| The long-term use of implanted medical devices is impeded by bacterial infection on their surfaces. When such devices are inserted in the body, plasma proteins adsorb rapidly onto the material, providing a substrate that facilitates the binding and subsequent colonization of various microorganisms including Staphylococcus aureus, a common pathogen found in hospitals. The attachment of a single bacterium or small clusters of bacteria to biomaterial surfaces is the necessary and often the rate-limiting step in the pathogenesis of a device centered infection. Because microbes are present in almost every aqueous environment, adhesion to other man-made surfaces (water-purification filters, air conditioning condensers, etc.) can lead to troublesome and costly problems for a variety of industries.; An investigation of both the role of both non-specific forces (i.e., electrostatic) and specific interactions (i.e., pair-wise bond formation) in the promotion of bacterial adhesion was performed through direct force measurements. In particular, the effect of MSCRAMM (microbial surface component recognizing adhesive matrix molecules) length on attachment kinetics was investigated. Experiments were performed to (1) measure the interaction forces involved in S. aureus attachment and (2) determine the kinetic rate constant of S. aureus to various surfaces under well-defined flow conditions. These parameters were controlled through the use of specifically engineered S. aureus mutants that express the membrane protein “clumping factor” (ClfA) at varying lengths extending out from the cell surface.; The measurement of interaction forces and dynamics of S. aureus attachment was performed using a new technique that makes the uses evanescent wave light scattering (EWLS) in conjunction with 3-dimensional optical trapping. To validate the technique, measurements were first performed in simplified systems, where bare surfaces and low electrolyte conditions allowed for direct comparisons with established theories. Further investigations of the effect of MSCRAMM length on binding forces revealed the length scales at which specific interactions can act in physiological conditions as well as the magnitude of the forces involved. The corresponding measurement of kinetic rate constants of S. aureus attachment to surfaces was made using a parallel-plate flow chamber with the aid of automated video-microscopy, the results of which were correlated with direct force measurements. |
