Salmonella surface characterization and adhesion to food and other surfaces

Samantha BegnocheAdvisor: Dr. Sharon WalkerBioengineering Research Institute for Technical ExcellenceAugust 20, 2009

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.

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

Goals Short-term work:

Characterize strains’ surface chemistries

Three strains focused on: SGSC 4910 (newport) SGSC 2377 (enteritidis) SA 5983 (typhimurium)

S. typhimurium respresentationProvided by Berat Haznedaroglu

Methods Bacteria Preparation- Inoculate bacteria night before- Preculture in LB media for specified time- Harvest – centrifuge, wash in KCl twice,

resuspend for stock solution

Incubation CentrifugeInoculation

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.

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.

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

Fluorescent light

Fluorescent light

Parallel plate flow chamber system

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%

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

Hydrophobicity ResultsHydrophobicity of SGSC 4910 at different ionic strength

0

10

20

30

40

50

60

70

80

90

100

1 10 100

Ionic Strength (mM)

% P

arti

tio

nin

g

SGSC 4910

GFP SGSC 4910

Hydrophobicity ResultsHydrophobicity of SGSC 2377 at different ionic strengths

0

10

20

30

40

50

60

70

80

90

100

1 10 100Ionic Strength (mM)

% P

art

itio

nin

g

SGSC 2377

GFP SGSC 2377

Hydrophobicity ResultsHydrobicity of SA 5983 at different ionic strength

0

10

20

30

40

50

60

70

80

90

100

1 10 100Ionic Strength (mM)

% P

art

itio

nin

g

Sa 5983

GFP Sa 5983

Electrophoretic Mobility ResultsElectrophoretic Mobility by Ionic Strength

SGSC 4910

-2.5 -2 -1.5 -1 -0.5 0

1

10

30

100

Ion

ic S

tren

gth

(m

M)

Electrophoretic Mobility

GFP SGSC 4910

SGSC 4910

Electrophoretic Mobility ResultsElectrophoretic Mobility by Ionic Strength

SGSC 2377

-2.5 -2 -1.5 -1 -0.5 0

1

10

30

100

Ion

ic S

tre

ng

th (

mM

)

Electrophoretic Mobility

GFP SGSC 2377

SGSC 2377

Electrophoretic Mobility ResultsElectrophoretic Mobility by Ionic Strength

SA 5983

-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5

1

10

30

100

Ion

ic S

tre

ng

th (

mM

)

Electrophoretic Mobility

Gfp Sa 5983

Sa 5983

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

Parallel Plate Flow Chamber ResultsFor 100 mM KCl 1.5 ml/min 2.0 ml/min

Length (um) 211 211

Width (um) 168 168

Microscope viewing area (m2) 3.5448E-08 3.5448E-08

average particle diameter (nm) 338 338

No of particles in inflow (particles/L) 500000000 500000000

Slope (particles/sec) 0.118 0.0487

Flux Jz (particles/sec-m2) 3328819.68 1373843.376

Kpp (m/s) 6.60E-06 2.75E-06

* Data is representative as work is ongoing

Closing Three strains are very different As of yet, no inferences can be drawn

connecting phenotypic nature and adhesion.

Further work 96 well plate coated to

mimic surface layer of foods

Ultimately: identify properties that inhibit adhesion and a solution to foodborne outbreaks

1

2

3

References1. http://www.clker.com/search/cartoon+food/32. http://drjean.org/html/monthly_act/

act_2005/09_Sep/pg04.html3. http://www.steveklotz.com/blog/?m=2006094. Yates, Walker, Bianchi. Ensuring Food Safety

from Pathogens: Farm to Fork.

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 Thomsen for 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.