Transcript Document

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 92507
Introduction
Methods
• 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.
•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.
Results
100
•ELECTROPHORETIC
MOBILITY
GFP SGSC 2377
% Partitioning
SGSC 4910
70
GFP SGSC 4910
60
-1
-0.5
0
Gfp Sa 5983
Sa 5983
100
SGSC 2377
30
10
100
30
10
60
1
1
50
40
-2.5
-2
-1.5
-1
-0.5
0
Electrophoretic Mobility
30
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
Electrophoretic Mobility
20
50
10
40
0
1
30
10
100
Ionic Strength (mM)
20
10
0
1
10
Hydrobicity of SA 5983 at different ionic strength
100
Ionic Strength (mM)
100
90
Sa 5983
80
GFP Sa 5983
70
% Partitioning
% Partitioning
90
-1.5
Electrophoretic Mobility by Ionic Strength
SA 5983
Electrophoretic Mobility by Ionic Strength
SGSC 2377
70
100
-2
Electrophoretic Mobility
Ionic Strength
Hydrophobicity of SGSC 4910 at different ionic strength
SGSC 2377
80
10
-2.5
Hydrophobicity of SGSC 2377 at different ionic strengths
90
30
1
GFP SGSC 2377
100
SGSC 4910
Ionic Strength (mM)
•VIABILITY
SA 5983
1 mM KCl: 0.815 ± 0.017 %
10 mM KCl: 0.874 ± 0.021 %
100 mM KCl: 0.897 ± 0.118 %
GFP SGSC 4910
Ionic Strength (mM)
•HYDROPHOBICITY
80
Conclusion and Future Work
Electrophoretic Mobility by Ionic Strength
SGSC 4910
•SIZE MEASUREMENT
SA 5983:
0.326 ± 0.113 µm
SGSC 4910: 0.368 ± 0.145 µm
SGSC 2377: 0.317 ± 0.067 µm
60
50
40
30
20
10
0
1
10
100
•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.
Ionic Strength
References:
1. FDA 2009. Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. 2. Chen et al. 2009 Langmuir 25 (3), 1620-1626
•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).
• 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.
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.