Detachment and successive re-attachment of multiple, reversibly-binding tethers result in irreversible bacterial adhesion to surfaces.

Bacterial adhesion to surfaces occurs ubiquitously and is initially reversible, though becoming more irreversible within minutes after first contact with a surface. We here demonstrate for eight bacterial strains comprising four species, that bacteria adhere irreversibly to surfaces through multiple, reversibly-binding tethers that detach and successively re-attach, but not collectively detach to cause detachment of an entire bacterium. Arguments build on combining analyses of confined Brownian-motion of bacteria adhering to glass and their AFM force-distance curves and include the following observations: (1) force-distance curves showed detachment events indicative of multiple binding tethers, (2) vibration amplitudes of adhering bacteria parallel to a surface decreased with increasing adhesion-forces acting perpendicular to the surface, (3) nanoscopic displacements of bacteria with relatively long autocorrelation times up to several seconds, in absence of microscopic displacement, (4) increases in Mean-Squared-Displacement over prolonged time periods according to tα with 0 < α ≪ 1, indicative of confined displacement. Analysis of simulated position-maps of adhering particles using a new, in silico model confirmed that adhesion to surfaces is irreversible through detachment and successive re-attachment of reversibly-binding tethers. This makes bacterial adhesion mechanistically comparable with the irreversible adsorption of high-molecular-weight proteins to surfaces, mediated by multiple, reversibly-binding molecular segments.

Implications for directionality of nanoscale forces in bacterial attachment.

Adhesion and friction are closely related and play a predominant role in many natural processes. From the wall-clinging feet of the gecko to bacteria forming a biofilm, in many cases adhesion is a necessity to survive. The direction in which forces are applied has shown to influence the bond strength of certain systems tremendously and can mean the difference between adhesion and detachment. The spatula present on the extension of the feet of the gecko can either attach or detach, based on the angle at which they are loaded. Certain proteins are known to unfold at different loads, depending on the direction at which the load is applied and some bacteria have specific receptors which increase their bond strength in the presence of shear. Bacteria adhere to any man-made surface despite the presence of shear forces due to running fluids, air flow, and other causes. In bacterial adhesion research, however, adhesion forces are predominantly measured perpendicularly to surfaces, whereas other directions are often neglected. The angle of shear forces acting on bacteria or biofilms will not be at a 90° angle, as shear induced by flow is often along the surface. Measuring at different angles or even lateral to the surface will give a more complete overview of the adhesion forces and mechanism, perhaps even resulting in alternative means to discourage bacterial adhesion or promote removal.

Residence-time dependent cell wall deformation of different Staphylococcus aureus strains on gold measured using surface-enhanced-fluorescence.

Bacterial adhesion to surfaces is accompanied by cell wall deformation that may extend to the lipid membrane with an impact on the antimicrobial susceptibility of the organisms. Nanoscale cell wall deformation upon adhesion is difficult to measure, except for Δpbp4 mutants, deficient in peptidoglycan cross-linking. This work explores surface enhanced fluorescence to measure the cell wall deformation of Staphylococci adhering on gold surfaces. Adhesion-related fluorescence enhancement depends on the distance of the bacteria from the surface and the residence-time of the adhering bacteria. A model is forwarded based on the adhesion-related fluorescence enhancement of green-fluorescent microspheres, through which the distance to the surface and cell wall deformation of adhering bacteria can be calculated from their residence-time dependent adhesion-related fluorescence enhancement. The distances between adhering bacteria and a surface, including compression of their extracellular polymeric substance (EPS)-layer, decrease up to 60 min after adhesion, followed by cell wall deformation. Cell wall deformation is independent of the integrity of the EPS-layer and proceeds fastest for a Δpbp4 strain.