1. Comparing Adhesive Properties of Streptococcus gordonii
in Constant and Pulsatile Flow
ABSTRACT METHODS
Acknowledgements: This research was made possible by the NIH Foundation (1R01 AI106987-01),
and NASA Space Grant Consortium.
Jasmine Hawkins, Jamie Nunez, Olga Yakovenko, Dr. Wendy Thomas
Summer Undergraduate Research Program 2015
Department of Bioengineering, University of Washington, Seattle, WA
BACKGROUND
What is anti-adhesion therapy?
• Can be used to treat bacterial infections.
• Alternative to antibiotics and avoids drug resistance.
• Targets the process of bacterial adhesion - a prerequisite for bacterial infections.
How do they cause infections in the human body?
• Fluid flow plays a significant role in weakening or enhancing bacterial adhesion.
• The flow in human bodies is pulsatile.
• Certain strains of Streptococcus have found methods to enhance binding under high flow
conditions, called shear-enhanced adhesion.
Understanding the effect of both constant and pulsatile flow on strains of
Streptococcus may help design inhibitors to bacterial adhesion.
What is the significance of Streptococcus?
• Certain species cause bacterial infections such as strep
throat, pneumonia, and meningitis.
• The viridans group streptococci are responsible for
many cases of bacterial endocarditis, an infection of
the inner lining of the heart.
• They can cause infections by binding to human
platelets, a component of human blood.
Figure 1: Streptococci "viridans" group
binding to host cell via glycoproteins.
(http://streptococcusviridans.org)
BACTERIAL ADHESION
Streptococcus gordonii has a serine-rich repeat (SRR) surface
Protein B known as GspB. This glycoprotein adhesin
binds through its interaction with sialoglycans, specifically
sialyl-Tantigen (sTa) on the platelet membrane receptor
GPIbα.
Strep demonstrate shear-enhanced adhesion. Under low
shear stress, Strep form short-lived bonds and exhibit
free-floating transient adhesion. Strep switch to a steady
rolling adhesion under medium shear stress that balances
between formation and dissociation of adhesive bonds.
Under high shear stress, Strep exhibit a robust stationary
mode. Bacteria are more resistant to soluble inhibitors
during the stationary mode of adhesion.
Platelet
GPIbα
Streptococcus
gordonii
sTa
GspB
Strep
Strep
Shear Stress Adhesive Mode
Low
Medium
High
Strep
A short-lived bonds
B rolling
C stationary
RESULTS AND CONCLUSION
0%
20%
40%
60%
80%
100%
0 0.5 1 1.5 2 2.5 3 3.5
PercentofBacteriaBoundRelative
toConstantPeakValue
Shear Stress (Pa)
Adhesion in Constant and Pulsatile Flow
Constant
Pulsatile
Our results indicate that Streptococcus
gordonii demonstrate shear-enhanced
adhesion, where the peak value of
bacteria binding in pulsatile flow is
10 fold that of constant flow. Both
constant and pulsatile flow follow
similar trends, with the exception of
pulsatile flow increasing the peak
shear stress value.
Further research may look to include
shear stresses that are physiologically
possible since bacterial lesions are
exposed to 2-8 Pa in the circulatory
system. Also, a higher frequency of
pulses are desired and more
physiologically relevant.
The purpose of our research is to understand and compare the
adhesion of Streptococcus gordonii to platelets in constant and
pulsatile flow. Our research focuses primarily on cases of
bacterial endocarditis, a life-threatening infection of the
interior of the heart, which can be caused by strains of
Streptococcus. In our model system, we utilize a flow chamber to
mimic pulsatile flow conditions in heart valves, which oscillate
between a low and high flow, with a maximum shear stress of
2-8 Pa. We hypothesize that under pulsatile flow, S. gordonii will
preferentially attach to platelets under low flow conditions, and
will switch to a robust stationary mode when placed under
high flow conditions. We took videos to determine the number
of bacteria that bind to platelets under constant and pulsatile
flows, and analyzed these to determine the conditions that
enhance adhesion. In constant flow, our results indicate that S.
gordonii bind best at a low shear stress. The peak of bacteria
binding in pulsatile flow is nearly 10 fold that of constant flow.
This suggests that S. gordonii binding to platelets is enhanced by
pulsatile flow. Both constant and pulsatile flow indicate similar
trends in the number binding as the shear stress increases to
high values. By understanding the adhesive properties of these
strains, our research has the potential to provide significant
insights into understanding shear-enhanced adhesion.
Furthermore, our research may contribute to the development
of anti-adhesive therapies to treat bacterial endocarditis.
Figure 2: Flow chamber
attached to gasket with
vacuum seal, placed within
plate coated with sTa.
Input Output
Vacuum
Gasket
Plate
Figure 3: Data taken of S. gordonii adhesion to sTa. All values normalized to constant flow peak.
Bacterial culture: Strain M99 of Streptococcus gordonii was
grown and incubated anaerobically.
Preparation of sTa-coated surfaces: Corningwire plates
were coated with biotinylated bovine serum albumin,
streptavidin, and sialyl-Tantigen respectively.
Binding in flow: A glycotechTM flow chamber was positioned
on microscope, and attached to a 50 mL syringe filled with
bacteria. Syringe pump was programmed at both constant and
pulsatile flow rates to achieve necessary shear stresses. Pulsatile
flow turned on and off every 2 seconds for 3 minutes.
Analyzing properties of adhesion: Videos of bacteria
analyzed using ImageJ. To determine the number of bacteria
that bind to the plate, the last shot was subtracted from the
first shot of the video.