Salmonella surface characterization and adhesion to food and other surfaces

1. Salmonella surface characterization and adhesion to food and other surfaces Samantha Begnoche Advisor: Dr. Sharon Walker Bioengineering Research Institute for Technical Excellence August 20, 2009

2. Background • From 1988 – 1995 reported cases varied between 40,000 and 50,000 • Previous projects limited to genotypic nature • Objective: Understand surface chemistries and transport kinetics of Salmonella to keep future outbreaks to a minimum.

3. Some Facts • Still, approximately 40,000 cases of Salmonella poisoning reported annually • ~400 deaths annually are result of Salmonella • Children, elderly, and immune diseased are most vulnerable

4. Goals • Short-term work: Characterize strains’ surface chemistries • Three strains focused on: • SGSC 4910 (newport) • SGSC 2377 (enteritidis) • SA 5983 (typhimurium) S. typhimurium respresentation Provided by Berat Haznedaroglu

5. Incubation Centrifuge Inoculation Methods • Bacteria Preparation - Inoculate bacteria night before - Preculture in LB media for specified time - Harvest – centrifuge, wash in KCl twice, resuspend for stock solution

6. Characterization methods • Viability • Mix stock solution and dye. Vortex and wait 15 minutes. Count live (green) and dead (red). • Size measurement • Measure length and width using images taken with phase contrast microscope. Calculate spherical radii.

7. Characterization methods • Hydrophobicity measurement • Using MATH test with n-dodecane, measure the percentage of cells that choose the hydrocarbon versus the electrolyte condition. <40% is hydrophilic • Electrophoretic mobility measurement • Using ZetaPALS, calculate the electrophoretic mobility and zeta potential from a solution diluted to an optical density of .200 to .225 to result in a concentration of about 107 cells.

8. Parallel plate flow chamber methods • Parallel plate flow chamber • Take picture of cells flowing through at 20 second intervals for set amount of time • Number of cells depositing on the surface is counted and plotted as a function of time • Adhesion rate is calculated from slope

9. Fluorescent light Parallel plate flow chamber system Fluorescent light

10. Viability Results • SA 5983: • 1 mM KCl: 81.5 ± 1.7% • 10 mM KCl: 87.4 ± 2.1% • 100 mM KCl: 89.7 ± 11.8%

11. Size Measurement Results • SA 5983: 0.326 ± 0.113 µm • SGSC 4910: 0.368 ± 0.145 µm • SGSC 2377: 0.317 ± 0.067 µm

12. Hydrophobicity Results

13. Hydrophobicity Results

14. Hydrophobicity Results

15. Electrophoretic Mobility Results

16. Electrophoretic Mobility Results

17. Electrophoretic Mobility Results

18. Parallel Plate Results • SA 5983 exhibits no attachment for 1 mM KCl solution. • Amount of deposition is expected to increase with increasing ionic strength • Also expected to increase at lower rates

19. Parallel Plate Flow Chamber Results * Data is representative as work is ongoing

20. Closing • Three strains are very different • As of yet, no inferences can be drawn connecting phenotypic nature and adhesion.

21. Further work 1 • 96 well plate coated to mimic surface layer of foods • Ultimately: identify properties that inhibit adhesion and a solution to foodborne outbreaks 2 3

22. References • http://www.clker.com/search/cartoon+food/3 • http://drjean.org/html/monthly_act/act_2005/09_Sep/pg04.html • http://www.steveklotz.com/blog/?m=200609 • Yates, Walker, Bianchi. Ensuring Food Safety from Pathogens: Farm to Fork.

23. Acknowledgments • Thank you, especially, Dr. Sharon Walker and Olgun Zorlu for teaching me everything that you have and making it run so smoothly. • Thank you very much, Dr. Sharon Walker’s lab group, including Gexin Chen, Indranil Chowdury, Amy Gong, Berat Haznedaroglu, Ian Marcus, Brian Perez, and Chad Thomsenfor all the help in the lab. • Thank you, Jun Wang, for all the work done to make this a great summer for all of us. • Thank you, National Science Foundation for funding opportunities like this one for college students like myself.