Staphylococcus epidermidis Has Growth Phase Dependent Affinity for Fibrinogen and Resulting Fibrin Clot Elasticity

Abstract

Bacterial infection and thrombosis are highly correlated, especially in patients with indwelling medical devices. Coagulase-negative staphylococci, typified by Staphylococcus epidermidis, are a common cause of medical device infections owing to their biofilm forming capacity which provides protection from antibiotics and host immune response. Attention has been drawn to the interaction between S. epidermidis and host proteins, specifically fibrinogen. However, little is known regarding the impact of the transition from planktonic to biofilm forming phenotype on this interaction. Here we investigate the growth phase dependence of bacteria-fibrinogen interaction and the resulting effect on fibrin clot formation, structure, and mechanics. Flow cytometry demonstrated growth phase dependent affinity for fibrinogen. To mimic intravascular device seeding, we quantified the adhesion of S. epidermidis to a fibrinogen coated surface under continuous flow conditions in vitro. The bacterial deposition rate onto fibrinogen was significantly greater for stationary (5,360 ± 1,776 cells/cm²s) versus exponential phase (2,212 ± 264, cells/cm² s). Furthermore, the expression of sdrG-a cell surface adhesion protein with specificity for fibrinogen-was upregulated ∼twofold in the stationary versus the exponential phase. Rheometry and confocal microscopy demonstrated that stationary phase S. epidermidis slows clot formation and generates a more heterogeneous fibrin network structure with greater elasticity (G' = 5.7 ± 1.0 Pa) compared to sterile fibrinogen (G' = l.5 ± 0.2 Pa), while exponential phase cells had little effect. This work contributes to the current understanding of the growth phase dependent regulation of bacterial virulence factors and the correlation between bacterial infection and thrombosis.

Keywords: SSPA; SdrG; bacterial adhesion; biofilm; rheometry; thrombosis; virulence.

FIGURE 1

Flow cytometry of Staphylococcus epidermidis incubated with fluorescently labeled fibrinogen. (A) Example density plot of fibrinogen fluorescence for a single flow cytometry experiment. (B) Standard box (IQR) and whiskers (1.5*IQR) plot of mean fluorescence of S. epidermidis in exponential versus stationary phase. Bacterial cell concentration was 4 × 10⁹ cells/mL and fibrinogen concentration was 0.5 mg/mL. N = 9 paired experiments with triplicate measurements.

FIGURE 2

Adhesion of S. epidermidis to a fibrinogen coated surface under continuous flow. (A–E) Representative sequential images of fluorescently labeled S. epidermidis (green) captured every 3 s with absence of a cell in white frame in panel (A), adhesion event demonstrated in frames (B–D) and subsequent absence in panel (E) representing a release event. Time between frames (B–E) represents the residence time of that single cell. Scale bar = 20μm. (F) Graphical representation of a single experiment of adhesion events over time for exponential (green) versus stationary phase (red). (G) Deposition rate of exponential versus stationary phase S. epidermidis for N = 10 paired experiments with duplicate measures. (H) Residence time probability distribution of exponential versus stationary phase S. epidermidis for all 10 paired experiments with duplicate measures.

FIGURE 3

Staphylococcus epidermidis alters fibrin elasticity and structure in a growth phase dependent manner. (A) Time sweep of elastic modulus, G′, of pure fibrin clot (0.5 mg/mL) (black) and fibrin clots infected by stationary and (red), exponential phase S. epidermidis (green). Error bars denote standard deviation with N = 3. (B) Mesh size characterization of steady-state pure fibrin clot and fibrin clots infected with stationary and exponential phase S. epidermidis. Error bars denote standard deviations with N = 5. Note that the stack bars (gray and red) in the stationary phase condition indicate two characteristic mesh sizes. (C) Axial projections of a pure fibrin clot, a fibrin clot infected with (D) exponential and (E) stationary phase S. epidermidis. Scale bar = 10 m. Insets with higher magnification, scale bars = 2.5 μm. Fibrin is red and cells are blue. In all cases, cell concentrations are all 4 × 10⁹ cells/mL and fibrin concentration is 0.5 mg/mL.

FIGURE 4

Hypothesized mechanism for the observed phenomenon in Figure 3. When S. epidermidis cells (blue circles) have sufficient affinity for fibrinogen (red lines), the attraction between cells and fibrinogen generate a heterogeneous fibrinogen distribution that results in a heterogeneous fibrin network structure. This has profound implications on fibrin network elasticity and ultimately potential for embolization.