: ± % 100 mM KCl: ± % ELECTROPHORETIC MOBILITY