(28g) A Matlab-Based Analysis of the Dynamics and Organization of Staphylococcus Aureus Surface Adhesion in a Bioflux 200 System

Staphylococcus aureus is a gram-positive bacterial pathogen that can form biofilms on a variety of surfaces, including medical implants and host tissues. Adhesion to host tissues is a critical step in the pathogenesis of S. aureus infections. Since most of these biofilms develop in wet environments, shear stress forces generated by fluid flow affect bacterial adhesion dynamics. In this study, we investigated the kinetics and spatial organization of S. aureus cells under varied levels of shear stress using the BioFlux 200 microfluidic system. The overall hypothesis is that hydrodynamic conditions may impact the three-dimensional architecture and strength of biofilm structure by governing the initial patterns of adhesion of bacterial cells to surfaces.

Cell adhesion assays are performed under hydrodynamic stress at 37°C using 48-microwell Low Shear plates (maximum shear stress up to 20 dyn/cm²) with flow channel dimensions of 75μm depth and 350μm width. The system is connected to an inverted fluorescence microscope. To understand the effect of shear stress on the overall organization and pattern of bacterial adhesion, MATLAB coding is used in conjunction with OpenCFU to process time-lapsed microscope images. OpenCFU is an open-sourced application designed to count bacterial cell colonies from images. The MATLAB code retrieves x and y coordinates of bacterial cells from an Excel file and calculates the distances between all pairs of points using the Euclidean distance metric. Then it generates matrices that output both the average distance between all cells in the image and the average minimum distance between these cells. A histogram showing the distribution of the range of distances between all cells is also generated. This program enables us to automate the distance calculations between numerous cells in each image.

Data suggests that increasing shear stress from 1 dyn/cm² to 5 dyn/cm² leads to a 64% decrease in S. aureus surface concentration after one hour and a 49% decrease in the maximum rate of adhesion, which may be explained by the lower residence time of the bacteria within the microfluidic system. In comparison, analysis of the surface adhesion kinetics of polystyrene FluoSpheres under the same hydrodynamic conditions reveal that increasing shear stress from 1 dyn/cm² to 5 dyn/cm² leads to a 55% decrease in FluoSpheres surface concentration after one hour. However, under similar circumstances, the observed surface concentration of FluoSpheres may be nearly 94% less than that of S. aureus cells.

Further studies are ongoing to determine the extent of hydrodynamic influence on adhesion compared to the chemical and biological properties of bacteria which can lead to an enhanced understanding and visualization of the biofilm formation process and architecture. Future studies will further explore the molecular mechanisms underlying the observed adhesion dynamics and investigate the impact of shear stress on biofilm formation in in-vivo and clinical settings. Ultimately, these insights could inform the development of novel therapeutic approaches to prevent or treat S. aureus infections, particularly those associated with medical devices or implanted tissues.

Funder Acknowledgment(s): This study was supported by an NSF CMMI Award # 2000330 to Dr. Patrick Ymele-Leki.