Bacterial Adhesion to Tissues Used for RVOT Reconstruction under Static and Shear Stress Conditions

Introduction: Patients after repair of congenital heart disease (e.g., Tetralogy of Fallot) frequently develop dysfunction of the right ventricular outflow tract (RVOT). Implantation of cryopreserved pulmonary homografts, Contegra conduit or stent-mounted Melody valves are often used for reconstruction. This improves patients' quality of life but inherits an increased risk of infective endocarditis (IE). In this work, we focus on the susceptibility of cryopreserved pulmonary homograft and Contegra wall and leaflets to Staphylococcus aureus and Streptococcus sanguinis adherence.

Methods: Standardized tissue pieces prepared as for clinical use were mounted in a 6-well plate and incubated for 1h at 37°C with 10⁷ CFU/mL of bacteria (labeled with carboxy-fluorescein) for static adhesion and adherence under laminar shear stress of 10 dyne/cm² in a flow chamber. Bacterial adhesion was confirmed using the fluorescence microscope IN Cell Analyzer 2000 (GE Helathcare) and quantified by CFU count on blood agar plates. Tissue pieces were sonicated in 1 mL of 0.9% NaCl for bacterial detachment and serial dilution were spotted on blood agar plates. Immunohistochemistry and scan electronic microscopy (SEM) was also performed after fixation.

Results: The adhesion ability of S. aureus was not significantly different comparing the homograft and Contegra^® conduit under static and flow conditions (P< 0.05). Shear stress led to a significant increase of S. aureus adhesion on all tissues compared with the static condition. S. sanguinis shows equal adhesion to homograft and Contegra conduit in static and flow condition (p < 0.05). Interestingly, flow conditions significantly increased the bacterial adhesion (p < 0.05) to the homograft but not to both, Contegra wall and leaflet (p > 0.05). These findings were confirmed using the IN Cell Analyzer 2000. The immunohistochemistry and SEM revealed a “single-cell like” bacterial attachment in static conditions and a “cluster like” pattern under shear stress.

Conclusion: Our results indicate that all graft tissues tested have similar susceptibility to be colonized by bacteria. The important influence of shear stress in bacterial adhesion is verified. It seems that the tissue surface itself has no significant influence on bacterial adhesion; however, it might modulate endothelial cell adhesion and inflammatory responses that are crucial for IE development. Our following studies will focus on these aspects.