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 2015Department of Bioengineering, University of Washington,

Seattle, WA BACKGROUNDWhat 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 0.5 1 1.5 2 2.5 3 3.50%

20%

40%

60%

80%

100%

Adhesion in Constant and Pulsatile Flow

ConstantPulsatile

Shear Stress (Pa)

Perc

ent o

f Bac

teri

a B

ound

R

elat

ive

to C

onst

ant P

eak

Val

ueOur 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.